DISEASE PREVENTION AND VACCINATION FOLLOWING THYMIC REACTIVATION
FIELD OF THE INVENTION
The present disclosure is in the field of immunology. In particular this invention is in the field of thymus regeneration. The present invention is in the filed of stimulation and/or modification of a patient's immune system for the prevention or treatment of disease and/or for improved responsiveness to vaccine antigens, foreign antigens, or self antigens, with optional gene therapy utilizing hematopoietic stem cells (HSC), hematopoietic progenitor cells, epithelial stem cells or bone marrow.
BACKGROUND
THE IMMUNE SYSTEM
The major function of the immune system is to distinguish "foreign" (i.e., derived from any source outside the body) antigens from "self (i.e., derived from within the body) and respond accordingly to protect the body against infection. In more practical terms, the immune response has also been described as responding to danger signals. These danger signals may be any change in the property of a cell or tissue which alerts cells of the immune system that this cell/tissue in question is no longer "normal." Such alerts may be very important in causing, for example, rejection of foreign agents such as viral, bacterial, parasitic and fungal infections; they may also be used to induce anti-tumor responses. However, such danger signals may also be the reason why some autoimmune diseases start, due to either inappropriate cell changes in the self cells which are then become targeted by the immune system (e.g., the pancreatic β-islet cells in diabetes mellitus) Alternatively, inappropriate stimulation of the immune cells themselves, can lead to the destruction of normal self cells, in addition to the foreign cell or microorganism which induced the initial response.
In normal immune responses, the sequence of events involves dedicated antigen presenting cells (APC) capturing foreign antigen and processing it into small peptide fragments which are then presented in clefts of major histocompatibility complex (MHC)
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molecules on the APC surface. The MHC molecules can either be of class I expressed on all nucleated cells (recognized by cytotoxic T cells (Tc)) or of class II expressed primarily by cells of the immune system (recognized by helper T cells (Th)). Th cells recognize the MHC II/peptide complexes on APC and respond. Factors released by these cells then promote the activation of either of both Tc cells or the antibody producing B cells which are specific for the particular antigen. The importance of Th cells in virtually all immune responses is best illustrated in HIV/AIDS where their absence through destruction by the virus causes severe immune deficiency eventually leading to death due to opportunistic infections. Inappropriate development of Th (and to a lesser extent Tc) can lead to a variety of other diseases such as allergies, cancer and autoimmunity.
The inappropriate development of such cells may be due to an abnormal thymus in which the structural organization is markedly altered e.g., in many autoimmune diseases, the medullary epithelial cells, which are required for development of mature thymocytes, are ectopically expressed in the cortex where immature T cells normally reside. This could mean that the developing immature T cells prematurely receive late stage maturation signals and in doing so become insensitive to the negative selection signals that would normally delete potentially autoreactive cells. Indeed this type of thymic abnormality was found in NZB mice which develop Lupus-like symptoms (Takeoka et al., (1999) Clin. Immunol. 90:388) and more recently NOD mice which develop type I diabetes (Thomas- Vaslin et al., (1997) P.N.A.S. USA 94:4598; Atim-G&p r et al.,(l999) Autoimmunity 3:249-260). It is not known how or when these forms of thymic abnormality develop, but it could be through the natural aging process or from destructive agents such as viral infections (changes in the thymus have been described in AIDS patients), stress, chemotherapy and radiation therapy (Mackall et al., (1995) N. Eng. J. Med. 332: 143; Heitger et al, (1997) Blood 99:4053; Mackall and Gress, (1997) Immunol. Rev. 160:91). It is also possible that the defects are present at birth.
The ability to recognize antigen is encompassed in a plasma membrane receptor in T and B lymphocytes. These receptors are generated randomly by a complex series of rearrangements of many possible genes, such that each individual T or B cell has a unique antigen receptor. This enormous potential diversity means that for any single antigen the body might encounter, multiple lymphocytes will be able to recognize it with varying degrees of binding strength (affinity) and respond to varying degrees. Since the antigen receptor specificity arises by chance, the problem thus arises as to why the body does not "self
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destruct" through lymphocytes reacting against self antigens. Fortunately there are several mechanisms which prevent the T and B cells from doing so, and collectively they create a situation where the immune system is tolerant to self.
The most efficient form of self tolerance is to physically remove or kill any potentially reactive lymphocytes at the sites where they are produced. These sites include the thymus for T cells and the bone marrow for B cells. This is called central tolerance. An important, additional method of tolerance is through regulatory Th cells which inhibit autoreactive cells either directly or via the production of cytokines. Given that virtually all immune responses require initiation and regulation by T helper cells, a major aim of any tolerance induction regime would be to target these T helper cells. Similarly, since Tc's are very important effector cells, their production is a major aim of strategies for, e.g., anti- cancer and anti-viral therapy. In addition, T regulatory cells (Tregs), such as CD4+CD25+ and NKT cells, provide a means whereby they can suppress potentially autoreactive cells.
THE THYMUS
The thymus essentially consists of developing (T lymphocytes within the thymus) interspersed within the diverse stromal cells (predominantly epithelial cell subsets) which constitute the microenvironment and provide the growth factors (GF) and cellular interactions necessary for the optimal development of the T cells.
The thymus is an important organ in the immune system because it is the primary site of production of T lymphocytes. Its role is to attract appropriate bone marrow-derived precursor cells from the blood, and induce their commitment to the T cell lineage including the gene rearrangements necessary for the production of the T cell receptor for antigen (TCR). Each T cell has a single TCR type and is unique in its specificity. Associated with this TCR production is cell division, which expands the number of T cells with that TCR type and hence increases the likelihood that every foreign antigen will be recognized and eliminated. However, a unique feature of T cell recognition of antigen is that, unlike B cells, the TCR only recognizes peptide fragments physically associated with MHC molecules. Normally this is self MHC, and the ability of a TCR to recognize the self MHC/peptide complex is selected for in the thymus. This process is called positive selection and is an exclusive feature of cortical epithelial cells. If the TCR fails to bind to the self MHC/peptide complexes, the T cell dies by "neglect" because the T cells needs some degree of signalling through the TCR for its continued survival and maturation.
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Since the outcome of the TCR gene rearrangements is a random event, some T cells will develop which, by chance, can recognize self MHC/peptide complexes with high affinity. Such T cells are thus potentially self-reactive and could be involved in autoimmune diseases, such as multiple sclerosis (MS), rheumatoid arthritis (RA), diabetes, thyroiditis and systemic lupus erythematosus (SLE). Fortunately, if the affinity of the TCR to self
MHC/peptide complexes is too high, and the T cell encounters this specific complex in the thymus, the developing thymocyte is induced to undergo a suicidal activation and dies by apoptosis, a process called negative selection. This process is also called central tolerance. Such "high affinity for self T cells die rather than respond because in the thymus they are still immature. The most potent inducers of this negative selection in the thymus are APC called dendritic cells (DC). DC deliver the strongest signal to the T cells, which causes deletion in the thymus. However, in the peripheral lymphoid organs where the T cells are more mature, the DC presenting the same MHC/peptide complex to the same TCR would cause activation of that T cell bearing the TCR.
THYMUS ATROPHY AND AGE
While the thymus is fundamental for a functional immune system, releasing about 1% of its T cell content into the bloodstream per day, one of the apparent anomalies of mammals and other animals is that this organ undergoes severe atrophy as a result of sex steroid production. This atrophy occurs gradually over a period of about 5-7 years, with the nadir level of T cell output being reached around 20 years of age (Douek et al., Nature (1998)
396:690-695) and is in contrast to the reversible atrophy induced during a stress response to corticosteroids. Structurally, the thymic atrophy involves a progressive loss of lymphocyte content, a collapse of the cortical epithelial network, an increase in extracellular matrix material, and an infiltration of the gland with fat cells (adipocytes) and lipid deposits (Haynes et al., (1999) J. Clin. Invest. 103: 453). This process may even begin in young children (e.g., around five years of age; Mackall et al., (1995) N. Eng. J. Med. 332:143), but it is profound from the time of puberty when sex steroid levels reach a maximum. For normal healthy individuals this loss of production and release of new T cells does not always have clinical consequences, although immune-based disorders such as general immunodeficiency and poor responsiveness to vaccines and an increase in the frequency of autoimmune diseases such as multiple sclerosis, rheumatoid arthritis and lupus (Doria et al, (1997) Mech. Age. Dev. 95: 131-142; Weyand etα/., (\99 ) Mech. Age. Dev. 102: 131-147; Castle, (2000) Clin Infect Dis 31(2): 578-585; Murasko et al., (2002) Exp. Gerontol. 37:427-439) increase in incidence and
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severity with age. When there is a major loss of T cells, e.g., in AIDS and following chemotherapy or radiotherapy, the patients are highly susceptible to disease because all these conditions involve a loss of T cells (especially Th in HIV infections) or all blood cells including T cells in the case of chemotherapy and radiotherapy. As a consequence these patients lack the cells needed to respond to infections and they become severely immune suppressed (Mackall et al., (1995) N. Eng. J. Med. 332:143; Heitger et al, (2002) Blood 99:4053).
Since the thymus is the primary site for the production and maintenance of the peripheral T cell pool, this atrophy has been widely postulated as being the primary cause of the increased incidence of immune-based disorders in the elderly. In particular, conditions, such as general immunodeficiency, poor responsiveness to opportunistic infections and vaccines, and an increase in the frequency of autoimmune diseases, such as multiple sclerosis, rheumatoid arthritis, and lupus (Doria et al, (1997) Mech. Age. Dev. 95: 131-142; Weyand et al., (1998) Mech. Age. Dev. 102: 131-147; Castle, (2000) Clin Infect Dis 31(2): 578-585; Murasko et al., (2002) Exp. Gerontol. 37:427-439), increase in incidence and severity with age. Such deficiencies of the immune system, are often illustrated by a decrease in T cell dependent immune functions (e.g., cytolytic T cell activity and mitogenic responses). While homeostatic mechanisms maintain T cell numbers in healthy individuals, when there is a major loss of T cells, e.g., in AIDS, and following chemotherapy or radiotherapy, adult patients are highly susceptible to opportunistic infections because all these conditions involve a loss of T cells and/or other blood cells (see below). Lymphocyte recovery is also severely retarded. The atrophic thymus is unable to reconstitute CD4+ T cells that are lost during HIV infection (Douek et al. Nature (1998) 396:690-695) and CD4+ T cells take three to four times longer to return to normal levels following chemotherapy in post-pubertal patients as compared to pre-pubertal patients (Mackall et al. (1995) N Engl. J. Med. 332:143-149). As a consequence these patients lack the cells needed to respond to infections, and they become severely immune suppressed (Mackall et al, (1995) N. Eng. J. Med. 332:143; Heitger et al., (2002) Blood 99:4053). There is also an increase in cancers and tumor load in later life (Hirokawa, (1998) "Immunity and Ageing," in PRINCIPLES AND PRACTICE OF GERIATRIC MEDICINE, (M. Pathy, ed.) John Wiley and Sons Ltd; Doria et al, (1997) Mech. Age. Dev. 95: 131; Castle, (2000) Clin. Infect. Dis. 31:578).
The impact of thymus atrophy is reflected in the periphery, with reduced thymic input to the T cell pool, which results in a less diverse TCR repertoire (as this can only be provided
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by the new naive T cells). Altered cytokine profiles (Hobbs et al., (1993) J. Immunol. 150:3602; Kurashima et al., (1995) Int. Immunol. 7:97), changes in CD4+ and CD8+ subsets, biases towards memory as opposed to naive T cells (Mackall et al., (1995) N. Engl. J. Med. 332: 143), and a reduced ability to response to antigenic or mitogenic stimulation are also observed. Furthermore, the efficiency of thymopoiesis is impaired with age such that the ability of the immune system to regenerate normal T cell numbers after T cell depletion is eventually lost (Mackall et al. (1995) N. Engl. J. Med. 332:143-149).
However, recent work by Douek et al., ((1998) Nature 396:690) has shown thymic output occurs even if only very slight (about 5% of the young levels), in older humans (e.g., even sixty-five years old and above, and after anti-retroviral treatment in older HIV patients). This was exemplified by the presence of T cells with T Cell Receptor Excision Circles (TRECs); TRECs are formed as part of the generation of the TCR for antigen and are only found in newly produced T cells). Furthermore Timm and Thoman ((1999) J. Immunol. 162:711) have shown that although CD4+ T cells are regenerated in old mice post-bone marrow transplant (BMT), they appear to show a bias towards memory cells due to the aged peripheral microenvironment coupled to poor thymic production of naive T cells.TREC levels has also been analysed following hematopoietic stem cell transplantation (Douek et al, (2000) Lancet 355:1875).
THYMUS AND THE NEUROENDOCRLNE AXIS
The thymus is influenced to a great extent by its bidirectional communication with the neuroendocrine system (Kendall, (1988) "Anatomical and physiological factors influencing the thymic microenvironment," in THYMUS UPDATE I, Vol. 1. (M. D. Kendall, and M. A. Ritter, eds.) Harwood Academic Publishers, p. 27). Of particular importance is the inteφlay between the pituitary, adrenals, and gonads on thymic function, including both trophic (thyroid stimulating hormone or TSH, and growth hormone or GH) and atrophic effects (luteinizing hormone or LH, follicle stimulating hormone or FSH, and adrenocorticotropic hormone or ACTH) (Kendall, (1988) "Anatomical and physiological factors influencing the thymic microenvironment," in THYMUS UPDATE I, Vol. 1. (M. D. Kendall, and M. A. Ritter, eds.) Harwood Academic Publishers, p. 27; Homo-Delarche et al., (1993) Springer Sem.
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Immunopathol. 14:221). Indeed, one of the characteristic features of thymic physiology is the progressive decline in structure and function, which is commensurate with the increase in circulating sex steroid production around puberty, which in humans generally occurs from the age of 12-14 onwards (Hirokawa and Makinodan, (1975) /. Immunol. 114:1659; Tosi et al, (1982) Clin. Exp. Immunol. 47:497; and Hirokawa, et al, (1994) Immunol. Lett. 40:269).
The thymus essentially consists of developing thymocytes interspersed within the diverse stromal cells (predominantly epithelial cell subsets) which constitute the microenvironment and provide the growth factors and cellular interactions necessary for the optimal development of the T cells. The precise target of the hormones, as well as the mechanism by which they induce thymus atrophy and improved immune responses, has yet to be determined. Examination of testicular feminised mutant mice, however, indicates that functional sex steroid receptors must be expressed on the stromal cells of the thymus for atrophy to occur. The symbiotic developmental relationship between thymocytes and the epithelial subsets that controls their differentiation and maturation (Boyd et al, (1993) Immunol. Today 14:445) means that sex-steroid inhibition could occur at the level of either cell type, which would then influence the status of the other cell type. Bone marrow stem cells are reduced in number and are qualitatively different in aged patients. HSC are able to repopulate the thymus, although to a lesser degree than in the young. Thus, the major factor influencing thymic atrophy is appears to be intrathymic. Furthermore, thymocytes in older aged animals (e.g., those >18 months) retain their ability to differentiate to at least some degree (George and Ritter, (1996) Immunol. Today 17:267; Hirokawa et al, (1994) Immunology Letters 40:269; Mackall et al, (1998) Ear. J. Immunol. 28: 1886). However, recent work by Aspinall has shown that in aged mice there is a defect in thymocyte production, which is manifested as a block within the precursor triple negative population, namely the CD44+CD25+ (TN2) stage. (Aspinall et al, (1997) J. Immunol. 158:3037).
In the particular case for AIDS, the primary defect in the immune system is the destruction of CD4+ cells and to a lesser extent the cells of the myeloid lineages of macrophages and dendritic cells (DC). Without these the immune system is paralysed and the patient is extremely susceptible to opportunistic infection with death a common consequence. The present treatment for AIDS is based on a multitude of anti-viral drugs to kill or deplete the HIV virus. Such therapies are now becoming more effective with viral loads being reduced dramatically to the point where the patient can be deemed as being in remission. The major problem of immune deficiency still exists, however, because there are
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still very few functional T cells, and those which do recover, do so very slowly. The period of immune deficiency is thus still a very long time and in some cases immune defense mechanisms may never recover sufficiently. The reason for this is that in post-pubertal people the thymus is atrophied.
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SUMMARY OF THE INVENTION
The present inventors have demonstrated that thymic atrophy (age induced, or as a consequence of conditions such as chemotherapy or radiotherapy) can be profoundly reversed by inhibition of sex steroid production, with virtually complete restoration of thymic structure and function. The present inventors have also found that the basis for this thymus regeneration is in part due to the initial expansion of precursor cells which are derived both intrathymically and via the blood stream.
These findings have led to the discovery that is possible to seed the thymus with exogenous hematopoietic stem cells (HSC) which have been injected into the subject. The ability to seed the thymus with genetically modified or exogenous HSC by disrupting sex steroid signaling, means that gene therapy in the HSC may be used more efficiently to treat T cell (and myeloid cells which develop in the thymus) disorders. HSC stem cell therapy has met with little or no success to date because the thymus is dormant and incapable of taking up many if any HSC, with T cell production less than 1% of normal levels.
The present disclosure concerns methods of gene therapy utilizing genetically modified HSC, lymphoid or myeloid progenitor cells, epithelial stem cells, or combinations thereof (the group and each member herein referred to as "GM cells"), delivered to a reactivating thymus to create particular immunities. In this context, a reactivating thymus is one in which the patient has been depleted of sex steroids via castration, GnRH, LHRH, or other sex steroid analogs. Thus, in one aspect, a method of gene therapy is provided, the method comprising disrupting sex steroid mediated signaling in the patient. In one embodiment, the atrophic thymus in an aged (post-pubertal) patient is reactivated and the functional status of the peripheral T cells is improved.
The present disclosure also provides methods for preventing, diminishing the risk, or treating a disease or illness in a patient by disrupting sex steroid mediated signaling and causing the patient's thymus to reactivate. In one embodiment, the disease is a T cell disorder. In another embodiment, the disease is an autoimmune disease or allergy. Additionally, the present disclosure also provides methods for improving a patient's immune response to a vaccine antigen (e.g., that of an agent) by disrupting sex steroid mediated signaling and causing the thymus to reactivate. In both cases, the functional status of the peripheral T cells may be improved and may be accomplished by quantitatively and
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qualitatively restoring the peripheral T cell pool, particularly at the level of naive T cells. These naive T cells are then able to respond to a greater degree to presented foreign antigen.
The thymus begins to increase the rate of proliferation of the early precursor cells (CD3 CD4 CD8" cells) and to convert them into CD4+CD8+, and subsequently new mature CD3hiCD4+CD8" (T helper (Th) lymphocytes) or CD3hiCD4"CD8+ (T cytotoxic lymphocytes (CTL)). The rejuvenated thymus also increases its uptake of hematopoietic stem cells (HSC) HSC, or other stem cells or progenitor cells capable of forming into T cells, from the blood stream and converts them into new T cells and intrathymic dendritic cells. The increased activity in the thymus resembles that found in a normal younger thymus (prior to the atrophy at about 20 years of age) caused increased levels of sex steroids. The result of this renewed thymic output is increased levels of naive T cells (those T cells which have not yet encountered antigen) in the blood. There is also an increase in the ability of the blood T cells to respond to stimulation, e.g., by using anti-CD28 Abs, cross-linking the TCR with, e.g., by cross-linking with anti-CD28 Abs, or by TCR stimulation with, e.g., anti-CD3 antibodies, or stimulation with mitogens, such as pokeweed mitogen (PWM). This combination of events results in the body becoming better able to defend against infection and other immune system challenges (e.g., cancers), becoming better able to respond to vaccine antigens, or becoming better able to recover from chemotherapy and radiotherapy.
As herein defined, "prevention" of or "preventing" a disease refers to complete as well as partial protection including, without limitation, reduced severity of clinical symptoms than would have otherwise occurred in a patient from disease or illness caused by an infectious agent, cancer, etc. With an improved immune system the individual will have a reduced likelihood of succumbing to a tumor or cancer, a prevailing infection (e.g., viral, bacterial, fungal, or parasitic), and will show better responses to a vaccination (e.g., increased levels of antibody (Ab) specific to that vaccine or antigen, and development of effector T cells). For example, the methods of this invention would be applicable to prevention of viral infections, such as In some embodiments, the methods of the invention are used to prevent or treat viral infections, such as HIV, heφes, influenza, and hepatitis. In other embodiments, the methods of the invention are used to prevent or treat bacterial infections, such as pneumonia and tuberculosis (TB). In yet other embodiments, the methods of the invention are used to prevent or treat fungal infections, parasitic infections, allergies, and/or tumors and other cancers, whether malignant or benign., and prevention of bacterial infections, such as pneumonia and tuberculosis (TB).
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In certain embodiments, castration, GnRH analogs, LHRH analogs, or other sex steroid analogs (including agonists and antagonists thereto) are used to disrupt sex steroid- mediated signaling. In another embodiment, GnRH analogs, LHRH analogs, or other sex steroid analogs directly stimulate (i.e., directly increase the functional activity of) the thymus, bone marrow, and pre-existing cells of the immune system, such as T cells, B cells, and dendritic cells (DC).
In one embodiment the atrophic thymus in an aged (post-pubertal) patient is reactivated. The reactivating thymus becomes capable of taking up HSC, BM cells from the blood, and other appropriate progenitors, and converting them in the thymus to both new T cells and DC.
In certain embodiments, the patient receives genetically modified HSC (or other GM cell) transplantation. In another embodiment, the patient receives non-genetically modified HSC transplantation.
In some embodiments, bone marrow or HSC are transplanted into the patient to provide a reservoir of precursor cells for the renewed thymic growth. These HSC have the capability of turning into DC, which may have the effect of providing better antigen presentation to the T cells and therefore a better immune response (e.g., increased antibody (Ab) production and effector T cells number and/or function).
In certain embodiments, inhibition of sex steroid production is achieved by either castration or administration of a sex steroid analogue(s). Non-limiting sex steroid analogs include eulexin, goserelin, leuprolide, dioxalan derivatives such as triptorelin, meterelin, buserelin, histrelin, nafarelin, lutrelin, leuprorelin, and luteinizing hormone-releasing hormone analogues. In some embodiments, the sex steroid analog is an analog of luteinizing hormone-releasing hormone. In certain embodiments, the luteinizing hormone-releasing hormone analog is deslorelin.
In another aspect, the present disclosure provides for the reactivation of the thymus by disrupting sex steroid mediated signaling. In one embodiment, castration is used to disrupt the sex steroid mediated signaling. In one embodiment chemical castration is used. In another embodiment surgical castration is used. Castration reverses the state of the thymus towards its pre-pubertal state, thereby reactivating it. Both of these processes result in a loss
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of sex steroids, but may also induce increases in other molecules which increase immune responsiveness.
In certain embodiments, sex steroid mediated signaling may be directly or indirectly blocked (e.g., inhibited, inactivated or made ineffectual) by the administration of modifiers of sex hormone production, action, binding or signaling, including but not limited to agents which bind a sex hormone or its receptor, agonists or antagonists of sex hormones, including, but not limited to, GnRH/LHRH, anti-estrogenic and anti-androgenic agents, SERMs, SARMs, anti-estrogen antibodies, anti-androgen ligands, anti-estrogen ligands, LHRH ligands, passive (antibody) or active (antigen) anti-LHRH (or other sex steroid) vaccinations, or combinations thereof ("blockers"). In one embodiment, one or more blocker is used. In some embodiments, the one or more blocker is administered by a sustained peptide-release formulation. Examples of sustained peptide-release formulations are provided in WO 98/08533, the entire contents of which are incoφorated herein by reference.
In another aspect, the present disclosure provides methods for preventing, diminishing the risk of, or treating infection or other disease caused by an agent. The GnRH induces both thymic regrowth and the production of new T cells, as well as increases the activity of the T cells to immune stimulation. For instance, transplantation of GM cells that have been genetically modified to resist or prevent infection, activity, replication, and the like, and combinations thereof, of the infectious agent are injected into a patient concurrently or after with thymic reactivation.
In one embodiment, the present disclosure provides methods for preventing or treating infection by an infectious agent such as HIV. In a certain embodiment, HSC are genetically modified to create resistance to HIV in the T cells formed during and after thymic reactivation. For example, the HSC are modified to include a gene whose product will interfere with HIV infection, function and or replication in the T cells (and/or other HSC- derived cells) of the patient. GM that have been genetically modified to resist or prevent infection, activity, replication, and the like, and combinations thereof, of the infectious agent are injected into a patient concurrently with thymic reactivation. In another embodiment, HSC are genetically modified to create resistance (complete or partial) to HIV in the T cells formed during and after thymic reactivation. For example, the HSC are modified to include a gene whose product will interfere with HIV infection, function and/or replication in the T cell. In one embodiment, HSC are genetically modified with the RevMlO gene (see, e.g.,
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Bonyhadi et al, (1997) J. Virol. 71:4707) or the CXCR4 or PolyTAR genes (Strayer et al, (2002) Mol. Ther. 5:33). This confers a degree of resistance to the virus, thereby preventing or treating disease caused by the virus.
In yet another embodiment, genetically modified HSC are transplanted into the patient, in an embodiment just before, at the time of, or after reactivation of the thymus, thereby creating a new population of genetically modified T cells.
In another embodiment, the method comprises transplanting enriched HSC into the subject. The HSC may be autologous or heterologous. The HSC may be genetically modified or may not be genetically modified.
In cases where the subject is infected with HIV, the HSC may be genetically modified such that they and their progeny, in particular T cells, macrophages and dendritic cells, are resistant to infection and / or destruction with the HIV virus. The genetic modification may involve introduction into HSC one or more nucleic acid molecules which prevent viral replication, assembly and/or infection. The nucleic acid molecule may be a gene which encodes an antiviral protein, an antisense construct, a ribozyme, a dsRNA and a catalytic nucleic acid molecule.
In some embodiments, the patient has AIDS and has had (or is having) the viral load reduced by anti-viral treatment.
The method of the present invention is particularly useful for the treatment of AIDS, where the treatment preferably involves reduction of viral load, reactivation of thymic function through inhibition of sex steroids and transfer into the patients of HSC (autologous or from a second party donor) which have been genetically modified such that all progeny (especially T cells, DC) are resistant to further HIV infection. This means that not only will the patient be depleted of HIV virus and no longer susceptible to general infections because the T cells have returned to normal levels, but the new T cells being resistant to HTV will be able to remove any remnant viral infected cells. In principle a similar strategy could be applied to gene therapy in HSC for any T cell defect or any viral infection which targets T cells.
In another embodiment, the disease is a T cell disorder selected from the group consisting of viral infections (such as human immunodeficiency virus (HIV)), T cell functional disorders, and any other disease or condition that reduces T cells numerically or
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functionally, either directly or indirectly, or causes T cells to function in a manner which is harmful to the individual.
In cases where the subject has defective T cells, the HSC may be genetically modified to normalize the defect. For diseases such as T cell leukaemias, the modification may include the introduction of nucleic acid constructs or genes which normalize the HSC and inhibit or reduce its likelihood of becoming a cancer cell.
In one embodiment, the disease is one that has a defined genetic basis, such as that caused by a genetic defect. These genetic diseases are well known to those in the art, and include autoimmune diseases, diseases resulting from the over- or under-production of certain proteins, tumors and cancers, etc. The disease-causing genetic defect is repaired by insertion of the normal gene into the HSC, and, using the methods of the invention, every cell produced from this HSC will then carry the gene correction. One method involves reactivating thymic function through inhibition of sex steroids to increase the uptake of blood-borne hematopoietic stem cells (HSC). In general, after the onset of puberty, the thymus undergoes severe atrophy under the influence of sex steroids, with its cellular production reduced to less than 1% of the pre-pubertal thymus. The present invention is based on the finding that the inhibition of production of sex steroids releases the thymic inhibition and allows a full regeneration of its function, including increased uptake of blood- derived HSC. The origin of the HSC can be directly from injection or from the bone marrow following prior injection. It is envisaged that blood cells derived from modified HSC will pass the genetic modification onto their progeny cells, including HSC derived from self- renewal, and that the development of these HSC along the T cell and dendritic cell lineages in the thymus is greatly enhanced if not fully facilitated by reactivating thymic function through inhibition of sex steroids.
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DESCRIPTION OF THE FIGURES
Figure 1 A, IB, and IC: Castration rapidly regenerates thymus cellularity. Figure 1A-1C are graphic representations showing that the changes in thymus weight and thymocyte number pre- and post-castration. Thymus atrophy results in a significant decrease in thymocyte numbers with age, as measured by thymus weight (Fig. IA) or by the number of cells per thymus (Figs. IB and IC). For these studies, aged (i.e., 2-year old) male mice were surgically castrated. Thymus weight in relation to body weight (Fig. 1 A) and thymus cellularity (Figs. IB and IC) were analyzed in aged (1 and 2 years) and at 2-4 weeks post- castration (post-cx) male mice. A significant decrease in thymus weight and cellularity was seen with age compared to young adult (2-month) mice. This decrease in thymus weight and cell number was restored by castration, although the decrease in cell number was still evident at 1 week post-castration (see Fig. IC). By 2 weeks post-castration, cell numbers were found to increase to approximately those levels seen in young adults (Figs. IB and IC). By 3 weeks post-castration, numbers have significantly increased from the young adult and these were stabilized by 4 weeks post-castration (Figs. IB and IC). Results are expressed as mean ±ISD of 4-8 mice per group (Figs. IA and IB) or 8-12 mice per group (Fig. IC). **= p<0.01; *** = p≤O.OOl compared to young adult (2 month) thymus and thymus of 2-6 wks post-castrate mice.
Figure 2 A-F: Castration restores the CD4:CD8 T cell ratio in the periphery. For these studies, aged (2-year old) mice were surgically castrated and analyzed at 2-6 weeks post-castration for peripheral lymphocyte populations. Figs. 2A and 2B show the total lymphocyte numbers in the spleen. Spleen numbers remain constant with age and post- castration because homeostasis maintains total cell numbers within the spleen (Figs. 2A and 2B). However, cell numbers in the lymph nodes in aged (18-24 months) mice were depleted (Fig. 2B). This decrease in lymph node cellularity was restored by castration (Fig. 2B). Figs. 2C and 2D show that the ratio of B cells to T cells did not change with age or post-castration in either the spleen or lymph node, as no change in this ratio was seen with age or post- castration. However, a significant decrease (p<0.001) in the CD4+:CD8+ T cell ratio was seen with age in both the (pooled) lymph node and the spleen (Figs. 2E and 2F). This decrease was restored to young adult (i.e., 2 month) levels by 4-6 weeks post-castration (Figs. 2E and 2F).
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Results are expressed as mean±lSD of 4-8 (Figs. 2A, 2C, and 2E) or 8-10 (Figs. 2B, 2D, and 2F) mice per group. * = p<0.05; ** = p≤O.Ol; *** = p≤O.OOl compared to young adult (2- month) and post-castrate mice.
Figure 3: Thymocyte subpopulations are retained in similar proportions despite thymus atrophy or regeneration by castration. For these studies, aged (2-year old) mice were castrated and the thymocyte subsets analyzed based on the markers CD4 and CD8. Representative Fluorescence Activated Cell Sorter (FACS) profiles of CD4 (X-axis) vs. CD8 (Y-axis) for CD4-CD8-DN, CD4+CD8+DP, CD4+CD8- and CD4-CD8+ SP thymocyte populations are shown for young adult (2 months), aged (2 years) and aged, post-castrate animals (2 years, 4 weeks post-cx). Percentages for each quadrant are given above each plot. No difference was seen in the proportions of any CD4/CD8 defined subset with age or post- castration. Thus, subpopulations of thymocytes remain constant with age and there was a synchronous expansion of thymocytes following castration.
Figure 4: Regeneration of thymocyte proliferation by castration. Mice were injected with a pulse of BrdU and analyzed for proliferating (BrdU+) thymocytes. Figs. 4A and 4B show representative histograms of the total % BrdU+ thymocytes with age and post-cx. Fig. 4C shows the percentage (left graph) and number (right graph) of proliferating cells at the indicated age and treatment (e.g., week post-cx). For these studies, aged (2-year old) mice were castrated and injected with a pulse of bromodeoxyuridine (BrdU) to determine levels of proliferation. Representative histogram profiles of the proportion of BrdU-i- cells within the thymus with age and post-castration are shown (Figs. 4A and 4B). No difference was observed in the total proportion of proliferation within the thymus, as this proportion remains constant with age and following castration (Figs. 4A, 4B, and left graph in Fig. 4C).
However, a significant decrease in number of BrdU+ cells was seen with age (Fig. 4C, right graph). By 2 weeks post-castration, the number of BrdU+ cells increased to a number that similar to seen in young adults (i.e., 2 month) (Fig. 4C, right graph). Results are expressed as mean±lSD of 4-14 mice per group. ***=p<0.001 compared to young adult (2-month) control mice and 2-6 weeks post-castration mice.
Figures 5A-K: Castration enhances proliferation within all thymocyte subsets. For these studies, aged (2-year old) mice were castrated and injected with a pulse of bromodeoxyuridine (BrdU) to determine levels of proliferation. Analysis of proliferation within the different subsets of thymocytes based on CD4 and CD8 expression within the
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thymus was performed. Fig. 5A shows that the proportion of each thymocyte subset within the BrdU+ population did not change with age or post-castration. However, as shown in Fig. 5B, a significant decrease in the proportion of DN (CD4-CD8-) thymocytes proliferating was seen with age. A decrease in the proportion of TN (i.e., CD3 CD4 CD8") thymocytes was also seen with age (data not shown). Post-castration, this was restored and a significant increase in proliferation within the CD4-CD8+ SP thymocytes was observed. Looking at each particular subset of T cells, a significant decrease in the proportion of proliferating cells within the CD4-CD8- and CD4-CD8+ subsets was seen with age (Figs. 5C and 5E). At 1 and 2 weeks post-castration, the percentage of BrdU-i- cells within the CD4-CD8+ population was significantly increased above the young control group (Fig. 5E). Fig. 5F shows that no change in the total proportion of BrdU+ cells (i.e., proliferating cells) within the TN subset was seen with age or post-castration. However, a significant decrease in proliferation within the TNI (CD44+CD25-CD3-CD4-CD8-) subset (Fig. 5H) and significant increase in proliferation within TN2 (CD44+CD25+CD3-CD4-CD8-) subset (Fig. 51) was seen with age. This was restored post-castration (Figs. 5G, 5H, and 51). Results are expressed as mean±lSD of 4-17 mice per group. *=p<0.05; ** =p≤0.01 (significant) ; *** = p≤O.OOl (highly significant) compared to young adult (2-month) mice; Λ = significantly different from 1-6 weeks post-castrate mice (Figs. 5C-5E) and 2-6 weeks post-castrate mice (Figs. 5H-5K).
Figures 6A-6C: Castration increases T cell export from the aged thymus. For these studies, aged (2-year old) mice were castrated and were injected intrathymically with FITC to determine thymic export rates. The number of FITC+ cells in the periphery was calculated 24 hours later. As shown in Fig. 6A, a significant decrease in recent thymic emigrant (RTE) cell numbers detected in the periphery over a 24 hours period was observed with age. Following castration, these values had significantly increased by 2 weeks post-cx. As shown in Fig. 6B, the rate of emigration (export/total thymus cellularity) remained constant with age, but was significantly reduced at 2 weeks post-castration. With age, a significant increase in the ratio of CD4+ to CD8+ RTE was seen; this was normalized by 1- week post-cx (Fig. 6C).
Results are expressed as mean±lSD of 4-8 mice per group. * = p<0.05; ** = p≤O.Ol; *** = p≤O.001 compared to young adult (2-month) mice for (Fig. 6A) and compared to all other groups (Figs. 6B and 6C). Λ = p<0.05 compared to aged (1- and 2-year old) non-cx mice and compared to 1-week post-cx, aged mice.
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Figures 7A and 7B: Castration enhances thymocyte regeneration following T cell depletion. 3-month old mice were either treated with cyclophosphamide (intraperitoneal injection with 200mg/kg body weight cyclophosphamide, twice over 2 days) (Fig. 7A) or exposed to sublethal irradiation (625 Rads) (Fig. 7B). For both models of T cell depletion studied, castrated (Cx) mice showed a significant increase in the rate of thymus regeneration compared to their sham-castrated (ShCx) counteφarts. Analysis of total thymocyte numbers at 1 and 2-weeks post-T cell depletion (TCD) showed that castration significantly increases thymus regeneration rates after treatment with either cyclophosphamide or sublethal irradiation (Figs. 7A and 7B, respectively). Data is presented as mean±lSD of 4-8 mice per group. For Fig. 7A, *** = p≤O.OOl compared to control (age-matched, untreated) mice; Λ = p≤O.OOl compared to both groups of castrated mice. For Fig. 7B, *** = p≤O.OOl compared to control mice; Λ = p≤O.OOl compared to mice castrated 1-week prior to treatment at 1-week post-irradiation and compared to both groups of castrated mice at 2-weeks post-irradiation.
Figures 8A-8C: Changes in thymus (Fig. 8A), spleen (Fig. 8B) and lymph node (Fig. 8C) cell numbers following treatment with cyclophosphamide and castration. For these studies, (3 month old) mice were depleted of lymphocytes using cyclophosphamide (intraperitoneal injection with 200mg/kg body weight cyclophosphamide, twice over 2 days) and either surgically castrated or sham-castrated on the same day as the last cyclophosphamide injection. Thymus, spleen and lymph nodes (pooled) were isolated and total cellularity evaluated. As shown in Fig. 8A, significant increase in thymus cell number was observed in castrated mice compared to sham-castrated mice. Note the rapid expansion of the thymus in castrated animals when compared to the non-castrate (cyclophosphamide alone) group at 1 and 2 weeks post-treatment. Fig. 8B shows that castrated mice also showed a significant increase in spleen cell number at 1-week post-cyclophosphamide treatment. A significant increase in lymph node cellularity was also observed with castrated mice at 1- week post-treatment (Fig. 8C). Thus, spleen and lymph node numbers of the castrate group were well increased compared to the cyclophosphamide alone group at one week post- treatment. By 4 weeks, cell numbers are normalized. Results are expressed as mean±lSD of 3-8 mice per treatment group and time point. *** = p<0.001 compared to castrated mice.
Figures 9A-B: Total lymphocyte numbers within the spleen and lymph nodes post- cyclophosphamide treatment. Sham-castrated mice had significantly lower cell numbers in the spleen at 1 and 4-weeks post-treatment compared to control (age-matched, untreated) mice (Fig. 9A). A significant decrease in cell number was observed within the lymph nodes
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at 1 week post-treatment for both treatment groups (Fig. 9B). At 2-weeks post-treatment, Cx mice had significantly higher lymph node cell numbers compared to ShCx mice (Fig. 9B). Each bar represents the mean±lSD of 7-17 mice per group. * = p≤0.05; ** = p≤O.Ol compared to control (age-matched, untreated). Λ=p≤0.05 compared to castrate mice.
Figure 10: Changes in thymus (open bars), spleen (gray bars) and lymph node (black bars) cell numbers following treatment with cyclophosphamide, a chemotherapy agent, and surgical or chemical castration performed on the same day. Note the rapid expansion of the thymus in castrated animals when compared to the non-castrate (cyclophosphamide alone) group at 1 and 2 weeks post-treatment. In addition, spleen and lymph node numbers of the castrate group were well increased compared to the cyclophosphamide alone group, (n = 3-4 per treatment group and time point). Chemical castration is comparable to surgical castration in regeneration of the immune system post-cyclophosphamide treatment.
Figures 11A-C: Changes in thymus (Fig. 11 A), spleen (Fig. 1 IB) and lymph node (Fig. 11C) cell numbers following irradiation (625 Rads) one week after surgical castration. For these studies, young (3-month old) mice were depleted of lymphocytes using sublethal (625 Rads) irradiation. Mice were either sham-castrated or castrated 1-week prior to irradiation. A significant increase in thymus regeneration (i.e., faster rate of thymus regeneration) was observed with castration (Fig. 11 A). Note the rapid expansion of the thymus in castrated animals when compared to the non-castrate (irradiation alone) group at 1 and 2 weeks post-treatment, (n = 3-4 per treatment group and time point). No difference in spleen (Fig. 11B) or lymph node (Fig. 11C) cell numbers was seen with castrated mice. Lymph node cell numbers were still chronically low at 2-weeks post-treatment compared to control mice (Fig. 1 IC). Results are expressed as mean±lSD of 4-8 mice per group. * = p≤0.05; ** = p≤O.01 compared to control mice; *** = p≤O.001 compared to control and castrated mice.
Figures 12A-C: Changes in thymus (Fig. 12A), spleen (Fig. 12B) and lymph node (Fig. 12C) cell numbers following irradiation and castration on the same day. For these studies, young (3-month old) mice were depleted of lymphocytes using sublethal (625 Rads) irradiation. Mice were either sham-castrated or castrated on the same day as irradiation. Castrated mice showed a significantly faster rate of thymus regeneration compared to sham- castrated counteφarts (Fig. 12A). Note the rapid expansion of the thymus in castrated animals when compared to the non-castrate group at 2 weeks post-treatment. No difference
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in spleen (Fig. 12B) or lymph node (Fig. 12C) cell numbers was seen with castrated mice. Lymph node cell numbers were still chronically low at 2-weeks post-treatment compared to control mice (Fig. 12C). Results are expressed as mean±lSD of 4-8 mice per group. * = p≤0.05; ** = p≤O.01 compared to control mice; *** = p≤O.OOl compared to control and castrated mice.
Figure 13A-13B: Total lymphocyte numbers within the spleen and lymph nodes post- irradiation treatment. 3-month old mice were either castrated or sham-castrated 1-week prior to sublethal irradiation (625Rads). Severe lymphopenia was evident in both the spleen (Fig. 13 A) and (pooled) lymph nodes (Fig. 13B) at 1-week post-treatment. Splenic lymphocyte numbers were returned to control levels by 2-weeks post-treatment (Fig. 13 A), while lymph node cellularity was still significantly reduced compared to control (age-matched, untreated) mice (Fig. 13B). No differences were observed between the treatment groups. Each bar represents the mean±lSD of 6-8 mice per group. ** = p≤O.Ol; *** = p≤O.OOl compared to control mice.
Figures 14A and 14B: Figure 14A shows the lymph node cellularity following footpad immunization with Heφes Simplex Virus- 1 (HSV-1). Note the increased cellularity in the aged post-castration as compared to the aged non-castrated group. Figure 14B illustrates the overall activated cell number as gated on CD25 vs. CD8 cells by FACS (i.e., the activated cells are gated on CD8+CD25+ cells).
Figures 15A-15C: VD 10 expression (HSV-specific) on CTL (cytotoxic T lymphocytes) in activated LN (lymph nodes) following HSV-1 inoculation. Despite the normal VβlO responsiveness in aged (i.e., 18 months) mice overall, in some mice a complete loss of VβlO expression was observed. Representative histogram profiles are shown. Note the diminution of a clonal response in aged mice and the reinstatement of the expected response post-castration.
Figure 16: Castration restores responsiveness to HSV-1 immunization. Mice were immunized in the hind foot-hock with 4xl05 pfu of HSV. On Day 5 post-infection, the draining lymph nodes (popliteal) were analyzed for responding cells. Aged mice (i.e., 18 months-2 years, non-cx) showed a significant reduction in total lymph node cellularity post- infection when compared to both the young and post-castrate mice. Results are expressed as mean±lSD of 8-12 mice. **=p≤0.01 compared to both young (2-month) and castrated mice.
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Figures 17A-B: Castration enhances activation following HSV-1 infection. Figure 17A shows representative FACS profiles of activated (CD8+CD25+) cells in the LN of HSV-1 infected mice. No difference was seen in proportions of activated CTL with age or post- castration. As shown in Fig. 17B, the decreased cellularity within the lymph nodes of aged mice was reflected by a significant decrease in activated CTL numbers. Castration of the aged mice restored the immune response to HSV-1 with CTL numbers equivalent to young mice. Results are expressed as mean±lSD of 8-12 mice. **=p≤0.01 compared to both young (2-month) and castrated mice.
Figure 18: Specificity of the immune response to HSV-1. Popliteal lymph node cells were removed from mice immunized with HSV-1 (removed 5 days post-HSV-1 infection), cultured for 3-days, and then examined for their ability to lyse HSV peptide pulsed EL 4 target cells. CTL assays were performed with non-immunized mice as control for background levels of lysis (as determined by Cr-release). Aged mice showed a significant (p≤O.01, **) reduction in CTL activity at an E:T ratio of both 10:1 and 3:1 indicating a reduction in the percentage of specific CTL present within the lymph nodes. Castration of aged mice restored the CTL response to young adult levels since the castrated mice demonstrated a comparable response to HSV-1 as the young adult (2-month) mice. Results are expressed as mean of 8 mice, in triplicate ±1 SD. ** = p≤O.Ol compared to young adult mice; Λ = significantly different to aged control mice (p<0.05 for E:T of 3:1; p≤O.Ol for E:T of 0.3: 1).
Figures 19A and B: Analysis of VD TCR expression and CD4+ T cells in the immune response to HSV-1. Popliteal lymph nodes were removed 5 days post-HSV-1 infection and analyzed ex-vivo for the expression of CD25, CD8 and specific TCRVD markers (Fig. 19A) and CD4/CD8 T cells (Fig. 19B). The percentage of activated (CD25+) CD8+ T cells expressing either VD 10 or VD8.1 is shown as mean ±ISD for 8 mice per group in Fig. 19A. No difference was observed with age or post-castration. However, a decrease in CD4/CD8 ratio in the resting LN population was seen with age (Fig. 19B). This decrease was restored post-castration. Results are expressed as mean±lSD of 8 mice per group. *** = p≤O.001 compared to young and post-castrate mice.
Figures 20A-D: Castration enhances regeneration of the thymus (Fig. 20A, spleen
(Fig. 20B) and bone marrow (Fig. 20D), but not lymph node (Fig. 20C) following bone marrow transplantation (BMT) of Ly5 congenic mice. 3 month old, young adults, C57/BL6
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Ly5.1+ (CD45.1+) mice were irradiated (at 6.25 Gy), castrated, or sham-castrated 1 day prior to transplantation with C57/BL6 Ly5.2+ (CD45.2+) adult bone marrow cells (106 cells). Mice were killed 2 and 4 weeks later and the), thymus (Fig. 20A), spleen (Fig. 20B), lymph node (Fig. 20C) and BM (Fig. 20D) were analyzed for immune reconstitution. Donor/Host origin was determined with anti-CD45.2 (Ly5.2), which only reacts with leukocytes of donor origin. There were significantly more donor cells in the thymus of castrated mice 2 and 4 weeks after BMT compared to sham-castrated mice (Fig. 20A). Note the rapid expansion of the thymus in castrated animals when compared to the non-castrate group at all time points post-treatment. There were significantly more cells in these spleen and BM of castrated mice 2 and 4 weeks after BMT compared to sham-castrated mice (Figs. 20B and 20D). There was no significant difference in lymph node cellularity 2, 4, and 6 weeks after BMT (Fig. 20C). Castrated mice had significantly increased congenic (Ly5.2) cells compared to non-castrated animals (data not shown). Data is expressed as mean+lSD of 4-5 mice per group. *=p≤0.05; **=p≤0.01.
Figures 21A and 21B: Changes in thymus cell number in castrated and noncastrated mice after fetal liver (E14, 106 cells) reconstitution. (n = 3-4 for each test group.) Fig. 21A shows that at two weeks, thymus cell number of castrated mice was at normal levels and significantly higher than that of noncastrated mice (*p< 0.05). Hypertrophy was observed in thymuses of castrated mice after four weeks. Noncastrated cell numbers remain below control levels. Fig. 21B shows the change in the number of CD45.2+ cells. CD45.2+ (Ly5.2+) is a marker showing donor derivation. Two weeks after reconstitution, donor- derived cells were present in both castrated and noncastrated mice. Four weeks after treatment approximately 85% of cells in the castrated thymus were donor-derived. There were no or very low numbers of donor-derived cells in the noncastrated thymus.
Figure 22: FACS profiles of CD4 versus CD8 donor derived thymocyte populations after lethal irradiation and fetal liver reconstitution, followed by surgical castration. Percentages for each quadrant are given to the right of each plot. The age matched control profile is of an eight month old Ly5.1 congenic mouse thymus. Those of castrated and noncastrated mice are gated on CD45.2+ cells, showing only donor derived cells. Two weeks after reconstitution, subpopulations of thymocytes do not differ proportionally between castrated and noncastrated mice demonstrating the homeostatic thymopoiesis with the major thymocyte subsets present in normal proportions.
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Figures 23A and 23B: Castration enhances dendritic cell generation in the thymus following fetal liver reconstitution. Myeloid and lymphoid dendritic cell (DC) number in the thymus after lethal irradiation, fetal liver reconstitution and castration. (n= 3-4 mice for each test group.) Control (white) bars on the graphs are based on the normal number of dendritic cells found in untreated age matched mice. Fig. 23A shows donor-derived myeloid dendritic cells. Two weeks after reconstitution, donor-derived myeloid DC were present at normal levels in noncastrated mice. There were significantly more myeloid DC in castrated mice at the same time point. (*p< 0.05). At four weeks myeloid DC number remained above control levels in castrated mice. Fig. 23B shows donor-derived lymphoid dendritic cells. Two weeks after reconstitution, donor-derived lymphoid DC numbers in castrated mice were double those of noncastrated mice. Four weeks after treatment, donor-derived lymphoid DC numbers remained above control levels.
Figures 24A and 24B: Changes in total and donor CD45.2+ bone marrow cell numbers in castrated and noncastrated mice after fetal liver reconstitution. n=3-4 mice for each test group. Fig. 24A shows the total number of bone marrow cells. Two weeks after reconstitution, bone marrow cell numbers had normalized and there was no significant difference in cell number between castrated and noncastrated mice. Four weeks after reconstitution, there was a significant difference in cell number between castrated and noncastrated mice (*p≤ 0.05). Indeed, four weeks after reconstitution, cell numbers in castrated mice were at normal levels. Fig. 24B shows the number of CD45.2+ cells (i.e., donor-derived cells). There was no significant difference between castrated and noncastrated mice with respect to CD45.2+ cell number in the bone marrow two weeks after reconstitution. CD45.2+ cell number remained high in castrated mice at four weeks; however, there were no donor-derived cells in the noncastrated mice at the same time point. The difference in BM cellularity was predominantly due to a lack of donor-derived BM cells at 4-weeks post-reconstitution in sham-castrated mice. Data is expressed as mean±lSD of 3- 4 mice per group. *=p≤0.05.
Figures 25A-25C: Changes in T cells and myeloid and lymphoid derived dendritic cells (DC) in bone marrow of castrated and noncastrated mice after fetal liver reconstitution. (n=3-4 mice for each test group.) Control (white) bars on the graphs are based on the normal number of T cells and dendritic cells found in untreated age matched mice. Fig. 25 A shows the number of donor-derived T cells. As expected, numbers were reduced compared to normal T cell levels two and four weeks after reconstitution in both castrated and
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noncastrated mice. By 4 weeks there was evidence of donor-derived T cells in the castrated but not control mice. Figure 25B shows the number of donor-derived myeloid dendritic cells (i.e., CD45.2+). Two weeks after reconstitution, donor myeloid DC cell numbers were normal in both castrated and noncastrated mice. At this time point there was no significant difference between numbers in castrated and noncastrated mice. However, by 4 weeks post- reconstitution, only the castrated animals have donor-derived myeloid dendritic cells. Fig. 25C shows the number of donor-derived lymphoid dendritic cells. Numbers were at normal levels two and four weeks after reconstitution for castrated mice but by 4 weeks there were no donor-derived DC in the sham-castrated group.
Figures 26A and 26B: Changes in total and donor (CD45.2+) lymph node cell numbers in castrated and non-castrated mice after fetal liver reconstitution. Control (striped) bars on the graphs are based on the normal number of lymph node cells found in untreated age matched mice. As shown in Fig. 26A, two weeks after reconstitution, cell numbers in the lymph node were not significantly different between castrated and sham-castrated mice. Four weeks after reconstitution, lymph node cell numbers in castrated mice were at control levels. Fig. 26B shows that there was no significant difference between castrated and non-castrated mice with respect to donor-derived CD45.2+ cell number in the lymph node two weeks after reconstitution. CD45.2+ cell numbers remained high in castrated mice at four weeks. There were no donor-derived cells in the non-castrated mice at the same point. Data is expressed as mean±lSD of 3-4 mice per group.
Figures 27A and 27B: Change in total and donor (CD45.2+) spleen cell numbers in castrated and non-castrated mice after fetal liver reconstitution. Control (white) bars on the graphs are based on the normal number of spleen cells found in untreated age matched mice. As shown in Fig. 27A, two weeks after reconstitution, there was no significant difference in the total cell number in the spleens of castrated and non-castrated mice. Four weeks after reconstitution, total cell numbers in the spleen were still approaching normal levels in castrated mice but were very low in non-castrated mice. Fig. 27B shows the number of donor (CD45.2+) cells. There was no significant difference between castrated and non-castrated mice with respect to donor-derived cells in the spleen, two weeks after reconstitution. However, four weeks after reconstitution, CD45.2+ cell number remained high in the spleens of castrated mice, but there were no donor-derived cells in the noncastrated mice at the same time point. Data is expressed as mean±lSD of 3-4 mice per group. *=p≤0.05
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Figures 28A-28C: Castration enhances DC generation in the spleen after fetal liver reconstitution. Control (white) bars on the graphs are based on the normal number of splenic T cells and dendritic cells found in untreated age matched mice. As shown in Fig. 28A, total T cell numbers were reduced in the spleen two and four weeks after reconstitution in both castrated and sham-castrated mice. Fig. 28B shows that at 2-weeks post- reconstitution, donor-derived (CD45.2+) myeloid DC numbers were normal in both castrated and sham- castrated mice. Indeed, at two weeks there was no significant difference between numbers in castrated and non-castrated mice. However, no donor-derived DC were evident in sham- castrated mice at 4- weeks post-reconstitution, while donor-derived (CD45.2+) myeloid DC were seen in castrated mice. As shown in Fig. 28C, donor-derived lymphoid DC were also at normal levels two weeks after reconstitution. At two weeks there was no significant difference between numbers in castrated and non-castrated mice. Again, no donor-derived lymphoid DC were seen in sham-cx mice at 4-weeks compared to cx mice. Data is expressed as mean±lSD of 3-4 mice per group. *=p≤0.05.
Figures 29A-29C: Changes in T cells and myeloid and lymphoid derived dendritic cells (DC) in the mesenteric lymph nodes of castrated and non-castrated mice after fetal liver reconstitution. (n=3-4 mice for each test group.) Control (striped) bars are the number of T cells and dendritic cells found in untreated age matched mice. Mesenteric lymph node T cell numbers were reduced two and four weeks after reconstitution in both castrated and noncastrated mice (Fig. 29 A). Donor derived myeloid dendritic cells were normal in the mesenteric lymph node of both castrated and noncastrated mice, while at four weeks they were decreased (Fig. 29B). At two weeks there was no significant difference between numbers in castrated and noncastrated mice. Fig. 29C shows donor-derived lymphoid dendritic cells in the mesenteric lymph node of both castrated and noncastrated mice. Numbers were at normal levels two and four weeks after reconstitution in castrated mice but were not evident in the control mice.
Figures 30A-30C: Castration Increases Bone Marrow and Thymic Cellularity following
Congenic BMT. As shown in Fig. 30A, there are significantly more cells in the BM of castrated mice 2 and 4 weeks after BMT. BM cellularity reached untreated control levels (1.5xl07± 1.5xl06) in the sham-castrates by 2 weeks. BM cellularity is above control levels in castrated mice 2 and 4 weeks after congenic BMT. Fig. 30b shows that there are
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significantly more cells in the thymus of castrated mice 2 and 4 weeks after BMT. Thymus cellularity in the sham-castrated mice is below untreated control levels (7.6x10 ± 5.2x10 ) 2 and 4 weeks after congenics BMT. 4 weeks after congenic BMT and castration thymic cellularity is increased above control levels. Fig. 30C shows that there is no significant difference in splenic cellularity 2 and 4 weeks after BMT. Spleen cellularity has reached control levels (8.5xl07 ± l.lxlO7) in sham-castrated and castrated mice by 2 weeks. Each group contains 4 to 5 animals. \ indicates sham-castration; |, castration.
Figure 31: Castration increases the proportion of Hematopoietic Stem Cells following Congenic BMT. There is a significant increase in the proportion of donor-derived HSCs following castration, 2 and 4 weeks after BMT.
Figures 32A and 32B: Castration increases the proportion and number of Hematopoietic Stem Cells following Congenic BMT. As shown in Fig. 32A, there was a significant increase in the proportion of HSCs following castration, 2 and 4 weeks after BMT (* p<0.05). Fig. 32B shows that the number of HSCs is significantly increased in castrated mice compared to sham-castrated controls, 2 and 4 weeks after BMT (* p<0.05 ** p<0.01). Each group contains 4 to 5 animals. Q indicates sham-castration; |, castration.
Figures 33A and 33B: There are significantly more donor-derived B cell precursors and B cells in the BM of castrated mice following BMT. As shown in Fig. 33A, there were significantly more donor-derived CD45.1+B220+IgM"B cell precursors in the bone marrow of castrated mice compared to the sham-castrated controls (* p<0.05). Fig. 33B shows that there were significantly more donor-derived B220+IgM+ B cells in the bone marrow of castrated mice compared to the sham-castrated controls (* p<0.05). Each group contains 4 to 5 animals. Q indicates sham-castration; |, castration.
Figure 34: Castration does not effect the donor-derived thymocyte proportions following congenic BMT. 2 weeks after sham-castration and castration there is an increase in the proportion of donor-derived double negative (CD45.1+CD4"CD8 ) early thymocytes. There are very few donor-derived (CD45.1+) CD4 and CD8 single positive cells at this early time point. 4 weeks after BMT, donor-derived thymocyte profiles of sham-castrated and castrated mice are similar to the untreated control.
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Figure 35: Castration does not increase peripheral B cell proportions following congenic BMT. There is no difference in splenic B220 expression comparing castrated and sham-castrated mice, 2 and 4 weeks after congenic BMT.
Figure 36: Castration does not increase peripheral B cell numbers following congenics BMT. There is no significant difference in B cell numbers 2 and 4 weeks after BMT. 2 weeks after congenic BMT B cell numbers in the spleen of sham-castrated and castrated mice are approaching untreated control levels (5.0 x 107 ± 4.5x10 ). Each group contains 4 to 5 animals. [V indicates sham-castration; |, castration.
Figure 37: Donor-derived Triple negative, double positive and CD4 and CD8 single positive thymocyte numbers are increased in castrated mice following BMT. Fig. 37A shows that there were significantly more donor-derived triple negative (CD45.1+CD3~CD4"CD8~) thymocytes in the castrated mice compared to the sham-castrated controls 2 and 4 weeks after BMT (* p<0.05 **p<0.01). Fig. 37B shows there were significantly more double positive (CD45.1+CD4+CD8+) thymocytes in the castrated mice compared to the sham-castrated controls 2 and 4 weeks after BMT (* p<0.05 **p<0.01). As shown in Fig. 37C, there were significantly more CD4 single positive (CD45.1+CD3+CD4+CD8~) thymocytes in the castrated mice compared to the sham-castrated controls 2 and 4 weeks after BMT (* p<0.05 **p<0.01). Fig. 37D shows there were significantly more CD8 single positive (CD45.1+CD3+CD4"CD8+) thymocytes in the castrated mice compared to the sham-castrated controls 4 weeks after BMT (* p<0.05 **p<0.01). Each group contains 4 to 5 animals. indicates sham-castration; |, castration.
Figure 38: There are very few donor-derived, peripheral T cells 2 and 4 weeks after congenic BMT. As shown in Fig. 38A, there was a very small proportion of donor-derived CD4+ and CD8+ T cells in the spleens of sham-castrated and castrated mice 2 and 4 weeks after congenic BMT. Fig. 38B shows that there was no significant difference in donor- derived T cell numbers 2 and 4 weeks after BMT. 4 weeks after congenics BMT there are significantly less CD4+ and CD8+ T cells in both sham-castrated and castrated mice compared to untreated age-matched controls (CD4+-l.lxl07 ± 1.4xl06, CD8+ - 6.0xl06 ± 1.0x10s) Each group contains 4 to 5 animals. Q indicates sham-castration; |, castration.
Figure 39: Castration increases the number of donor-derived dendritic cells in the thymus 4 weeks after congenics BMT. As shown in Fig. 39A, donor-derived dendritic cells were CD45.1+CDl lc+ MHClf. Fig. 39B shows there were significantly more donor-derived
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thymic DCs in the castrated mice 4 weeks after congenic BMT (* p<0.05). Dendritic cell numbers are at untreated control levels 2 weeks after congenic BMT (1.4xl05 ± 2.8xl04). 4 weeks after congenic BMT dendritic cell numbers are above control levels in castrated mice. Each group contains 4 to 5 animals. Q indicates sham-castration; |, castration.
Figure 40: The phenotypic composition of peripheral blood lymphocytes was analyzed in human patients (all >60 years) undergoing LHRH agonist treatment for prostate cancer. Patient samples were analyzed before treatment and 4 months after beginning LHRH agonist treatment. Total lymphocyte cell numbers per ml of blood were at the lower end of control values before treatment in all patients. Following treatment, 6/9 patients showed substantial increases in total lymphocyte counts (in some cases a doubling of total cells was observed). Correlating with this was an increase in total T cell numbers in 6/9 patients. Within the CD4+ subset, this increase was even more pronounced with 8/9 patients demonstrating increased levels of CD4 T cells. A less distinctive trend was seen within the CD8+ subset with 4/9 patients showing increased levels, albeit generally to a smaller extent than CD4+ T cells.
Figure 41: Analysis of human patient blood before and after LHRH-agonist treatment demonstrated no substantial changes in the overall proportion of T cells, CD4 or CD8 T cells, and a variable change in the CD4:CD8 ratio following treatment. This indicates the minimal effect of treatment on the homeostatic maintenance of T cell subsets despite the substantial increase in overall T cell numbers following treatment. All values were comparative to control values.
Figure 42: Analysis of the proportions of B cells and myeloid cells (NK, NKT and macrophages) within the peripheral blood of human patients undergoing LHRH agonist treatment demonstrated a varying degree of change within subsets. While NK, NKT and macrophage proportions remained relatively constant following treatment, the proportion of B cells was decreased in 4/9 patients.
Figure 43: Analysis of the total cell numbers of B and myeloid cells within the peripheral blood of human patients post-treatment showed clearly increased levels of NK (5/9 patients), NKT (4/9 patients) and macrophage (3/9 patients) cell numbers post-treatment. B cell numbers showed no distinct trend with 2/9 patients showing increased levels; 4/9 patients showing no change and 3/9 patients showing decreased levels.
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Figures 44A and 44B: The major change seen post-LHRH agonist treatment was within the T cell population of the peripheral blood. White bars represent pre-treatment; black bars represent 4 months post-LHRH-A treatment. Shown are representative FACS histograms (using four color staining) from a single patient. In particular there was a selective increase in the proportion of naive (CD45RA+) CD4+ cells, with the ratio of naive (CD45RA+) to memory (CD45RO+) in the CD4+ T cell subset increasing in 6/9 of the human patients.
Figure 45 is a line graph showing that while 60% of the sham-operated mice had diabetes, fewer than 20% of the castrated group had diabetes.
Figure 46 is a bar graph showing that castrated NOD mice had a marked increase in total thymocyte number but no differences in total spleen cells.
Figures 47A-47C are bar graphs showing that there was a significant increase in all thymocyte subclasses (Fig. 47A) in castrated NOD mice. There no change in B cells compared to sham-castrated NOD mice (Fig. 47C) nor in the total T or B cells in the spleen (Fig. 47B).
Figures 48A and 48B show a marked in total thymocytes (Fig. 48A) and spleen cells (Fig. 48B) in castrated NZB mice.
Figure 49 is a graph showing decreased tumor incidence in mice that have been castrated and immunized as compared to controls.
Figures 50A-C are bar graphs showing that castrated and immunized mice have increased splenic cellularity as compared to controls.
Figures 51A-B are graphs showing increased γlFN production in mice that have castrated and immunized as compared to controls.
Figures 52A-B are graphs showing that castrated and immunized mice exhibit enhanced antigen-specific CTL responses as compared to controls.
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DETAILED DESCRIPTION OF THE INVENTION
The patent and scientific literature referred to herein establishes knowledge that is available to those with skill in the art. The issued U.S. patents, allowed applications, published foreign applications, and references, including GenBank database sequences, that are cited herein are hereby incoφorated by reference to the same extent as if each was specifically and individually indicated to be incoφorated by reference.
The phrase "modifying the T cell population makeup" refers to altering the nature and/or ratio of T cell subsets defined functionally and by expression of characteristic molecules. Examples of these characteristic molecules include, but are not limited to, the T cell receptor, CD4, CD8, CD3, CD25, CD28, CD44, CD45, CD62L and CD69.
The phrase "increasing the number of T cells" refers to an absolute increase in the number of T cells in a subject in the thymus and/or in circulation and/or in the spleen and/or in the bone marrow and/or in peripheral tissues such as lymph nodes, gastrointestinal, urogenital and respiratory tracts. This phrase also refers to a relative increase in T cells, for instance when compared to B cells.
A "subject having a depressed or abnormal T cell population or function" includes an individual infected with the human immunodeficiency virus, especially one who has AIDS, or any other, virus or infection which attacks T cells or any T cell disease for which a defective gene has been identified. Furthermore, this phrase includes any post-pubertal individual, especially an aged person who has decreased immune responsiveness and increased incidence of disease as a consequence of post-pubertal thymic atrophy.
Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.
To generate new T lymphocytes, the thymus requires precursor cells; these can be derived from within the organ itself for a short time, but by 3-4 weeks, such cells are depleted and new hematopoietic stem cells (HSC) must be taken in (under normal circumstances this would be from the bone marrow via the blood). However, even in a normal functional young thymus, the intake of such cells is very low (sufficient to maintain T cell production at homeostatically regulated levels). Indeed the entry of cells into the thymus is extremely
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limited and effectively restricted to HSC (or at least prothymocytes which already have a preferential development along the T cell lineage). In the case of the thymus undergoing rejuvenation due a loss of sex steroid inhibition, this organ has been demonstrated to now be very receptive to new precursor cells circulating in the blood, such that the new T cells which develop from both intrathymic and external precursors. By increasing the level of the blood precursor cells, the T cells derived from them will progressively dominate the T cell pool. This means that any gene introduced into the precursors (HSC) will be passed onto all progeny T cells and eventually be present in virtually all of the T cell pool. The level of dominance of these cells over those derived from endogenous host HSC can be easily increased to very high levels by simply increasing the number of transferred exogenous HSC.
The present disclosure provides methods for preventing or treating an illness or disease in a patient. In one embodiment, the disease is a T cell disorder. In another embodiment, the disease is an autoimmune disease or allergy. Additionally, the present disclosure also comprises methods for improving a patient' immune response to a vaccine. Each may be accomplished by quantitatively and qualitatively restoring the peripheral T cell pool, particularly at the level of naive T cells. Na ve T cells are those that have not yet contacted antigen and therefore have broad based specificity, i.e., are able to respond to any one of a wide variety of antigens. As a result of the procedures of this invention a large pool of naive T cells becomes available to respond to a disease antigen of an infectious agent, cancer, etc. or when administered in a vaccine.
As described above, the aged (post-pubertal) thymus causes the body's immune system to function at less than peak levels (such as that found in the young, pre-pubertal thymus). "Post-pubertal" is herein defined as the period in which the thymus has reached substantial atrophy. In humans, this occurs by about 20-25 years of age, but may occur earlier or later in a given individual. "Pubertal" is herein defined as the time during which the thymus begins to atrophy, but may be before it is fully atrophied. In humans this occurs from about 10-20 years of age, but may occur earlier or later in a given individual. "Pre- pubertal" is herein defined as the time prior to the increase in sex steroids in an individual. In humans, this occurs at about 0-10 years of age, but may occur earlier or later in a given individual.
The present disclosure uses reactivation of the thymus to improve immune system function, as exemplified by increased functionality of T lymphocytes (e.g., Th and CTL)
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including, but not limited to, better killing of target cells; increased release of cytokines, interleukins and other growth factors; increased levels of Ab in the plasma; and increased levels of innate immunity (e.g., natural killer (NK) cells, DC, neutrophils, macrophages, etc.) in the blood, all of which can combat disease and infection, thereby increasing resistance to, and preventing or treating infection by, various foreign agents, and/or can be beneficial in increasing the response to vaccine antigens.
"Recipient," "patient" and "host" are used interchangeably and are herein defined as a subject receiving sex steroid ablation therapy and/or therapy to interrupt sex steroid mediated signaling and or, when appropriate, the subject receiving the HSC transplant. "Donor" is herein defined as the source of the transplant, which may be syngeneic, allogeneic or xenogeneic. In some instances, the patient may provide, e.g., his or her own autologous cells for transplant into the patient at a later time point Allogeneic HSC grafts may be used, and such allogeneic grafts are those that occur between unmatched members of the same species, while in xenogeneic HSC grafts the donor and recipient are of different species. Syngeneic HSC grafts, between matched animals, may also be used. The terms "matched,"
"unmatched," "mismatched," and "non-identical" with reference to HSC grafts are herein defined as the MHC and/or minor histocompatibility markers of the donor and the recipient are (matched) or are not (unmatched, mismatched and non-identical) the same.
The terms "vaccinating," "vaccination," "vaccine, " "immunizing," "immunization," are herein defined as administration to a patient of a preparation to elicit an immune response to an antigen. Vaccination may include both prophylactic and therapeutic vaccines. As will be understood by those skilled in the art, infection by, e.g., a virus or other agent is also a method of vaccinating an individual.
The terms "improving," "enhancing," or "increasing" "vaccine responses" or "vaccine responsiveness" in a patient or "improving," "enhancing," or "increasing" the "immune responsiveness of a patient to a vaccine" and other similar language is used interchangeably and herein defined as meaning that a patient's immune response to the vaccine or vaccine antigen is improved compared to the immune response which would have otherwise occurred in a patient without disruption of sex steroid signaling.
"Illness" and "disease" are used interchangeably and are herein defined as any disease, infection or medical condition (symptomatic or asymptomatic) in which an immune response, defense or modified immune system would be beneficial to the patient. The illness
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may be caused by an infectious agent, cancer, drug treatment (e.g„ chemotherapy), irradiation, chemical poisoning, genetic defect or other disorder.
"Prevention" of or "preventing" an illness is herein defined as complete as well as partial protection including without limitation reduced severity of clinical symptoms than would have otherwise occurred in the patient. With an improved or modified immune system the individual will have a reduced likelihood of succumbing to, or suffering from, a tumor or cancer, allergy, autoimmune diseases, a prevailing infection (e.g., viral, bacterial, fungal, or parasitic) or illness, and/or will show better responses to a vaccination (e.g., increased levels of antibody (Ab) specific to that vaccine or antigen, and development of effector T cells). Prevention of an illness may occur by activating or modifying immune defense mechanisms to inhibit or reduce the development of clinical symptoms, such as to a point where only reduced or minimal medical care is required. Preventing an infection also encompasses defending the body against infectious agents, such as viruses, bacteria, parasites, fungi, etc. or against non-infectious agents. This may take the form of preventing such agents from entering the cells in the body and/or the efficient removal of the agents by cells of the broad immune system (e.g., NK, DC, macrophages, neutrophils, etc.). In some instances complete prevention of illness is not achieved, and instead partial prevention is achieved in which a stronger, more resilient or more effective immune system will aid the body in decreasing the extent, severity and duration of illness or clinical symptoms of illness or recovery time or delay the onset of clinical symptoms.
"Treatment" of or "treating" an illness encompasses completely or partially reducing the symptoms of the illness in the patients, as compared to those symptoms that would have otherwise occurred in the patient without sex steroid ablation or interruption of sex steroid mediated signaling. Treatment of an illness may occur by activating immune defense mechanisms to inhibit, delay or reduce the development of clinical symptoms. In one example, the patient has already contacted the agent, or is at a high risk of doing so.
The ability to respond better to, or to overcome, a new (by prevention) or existing (by treatment) illness involves increasing the immune defense of the body, which includes increasing the functionality and/or the number of cells involved in immune defense. Activation of the immune system also increases the number of lymphocytes capable of responding to the antigen of the agent in question, which will lead to the elimination (complete or partial) of the antigen or agent creating a situation where the host is treated for
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or resistant to the infection or illness. With an improved or modified immune system the individual will have a reduced likelihood of succumbing to or suffering from a tumor or cancer, allergy, autoimmune diseases, a prevailing infection (e.g., viral, bacterial, fungal, or parasitic) or illness, and/or will show better responses to a vaccination (e.g., increased levels of antibody (Ab) specific to that vaccine or antigen, and development of effector T cells).
This increase is exemplified in the dramatic improvement of aged mice to the human heφes simplex virus infection (see Example 3, and Figures 14-19). The castrated aged mice initially show a marked increase of lymphocyte infiltration into the draining lymph node. This infiltration is the first step in an immune response, and is generally required to increase the likelihood of the antigen-specific lymphocyte contacting the antigen. The next step is the activation of the lymphocytes by antigen and the development of Ab and/or CTL and release of cytokines from lymphocytes, all of which combine to destroy the agent.
"Infectious agents," "foreign agents," and "agents" are used interchangeably and include any cause of disease or illness in an individual. Agents include, but are not limited to viruses, bacteria, fungi, parasites, prions, cancers, precancerous cells, chemical or biological toxins, allergens, asthma-inducing agents, self proteins and antigens which contribute to autoimmune disease, etc.
In one case, the agent is a virus, bacteria, fungi, or parasite e.g., from the coat protein of a human papilloma virus (HPV), which causes uterine cancer; or an influenza peptide (e.g., hemagglutinin (HA), nucleoprotein (NP), or neuraminidase (N)).
Nonlimiting examples of infectious viruses include: Retroviridae (e.g., human immunodeficiency viruses, such as HIV-1 (also referred to as HTLV-III, LAV or HTLV- III/LAV), or HIV-III; and other isolates, such as HIV-LP; Picornaviridae (e.g., polio viruses, hepatitis A virus; enteroviruses, human coxsackie viruses, rhinoviruses, echoviruses); Calciviridae (e.g., strains that cause gastroenteritis); Togaviridae (e.g., equine encephalitis viruses, rubella viruses); Flaviridae (e.g., dengue viruses, encephalitis viruses, yellow fever viruses); Coronaviridae (e.g., coronaviruses, severe acute respiratory syndrome (SARS) virus); Rhabdoviridae (e.g., vesicular stomatitis viruses, rabies viruses); Filoviridae (e.g., ebola viruses); Paramyxoviridae (e.g., parainfluenza viruses, mumps virus, measles virus, respiratory syncytial virus); Orthomyxoviridae (e.g., influenza viruses); Bungaviridae (e.g., Hantaan viruses, bunga viruses, phleboviruses and Nairo viruses); Arena viridae (hemorrhagic fever viruses); Reoviridae (e.g., reoviruses, orbiviurses and rotaviruses);
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Birnaviridae; Hepadnaviridae (e.g, Hepatitis B virus); Parvoviridae (parvoviruses); Papovaviridae (papilloma viruses, polyoma viruses); Adenoviridae (most adenoviruses); Heφesviridae (e.g., heφes simplex virus (HSV) 1 and 2, varicella zoster virus, cytomegalovirus (CMV), heφes viruses); Poxviridae (e.g., variola viruses, vaccinia viruses, pox viruses); and Iridoviridae (e.g., African swine fever virus); and unclassified viruses (e.g., the etiological agents of Spongiform encephalopathies, the agent of delta hepatitis (thought to be a defective satellite of hepatitis B virus), the agents of non-A, non-B hepatitis (class l=internally transmitted; class 2=parenterally transmitted (i.e., Hepatitis C); Norwalk and related viruses, and astroviruses).
Nonlimiting examples of infectious bacteria include: Helicobacter pylons, Borelia burgdorferi, Legionella pneumophilia, Mycobacteήa sporozoites (sp.) (e.g. M. tuberculosis, M. avium, M. intracellulare, M. kansaii, M. gordonae), Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitidis, Listeria monocytogenes, Streptococcus pyogenes (Group A Streptococcus), Streptococcus agalactiae (Group B Streptococcus), Streptococcus (viridans group), Streptococcus faecalis, Streptococcus bovis, Streptococcus (anaerobic sps.), Streptococcus pneumoniae, pathogenic Campylobacter sp., Enterococcus sp., Haemophilus influenzae, Bacillus antracis, Corynebacterium diphtheriae, Corynebacterium sp., Erysipelothrix rhusiopathiae, Clostridium perfringens, Clostridium tetani, Enterobacter aerogenes, Klebsiella pneumoniae, Pasturella multocida, Bacteroides sp., Fusobacterium nucleatum, Streptobacillus moniliformis, Treponema pallidium, Treponema pertenue, Leptospira, and Actinomyces israelli.
Nonlimiting examples of infectious fungi include: Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Chlamydia trachomatis, Candida albicans.
Other infectious organisms (i.e., protists) include: Plasmodium falciparum and Toxoplasma gondii.
In another case the agent is an allergen. Allergic conditions include, but are not limited to, eczema, allergic rhinitis or coryza, hay fever, bronchial asthma, urticaria (hives) and food allergies, and other atopic conditions.
In yet another embodiment, the agent is a cancer or tumor. The cancer or tumor may be malignant or non-malignant. As used herein, a tumor or cancer includes, e.g., tumors of
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the brain, lung (e.g., small cell and non-small cell), and pleura, gynecological, urogenital and endocrine system, ( e.g., cervix, uterus, endometrium, bladder, renal organs, ovary, breast, and/or prostate), gastrointestinal tract (e.g., anal, bile duct, carcinoid tumor, gallbladder, gastric or stomach, liver, esophagus, pancreas, rectum, small intestine, and/or colon), as well as other carcinomas, and bone, skin and connective tissue (e.g., melanomas and/or sarcomas), and or the haematological system (e.g., blood, myelodysplastic syndromes, myeloproliferative disorders, plasma cell neoplasm, lymphomas and/or leukemias).
Recombinant gene expression vectors may be used for the vaccination methods of the invention. The recombinant vectors may be plasmids or cosmids, which include the antigen coding polynudeotides of the invention, but may also be viruses or retroviruses. The vectors used in the polynucleotide vaccines may be "naked" (i.e., not associated with a delivery vehicle such as liposomes, colloidal particles, etc.). For convenience, the term "plasmid" as used in this disclosure will refer to plasmids or cosmids, depending on which is appropriate to use for expression of the peptide of interest (where the choice between the two is dictated by the size of the gene encoding the peptide of interest). A commonly used plasmid vector which may be used is pBR322.
Various viral vectors that can be utilized in the vaccination methods of the invention include adenovirus, heφes virus, vaccinia, or an RNA virus such as a retrovirus. Retroviral vectors may be derivatives of a murine or avian retrovirus. Examples of retroviral include, but are not limited to: Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuS-V), murine mammary tumor virus (MuMTV), and Rous Sarcoma Virus (RSV).
Other plasmids and viral vectors useful in the vaccination methods of the invention are well known in the vaccine art.
The present disclosure also comprises methods for gene therapy using genetically modified hematopoietic stem cells, lymphoid progenitor cells, myeloid progenitor cells, epithelial stem cells, or combinations thereof (GM cells). Previous attempts by others to deliver such cells as gene therapy have been unsuccessful, resulting in negligible levels of the modified cells. The present disclosure provides a new method for delivery of these cells which promotes uptake and differentiation of the cells into the desired T cells. The modified cells are injected into a patient whose thymus is being reactivated by the methods of this invention. The modified stem and progenitor cells are taken up by the thymus and converted
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into T cells, dendritic cells, and other cells produced in the thymus. Each of these new cells contains the genetic modification of the parent stem progenitor cell.
In one embodiment, the methods of the invention use genetically modified HSC, lymphoid progenitor cells, myeloid progenitor cells, epithelial stem cells or combinations thereof (collectively referred to as GM cells) to produce an immune system resistant to attack by particular antigens.
An appropriate gene or polynucleotide (i.e., the nucleic acid sequence defining a specific protein) that will create or induce resistance to one or more infectious or other agents is engineered into the stem and/or progenitor cells. By introducing the specific gene into the HSC, the cell differentiates into, e.g., an APC, it will express the protein as a peptide expressed in the context of MHC class I or II. This expression will greatly increase the number of APC "presenting" the desired antigen than would normally occur, thereby increasing the chance of the appropriate T cell recognizing the specific antigen and responding.
Many antineoplastic or cytotoxic anticancer drugs used in the clinic today cause moderate to severe bone marrow toxicity (e.g., vinblastine, cisplatin, methotrexate, alkylating agents, anti-folate, a vinca alkaloid and anthracyclines). In another embodiment of the present invention, drug resistance genes can be introduced into HSC to confer resistance to anticancer drugs. Such genes include for example dihydrofolate reductase. The use of the present invention to provide HSC which are resistant to the cytotoxic effects of these chemotherapeutics may allow for the greater use of these drugs and/or less side effects by reducing the incidence and severity of myelosupporession. (Podda et al, (1992) Proc. Natl. Acad. Sci. USA 89:9676; Banerjee et al, (1994) Stem Cells 12:378)
The modified stem and progenitor cells are taken up by the thymus and converted into T cells, DC, and other cells produced in the thymus. Each of these new cells contains the genetic modification of the parent stem/progenitor cell, and is thereby completely or partially resistant to infection or damage by the agent or agents. B cells are also increased in number in the bone marrow, blood and peripheral lymphoid organs, such as the spleen and lymph nodes, within e.g., two weeks of castration.
In one embodiment, an agent has already been in contact with a person, or is at a high risk of doing so. The person may be given GnRH to activate their thymus, and also to
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improve their bone marrow function, which includes the increased ability to take up and produce HSC. The person may be injected with their own HSC, or may be injected with HSC from an appropriate donor, which has, e.g., treatment with G-CSF for 3 days (2 injections, subcutaneously per day) followed by collection of HSC from the blood on days 4 and 5. The HSC may be transfected or transduced with a gene (e.g., encoding the protein, peptide, or antigen from the agent) to produce to the required protein or antigen. Following injection into the patient, the HSC enter the bone and bone marrow from the blood and then some exit back to the blood, and eventually some evolve into antigen presenting cells (APC) throughout the body. The antigen is expressed in the context of MHC class I and/or MHC class II molecules on the surface of these APC. By expressing the desired antigen, the APC improve the activation of T and B lymphocytes. The transplanted HSC may also enter the thymus, develop into DC, and present the antigen in question to developing T lymphocytes.
If present in low numbers (e.g., <0.1% of thymus cells) the DC can bias the selection of new T cells to those reactive to the antigen. If the particular DC are present in high numbers, the same principle can be used to delete the new T cells which are potentially reactive to the antigen, which may be used in the prevention or treatment of autoimmune diseases.
In the case of the HSC being GM for a specific trait, each of the new cells contains the genetic modification of the parent stem/progenitor cell, and is thereby completely or partially resistant to infection or damage by the agent or agents
In one embodiment, a patient is infected with HIV. In a specific embodiment, the method for treating this patient includes the following steps, which are provided in more detail below: (1) treatment with Highly Active Anti-Retrovirus Therapy (HAART) to lower the viral titer, which treatment continues throughout the procedure to prevent or reduce infection of new T cells; (2) ablation of T cells (immunosuppression); (3) blockage of sex steroid mediated signaling, for example, by administering an LHRH analog; (4) at the time the thymus begins reactivating, administration of GM cells that have been modified to contain a gene that expresses a protein that will prevent HIV infection, prevent HIV replication, disable the HIV virus, or other action that will stop the infection of T cells by HIV; (5) if the GM cells are not autologous, administration of the donor cells before, concurrently with, or after thymus reactivation will prime the immune system to recognize the donor cells as self; and (6) when the thymic chimera is established and the new
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cohort of mature T cells have begun exiting the thymus, reduction and eventual elimination of immunosuppression.
The inventions provided herein may be used with any animal species (including humans) having sex steroid driven maturation and an immune system, such as mammals and marsupials. In some embodiments, the invention is used with large mammals, such as humans.
DISRUPTION OF SEX STEROID MEDIATED SIGNALING
As used herein, "sex steroid ablation," "inhibition of sex steroid-mediated signaling," "sex steroid disruption" "interruption of sex steroid signaling" and other similar terms are herein defined as at least partial disruption of sex steroid (and/or other hormonal) production and/or sex steroid (and/or other hormonal) signaling, whether by direct or indirect action. In one embodiment, sex steroid signaling to the thymus is interrupted. As will be readily understood, sex steroid-mediated signaling can be disrupted in a range of ways well known to those of skill in the art, some of which are described herein. For example, inhibition of sex hormone production or blocking of one or more sex hormone receptors will accomplish the desired disruption, as will administration of sex steroid agonists and/or antagonists, or active (antigen) or passive (antibody) anti-sex steroid vaccinations.
A non-limiting method for creating disruption of sex steroid mediated signaling is through castration. Methods for castration include, but are not limited to, chemical castration and surgical castration.
"Castration" is herein defined as the reduction or elimination of sex steroid production, action and/or distribution in the body. This effectively eventually returns the patient to a pre-pubertal status when the thymus is more fully functioning than immediately prior to castration. Surgical castration removes the patient's gonads. Methods for surgical castration are well known to routinely trained veterinarians and physicians. One non-limiting method for castrating a male animal is described in the examples below. Other non-limiting methods for castrating human patients include a hysterectomy or ovariectomy procedure (to castrate women) and surgical castration to remove the testes (to castrate men). In some clinical cases, permanent removal of the gonads via physical castration may be appropriate.
Chemical castration is a less permanent version of castration. As herein defined,
"chemical castration" is the administration of a chemical for a period of time, which results
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in the reduction or elimination of sex steroid production, action and/or distribution in the body. A variety of chemicals are capable of functioning in this manner. Non-limiting examples of such chemicals are the sex steroid inhibitors and/or analogs described below. During the chemical delivery, and for a period of time afterwards, the patient's hormone production is turned off or reduced. The castration may be reversed upon termination of chemical delivery of the relevant sex hormones.
The terms thymus "regeneration," "reactivation" and "reconstitution" and their derivatives are used interchangeably herein, and are herein defined as the recovery of an atrophied or damaged (e.g., by chemicals, radiation, graft versus host disease, infections, genetic predisposition) thymus to its active state. "Active state" is herein defined as meaning a thymus in a patient whose sex steroid hormone mediated signaling has been disrupted, achieves an output of T cells that is at least 10%, or at least 20%, or at least 40%, or at least 60%, or at least 80%, or at least 90% of the output of a pre-pubertal thymus (i.e., a thymus in a patient who has not reached puberty).
The patient's thymus may be reactivated by disruption of sex steroid mediated signaling. This disruption reverses the hormonal status of the recipient. In certain embodiments, the recipient is post-pubertal. According to the methods of the invention, the hormonal status of the recipient is reversed such that the hormones of the recipient approach pre-pubertal levels. By lowering the level of sex steroid hormones in the recipient, the signalling of these hormones to the thymus is lowered, thereby allowing the thymus to be reactivated.
In one embodiment, blocking sex steroid mediated signaling creates this pool of naive T cells by reactivating the atrophied thymus. This disruption reverses the hormonal status of the recipient.
In one embodiment, during or after the castration step, hematopoietic stem or progenitor cells, or epithelial stem cells, from the donor may be transplanted into the recipient. These cells are accepted by the thymus as belonging to the recipient and become part of the production of new T cells and DC by the thymus. The resulting population of T cells recognize both the recipient and donor as self, thereby creating tolerance for a graft from the donor. The graft may be cells, tissues or organs of the donor, or combinations thereof.
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In another embodiment, thymic grafts can be used in the methods of the invention to improve engraftment of the donor cells or tolerance to the donor graft. In some embodiments, thymic grafts are when the patient is athymic, when the patient's thymus is resistant to regeneration, or to hasten regeneration. In certain embodiments, a thymic xenograft to induce tolerance is used (see .e.g., U.S. Patent No. 5,658,564). In other embodiments, an allogenic thymic graft is used.
As herein defined, the phrase "creating tolerance" or "inducing tolerance" in a patient, and other similar phrases, refers to complete, as well as partial tolerance induction (e.g.,, a patient may become either more tolerant, or completely tolerant, to the graft, as compared to a patient that has not been treated according to the methods of the invention). Tolerance induction can be tested, e.g., by an MLR reaction, using methods known in the art.
One method of reactivating the thymus is by blocking the direct and/or indirect stimulatory effects of LHRH on the pituitary, which leads to a loss of the gonadotrophins FSH and LH. These gonadotrophins normally act on the gonads to release sex hormones, in particular estrogens in females and testosterone in males; the release is blocked by the loss of FSH and LH. The direct consequences of this are an immediate drop in the plasma levels of sex steroids, and as a result, progressive release of the inhibitory signals on the thymus. The degree and kinetics of thymic regrowth can be enhanced by injection of CD34+ hematopoietic cells (ideally autologous).
In one embodiment, the patient's thymus is reactivated following a subcutaneous injection of a "depot" or "impregnated implant" containing about 30 mg of Lupron. A 30 mg Lupron injection is sufficient for 4 months of sex steroid ablation to allow the thymus to rejuvenate and export new na ve T cells into the bloodstream. The length of time of the GnRH treatment will vary with the degree of thymic atrophy and damage, and will be readily determined by those skilled in the art without undue experimentation. For example, the older the patient, or the more the patient has been exposed to T cell depleting reagents such as chemotherapy or radiotherapy, the longer it is likely that they will require GnRH. Four months is generally considered long enough to detect new T cells in the blood. Methods of detecting new T cells in the blood are known in the art. For instance, one method of T cell detection is by determining the existence of T cell receptor excision circles (TRECs), which are formed when the TCR is being formed and are lost in the cell after it divides or following apoptosis. Hence, TRECs are only found in new (naive) T cells. TREC levels are an
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indicator of thymic function in humans. These and other methods are described in detail in WO/00 230,256.
The terms "sex steroid analog," "sex steroid ablating agent," "sex steroid inhibitor," "inhibitor of sex steroid signalling," "modifier of sex steroid signalling," and other similar terms are herein defined as any one or more pharmaceutical agent that will decrease, disrupt, prevent, or abolish sex steroid (and or other hormone) mediated signalling. GnRH (also called LHRH or GnRH/LHRH herein), and analogs thereof, are nonlimiting exemplary inhibitors of sex steroid signalling used throughout this application. However, as will be readily understood by one skilled in the art, in practicing the inventions provided herein, GnRH/LHRH, or analogs thereof, may be replaced with any one (or more) of a number of substitute sex steroid inhibitors or analogs (or other blocker(s) or physical castration) which are described herein, without undue experimentation.
Any pharmaceutical drug, or other method of castration, that ablates sex steroids or interrupts sex steroid-mediated signaling may be used in the methods of the invention. For example, one nonlimiting method of, inhibiting sex steroid signaling and/or reactivating the thymus is by modifying the normal action of GnRH on the pituitary (i.e., the release of gonadotrophins, FSH and LH) and consequently reducing normal sex steroid production or release from the gonads. Thus, in one case, sex steroid ablation is accomplished by administering one or more sex hormone analogs, such as a GnRH analog. GnRH is a hypothalamic decapeptide that stimulates the secretion of the pituitary gonadotropins, leutinizing hormone (LH) and follicle-stimulating hormone (FSH). Thus, GnRH agonists (e.g., in the form of Synarel® or Lupron©) initially result in over stimulation of the receptor and through feedback mechanisms will suppress the pituitary production of FSH and LH by desensitization of LHRH Receptors. These gonadotrophins normally act on the gonads to release sex steroids, in particular estrogens in females and testosterone in males; the release of which is significantly reduced by the absence of FSH and LH. The direct consequences of this are a drop in the plasma levels of sex steroids, and as a result, progressive release of the inhibitory signals on the thymus. A more rapid drop in circulating sex steroid levels can be achieved for example by the use of a GnRH antagonist.
In some embodiments, the sex steroid mediated signaling is disrupted by administration of a sex steroid analog, such as an analog of leutinizing hormone-releasing hormone (LHRH). Sex steroid analogs and their use in therapies and chemical castration are
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well known. Sex steroid analogs are commercially and their use in therapies and chemical castration are well known. Such analogs include, but are not limited to, the following agonists of the LHRH receptor (LHRH-R): buserelin (e.g., buserelin acetate, trade names Suprefact® (e.g., 0.5-02 mg s.c./day), Suprefact Depot®, and Suprefact® Nasal Spray (e.g., 2 μg per nostril, every 8 hrs.), Hoechst, also described in U.S. Patent Nos. 4,003,884,
4,118,483, and 4,275,001); Cystorelin® (e.g., gonadorelin diacetate tetrahydrate, Hoechst); deslorelin (e.g., desorelin acetate, Deslorell®, Balance Pharmaceuticals); gonadorelin (e.g., gonadorelin hydrocholoride, trade name Factrel® (100 μg i.v. or s.α), Ayerst Laboratories); goserelin (goserelin acetate, trade name Zoladex®, AstraZeneca, Aukland, NZ, also described in U.S. Patent Nos. 4,100,274 and 4,128,638; GB 9112859 and GB 9112825); histrelin (e.g., histerelin acetate, Supprelin®, (s.c.,10 μg/kg.day), Ortho, also described in EP 217659); leuprolide (leuprolide acetate, trade name Lupron® or Lupron Depot®; Abbott/TAP, Lake Forest, IL, also described in U.S. Patent Nos. 4,490,291 3,972,859, 4,008,209, 4,992,421, and 4,005,063; DE 2509783); leuprorelin (e.g., leuproelin acetate, trade name Prostap SR® (e.g., single 3.75 mg dose s.c. or i.m./month), Prostap3® (e.g., single 11.25mg dose s.c. every 3 months), Wyeth, USA, also described in Plosker et al., (1994) Drugs 48:930); lutrelin (Wyeth, USA, also described in U.S. Patent No. 4,089,946); Meterelin® (e.g., Avorelina (e.g., 10-15 mg slow-release formulation), also described in EP 23904 and WO 91/18016); nafarelin (e.g., trade name Synarel® (i.n. 200-1800 μg/day), Syntex, also described in U.S. Patent No. 4,234,571; W0 93/15722; and EP 52510); and triptorelin (e.g., triptorelin pamoate; trade names Trelstar LA® (11.25 mg over 3 months), Trelstar LA Debioclip® (pre-filled, single dose delivery), LA Trelstar Depot® (3.75 mg over one month), and Decapeptyl®, Debiopharm S.A., Switzerland, also described in U.S. Patent Nos. 4,010,125, 4,018,726, 4,024,121, and 5,258,492; EP 364819). LHRH analogs also include, but are not limited to, the following antagonists of the LHRH-R: abarelix (trade name Plenaxis™ (e.g., 100 mg i.m. on days 1, 15 and 29, then every 4 weeks thereafter), Praecis Pharmaceuticals, Inc., Cambridge, MA) and cetrorelix (e.g., cetrorelix acetate, trade name Cetrotide™ (e.g., 0.25 or 3 mg s.c), Zentaris, Frankfurt, Germany). Additional sex steroid analogs include Eulexin® (e.g., flutamide (e.g., 2 capsules 2x/day, total 750 mg/day), Schering-Plough Corp., also described in FR 7923545, WO 86/01105 and PT 100899), and dioxane derivatives (e.g., those described in EP 413209), and other LHRH analogs such as are described in EP 181236, U.S. Patent Nos. 4,608,251, 4,656,247, 4,642,332, 4,010,149, 3,992,365, and 4,010,149. Combinations of agonists, combinations of antagonists, and combinations of agonists and antagonists are also included. One non-limiting analog of the
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invention is deslorelin (described in U.S. Patent No. 4,218,439). For a more extensive list, of analogs, see Vickery et al. (1984) LHRH AND ITS ANALOGS: CONTRACEPTIVE & THERAPEUTIC APPLICAΉONS (Vickery et al, eds.) MTP Press Ltd., Lancaster, PA. Each analog may also be used in modified form, such as acetates, citrates and other salts thereof, which are well known to those in the art.
One non-limiting example of administration of a sex steroid ablating agent is a subcutaneous/intradermal injection of a "slow-release" depot of GnRH agonist (e.g., one, three, or four month Lupron® injections) or a subcutaneous/intradermal injection of a "slow- release" GnRH-containing implant (e.g., one or three month Zoladex®, e.g., 3.6 mg or 10.8 mg implant). These could also be given intramuscular (i.m.), intravenously (i.v.) or orally, depending on the appropriate formulation. Another example is by subcutaneous injection of a "depot" or "impregnated implant" containing, for example, about 30 mg of Lupron® (e.g., Lupron Depot® ,(leuprolide acetate for depot suspension) TAP Pharmaceuticals Products, Inc., Lake Forest, IL). A 30 mg Lupron® injection is sufficient for four months of sex steroid ablation to allow the thymus to rejuvenate and export new na ve T cells into the blood stream.
Many of the mechanisms of inhibiting sex steroid signaling described herein are well known and some of these drugs, in particular the GnRH angonists, have been used for many years in the treatment of disorders of the reproductive organs, such as some hormone sensitive cancers including, breast and prostate cancer, endometriosis, reproductive disorders, hirsuitism, precocuis puberty, sexual deviancy and in the control of fertility.
In certain examples, the thymus of the patient is ultimately reactivated by sex steroid ablation and/or interruption or disruption of sex steroid-mediated signalling. In some cases, disruption reverses the hormonal status of the patient. According to the methods of the invention, the hormonal status of the recipient is reversed such that the hormones of the recipient approach pre-pubertal levels. By lowering the level of sex steroid hormones in the recipient, the signalling of these hormones to the thymus is lowered, thereby allowing the thymus to be reactivated. The patient may be pubertal or post-pubertal, or the patient has (or has had) a disease that at least in part atrophied the thymus. Alternatively, the patient has (or has had) a treatment of a disease, wherein the treatment of the disease at least in part atrophied the thymus of the patient. Such treatment may be anti-viral, immunosuppression, chemotherapy, and/or radiation treatment. In other embodiments, the patient is menopausal
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or has had sex steroid (or other hormonal levels) decreased by another means, e.g., trauma, drugs, etc.
In some embodiments, sex steroid ablation or inhibition of sex steroid signaling is accomplished by administering an anti-androgen such as an androgen blocker (e.g., bicalutamide, trade names Cosudex® or Casodex®, 5-500 mg, e.g., 50 mg po QJD,
AstraZeneca, Aukland, NZ), either alone or in combination with an LHRH analog or any other method of castration. Sex steroid ablation or interruption of sex steroid signaling may also be accomplished by administering cyproterone acetate (trade name, Androcor®, Shering AG, Germany; e.g., 10-1000 mg, 100 mg bd or tds, or 300 mg LM weekly, a 17- hydroxyprogesterone acetate, which acts as a progestin, either alone or in combination with an LHRH analog or any other method of castration. Other anti-androgens may be used (e.g., antifungal agents of the imidazole class, such as liarozole (Liazol® e.g., 150 mg/day, an aromatase inhibitor) and ketoconazole, flutamide (trade names Euflex® and Eulexin®, Shering Plough Coφ, N.J.; 50-500 mg e.g., 250 or 750 mg po QID), megestrol acetate (Megace® e.g., 480-840 mg/day or nilutamide (trade names Anandron®, and Nilandron®, Roussel, France e.g., orally, 150-300 mg/day)). Antiandrogens are often important in therapy, since they are commonly utilized to address flare by GnRH analogs. Some antiandrogens act by inhibiting androgen receptor translocation, which interrupts negative feedback resulting in increased testosterone levels and minimal loss of libido/potency. Another class of anti-androgens useful in the present invention are the selective androgen receptor modulators (SARMS) (e.g., quinoline derivatives, bicalutamide (trade name Cosudex® or Casodex®, as above), and flutamide (trade name Eulexin®, e.g., orally, 250 mg/day)). Other well known anti-androgens include 5 alpha reductase inhibitors (e.g., dutasteride,(e.g., po 0.5 mg/day)) which inhibits both 5 alpha reductase isoenzymes and results in greater and more rapid DHT suppression; finasteride (trade name Proscar®; 0.5-500 mg, e.g.,, 5 mg po daily), which inhibits 5alpha reductase 2 and consequent DHT production, but has little or no effect on testosterone or LH levels);
In other embodiments, sex steroid ablation or inhibition of sex steroid signaling is accomplished by administering anti-estrogens either alone or in combination with an LHRH analog or any other method of castration. Some anti-estrogens (e.g., anastrozole (trade name Arimidex®), and fulvestrant (trade name Faslodex®, 10-1000 mg, e.g., 250 mg LM monthly) act by binding the estrogen receptor (ER) with high affinity similar to estradiol and consequently inhibiting estrogen from binding. Faslodex® binding also triggers
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conformational change to the receptor and down-regulation of estrogen receptors, without significant change in FSH or LH levels. Other non-limiting examples of anti -estrogens are tamoxifen (trade name Nolvadex®); Clomiphene (trade name Clomid®) e.g., 50-250 mg/day, a non-steroidal ER ligand with mixed agonist/antagonist properties, which stimulates release of gonadotrophins; diethylstilbestrol ((DES), trade name Stilphostrol®) e.g., 1-3 mg/day, which shows estrogenic activity similar to, but greater than, that of estrone, and is therefore considered an estrogen agonist, but binds both androgen and estrogen receptors to induce feedback inhibition on FSH and LH production by the pituitary, diethylstilbestrol diphosphate e.g., 50 to 200 mg/day; as well as danazol, , droloxifene, and iodoxyfene, which each act as antagonists. Another class of anti-estrogens which may be used either alone or in combination with other methods of castration, are the selective estrogen receptor modulators (SERMS) (e.g., toremifene (trade name Fareston®, 5-1000 mg, e.g., 60 mg po QID), raloxofene (trade name Evista®), and tamoxifen (trade name Nolvadex®, 1-1000 mg, e.g., 20 mg po bd), which behaves as an agonist at estrogen receptors in bone and the cardiovascular system, and as an antagonist at estrogen receptors in the mammary gland). Estrogen receptor downregulators (ERDs) (e.g., tamoxifen (trade name, Nolvadex®)) may also be used in the present invention.
Other non-limiting examples of methods of inhibiting sex steroid signalling which may be used either alone or in combination with other methods of castration, include aromatase inhibitors and other adrenal gland blockers (e.g., Aminoglutethimide, formestane, vorazole, exemestane, anastrozole (trade name Arimidex®, 0.1-100 mg, e.g., 1 mg po QID), which lowers estradiol and increases LH and testosterone), letrozole (trade name Femara®, 0.2-500 mg, e.g., 2.5 mg po QID), and exemestane (trade name Aromasin®) 1-2000 mg, e.g., 25 mg/day); aldosterone antagonists (e.g., spironolactone (trade name, Aldactone®) e.g., 100 to 400 mg/day), which blocks the androgen cytochrome P-450 receptor;) and eplerenone, a selective aldosterone-receptor antagonist) antiprogestogens (e.g., medroxypregesterone acetate, e.g. 5 mg/day, which inhibits testosterone syntheses and LH synthesis); and progestins and anti-progestins such as the selective progesterone response modulators (SPRM) (e.g., megestrol acetate e.g., 160 mg/day, mifepristone (RU 486, Mifeprex®, e.g. 200 mg/day); and other compounds with estrogen/antiestrogenic activity, (e.g., phytoestrogens, flavones, isoflavones and coumestan derivatives, lignans, and industrial compounds with phenolic ring (e.g., DDT)). Also, anti-GnRH vaccines (see, e.g., Hsu et al, (2000) Cancer
Res. 60:3701; Talwar, (1999) Immunol. Rev. 171: 173-92), or any other pharmaceutical which mimics the effects produced by the aforementioned drugs, may also be used. In addition,
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steroid receptor based modulators, which may be targeted to be thymic and/or BM specific, may also be developed and used. Many of these mechanisms of inhibiting sex steroid signaling are well known. Each drugs may also be used in modified form, such as acetates, citrates and other salts thereof, which are well known to those in the art.
Because of the complex and interwoven feedback mechanisms of the hormonal system, administration of sex steroids may result in inhibition of sex steroid signalling. For example, estradiol decreases gonadotropin production and sensitivity to GnRH action. However, higher levels of estradiol result in gonadotropin surge. Likewise, progesterone influences frequency and amount of LH release. In men, testosterone inhibits gonadotropin production. Estrogen administered to men decreases LH and testosterone, and anti-estrogen increases LH.
In other embodiments, prolactin is inhibited in the patient. Another means of inhibiting sex steroid mediated signaling may be by means of direct or indirect modulation of prolactin levels. Prolactin is a single-chain protein hormone synthesized as a prohormone. The normal values for prolactin are males and nonpregnant females typically range from about 0 to 20 ng/ml, but in pregnancy the range is typically about 10 to 300 ng/ml . Overall, several hundred different actions have been reported for prolactin. Prolactin stimulates breast development and milk production in females. Abnormal prolactin is known to be involved in pituitary tumors, menstrual irregularities, infertility, impotence, and galactorrhea (breast milk production). A considerable amount of research is in progress to delineate the role of prolactin in normal and pathologic immune responses. It appears that prolactin has a modulatory role in several aspects of immune function, yet there is evidence to suggest that hypeφrolactinemia is immunosuppressive (Matera L, Neuroimmunomodulation. 1997 Jul- Aug;4(4): 171-80). Administration of prolactin in pharmacological doses is associated with a decreased survival and an inhibition of cellular immune functions in septic mice. (Oberbeck R , J Surg Res. 2003 Aug;l 13(2):248-56). There are also a large number of drugs which impair dopaminergic inhibition of prolactin and give rise to hypeφrolactinemia. Antidopaminergic agents include haloperidol, fluphenazine, sulpiride, metoclopramide and gastrointestinal prokinetics (e.g., bromopride, clebopride, domperidone and levosulpiride ) which have been exploited clinically for the management of motor disorders of the upper gastrointestinal tract.
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Inhibin A and B peptides made in the gonads in response to gonadotropins, down regulates the pituitary and suppress FSH. Activin normally up regulates GnRH receptors and stimulate FSH synthesis, however over production may shut down sex steroid production. Thus these hormones may also be the target of inhibition of sex steroid-mediated signalling.
In certain embodiments, an LHRH-R antagonist is delivered to the patient, followed by an LHRH-R agonist. For example, the antagonist can be administered as a single injection of sufficient dose to cause castration within 5-8 days (this is normal for, e.g., Abarelix). When the sex steroids have reached this castrate level, the agonist is given. This protocol abolishes or limits any spike of sex steroid production, before the decrease in sex steroid production, that might be produced by the administration of the agonist. In an alternate embodiment, an LHRH-R agonist that creates little or no sex steroid production spike is used, with or without the prior administration of an LHRH-R antagonist.
Inhibition of sex steroid signalling
Sex steroids comprise a large number of the androgen, estrogen and progestin family of hormone molecules. Non-limiting members of the progestin family of C21 steroids include progesterone, 17α-hydroxy progesterone, 20α-hydroxy progesterone, pregnanedione, pregnanediol and pregnenolone. Non-limiting members of the androgen family of C19 steroids include testosterone, androstenedione, dihydrotesterone (DHT), androstanedione, androstandiol, dehydroepiandrosterone and 17α-hydroxy androstenedione. Non-limiting members of the estrogen family of C17 steroids include estrone, estradiol- 17 α, and estradiol- 17β.
Signalling by sex steroids is the net result of complex outcomes of the components of the pathway that includes biosynthesis, secretion, metabolism, compartmentalization and action. Parts of this pathway are not fully understood; nevertheless, there are numerous existing and potential mechanisms for achieving inhibition of sex steroid signalling. In one aspect of the present invention, inhibition of sex steroid signalling is achieved by modifying the bioavailable sex steroid hormone levels at the cellular level, the so called 'free' levels, by altering biosynthesis or metabolism, the binding to sex steroid receptors on or in target cells, and/or intracellular signalling of sex steroids.
it is possible to influence the signalling pathways either directly or indirectly. The direct methods include methods of influencing sex steroid biosynthesis and metabolism,
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binding to the respective receptor and intracellular modification of the signal. The indirect methods include those methods known to influence sex steroid hormone production and action such as the peptide hormone and growth factors present in the pituitary gland and the gonad. The latter include but not be limited to follicle stimulating hormone (FSH), luteinizing hormone (LH) and activin made by the pituitary gland, and inhibin, activin and insulin-like growth factor- 1 (IGF-1) made by the gonad.
The person skilled in the art will appreciate that inhibition of sex steroid signaling may take place by making the aforementioned modifications at the level of the relevant hormone, enzyme, receptor, binding molecule and/or ligand, either by direct action upon that molecule or by action upon a precursor of that molecule, including a nucleic acid that encodes or regulates it, or a molecule that can modify the action of sex steroid.
Direct methods of inhibiting signaling
Biosynthesis
The rate of biosynthesis is the major rate determining step in the production of steroid hormones and hence the bioavailability of 'free' hormone in serum. Inhibition of a key enzyme such as P450 cholesterol side chain cleavage (P450scc), early in the pathway, will reduce production of all the major sex steroids. On the other hand, inhibition of enzymes later in the pathway, such as P450 aromatase (P450arom) that converts androgens to estrogens, or 5α-reductase that converts testosterone to DHT, will only effect the production of estrogens or DHT, respectively. Another important facet of sex steroid hormone biosynthesis is the family of oxidoreductase enzymes that catalyze the interconversion of inactive to bioactive steroids, for example, androstenedione to testosterone or estrone to estradiol- 17 Qby 17-hydroxysteroid dehydrogenase (17-HSD). These enzymes are tissue and cell specific and generally catalyze either the reduction or oxidation reaction e.g., 17β HSD type 3 is found exclusively in the Leydig cells of the testes, whereas 17β HSD type 1 is found in the ovary. They therefore offer the possibility of specifically reducing production of the active forms of androgens or estrogens.
There are many known inhibitors of the enzymes in the steroid biosynthesis pathway that are either already in clinical use or are under development. Some examples of these together with their treatment modalities are listed above. It is important that the action of these enzyme inhibitors does not unduly influence production of other steroids such as
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glucocorticoids and mineralocorticoids from the adrenal gland that are essential for metabolic stability. When using such inhibitors, it may be necessary to provide the patient with replacement glucocorticoids and sometimes mineralocorticoids.
Sex steroid biosynthesis occurs in varied sites and utilizing multiple pathways, predominantly produced the ovaries and testes, but there is some production in the adrenals, as well as synthesis of derivatives in other tissues, such as fat. Thus multiple mechanisms of inhibiting sex steroid signaling may be required to ensure adequate inhibition to achieve the present invention.
Metabolism and compartmentalization
Sex steroid hormones have a short half-life in blood, generally only several minutes, due to the rapid metabolism, particularly by the liver, and clearance by the kidney and fat. Metabolism includes conjugation by glycosylation and sulphation, as well as reduction. Some of these metabolites retain biological activity either as prohormones, for example estrone sulphate, or through intrinsic bioactivity such as the reduced androgens. Any interference in the rate of metabolism can influence the 'free' levels of sex steroid hormones., however methods of achieving this are not currently available as are methods of influencing biosynthesis.
Another method of reducing the level of 'free' sex steroid hormone is by compartmentalization by binding of the sex steroid hormone to proteins present in the serum such as sex hormone binding globulin, corticosteroid-binding globulin, albumin and testosterone-estradiol binding globulin. Binding to sex steroid ligands, such as carrier molecules may make sex steroids unavailable for receptor binding. Increased binding may result from increased levels of carriers, such as SHBG or introduction of other ligands which bind the sex steroids, such as soluble receptors. Alternatively decreased levels of carrier molecules may make sex steroids more susceptible to degradation.
Active or passive immunization against a particular sex steroid hormone is a form of compartmentalization. There are examples in the literature of this approach successfully increasing ovulation rates in animals after immunization against estrogen or androgen. Sex steroids are secreted from cells in secretory vesicles. Inhibition or modification of the secretory mechanism is another method of inhibiting sex steroid signaling
Receptors and intracellular signalling
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The sex steroids act on cells via specific receptors that can be either intracellular, or, as shown more recently, on the target cell membrane.
The intracellular receptors are members of the nuclear receptor superfamily. They are located in the cytoplasm of the cell and are transported to the nucleus after binding with the sex steroid hormone where they alter the transcription of specific genes. Receptors for the sex steroid hormones exist in several forms. Well known in the literature are two forms of the progesterone receptor, PRA and PRB, and three forms of the estrogen receptor, ERα, ERβl and ERβ2. Transcription of genes in response to the binding of the sex steroid hormone receptor to the steroid response element in the promoter region of the gene can be modified in a number of ways. Co-activators and co-repressors exist within the nucleus of the target cell that can modify binding of the steroid-receptor complex to the DNA and thereby effect transcription. The identity of many of these co-activators and co-repressors are known and methods of modifying their actions on steroid receptors are the topic of current research. Examples of the transcription factors involved in sex steroid hormone action are NF-1, SP1, Oct-land TFIID. These co-regulators are required for the full action of the steroids. Methods of modifying the actions of these nuclear regulators could involve the balance between activator and repressor by the use of antagonists or through control of expression of the genes encoding the regulators.
More recently, specific receptors for estrogens and progesterone have been identified on the membranes of cells whose structures are different from the intracellular PR. Unlike the classical steroid receptors that act on the genome, these receptors deliver a rapid non- genomic action via intracellular pathways that are not yet fully understood. One report suggests that estrogens interacting with membrane receptors activate the sphingosine pathway that is related to cell proliferation.
There are methods available or in development to alter the action of steroids via their cytoplasmic receptors. In this case, antiandrogens, antiestrogens and antiprogestins that interact with the specific steroid receptors, are well known in the literature and are in clinical use, as described above. Their action may be to compete for, or block the receptor, to modify receptor levels, sensitivity, conformation, associations or signaling. These drugs come in a variety of forms, steroidal and non-steroidal, competitive and non-competitive. Of particular interest are the selective receptor modulators, SARMS, SERMS and SPRM, which are targeted to particular tissues and are exemplified above.
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Down regulation of receptors can be achieved in 2 ways; first, by excess agonist (steroid ligand), and second, by inhibiting transcription of the respective gene that encodes the receptor. The first method can be achieved through the use of selective agonists such as tamoxifen. The second method is not yet in clinical use.
Indirect methods of inhibiting signaling
Biosynthesis
One of the indirect methods of inhibiting sex steroid signalling involves down regulation of the biosynthesis of the respective steroid by a modification to the availability or action of the pituitary gonadotrophins, FSH and LH, that are responsible for driving the biosynthesis of the sex steroid hormones in the gonad. One established inhibitor of FSH secretion is inhibin, a hormone produced by the gonads in response to FSH. Administration of inhibin to animals has been shown to reduce FSH levels in serum due to a decrease in the pituitary secretion of FSH. The best known way of accomplishing a reduction in both gonadotrophins is via the hypothalamic hormone, GnRH/LHRH, which drives the pituitary synthesis and secretion of FSH and LH. Agonists and antagonists of GnRH that reduce the secretion of FSH and LH, and hence gonadal sex steroid production, are now available for clinical use, as described herein.
Another indirect method of reducing the biosynthesis of sex steroid hormones is to modify the action of FSH and LH at the level of the gonad. This could be achieved by using antibodies directed against FSH and LH, or molecules designed to compete with FSH and LH for their respective receptors on gonadal cells that produce the sex steroid hormones. Another method of modifying the action of FSH and LH on gonadal cells is by a co-regulator of gonadotrophin action. For example, activin can reduce the capacity of the theca cells of the ovary and the Leydig cells of the testis to produce androgen in response to LH.
Modification may take place at the level of hormone precursors such as inhibition of cleavage of a signal peptide, for example the signal peptide of GnRH.
Receptors and intracellular signalling
indirect methods of altering the signalling action of the sex steroid hormones include down-regulation of the receptor pathways leading to the genomic or non-genomic actions of the steroids. An example of this is the capacity of progesterone to down regulate the level of
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ER in target tissues. Future methods include treatment with molecules known to influence the co-regulators of the receptors in the cell nucleus leading to a decrease in the capacity of the cell to respond to the steroid.
Additional factors
While the stimulus for the direct and indirect effects on thymic reactivation is fundamentally based on the inhibition of the effects of sex steroids and/or the direct effects of the LHRH analogs, it may be useful to include additional substances which can act in concert to enhance or increase (additive, synergistic, or complementary) the thymic, BM, and/or immune cell effects and functionality. Additional substances may or may not be used. Such compounds include, but are not limited to, cytokines and growth factors, such as interleukin-2 (TL-2; 100,000 to 1,000,000 IU, e.g., 600,000 IU/Kg every 8 hours by IV repeat doses), interleukin-7 (IL-7; lOng/kg/day to lOOmcg/kg/day subject to therapeutic discretion), interleukin-15 (IL-15; 0.1-20 mug/kg IL-15 per day), interleukin 11 (JX-11; 1-1000 μg/kg) members of the epithelial and fibroblast growth factor families, stem cell factor (SCF; also known as steel factor or c-kit ligand; 0.25-12.5 mg/ml), granulocyte colony stimulating factor (G-CSF; 1 and 15 μg/kg/day IV or SC), granulocyte macrophage stimulating factor (GM- CSF; 50-1000 μg/sq meter/day SC or IV), insulin dependent growth factor (IGF-1), and keratinocyte growth factor (KGF; 1 μg/kg to 100 mg/kg/day) (see, e.g., Sempowski et al, (2000) J. Immunol. 164:2180; Andrew and Aspinall, (2001) J. Immunol. 166: 1524-1530; Rossi et al, (2002) Blood 100:682); erythropoietin (EPO; 10-500units/kg IV or SC). A nonexclusive list of other appropriate hematopoietins, CSFs, cytokines, lymphokines, hematopoietic growth factors and interleukins for simultaneous or serial co-administration with the present invention includes, Meg-CSF (Megakaryocyte-Colony Stimulating Factor, more recently referred to as c-mpl ligand), MIF (Macrophage Inhibitory Factor), LIF (Leukemia Inhibitory Factor), TNF (Tumor Necrosis Factor), IGF, platelet derived growth factor (PDGF), M-CSF, TL-1 , IL-4, IL-5, IL-6, IL-8, JX-9, IL-10, IL-12, IL-13, LIF, flt3/flk2, human growth hormone, B-cell growth factor, B-cell differentiation factor and eosinophil differentiation factor, or combinations thereof.
One or more of these additional compound(s) may be given once at the initial LHRH analog (or other castration method) application. Each treatment may be given in combination with the agonist, antagonist or any other form of sex steroid disruption. Since the growth factors have a relatively rapid half-life (e.g., in the hours) they may need to be given each day
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(e.g., every day for 7 days or longer). The growth factors/cytokines may be given in the optimal form to preserve their biological activities, as prescribed by the manufacturer, e.g., in the form of purified proteins. However, additional doses of any one or combination of these substances may be given at any time to further stimulate the thymus. In certain cases, sex steroid ablation or interruption of sex steroid signalling is done concurrently with the administration of additional cytokines, growth factors, or combinations thereof. In other cases, sex steroid ablation or interruption of sex steroid signalling is done sequentially with the administration of additional cytokines, growth factors, or combinations thereof.
PHARMACEUTICAL COMPOSITIONS
The compounds used in this invention can be supplied in any pharmaceutically acceptable carrier or without a carrier. Formulations of pharmaceutical compositions can be prepared according to standard methods (see, e.g., Remington, The Science and Practice of Pharmacy, Gennaro A.R., ed., 20th edition, Williams & Wilkins PA, USA 2000). Non- limiting examples of pharmaceutically acceptable carriers include physiologically compatible coatings, solvents and diluents. For parenteral, subcutaneous, intravenous and intramuscular administration, the compositions may be protected such as by encapsulation. Alternatively, the compositions may be provided with carriers that protect the active ingredient(s), while allowing a slow release of those ingredients. Numerous polymers and copolymers are known in the art for preparing time-release preparations, such as various versions of lactic acid/glycolic acid copolymers. See, for example, U.S. Patent No. 5,410,016, which uses modified polymers of polyethylene glycol (PEG) as a biodegradable coating.
Formulations intended to be delivered orally can be prepared as liquids, capsules, tablets, and the like. These compositions can include, for example, excipients, diluents, and/or coverings that protect the active ingredient(s) from decomposition. Such formulations are well known (see, e.g., Remington, The Science and Practice of Pharmacy, Gennaro A.R., ed., 20th edition, Williams & Wilkins PA, USA 2000).
In any of the formulations of the invention, other compounds that do not negatively affect the activity of the LHRH analogs (i.e., compounds that do not block the ability of an LHRH analog to disrupt sex steroid hormone signaling) may be included. Examples are various growth factors and other cytokines as described herein.
Dose
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Doses of a sex steroid analog or inhibitor used, in according with the invention, to disrupt sex steroid hormone signaling, can be readily determined by a routinely trained physician or veterinarian, and may be also be determined by consulting medical literature (e.g., THE PHYSICIAN'S DESK REFERENCE, 52ND EDITION, Medical Economics Company, 1998).
The dosage regimen involved in a method for treating the above-described conditions will be determined by the attending physician considering various factors which modify the action of drugs, e.g., the condition, body weight, sex and diet of the patient, the severity of any illness, time of administration and other clinical factors. Progress of the treated patient can be monitored by periodic assessment of the hematological profile, e.g., differential cell count and the like.
The dosing recited above is adjusted to compensate for additional components in the therapeutic composition. These include co-administration with other CSF, cytokine, lymphokine, interleukin, hematopoietic growth factor; co-administration with chemotherapeutic drugs and/or radiation; and various patient-related issues as identified by the attending physician such as factors which modify the action of drugs, e.g., the condition, body weight, sex and diet of the patient, the severity of any illness, time of administration and other clinical factors.
In addition to dosing described above, for example, LHRH analogs and other sex steroid analogs can be administered in a one-time dose that will last for a period of time (e.g., 3 to 6 months). In certain cases, the formulation will be effective for one to two months. The standard dose varies with type of analog used, but is readily determinable by those skilled in the art without undue experimentation. In general, the dose is between about 0.01 mg/kg and about 10 mg/kg, or between about 0.01 mg/kg and about 5 mg/kg.
The length of time of sex steroid inhibition or LHRH/GnRH analog treatment varies with the degree of thymic atrophy and damage, and is readily determinably by those skilled in the art without undue experimentation. For example, the older the patient, or the more the patient has been exposed to T cell depleting reagents such as chemotherapy or radiotherapy, the longer it is likely that they will require treatment, for example with GnRH. Four months is generally considered long enough to detect new T cells in the blood. Methods of detecting new T cells in the blood are known in the art. For instance, one method of T cell detection is by determining the existence of T cell receptor excision circles (TRECs), which are formed
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when the TCR is being formed and are lost in the cell after it divides. Hence, TRECs are only found in new (na ve) T cells. TREC levels are an indicator of thymic function in humans. These and other methods are described in detail in WO/00 230,256.
Dose varies with the sex steroid inhibitor or, e.g. anti-sex steroid vaccine or other blocker used. In certain cases, a dose may be prepared to last as long as a periodic epidemic lasts. For example, "flu season" occurs usually during the winter months. A formulation of an LHRH analog can be made and delivered as described herein to protect a patient for a period of two or more months starting at the beginning of the flu season, with additional doses delivered every two or more months until the risk of infection decreases or disappears.
The formulation can be made to enhance the immune system. Alternatively, the formulation can be prepared to specifically deter infection by e.g., influenza (flu) viruses while also enhancing the immune system. This latter formulation may include genetically modified (GM) cells that have been engineered to create resistance to flu viruses (see below). The GM cells can be administered with the sex steroid analog or LHRH analog formulation or separately, both spatially and/or in time. As with the non-GM cells, multiple doses over time can be administered to a patient to create protection and prevent infection with the flu virus over the length of the flu season.
As will be understood by persons skilled in the art, at least some of the means for disrupting sex steroid signalling will only be effective as long as the appropriate compound is administered. As a result, an advantage of certain embodiments of the present invention is that once the desired immunological affects of the present invention have been achieved, (2-3 months) the treatment can be stopped and thee subjects reproductive system will return to normal.
Delivery Of Agents For Chemical Castration
Administration of sex steroid ablating agents may be by any method which delivers the agent into the body. Thus, the sex steroid ablating agent maybe be administered, in accordance with the invention, by any route including, without limitation, intravenous, subdermal, subcutaneous, intramuscular, topical, and oral routes of administration.
In addition to the methods described above, delivery of the compounds for use in the methods of this invention may be accomplished via a number of methods known to persons skilled in the art. One standard procedure for administering chemical inhibitors to inhibit sex
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steroid mediated signalling utilizes a single dose of an LHRH agonist that is effective for three months. For this a simple one-time i.v. or i.m. injection would not be sufficient as the agonist would be cleared from the patient's body well before the three months are over. Instead, a depot injection or an implant may be used, or any other means of delivery of the inhibitor that will allow slow release of the inhibitor. Likewise, a method for increasing the half-life of the inhibitor within the body, such as by modification of the chemical, while retaining the function required herein, may be used.
Useful delivery mechanisms include, but are not limited to, laser irradiation of the skin. This embodiment is described in more detail in co-owned, co-pending U.S. Serial No. 10/418,727 and also in U.S. Patent Nos. 4,775,361, 5,643,252, 5,839,446, 6,056,738,
6,315,772, and 6,251,099. Another useful delivery mechanism includes the creation of high pressure impulse transients (also called stress waves or impulse transients) on the skin. This embodiment is described in more detail in co-owned, co-pending U.S. Serial No. 10/418,727 and also U.S. Patent Nos. 5,614,502 and 5,658,822. Each method may be accompanied or followed by placement of the compound(s) with or without carrier at the same locus. One method of this placement is in a patch placed and maintained on the skin for the duration of the treatment.
Timing
In one case, the administration of agents (or other methods of castration) that ablate sex steroids or interrupt to sex steroid signaling occurs prior to a, e.g., a chemotherapy or radiation regimen that is likely to cause some BM marrow cell ablation and/or damage to circulating immune cells.
Cells
Injection of hematopoietic progenitor cells, e.g., broadly defined as CD34+ hematopoietic cells (ideally autologous) can enhance the degree and kinetics of thymic regrowth. HSC may also be further defined as Thy-1 low and CD38- ; CD34+CD38-; Thy-1 low cells also lack markers of other cell lineages (lin -ve) are the more primitive HSC being longer lasting or having longer-term repopulating capacity.
The methods of the various inventions described herein can be supplemented by the addition of, e.g., CD34+ HSC and/or epithelial stem cells. In one instance, these cells are autologous or syngeneic and have been obtained from the patient or twin prior to thymus
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reactivation. The HSC can be obtained by sorting CD34+ or CD3410 cells from the patient's blood and/or BM. The number of HSC can be enhanced in several ways, including (but not limited to) by administering G-CSF (Neupogen, Amgen) to the patient prior to collecting cells, culturing the collected cells in SCGF, and/or administering G-CSF to the patient after CD34+ cell supplementation. Alternatively, the CD34+ cells need not be sorted from the blood or BM if their population is enhanced by prior injection of G-CSF into the patient
HSC may be used for genetic modification. These may be derived from BM, peripheral blood, or umbilical cord, or any other source of HSC, and may be either autologous or nonautologous. Also useful are lymphoid and myeloid progenitor cells, mesenchymal stem cells also found in the bone marrow and epithelial stem cells, also either autologous or nonautologous. The stem cells may also include umbilical cord blood. They may also include stem cells which have the potential to form into many different cell types . e.g. embryonic stem cells and adult stem cells now found in may tissues, e.g., BM, pancreas, brain, and the olfactory system.
In the event that nonautologous (donor) cells are used, tolerance to these cells is created during or after thymus reactivation. During or after the initiation of blockage of sex steroid mediated signaling, the relevant (genetically modified (GM) or non-genetically modified) donor cells are transplanted into the recipient. These cells, ideally stem or progenitor cells, are incoφorated into and accepted by the thymus wherein they create tolerance to the donor by eliminating any newly produced T cells which by chance could be reactive against them. They are then "belonging to the recipient" and may become part of the production of new T cells and DC by the thymus. The resulting population of T cells recognize both the recipient and donor as self, thereby creating tolerance for a graft from the donor (see co-owned, co-pending U.S. Serial No. 10/419, 039 and PCT/IB 01/02740).
In another embodiment the administration of stem or precursor donor cells
(genetically modified or not genetically modified) comprises cells from more than one individual, so that the recipient develops tolerance to a range of MHC types, enabling the recipient to be considered a suitable candidate for a cell, tissue or organs transplant more easily or quickly, since they are an MHC match to a wider range of donors.
The present invention also provides methods for incoφoration of foreign DC into a patient's thymus. This may be accomplished by the administration of donor cells to a recipient to create tolerance in the recipient. The donor cells may be HSC, epithelial stem
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cells, adult or embryonic stem cells, or hematopoietic progenitor cells. The donor cells may be CD34+ HSC, lymphoid progenitor cells, or myeloid progenitor cells. In some cases, the donor cells are CD34+ or CD341o HSC. The donor HSC may develop into DC in the recipient. The donor cells may be administered to the recipient and migrate through the peripheral blood system to the reactivating thymus either directly or via the BM.
The uptake into the thymus of the hematopoietic precursor cells is substantially increased in the inhibition or absence of sex steroids. These cells become integrated into the thymus and produce DC, NK, NKT, and T cells in the same manner as do the recipient's cells. The result is a chimera of T cells, DC and the other cells. The incoφoration of donor DC in the recipient's thymus means that T cells produced by this thymus will be selected such that they are tolerant to donor cells. Such tolerance allows for a further transplant from the donor (or closely matched to the donor) of cells, tissues and organs with a reduced need for immonusuppressive drugs since the transplanted material will be recognized by the recipient's immune system as self.
Optional Genetic Modification Of Stem Or Progenitor Cells
The present disclosure also comprises methods for optionally altering the immune system of an individual and methods of gene therapy. This is accomplished by the administration of GM cells to a recipient and through disruption of sex steroid mediated signaling.
Methods for isolating and transducing stems cells and progenitor cells are well known to those skilled in the art. Examples of these types of processes are described, for example, in PCT Publication No. WO 95/08105, U.S. Patent No. 5,559,703, U.S. Patent No. 5,399,493, U.S. Patent No. 5,061,620, PCT Publication No. WO 96/33281, PCT Publication No. WO 96/33282, U.S. Patent No. 5,681,559 and U.S. Patent No. 5,199,942.
Antisense Polynu eotides
The term "antisense" is herein defined as a polynucleotide sequence which is complementary to a polynucleotide of the present invention. The polynucleotide may be DNA or RNA. Antisense molecules may be produced by any method, including synthesis by ligating the gene(s) of interest in a reverse orientation to a viral promoter which permits the synthesis of a complementary strand. Once introduced into a cell, this transcribed strand combines with natural sequences produced by the cell to form duplexes. These duplexes then
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block either the further transcription or translation. In this manner, mutant phenotypes may be generated.
Catalytic Nucleic Acids
The term "catalytic nucleic acid" is herein defined as a DNA molecule or DNA containing molecule (also known in the art as a "deoxyribozyme" or "DNAzyme") or an RNA or RNA-containing molecule (also known as a "ribozyme") which specifically recognizes a distinct substrate and catalyzes the chemical modification of this substrate. The nucleic acid bases in the catalytic nucleic acid can be bases A, C, G, T and U, as well as derivatives thereof. Derivatives of these bases are well known in the art.
Typically, the catalytic nucleic acid contains an antisense sequence for specific recognition of a target nucleic acid, and a nucleic acid cleaving enzymatic activity. The catalytic strand cleaves a specific site in a target nucleic acid. The types of ribozymes that are particularly useful in this invention are the hammerhead ribozyme (Haseloff and Gerlach (1988) Nature 334:585), Perriman et al, (1992) Gene 113:157) and the haiφin ribozyme (Shippy et al, (1999) Mol. Biotechnol 12: 117).
dsRNA
Double stranded RNA (dsRNA) is particularly useful for specifically inhibiting the production of a particular protein. Although not wishing to be limited by theory, one group has provided a model for the mechanism by which dsRNA can be used to reduce protein production (Dougherty and Parks, (1995), Curr. Opin. Cell Biol. 7:399). This model has more recently been modified and expanded (Waterhouse et al, (1998) Proc. Natl. Acad. Sci. USA 95:13959). This technology relies on the presence of dsRNA molecules that contain a sequence that is essentially identical to the mRNA of the gene of interest, in this case an mRNA encoding a polypeptide according to the first aspect of the invention. Conveniently, the dsRNA can be produced in a single open reading frame in a recombinant vector or host cell, where the sense and antisense sequences are flanked by an unrelated sequence which enables the sense and anti-sense sequences to hybridize to form the dsRNA molecule with the unrelated sequence forming a loop structure. The design and production of suitable dsRNA molecules for the present invention are well within the capacity of a person skilled in the art, particularly considering Dougherty and Parks, (1995), Curr. Opin. Cell Biol. 7:399;
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Waterhouse et al, (1998) Proc. Natl. Acad. Sci. USA 95:13959; and PCT Publication Nos. WO 99/32619, WO 99/53050, WO 99/49029, and WO 01/34815.
Anti-HIV Constructs
Those skilled in the art are able to develop suitable anti-HIV constructs for use in the present invention. Indeed, a number of anti-HIV antisense constructs and ribozymes have already been developed and are described, for example; in U.S. Patent No. 5,811,275, U.S. Patent No. 5,741,706, PCT Publication No. WO 94/26877, Australian Patent Application No. 56394/94 and U.S. Patent No. 5,144,019.
Genes
Useful genes and gene fragments (polynudeotides) for this invention include those that code for resistance to infection of T cells by a particular infectious agent or agents. Such infectious agents include, but are not limited to, HIV, T cell leukemia virus, and other viruses that cause lymphoproliferative diseases.
With respect to HIV/ AIDS, a number of genes and/or gene fragments may be used, including, but not limited to, the nef transcription factor; a gene that codes for a ribozyme that specifically cuts HIV genes, such as tat and rev (Bauer et al, (1997) Blood 89:2259); the trans-dominant mutant form of HIV-1 rev gene, RevMlO, which has been shown to inhibit HIV replication (Bonyhadi et al, (1997) J. Virol. 71:4707); an overexpression construct of the HIV-1 rev-responsive element (RRE) (Kohn et al, (1999) Blood 94:368); any gene that codes for an RNA or protein whose expression is inhibitory to HIV infection of the cell or replication; and fragments and combinations thereof.
These genes or gene fragments are used in a stably expressible form. These genes or gene fragments may be used in a stably expressible form. The term "stably expressible" is herein defined to mean that the product (RNA and/or protein) of the gene or gene fragment ("functional fragment") is capable of being expressed on at least a semi-permanent basis in a host cell after transfer of the gene or gene fragment to that cell, as well as in that cell's progeny after division and/or differentiation. This requires that the gene or gene fragment, whether or not contained in a vector, has appropriate signaling sequences for transcription of the DNA to RNA. Additionally, when a protein coded for by the gene or gene fragment is the active molecule that affects the patient's condition, the DNA will also code for translation signals.
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In most cases the genes or gene fragments are contained in vectors. Those of ordinary skill in the art are aware of expression vectors that may be used to express the desired RNA or protein.
Expression vectors are vectors that are capable of directing transcription of DNA sequences contained therein and translation of the resulting RNA. Expression vectors are capable of replication in the cells to be genetically modified, and include plasmids, bacteriophage, viruses, and minichromosomes. Alternatively the gene or gene fragment may become an integral part of the cell's chromosomal DNA. Recombinant vectors and methodology are in general well-known.
Expression vectors useful for expressing the proteins of the present disclosure may comprise an origin of replication. Suitably constructed expression vectors comprise an origin of replication for autonomous replication in the cells, or are capable of integrating into the host cell chromosomes. Such vectors may also contain selective markers, a limited number of useful restriction enzyme sites, a high copy number, and strong promoters. Promoters are DNA sequences that direct RNA polymerase to bind to DNA and initiate RNA synthesis; strong promoters cause such initiation at high frequency.
In one embodiment, the DNA vector construct comprises a promoter, enhancer, and a polyadenylation signal. The promoter may be selected from the group consisting of HIV, such as the Long Terminal Repeat (LTR), Simian Virus 40 (SV40), Epstein Barr virus, cytomegalovirus (CMV), Rous sarcoma virus (RSV), Moloney virus, mouse mammary tumor virus (MMTV), human actin, human myosin, human hemoglobin, human muscle creatine, human metalothionein. In one embodiment, an inducible promoter is used so that the amount and timing of expression of the inserted gene or polynucleotide can be controlled.
The enhancer may be selected from the group including, but not limited to, human actin, human myosin, human hemoglobin, human muscle creatine and viral enhancers such as those from CMV, RSV and EBV. The promoter and enhancer may be from the same or different gene.
The polyadenylation signal may be selected from the group consisting of: LTR polyadenylation signal and SV40 polyadenylation signal, particularly the SV40 minor polyadenylation signal among others.
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The expression vectors of the present disclosure may be operably linked to DNA coding for an RNA or protein to be used in this invention, i.e., the vectors are capable of directing both replication of the attached DNA molecule and expression of the RNA or protein encoded by the DNA molecule. Thus, for proteins, the expression vector must have an appropriate transcription start signal upstream of the attached DNA molecule, maintaining the correct reading frame to permit expression of the DNA molecule under the control of the control sequences and production of the desired protein encoded by the DNA molecule. Expression vectors may include, but are not limited to, cloning vectors, modified cloning vectors and specifically designed plasmids or viruses. An inducible promoter may be used so that the amount and timing of expression of the inserted gene or polynucleotide can be controlled.
One having ordinary skill in the art can produce DNA constructs which are functional in cells. In order to test expression, genetic constructs can be tested for expression levels in vitro using tissue culture of cells of the same type of those to be genetically modified.
Methods of Genetic Modification
Standard recombinant methods can be used to introduce genetic modifications into the cells being used for gene therapy. For example, retroviral vector transduction of cultured HSC is one successful method known in the art (Belmont and Jurecic (1997) "Methods for Efficient Retrovirus-Mediated Gene Transfer to Mouse Hematopoietic Stem Cells," in Gene Therapy Protocols (P.D. Robbins, ed.), Humana Press, pp.223-240; Bahnson et al, (1997)
"Method for Retrovirus-Mediated Gene Transfer to CD34+-Enriched Cells," in Gene Therapy Protocols (P.D. Robbins, ed.), Humana Press, pp.249-263). Additional vectors include, but are not limited to, those that are adenovirus derived or lentivirus derived, and Moloney murine leukemia virus-derived vectors.
Also useful for genetic modification of HSC are the following methods: particle- mediated gene transfer such as with the gene gun (Yang, N.-S. and P. Ziegelhoffer, (1994) "The Particle Bombardment System for Mammalian Gene Transfer," In PARTICLE BOMBARDMENT TECHNOLOGY FOR GENE TRANSFER (Yang, N.-S. and Christou, P., eds.), Oxford University Press, New York, pp. 117-141), liposome-mediated gene transfer (Nabel et al, (1992) Hum. Gene Ther. 3:649), coprecipitation of genetically modified vectors with calcium phosphate (Graham and Van Der Eb, (1973) Virol. 52:456), electroporation (Potter et al, (1984) Proc. Natl. Acad. Sci. USA 81:7161), and microinjection (Capecchi, (1980) Cell
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22:479), as well as any other method that can stably transfer a gene or oligonucleotide, which may be in a vector, into the HSC and other cells to be genetically modified such that the gene will be expressed at least part of the time.
Gene Therapy
The present disclosure provides methods for gene therapy through reactivation of a patient's thymus. This is accomplished by the administration of GM cells to a recipient and through disruption of sex steroid mediated signaling. By the methods described herein, the sex steroid-induced atrophic thymus is dramatically restored structurally and functionally to approximately its optimal pre-pubertal capacity in all currently definable terms. This includes the number, type and proportion of all T cell subsets. Also included are the complex stromal cells and their three dimensional architecture which constitute the thymic microenvironment required for producing T cells. The newly generated T cells emigrate from the thymus and restore peripheral T cell levels and function.
At this stage, the patient's immune system is rejuvenated and reactivated, thereby increasing its response to foreign antigens such as viruses and bacteria. This is shown, for example, in Figures 14-19, which show the effects of thymic reactivation on the mouse immune system, as demonstrated with viral (HSV) challenge. The mice having prior reactivation of the thymus demonstrate resistance to HSV infection, while those not having thymic reactivation (aged thymus) have higher levels of HSV infection. It is well known that the mouse immune system is very similar to the human immune system, and results in mice can be projected to show human responses. This is reinforced by the data showing the effects of thymic reactivation in humans.
The reactivation of the thymus can be supplemented by the addition of CD34+ hematopoietic stem cells (HSC) and/or epithelial stem cells slightly before or at the time the thymus begins to regenerate. Ideally these cells are autologous or syngeneic and have been obtained from the patient or twin prior to thymus reactivation. The HSC can be obtained by sorting CD34+ cells from the patient's blood and or bone marrow. The number of HSC can be enhanced in several ways, including (but not limited to) by administering G-CSF (Neupogen, Amgen) to the patient prior to collecting cells, culturing the collected cells in Stem Cell Growth Factor, and or administering G-CSF to the patient after CD34+ cell supplementation. Alternatively, the CD34+ cells need not be sorted from the blood or BM if their population is enhanced by prior injection of G-CSF into the patient.
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In one embodiment, hematopoietic cells are supplied to the patient during thymic reactivation, which increases the immune capabilities of the patient's body. The hematopoietic cells may or may not be genetically modified.
The genetically modified cells may be HSC, epithelial stem cells, embryonic or adult stem cells, or myeloid or lymphoid progenitor cells. In one embodiment, the genetically modified cells are CD34+ or CD341o HSC, lymphoid progenitor cells, or myeloid progenitor cells. In another embodiment, the genetically modified cells are CD34+ HSC. The genetically modified cells are administered to the patient and migrate through the peripheral blood system to the thymus. The uptake into the thymus of these hematopoietic precursor cells is substantially increased in the absence of sex steroids. These cells become integrated into the thymus and produce dendritic cells and T cells carrying the genetic modification from the altered cells. The results are a population of T cells with the desired genetic change that circulate in the peripheral blood of the recipient, and the accompanying increase in the population of cells, tissues and organs caused by reactivation of the patient's thymus.
Within 3-4 weeks of the start of blockage of sex steroid mediated signaling
(approximately 2-3 weeks after the initiation of LHRH treatment), the first new T cells may be present in the blood stream. Full development of the T cell pool, however, may take 3-4 (or more) months.
Skewing Of Developing TCR Repertoire Towards, Or Away From, Specific Antigens: Allergies And Autoimmune Diseases
The ability to enhance the uptake into the thymus of hematopoietic stem cells means that the nature and type of dendritic cells can be manipulated. For example the stem cells could be transfected with specific gene(s) which eventually become expressed in the dendritic cells in the thymus (and elsewhere in the body). Such genes could include those which encode specific antigens for which an immune response would be detrimental, as in autoimmune diseases and allergies.
This aspect of the invention stems from the discovery that reactivation of the thymus of an autoimmune patient will facilitate in overcoming an autoimmune disease suffered by that patient. This same principle also applies to patients suffering from allergies. Once the thymus is reactivated, a new immune system is created, one that no longer recognizes and/or responds to a self antigen.
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In accordance with the invention, the following protocol may be applied. A patient diagnosed with an autoimmune disease (e.g., type I diabetes) is first immunosuppressed to stop disease progression. This may be done by administering an immunosuppressant (e.g., cyclosporine or rapamycin) alone or together with anti-T and B cell antibodies, such as anti- CD3 or anti-T cell gamma globulin to get rid of T cells and anti-CD 19, CD20, or CD21 to get rid of B cells. At the same time that the patient is being immunosuppressed, his thymus may be reactivated by administering GnRH to him. His own T cells may then be mobilized with GCSF. If his autoimmunity arose as a result of a cross-reaction of his T cells with a pathogen he had previously encountered, the ablation of the T cells will remove the auto-reactive T cells, and the newly developed T cells will not continue to recognize his cells (e.g., his D- islet cells) as foreign. In this manner, his autoimmune disease is alleviated. Moreover, once his autoimmune disease has been alleviated, the sex steroid ablation therapy can be stopped, thereby restoring the patient's fertility.
In another non-limiting example of the invention, the autoimmune patient is reconstituted with allogeneic stem cells. In some embodiments, these allogeneic stem cells are umbilical cord blood cells, which do not include mature T cells.
In some embodiments, the transplanted HSC may follow full myeloablation or myelodepletion, and thus result in a full HSC transplant (e.g., 5xl06 cells/kg body weight per transplant). In some embodiments, only minor myeloablation need be achieved, for example, 2-3 Gy irradiation (or 300 rads) followed by administration of about 3-4 xlO5 cells/kg body weight. In some embodiments, T cell depletion (TCD), or another method of immune cell depletion, is used (see, e.g., Example 2). It may be that as little as 10% chimerism may be sufficient to alleviate the symptoms of the patient's allergy or autoimmune disease. In some embodiments, the donor HSC are from umbilical cord blood (e.g., 1.5x107 cells/kg for recipient engraftment).
The ability of the HSC to first colonise the BM and convert to blood cells (engraftment) is directly linked to the absolute number and quality of the HSC injected, and the functional capacity of the recipient bone marrow microenvironment and the HSC niches .The methods of the present invention either alone or in combination (concurrently or sequentially) with the administration of HSC mobilizing agents, such as cytokines (e.g., G- CSF or GM-CSF), or drugs (e.g., cyclophosphamide), allow faster and/or better engraftment
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and may also allow chemotherapy and radiation therapy to be given at higher doses and/or more frequently.
In other embodiments, patients begin to receive Lupron up to 45 days before myelo- ablative chemotherapy and continue on the Lupron concurrently with the BMT such that the total length of exposure to the drug is around 9 months (equivalent to 3 injections as each Lupron injection delivers drug over a 3 month period). At various intervals over the course of study, blood samples are collected for analysis of T cell numbers (particularly of new thymic emigrants) and functions (specifically, response to T cell stimuli in vitro). This embodiment is also generally applicable to HSCT for other purposes described herein (e.g., HSCT following cancer radiation or chemotherapy).
In other embodiments, the transplanted HSC may follow lymphoablation. In some embodiments, T cells and/or B cells may be selectively ablated, to remove cells, as needed (e.g., those cells involved in autoimmunity or allergy). The selection can involve deletion of cells that are activated, or of a cell type involved in the autoimmune or allergic response. The cells may be selected based upon cell surface markers, such as CD4, CD8, B220, thyl, TCR , CD3, CD5, CD7, CD25, CD26, CD23, CD30, CD38, CD49b, CD69, CD70, CD71, CD95, CD96, antibody specificity or Ig chain, or upregulated cytokine receptors e.g., IL2-R B chain, TGFβ. One well known method for depletion is the use of antilymphocyte globulin. Other methods of selecting and sorting cells are well known and include magnetic and fluorescent cell separation, centrifugation, and more specifically, hemapheresis, leukopheresis, and lymphopheresis.
In some embodiments, HSCT is performed without myeloablation, myelodepletion, lymphodepletion, T cell ablation, and/or other selective immune cell ablation.
In other embodiments, the methods of the invention further comprise immunosuppressing the patient by e.g., administration of an immunosuppressing agent (e.g., cyclosporin, prednisone, ozothioprine, FK506, Imunran, and/or methotrexate) (see, e.g., U.S. Patent No. 5,876,708). In an embodiment, immunosuppression is performed in the absence of HSCT. In one embodiment, immunosuppression is performed in conjunction with (e.g., prior to, concurrently with, or after) HSCT. In another embodiment, immunosuppression is performed in the absence of myeloablation, lymphoablation, T cell ablation and/or other selective immune cell ablation, deletion, or depletion. In yet another embodiment, immunosuppression is performed in conjunction with (e.g., prior to, concurrently with, or
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after) myeloablation, lymphoablation, T cell ablation, and/or other selective immune cell ablation, deletion, or depletion.
As described above, myeloablation, myelodepletion, lymphoablation, immunosuppression, T cell ablation, and/or other selective immune cell ablation, are nonlimiting exemplary types of immune cell ablation, which are used throughout this application. The general term "immune cell depletion" is defined herein as encompassing each of these methods, i.e., myeloablation, myelodepletion, lymphoablation, T cell ablation, and/or other selective immune cell ablation (e.g., B cell or NK cell depletion). As will be readily understood by one skilled in the art, in practicing the inventions provided herein, any one of these "depletion" methods may be replaced with any one (or more) of the other "depletion" methods.
In one embodiment, NK cells are depleted. NK antibodies useful for depleting the NK populations are known in the art. For example, one source of anti-NK antibody is anti- human thymocyte polyclonal anti-serum. U.S. Patent No. 6,296,846 describes NK and T cell depletion methods, as well as non-myeloablative therapy and formation of a chimeric lymphohematopoietic population, all of which may be used in the methods of the invention.
In some embodiments, the methods of the invention further comprise, e.g., prior to HSCT, absorbing natural antibodies from the blood of the recipient by hemoperfusing an organ (e.g.., the liver or kidney) obtained from the donor.
Hematopoietic stem cell transplantation (HSCT) - also commonly known as bone marrow transplantation (BMT)) - is a treatment used to enhance the recovery of the immune system in, e.g., certain critical cancer conditions. "HSCT" and "BMT" and "transplant" are used interchangeably and are herein defined as a transplant into a recipient, containing or enriched for HSC, BM cells, stem cells, and/or any other cells which gives rise to blood, thymus, BM and/ or any other immune cells, including, but not limited to, HSC, epithelial cells, common lymphoid progenitors (CLP), common myelolymphoid progenitors (CMLP), multilineage progenitors (MLP), and or mesenchymal stem cells in the BM. In some embodiments, the transplant may be a peripheral blood stem cell transplant (PBSCT). The HSC maybe be mobilized from the BM and then harvested from the blood, or contained within BM physically extracted from the donor. The HSC may be either purified, enriched, or simply part of the collected BM or blood, and are then injected into a recipient. Transplants
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may be allogeneic, autologous, syngeneic, or xenogenic, and may involve the transplant of any number of cells, including "mini-transplants," which involve smaller numbers of cells.
HSC is a nonlimiting exemplary type of cell, which may be transplanted and/or genetically modified, as used throughout this application. However, as will be readily understood by one skilled in the art, in practicing the inventions provided herein, HSC may be replaced with any one (or more) of a number of substitute cell types without undue experimentation, including, but not limited to BM cells, stem cells, and/or any other cell which gives rise to blood, thymus, BM and/ or any other immune cells, including, but not limited to, HSC, epithelial stem cells, CLP, CMLP, MLP, and/or mesenchymal stem cells in the BM. In some embodiments, HSC are derived from a fetal liver and/or spleen.
In yet further embodiments, where the antigen is not an auto-antigen but, rather, an external antigen (e.g., pollen or seafood), similar strategies can be employed. If the allergy arose from some chance activation of an aberrant T or B cell clone, immunosuppression to remove T cells and B cells, followed by (or concurrent with) thymus regeneration will remove the cells causing the allergic response. Since the allergy arose from the chance activation of an aberrant T or B cell clone, it is unlikely to arise again and, the newly regenerated thymus may also create regulatory T cells. While there may be auto-reactive IgE still circulating in the patient, these will eventually disappear, since the cells secreting them are effectively depleted. Once the immune system has been re-established, the sex steroid ablation therapy can be stopped, and the patient's fertility restored.
The present invention provides methods for treating autoimmune disease without a BMT, with BMT, or with GM cells as described herein. The methods of the invention may further comprise an organ or cell transplant to repair or replace damaged cells, tissues or organs. For example, in IDDM, a patient may require an islet cell transplant to replace islet cells damaged. Prevention of clinical symptoms of autoimmune disease may be achieved using the methods of the present invention, where a patient has pre-clinical symptoms or familial predisposition.
In further embodiments of the invention, genetic modification of the HSC may be employed if the antigen involved in the autoimmune disease or allergy is known. For example, in multiple sclerosis, the antigen may be myelinglycoprotein (MOG) myelin oligodendroglial protein, myelin basic protein or proteolipid protein. In pernicious anemia, the antigen may be the gastric proton pump. In type I diabetes, the antigen may be pro-
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insulin ( J Clin Invest. (2003) 111: 1365., GAD or an islet cell antigen. T cell epitopes of type II collagen have been described with rheumatoid arthritis in (Ohnishi et al. (2003) Int. J. Mol. Med. 1:331). For Hashimoto's thyroiditis, an antigen is thyroid peroxidase, and for Graves disease an antigen is the thyroid-stimulating hormone receptor, (Dawe et al, (1993) Springer Semin. Immunopathol 14:285. Systemic lupus erythematosus antigens include DNA, histones, ribosomes, snRNP, scRNP e.g., HI histone protein. In particular Ro (SS-A) and La (SS-B) ribonucleo-protein antigens (e.g., Ro60 and Ro52).are associated with patients systemic lupus erythematosus (SLE) and rheumatoid arthritis. Myasthenia gravis antigens include acetylcholine receptor alpha chain, and some T cell epitopes are described in Atassi et al, (2001) Crit. Rev. Immunol. 21:1. Likewise, certain allergic reactions are in response to known antigens (e.g., allergy to feline saliva antigen in cat allergies). In these situations, the donor HSC may first be genetically modified to express the antigen prior to being administered to the recipient. HSC may be isolated based on their expression of CD34. These cells can then be administered to the patient together with inhibitors of sex steroid mediated signaling, such as GnRH analogs, which enhances the functionality of the BM. Accordingly, the genetically-modified HSC not only develop into DC, and so tolerize the newly formed T cells, but they also enter the BM as DC and delete new, autoreactive or allergic B cells. Thus, central tolerance to the auto-antigen or allergen is achieved in both the thymus and the bone marrow, thereby alleviating the patient's autoimmune disease or allergic symptoms. In some embodiments, immune cell depletion or suppression is also used.
In another example for the depletion of hyperreactive T cells, for which the target antigen is known, thymic epithelial stem cells (e.g., autologous epithelial stem cells) can be transfected with the gene encoding the specific antigen for which tolerance is desired. Thymic epithelial progenitor cells can be isolated from the thymus itself (especially in the embryo) by their labeling with the Ab MTS 24 or its human counteφart (see Gill et al, (2002) Nat. Immunol. 3:635).
Thus, in accordance with the invention, the basic principle is stop ongoing autoimmune disease or prevent one developing in highly predictive cases (e.g., in familial distribution) with T cell and/or B cell, as appropriate, depletion followed by rebuilding a new tolerant immune system. First, the autoimmune disease is diagnosed, and a determination is made as to whether or not there is a familial (genetic) predisposition. Next, a determination is made as to whether or not there had been a recent prolonged infection in the patient which may have lead to the autoimmune disease through antigen mimicry or inadvertent clonal
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activation. In practice it may be impossible to determine the cause of the disease. Next, T cell depletion is performed and, as appropriate, B cell depletion is performed (and/or another method of immune cell depletion), combined with chemotherapy, radiation therapy and/or anti-B cell reagents (e.g., CD19, CD20, and CD21) or antibodies to specific Ig subclasses (anti IgE). The thymus and bone marrow function is then reactivated by administering GnRH to the patient. Simultaneous with this reactivation of the thymus and bone marrow is the injection of HSC which have been in vitro transfected with a gene encoding the autoantigen to enter the rejuvenating thymus and convert to DC for presentation of the autoantigen to developing T cells, thereby inducing tolerance. The transfected HSC also produce the antigen in the bone marrow, and present the antigen to developing immature B cells, thereby causing their deletion, similar to that occurring to T cells in the thymus. Use of the immunosuppressive regimes (anti-T, -B therapy) overcomes any untoward activation of preexisting potentially autoreactive T and B cells. Moreover, in the case of non-obvious genetic predisposition, GnRH may be combined with G-CSF injection to increase blood levels of autologous HSC to enhance the thymic regrowth.
In some embodiments, hematopoietic or lymphoid stem and/or progenitor cells from a donor (e.g., an MHC-matched donor) are transplanted into the recipient to increase the speed of regeneration of the thymus. In another embodiment these cells are transplanted from a healthy donor, without autoimmune disease or allergies, to replace aberrant stem and/or progenitor cells in the patient.
In one embodiment, a patient's autoimmune disease is eliminated at least in part by clearance of the patient's T cell population. Sex steroid mediated signaling is disrupted. Upon repopulation of the peripheral blood with new T cells, the aberrant T cells that failed tolerance induction to self remain eliminated from the T cell population.??
In another embodiment, a patient's immune system cells causing allergies are eliminated by the same lymphocyte ablation treatments accompanied by disruption of sex steroid mediated signaling to enhance thymic T cell development, to allow repopulation of the peripheral blood stream with a "clean" population of T cells.
Prevention
The present disclosure provides methods for preventing, increasing resistance to, or treating infection of a patient through reactivation of a patient's thymus. This is
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accomplished through disruption of sex steroid mediated signaling. By the methods described herein, the sex steroid-induced atrophic thymus is dramatically restored structurally and functionally to approximately its optimal pre-pubertal capacity in all currently definable terms. This includes the number, type and proportion of all T cell subsets. Also included are the complex stromal cells and their three dimensional architecture which constitute the thymic microenvironment required for producing T cells. The newly generated T cells emigrate from the thymus and restore peripheral T cell levels and function.
At this stage, the patient's immune system is enhanced, rejuvenated and reactivated, thereby increasing its response to foreign antigens such as viruses and bacteria. This is shown, for example, in Figures 14-19, which show the effects of thymic reactivation on the mouse immune system, as demonstrated with viral (HSV) challenge. The mice having prior reactivation of the thymus demonstrate resistance to HSV infection, while those not having thymic reactivation (aged thymus) have higher levels of HSV infection. It is well known that the mouse immune system is very similar to the human immune system, and results in mice can be projected to show human responses. This is reinforced by the data showing the effects of thymic reactivation in humans.
The reactivation of the thymus can be supplemented by the addition of CD34+ hematopoietic stem cells (HSC) and/or epithelial stem cells slightly before or at the time the thymus begins to regenerate. Ideally these cells are autologous or syngeneic and have been obtained from the patient or twin prior to thymus reactivation. The HSC can be obtained by sorting CD34+ cells from the patient's blood and/or bone marrow. The number of HSC can be enhanced in several ways, including (but not limited to) by administering G-CSF (Neupogen, Amgen) to the patient prior to collecting cells, culturing the collected cells in Stem Cell Growth Factor, and/or administering G-CSF to the patient after CD34+ cell supplementation. Alternatively, the CD34+ cells need not be sorted from the blood or BM if their population is enhanced by prior injection of G-CSF into the patient.
In one embodiment, hematopoietic cells are supplied to the patient during thymic reactivation, which increases the immune capabilities of the patient's body. The hematopoietic cells may or may not be genetically modified.
In another embodiment, the immune system is made to react specifically against various antigens by administering genetically modified cells to a recipient. The genetically modified cells may be hematopoietic stem cells (HSC), epithelial stem cells, or hematopoietic
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progenitor cells. The genetically modified cells may be CD34+ HSC, lymphoid progenitor cells, or myeloid progenitor cells. In an embodiment, the genetically modified cells are CD34+ or CD341o HSC. The genetically modified cells are administered to the patient and migrate through the peripheral blood system to the thymus. The uptake into the thymus of these hematopoietic precursor cells is substantially increased in the absence of sex steroids. These cells become integrated into the thymus and produce dendritic cells and T cells carrying the genetic modification from the altered cells. The results are a population of T cells with the desired genetic change that circulate in the peripheral blood of the recipient, and the accompanying increase in the population of cells, tissues and organs caused by reactivation of the patient's thymus, which are capable of rapid, specific responses to antigen.
Within 3-4 weeks of the start of blockage of sex steroid mediated signaling (approximately 2-3 weeks after the initiation of LHRH treatment), the first new T cells may be present in the blood stream. Full development of the T cell pool, however, may take 3-4 (or more) months.
Improvement of Vaccine Response
The present disclosure is in the field of "active vaccinations," where an antigen is administered to a patient whose immune system then responds to the antigen by forming an immune response against the antigen. Vaccination may include both prophylactic and therapeutic vaccines. As will be appreciated by those in the art, the methods of the invention may be used with virtually any method of vaccination in combination with sex steroid inhibition without undue experimentation.
In one embodiment, the vaccine is a killed or inactivated vaccine (e.g., by heat or other chemicals). In another embodiment, the vaccine is an attenuated vaccine (e.g., poliovirus and smallpox vaccines). In an embodiment, the vaccine is a subunit vaccine (e.g., hepatitis B vaccine, in which hepatitis B surface antigen (HBsAg) is the agent-specific protein).
In another embodiment, the vaccine is a recombinant vaccine. One type of recombinant vaccine is an attenuated vaccine in which the agent (e.g., a virus) has specific virulence-causing genes deleted, which renders the virus non-virulent. Another type of recombinant vaccine employs the use of infective, but non- virulent, vectors which are
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genetically modified to insert a gene encoding target antigens. Examples of a recombinant vaccines is a vaccinia virus vaccines.
In yet another embodiment, the vaccine is a DNA vaccine. DNA-based vaccines generally use bacterial plasmids to express protein immunogens in vaccinated hosts. Recombinant DNA technology is used to clone cDNAs encoding immunogens of interest into eukaryotic expression vectors. Vaccine plasmids are then amplified in bacteria, purified, and directly inoculated into the hosts being vaccinated. DNA can be inoculated by a needle injection of DNA in saline, or by a gene gun device which delivers DNA-coated gold beads into the skin. Methods for preparation and use of such vaccines will be well-known to, or may be readily ascertained by, those of ordinary skill in the art.
There are several parameters that can influence the nature and extent of immune responses: the level and type of antigen, the site of vaccination, the availability of appropriate APC, the general health of the individual, and the status of the T and B cell pools. Of these, T cells are the most vulnerable because of the marked sex steroid-induced shutdown in thymic export that becomes profound from the onset of puberty and the global suppression of T cell responses by sex steroids. Any vaccination program should therefore only be logically undertaken when the level of potential responder T cells is optimal with respect to both the existence of na ve T cells representing a broad repertoire of specificity, and the presence of normal ratios of Thl to Th2 cells and Th to Tc cells. The type of T cell help that supports an immune response determines whether the raised antibody will be C -dependent and phagocyte-mediated defenses will be mobilized (a type 1 response), or whether the raised antibody will be C -independent and phagocyte-independent defenses will be mobilized (a type 2 response) (for reviews, see Fearon and Locksley (1996) Science 272:50; Seder and Paul (1994) Annu. Rev. Immunol. 12:635). Historically type 1 responses have been associated with the raising of cytotoxic T cells and type 2 responses with the raising of antibody. Thus, the level and type of cytokines generated may also be manipulated to be appropriate for the desired response (e.g., some diseases require Thl responses, and some require Th2 responses, for protective immunity). This includes codelivery of Thl- or Th2- type cytokines (e.g., delivery of recombinant cytokines or DNA encoding cytokines) to shift the immune response patterns in the patient. Immunostimulatory CpG oligonucletides have also been utilized to shift immune response to various vaccine formulations to a more Thl- type response.
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The ability to reactivate the atrophic thymus through inhibition of sex steroid production, for example at the level of leutinizing hormone releasing hormone (LHRH) signaling to the pituitary, provides a potent means of generating a new cohort of naive T cells with a diverse repertoire of TCR types. This process effectively reverts the thymus towards its pre-pubertal state, and does so by using the normal regulatory molecules and pathways which lead to optimal thymopoiesis.
By the methods described herein, the sex steroid-induced atrophic thymus is dramatically restored structurally and functionally to approximately its optimal pre-pubertal capacity in all currently definable terms. This includes the number, type and proportion of all T cell subsets. Also included are the complex stromal cells and their three dimensional architecture which constitute the thymic microenvironment required for producing T cells. The newly generated T cells emigrate from the thymus and restore peripheral T cell levels and function.
At this stage, the patient's immune system is rejuvenated and reactivated, thereby increasing its response to foreign antigens such as viruses and bacteria. This is shown, for example, in Figures 14-19, which show the effects of thymic reactivation on the mouse immune system, as demonstrated with viral (HSV) challenge. The mice having prior reactivation of the thymus demonstrate resistance to HSV infection, while those not having thymic reactivation (aged thymus) have higher levels of HSV infection. It is well known that the mouse immune system is very similar to the human immune system, and is used as a model for human disease. Thus, results in mice can be projected to predict human responses. This is reinforced by the data showing the effects of thymic reactivation in humans.
The reactivation of the thymus can be supplemented by the addition of CD34+ hematopoietic stem cells (HSC) and/or epithelial stem cells slightly before or at the time the thymus begins to regenerate. Ideally these cells are autologous or syngeneic and have been obtained from the patient or twin prior to thymus reactivation. The HSC can be obtained by sorting CD34+ cells from the patient's blood and/or bone marrow. The number of HSC can be enhanced in several ways, including (but not limited to) by administering G-CSF (Neupogen, Amgen) to the patient prior to collecting cells, culturing the collected cells in Stem Cell Growth Factor, and/or administering G-CSF to the patient after CD34+ cell supplementation. Alternatively, the CD34+ cells need not be sorted from the blood or BM if their population is enhanced by prior injection of G-CSF into the patient.
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In one embodiment, hematopoietic cells are supplied to the patient during thymic reactivation, which increases the immune capabilities of the patient's body.
The HSC are administered to the patient and migrate through the peripheral blood system to the thymus. The uptake into the thymus of these hematopoietic precursor cells is substantially increased in the absence of sex steroids. These cells become integrated into the thymus and produce dendritic cells and T cells. The results are a population of T cells and other immune cells that circulate in the peripheral blood of the recipient, and the accompanying increase in the population of cells, tissues and organs caused by reactivation of the patient's thymus, which are capable improved responses to the vaccine antigen.
Within 3-4 weeks of the start of blockage of sex steroid mediated signaling
(approximately 2-3 weeks after the initiation of LHRH treatment), the first new T cells may be present in the blood stream. Full development of the T cell pool, however, may take 3-4 (or more) months. Vaccination may begin soon after the appearance of the newly produced na ve cells; however, the wait may be 4-6 weeks after the initiation of LHRH therapy to begin vaccination, when enough new T cells to create a strong response will have been produced and will have undergone any necessary post-thymic maturation
This procedure can be combined with any other form of immune system stimulation, including adjuvant, accessory molecules, and cytokine therapies. For example, useful cytokines include but are not limited to interleukin 2 (IL-2) and IL-15 as a general immune growth factor, IL-4 to skew the response to Th2 (humoral immunity), and IFNγ to skew the response to Thl (cell mediated, inflammatory responses), IL 12 to promote Thl and E 10 to promote Th2 cells. Accessory molecules include but are not limited to inhibitors of CTLA4, which enhance the general immune response by facilitating the CD28/B7.1,B7.2 stimulation pathway, which is normally inhibited by CTLA4.
Effects On The Bone Marrow And HSC
The present disclosure provides methods for increasing the production of bone marrow in a patient, including increasing production of HSC. These methods are useful in a number of applications. For example, one of the difficult side effects of chemotherapy or radiotherapy, whether given for cancer or for another puφose, can be its negative impact on the patient's bone marrow. Depending on the dose of chemotherapy, the BM may be damaged or ablated and production of blood cells may be impeded. Administration of a dose
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of a sex steroid analog (such as an LHRH analog) according to this invention after chemotherapy treatment aids in recovery from the damage done by the chemotherapy to the BM and blood cells. Alternatively, administration of the LHRH analog in the weeks prior to delivery of chemotherapy increases the population of HSC and other blood cells so that the impact of chemotherapy is decreased.
The methods described herein are useful to repair damage to the BM and/or assist in the replacement of blood cells that may have been injured or destroyed by various therapies (e.g., cancer chemotherapy drugs, radiation therapy ) or diseases (e.g., HIV, chronic renal failure).
In some chemotherapy regimens, such as high dose chemotherapy to treat any of the blood cancers, ablation of the bone marrow is a desired effect. The methods of this invention may be used immediately after ablation occurs to stimulate the bone marrow and increase the production of HSC and their progeny blood cells, so as to decrease the patient's recovery time. Following administration of the chemotherapy, usually allowing one or more days for the chemotherapy to clear from the patient's body, a dose of LHRH analog according to the methods described herein is administered to the patient. This can be in conjunction with the administration of autologous or heterologous bone marrow or hematopoietic stem or progenitor cells, as well as other factors such as colony stimulating factors (CSFs) and stem cell factor (SCF).
Alternatively, a patient may have suboptimal (or "tired) BM function and may not be producing sufficient or normal numbers of HSC and other blood cells. This can be caused by a variety of conditions, including normal ageing, prolonged infection, post-chemotherapy, post-radiation therapy, chronic disease states including cancer, genetic abnormalities, and immunosuppression induced in transplantation. Further, radiation, such as whole-body radiation, can have a major impact on the BM productivity. These conditions can also be either pre-treated to minimize the negative effects (such as for chemotherapy and/or radiation therapy, or treated after occurrence to reverse the effects.
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Effects on DC
DC are important antigen presenting cells and increased numbers and/or function may be useful in improving tolerance to a donor graft following transplantation of donor HSC or genetically modified HSC as described above.
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EXAMPLES
The following Examples provide specific examples of methods of the invention, and are not to be construed as limiting the invention to their content.
EXAMPLE 1 REVERSAL OF AGED-INDUCED THYMIC ATROPHY
Materials and Methods
Animals. CBA/CAH and C57B16/J male mice were obtained from Central Animal Services, Monash University and were housed under conventional conditions. C57B16/J Ly5.1+ were obtained from the Central Animal Services Monash University, the Walterand Eliza Hall institute for Medical Research (Parkville, Victoria) and the A.R.C. (Perth, Western Australia) and were housed under conventional conditions. Ages ranged from 4-6 weeks to 26 months of age and are indicated where relevant.
Surgical castration. Animals were anesthetized by intraperitoneal injection of 0.3 ml of 0.3 mg xylazine (Rompun; Bayer Australia Ltd., Botany NSW, Australia) and 1.5 mg ketamine hydrochloride (Ketalar; Parke-Davis, Caringbah, NSW, Australia) in saline.
Surgical castration was performed by a scrotal incision, revealing the testes, which were tied with suture and then removed along with surrounding fatty tissue. The wound was closed using surgical staples. Sham-castration followed the above procedure without removal of the testes and was used as controls for all studies.
Bromodeoxyuridine (BrdU) incorporation. Mice received two intraperitoneal injections of BrdU (Sigma Chemical Co., St. Louis, MO) at a dose of 100 mg/kg body weight in lOOμl of PBS, 4-hours apart (i.e., at 4 hour intervals). Control mice received vehicle alone injections. One hour after the second injection, thymuses were dissected and either a cell suspension made for FACS analysis, or immediately embedded in Tissue Tek (O.C.T. compound, Miles LNC, Indiana), snap frozen in liquid nitrogen, and stored at -70°C until use.
Flow Cytometric analysis. Mice were killed by CO2 asphyxiation and thymus, spleen, and mesenteric lymph nodes were removed. Organs were pushed gently through a 200μm sieve in cold PBS/1% FCS/0.02% Azide, centrifuged (650g, 5 min, 4°C), and resuspended in either PBS/FCS/Az. Spleen cells were incubated in red cell lysis buffer (8.9g/liter ammonium chloride) for 10 min at 4°C, washed and resuspended in PBS/FCS/Az.
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Cell concentration and viability were determined in duplicate using a hemocytometer and ethidium bromide/acridine orange and viewed under a fluorescence microscope (Axioskop; Carl Zeiss, Oberkochen, Germany).
For 3-color immunofluorescence, cells were labeled with anti-αβTCR-FITC, anti- CD4-PE and anti-CD8-APC (all obtained from Pharmingen, San Diego, CA) followed by flow cytometry analysis. Spleen and lymph node suspensions were labeled with either αβTCR-FITC/CD4-PE/CD8-APC or B220-B (Sigma) with CD4-PE and CD8-APC. B220-B was revealed with streptavidin-Tri-color conjugate purchased from Caltag Laboratories, Inc., Burlingame, CA.
For BrdU detection of cells, cells were surface labeled with CD4-PE and CD8-APC, followed by fixation and permeabilization as previously described (Carayon and Bord, (1989) J. Imm. Meth. 147:225). Briefly, stained cells were fixed overnight at 4°C in 1% paraformaldehyde (PFA )/0.01% Tween-20. Washed cells were incubated in 500μl DNase (100 Kunitz units, Roche, USA) for 30 mins at 37°C in order to denature the DNA. Finally, cells were incubated with anti-BrdU-FITC (Becton-Dickinson) for 30min at room temperature, washed and resuspended for FACS analysis.
For BrdU analysis of TN subsets, cells were collectively gated out on Lin- cells in APC, followed by detection for CD44-biotin and CD25-PE prior to BrdU detection. All antibodies were obtained from Pharmingen, USA.
For 4-color Immunofluorescence, thymocytes were labeled for CD3, CD4, CD8,
B220 and Mac-1, collectively detected by anti-rat Ig-Cy5 (Amersham, U.K.), and the negative cells (TN) gated for analysis. They were further stained for CD25-PE (Pharmingen) and CD44-B (Pharmingen) followed by Streptavidin-Tri-color (Caltag, CA) as previously described (Godfrey and Zlotnik, (1993) Immunol. Today 14:547). BrdU detection was then performed as described above.
Samples were analyzed on a FacsCalibur (Becton-Dickinson). Viable lymphocytes were gated according to 0° and 90° light scatter profiles and data was analyzed using Cell quest software (Becton-Dickinson).
Immunohistology. Frozen thymus sections (4μm) were cut using a cryostat (Leica) and immediately fixed in 100% acetone.
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For two-color immunofluorescence, sections were double-labeled with a panel of monoclonal antibodies: MTS6, 10, 12, 15, 16, 20, 24, 32, 33, 35 and 44 (Godfrey et al, (1990) Immunol. 70:66; Table 1) produced in this laboratory and the co-expression of epithelial cell determinants was assessed with a polyvalent rabbit anti-cytokeratin Ab (Dako, Caφinteria, CA). Bound mAb was revealed with FITC-conjugated sheep anti-rat Ig (Silenus Laboratories) and anti-cytokeratin was revealed with TRITC-conjugated goat anti-rabbit Ig (Silenus Laboratories).
For BrdU detection of sections, sections were stained with either anti-cytokeratin followed by anti-rabbit-TRITC or a specific mAb, which was then revealed with anti-rat Ig- Cγ3 (Amersham). BrdU detection was then performed as previously described (Penit et al, (1996) Proc. Natl. Acad. Sci, USA 86:5547). Briefly, sections were fixed in 70% Ethanol for 30 mins. Semi-dried sections were incubated in 4M HCl, neutralized by washing in Borate Buffer (Sigma), followed by two washes in PBS. BrdU was detected using anti-BrdU-FlTC (Becton-Dickinson).
For three-color immunofluorescence, sections were labeled for a specific MTS mAb together with anti-cytokeratin. BrdU detection was then performed as described above.
Sections were analyzed using a Leica fluorescent and Nikon confocal microscopes.
Migration studies (i.e., Analysis of recent thymic emigrants (RTE)). Animals were anesthetized by intraperitoneal injection of 0.3ml of 0.3mg xylazine (Rompun; Bayer Australia Ltd., Botany NSW, Australia) and 1.5mg ketamine hydrochloride (Ketalar; Parke- Davis, Caringbah, NSW, Australia) in saline.
Details of the FITC labeling of thymocytes technique are similar to those described elsewhere (Scollay et al, (1980) Eur. J. Immunol. 10:210; Berzins et al, (1998) J. Exp. Med. 187: 1839). Briefly, thymic lobes were exposed and each lobe was injected with approximately lOμm of 350 μg/ml FITC (in PBS). The wound was closed with a surgical staple, and the mouse was warmed until fully recovered from anesthesia. Mice were killed by CO2 asphyxiation approximately 24 hours after injection and lymphoid organs were removed for analysis.
After cell counts, samples were stained with anti-CD4-PE and anti-CD8-APC, then analyzed by flow cytometry. Migrant cells were identified as live-gated FITC+ cells expressing either CD4 or CD8 (to omit autofluorescing cells and doublets). The percentages
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of FITC+ CD4 and CD8 cells were added to provide the total migrant percentage for lymph nodes and spleen, respectively. Calculation of daily export rates was performed as described by Berzins et al, (1998) J. Exp. Med. 187:1839.
Data analyzed using the unpaired student 't' test or nonparametrical Mann-Whitney U- test was used to determine the statistical significance between control and test results for experiments performed at least in triplicate. Experimental values significantly differing from control values are indicated as follows: *p≤ 0.05, **p≤ 0.01 and ***p≤ 0.001.
Results
I. The effect of age on thymocyte populations.
(i) Thymic weight and thymocyte number
With increasing age there is a highly significant (p≤0.0001) decrease in both thymic weight (Fig. IA) and total thymocyte number (Figs. IB and IC) in mice. Relative thymic weight (mg thymus/g body) in the young adult has a mean value of 3.34 which decreases to 0.66 at 18-24 months of age (adipose deposition limits accurate calculation). The decrease in thymic weight can be attributed to a decrease in total thymocyte numbers: the 1-2 month (i.e., young adult) thymus contains -6.7 x 10 thymocytes, decreasing to -4.5 x 10 cells by 24 months. By removing the effects of sex steroids on the thymus by castration, thymocyte cell numbers are regenerated and by 4 weeks post-castration, the thymus is equivalent to that of the young adult in both weight (Fig. IA) and cellularity (Figs. IB and IC). Interestingly, there was a significant (p≤O.OOl) increase in thymocyte numbers at 2 weeks post-castration (1.2 x 108), which is restored to normal young levels by 4 weeks post-castration (Fig. IB).
The decrease in T cell numbers produced by the thymus is not reflected in the periphery, with spleen cell numbers remaining constant with age (Fig. 2A and 2B). Homeostatic mechanisms in the periphery were evident since the B cell to T cell ratio in spleen and lymph nodes was not affected with age and the subsequent decrease in T cell numbers reaching the periphery (Figs. 2C and 2D). However, the ratio of CD4+ to CD8+ T cell significantly decreased (p≤ 0.001) with age from 2: 1 at 2 months of age, to a ratio of 1 : 1 at 2 years of age (Figs. 2D and 2E). Following castration and the subsequent rise in T cell numbers reaching the periphery, no change in peripheral T cell numbers was observed: splenic T cell numbers and the ratio of B:T cells in both spleen and lymph nodes was not altered following castration (Figs. 2A-2D). The reduced CD4:CD8 ratio in the periphery
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with age was still evident at 2 weeks post-castration but was completely reversed by 4 weeks post-castration (Fig. 2E)
(ii) Thymocyte subpopulations with age and post-castration.
To determine if the decrease in thymocyte numbers seen with age was the result of the depletion of specific cell populations, thymocytes were labeled with defining markers in order to analyze the separate subpopulations. In addition, this allowed analysis of the kinetics of thymus repopulation post-castration. The proportion of the main thymocyte subpopulations was compared with those of the young adult (2-4 months) thymus (Fig. 3) and found to remain uniform with age. In addition, further subdivision of thymocytes by the expression of αβTCR revealed no change in the proportions of these populations with age (data not shown). At 2 and 4 weeks post-castration, thymocyte subpopulations remained in the same proportions and, since thymocyte numbers increase by up to 100-fold post- castration, this indicates a synchronous expansion of all thymocyte subsets rather than a developmental progression of expansion.
The decrease in cell numbers seen in the thymus of aged (2 year old) animals thus appears to be the result of a balanced reduction in all cell phenotypes, with no significant changes in T cell populations being detected. Thymus regeneration occurs in a synchronous fashion, replenishing all T cell subpopulations simultaneously rather than sequentially.
II. Proliferation of thymocytes
As shown in Figs. 4A-4C, 15-20% of thymocytes were proliferating at 2-4 months of age. The majority (-80%) of these are double positive (DP) (i.e., CD4+, CD8+) with the triple negative (TN) (i.e., CD3-CD4-CD8-) subset making up the second largest population at -6% (Figs. 5A). These TN cells are the most immature cells in the thymus and encompass the intrathymic precursor cells. Accordingly, most division is seen in the subcapsule and cortex by immunohistology (data not shown). Some division is seen in the medullary regions aligning with FACS analysis which revealed a proportion of single positive (i.e., CD4+CD8- or CD4-CD8+) cells (9% of CD4+ T cells and 25% of CD8+ T cells) in the young (2 months) thymus, dividing (Fig. 5B).
Although cell numbers were significantly decreased in the aged mouse thymus (2 years old), the total proportion of proliferating thymocytes remained constant (Figs. 4C and 5F), but there was a decrease in the proportion of dividing cells in the CD4-CD8- (Fig 5C)
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and proliferation of CD4-CD8+ T cells was also significantly (p< 0.001) decreased (Fig 5E). Immunohistology revealed the distribution of dividing cells at 1 year of age to reflect that seen in the young adult (2-4 months); however, at 2 years, proliferation is mainly seen in the outer cortex and surrounding the vasculature with very little division in the medulla (data not shown).
As early as one week post-castration there was a marked increase in the proportion of proliferating CD4-CD8- cells (Fig 5C) and the CD4-CD8+ cells (Fig 5E). Castration clearly overcomes the block in proliferation of these cells with age. There was a corresponding proportional decrease in proliferating CD4+CD8- cells post-castration (Fig 5D). At 2 weeks post-castration, although thymocyte numbers significantly increase, there was no change in the overall proportion of thymocytes that were proliferating, again indicating a synchronous expansion of cells (Figs. 4A, 4B, 4C and 5F). Immunohistology revealed the localization of thymocyte proliferation and the extent of dividing cells to resemble the situation in the 2- month-old thymus by 2 weeks post-castration (data not shown).
The DN subpopulation, in addition to the thymocyte precursors, contains D D DTCR +CD4-CD8- thymocytes, which are thought to have down-regulated both co-receptors at the transition to SP cells (Godfrey and Zlotnik, (1993) Immunol. Today 14:547). By gating on these mature cells, it was possible to analyze the true TN compartment (CD3 CD4 CD8 ) and their subpopulations expressing CD44 and CD25. Figures 5H, 51, 5J, and 5K illustrate the extent of proliferation within each subset of TN cells in young, old and castrated mice. This showed a significant (p<0.001) decrease in proliferation of the TNI subset (CD44+CD25" CD3 CD4 CD8"), from -10%% in the normal young to around 2% at 18 months of age (Fig. 5H) which was restored by 1 week post-castration.
III. Thymocyte emigration
Approximately 1% of T cells migrate from the thymus daily in the young mouse
(Scollay et al, (1980) Proc. Natl. Acad. Sci, USA 86:5547). Migration in castrated mice was found to occur at a proportional rate equivalent to the normal young mouse at 14 months and even 2 years of age, although significantly (p≤ 0.0001) reduced in number (Figs. 6A and 6B). There was an increase in the CD4:CD8 ratio of the recent thymic emigrants from -3: 1 at 2 months to -7:1 at 26 months (Fig. 6C). By 1 week post-castration, this ratio had normalized (Fig. 6C). By 2- weeks post-castration, cell number migrating to the periphery had
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substantially increased, with the overall rate of migration reduced to 0.4%, which reflected the expansion of the thymus (Fig. 6B).
By 2-weeks post-castration, a significant increase in RTE was observed (p≤O.Ol) compared to the aged mice. Despite the changes in cell numbers emigrating, the rate of emigration (RTE/total thymocytes) remained constant with age (Fig. 5b). However, at 2- weeks post-castration this had significantly decreased (p≤0.05), reflecting the increase in total thymocyte numbers at this time. Interestingly, there was an increase in the CD4:GD8 ratio of the RTE from -3:1 at 2 months to -7: 1 at 26 months (Fig. 6C). By 1 week post-castration, this ratio had normalized (Fig. 6C).
Discussion
It has been shown that aged thymus, although severely atrophic, maintains its functional capacity with age, with T cell proliferation, differentiation and migration occurring at levels equivalent to the young adult mouse. Although thymic function is regulated by several complex interactions between the neuro-endocrine-immune axes, the atrophy induced by sex steroid production exerts the most significant and prolonged effects illustrated by the extent of thymus regeneration post-castration.
Thymus weight is significantly reduced with age as shown previously (Hirokawa and Makinodan, (1975) J. Immunol. 114:1659, Aspinall, (1997) J. Immunol. 158:3037) and correlates with a significant decrease in thymocyte numbers. The stress induced by the castration technique, which may result in further thymus atrophy due to the actions of corticosteroids, is overridden by the removal of sex steroid influences with the 2-week castrate thymus increasing in cellularity by 20-30 fold from the pre-castrate thymus. By 3 weeks post-castration, the aged thymus shows a significant increase in both thymic size and cell number, suφassing that of the young adult thymus presumably due to the actions of sex steroids already exerting themselves in the 2 month old mouse.
The data presented herein confirms previous findings that emphasize the continued ability of thymocytes to differentiate and maintain constant subset proportions with age (Aspinall, 1997). In addition, thymocyte differentiation was found to occur simultaneously post-castration indicative of a synchronous expansion in thymocyte subsets. Since thymocyte numbers are decreased significantly with age, proliferation of thymocytes was analyzed to determine if this was a contributing factor in thymus atrophy.
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Proliferation of thymocytes was not affected by age-induced thymic atrophy or by removal of sex-steroid influences post-castration with -14% of all thymocytes proliferating. However, the localization of this division differed with age: the 2 month mouse thymus shows abundant division throughout the subcapsular and cortical areas (TN and DP T cells) with some division also occurring in the medulla. Due to thymic epithelial disorganization with age, localization of proliferation was difficult to distinguish but appeared to be less uniform in pattern than the young and relegated to the outer cortex. By 2 weeks post- castration, dividing thymocytes were detected throughout the cortex and were evident in the medulla with similar distribution to the 2 month thymus.
The phenotype of the proliferating population as determined by CD4 and CD8 analysis, was not altered with age or following castration. However, analysis of proliferation within thymocyte subpopulations, revealed a significant decrease in proliferation of both the TN and CD8+ cells with age. Further analysis within the TN subset on the basis of the markers CD44 and CD25, revealed a significant decrease in proliferation of the TNI (CD44+CD25 ) population which was compensated for by an increase in the TN2 (CD44 CD25+) population. These abnormalities within the TN population, reflect the findings by Aspinall (1997). Suφrisingly, the TN subset was proliferating at normal levels by 2 weeks post-castration indicative of the immediate response of this population to the inhibition of sex-steroid action. Additionally, at both 2 weeks and 4 weeks post-castration, the proportion of CD8+ T cells that were proliferating was markedly increased from the control thymus, possibly indicating a role in the reestablishment of the peripheral T cell pool.
Thymocyte migration was shown to occur at a constant proportion of thymocytes with age conflicting with previous data by Scollay et al, (1980) Proc. Natl. Acad. Sci, USA 86:5547 who showed a ten-fold reduction in the rate of thymocyte migration to the periphery. The difference in these results may be due to the difficulties in intrathymic FITC labelling of 2 year old thymuses or the effects of adipose deposition on FITC, uptake. However, the absolute numbers of T cells migrating was decreased significantly as found by Scollay resulting in a significant reduction in ratio of RTEs to the peripheral T cell pool. This will result in changes in the periphery predominantly affecting the T cell repertoire (Mackall et al. (1995) N Engl. J. Med. 332:143-149). Previous papers (Mackall et al. (1995) N Engl. J. Med. 332: 143-149) have shown a skewing of the T cell repertoire to a memory rather than naive T cell phenotype with age. The diminished T cell repertoire however, may not cope if the individual encounters new pathogens, possibly accounting for the rise in
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immunodeficiency in the aged. There is a need to reestablish the T cell pool in immunocompromised individuals. Castration allows the thymus to repopulate the periphery through significantly increasing the production of naive T cells.
In the periphery, T cell numbers remained at a constant level as evidenced in the B:T cell ratios of spleen and lymph nodes, presumably due to peripheral homeostasis (Mackall et al, (1995) N. Eng. J. Med. 332:143; Berzins et al, (1998) J. Exp. Med. 187:1839). However, disruption of cellular composition in the periphery was evident with the aged thymus showing a significant decrease in CD4:CD8 ratios from 2:1 in the young adult to 1:1 in the 2 year mouse, possibly indicative of the more susceptible nature of CD4+T cells to age or an increase in production of CD8+ T cells from extrathymic sources. By 2 weeks post- castration, this ratio has been normalized, again reflecting the immediate response of the immune system to surgical castration.
The above findings have shown firstly that the aged thymus is capable of functioning in a nature equivalent to the pre-pubertal thymus. In this respect, T cell numbers are significantly decreased but the ability of thymocytes to differentiate is not disturbed. Their overall ability to proliferate and eventually migrate to the periphery is again not influenced by the age-associated atrophy of the thymus. However, two important findings were noted. Firstly, there appears to be an adverse affect on the TΝ cells in their ability to proliferate, correlating with findings by Aspinall (1997). This defect could be attributed to an inherent defect in the thymocytes themselves. Yet the data presented herein and previous work has shown thymocyte differentiation, although diminished, still occurs and stem cell entry from the BM is also not affected with age (Hirokawa, (1998), "Immunity and Ageing," in PRINCIPLES AND PRACTICE OF GERIATRIC MEDICINE, (M. Pathy, ed.) John Wiley and Sons Ltd; Mackall and Gress, (1997) Immunol. Rev. 160:91). Secondly, the CD8+ T cells were significantly diminished in their proliferative capacity with age and, following castration, a significantly increased proportion of CD8+ T cells proliferated as compared to the 2 month mouse. The proliferation of mature T cells is thought to be a final step before migration (Suda and Zlotnik, (1991) J. Immunol. 146:3068), such that a significant decrease in CD8+ proliferation would indicate a decrease in their migrational potential. This hypothesis is supported by our finding that the ratio of CD4:CD8 T cells in RTEs increased with age, indicative of a decrease in CD8 T cells migrating. Alternatively, if the thymic epithelium is providing the key factor for the CD8 T cell maintenance, whether a lymphostromal molecule or cytokine influence, this factor may be disturbed with increased sex-steroid production. By
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removing the influence of sex-steroids, the CD8 T cell population can again proliferate optimally.
The defect in proliferation of the TNI subset which was observed indicates that loss of cortical epithelium affects thymocyte development at the crucial stage of TCR gene rearrangement whereby the cortical epithelium provides factors such as IL-7 and SCF necessary for thymopoiesis (Godfrey and Zlotnik, (1993) Immunol. Today 14:547; Aspinall, (1997) J. Immunol. 158:3037). Indeed, IL-77" and IL-7R'/" mice show similar thymic moφhology to that seen in aged mice (Wiles et al, (1992) EMr. J. Immunol. 22: 1037; Zlotnik and Moore, (1995) Curr. Opin. Immunol. 7:206); von Freeden-Jeffry, (1995) J. Exp. Med. 181:1519).
In conclusion, the aged thymus still maintains its functional capacity, however, the thymocytes that develop in the aged mouse are not under the stringent control by thymic epithelial cells as seen in the normal young mouse due to the lack of structural integrity of the thymic microenvironment. Thus the proliferation, differentiation and migration of these cells will not be under optimal regulation and may result in the increased release of autoreactive/immunodysfunctional T cells in the periphery. The defects within both the TN and particularly, CD8+ populations, may result in the changes seen within the peripheral T cell pool with age. Restoration of thymus function by castration will provide an essential means for regenerating the peripheral T cell pool and thus in re-establishing immunity in immunosuppressed, immunodeficient, or immunocompromised individuals.
EXAMPLE 2
REVERSAL OF CHEMOTHERAPY- OR RADIATION-INDUCED THYMIC
ATROPHY
Materials and Methods
Materials and methods were as described in Example 1. In addition, the following methods were used.
Bone Marrow reconstitution. Recipient mice (3-4 month-old C57BL6/J) were subjected to 5.5Gy irradiation twice over a 3-hour interval. One hour following the second irradiation dose, mice were injected intravenously with 5xl06 donor bone marrow cells. Bone marrow cells were obtained by passing RPMI-1640 media through the tibias and femurs of
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donor (2-month old congenic C57BL6/J Ly5.1+) mice, and then harvesting the cells collected in the media.
Irradiation. 3-4 month old mice were subjected to 625Rads of whole body D- irradiation.
T cell Depletion Using Cyclophosphamide. Old mice (e.g., 2 years old) were injected with cyclophosphamide (200mg/kg body wt over two days) and castrated.
Results
Castration enhanced regeneration following severe T cell depletion (TCD). For both models of T cell depletion studied (chemotherapy using cyclolphosphamide or sublethal irradiation using 625Rads), castrated (Cx) mice showed a significant increase in the rate of thymus regeneration compared to their sham-castrated (ShCx) counteφarts (Figs. 7A and 7B). By 1 week post-treatment castrated mice showed significant thymic regeneration even at this early stage (Figs. 7, 8, 10, 11, and 12). In comparison, non-castrated animals, showed severe loss of DN and DP thymocytes (rapidly-dividing cells) and subsequent increase in proportion of CD4 and CDS cells (radio-resistant). This is best illustrated by the differences in thymocyte numbers with castrated animals showing at least a 4-fold increase in thymus size even at 1 week post-treatment. By 2 weeks, the non-castrated animals showed relative thymocyte normality with regeneration of both DN and DP thymocytes. However, proportions of thymocytes are not yet equivalent to the young adult control thymus. Indeed, at 2 weeks, the vast difference in regulation rates between castrated and non-castrated mice was maximal (by 4 weeks thymocyte numbers were equivalent between treatment groups).
Thymus cellularity was significantly reduced in ShCx mice 1-week post- cyclophosphamide treatment compared to both control (untreated, aged-matched; p≤O.001) and Cx mice (p≤0.05) (Fig. 7A). No difference in thymus regeneration rates was observed at this time-point between mice castrated 1-week earlier or on the same day as treatment, with both groups displaying at least a doubling in the numbers of cells compared to ShCx mice (Figs. 7A and 8A). Similarly, at 2-weeks post-cyclophosphamide treatment, both groups of Cx mice had significantly (5-6 fold) greater thymocyte numbers (p≤O.OOl) than the ShCx mice (Fig. 7A). In control mice there was a gradual recovery of thymocyte number over 4 weeks but this was markedly enhanced by castration - even within one week (Fig. 8A).
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Similarly spleen and lymph node numbers were increased in the castrate mice after one week (Figs. 8B and 8C).
The effect of the timing of castration on thymic recovery was examined by castration one week prior to either irradiation (Fig. 11) or on the same day as irradiation (Fig. 12). When performed one week prior, castration had a more rapid impact on thymic recovery (Fig. 1 IA compared to Fig 12 A). By two weeks the same day castration had "caught up" with the thymic regeneration in mice castrated one week prior to treatment. In both cases there were no major effects on spleen or lymph nodes (Figs. 1 IB and 1 IC, and Figs. 12B and 12C) respectively.
Following irradiation treatment, both ShCx and mice castrated on the same day as treatment (SDCx) showed a significant reduction in thymus cellularity compared to control mice (p≤O.OOl) (Figs. 7B and 12A) and mice castrated 1-week prior to treatment (p≤O.Ol) (Fig. 7B). At 2 weeks post-treatment, the castration regime played no part in the restoration of thymus cell numbers with both groups of castrated mice displaying a significant enhancement of thymus cellularity post-irradiation (Plrr) compared to ShCx mice (p≤O.OOl) (Figs. 7B, 11 A, and 12A). Therefore, castration significantly enhances thymus regeneration post-severe T cell depletion, and it can be performed at least 1-week prior to immune system insult.
Interestingly, thymus size appears to 'overshoot' the baseline of the control thymus. Indicative of rapid expansion within the thymus, the migration of these newly derived thymocytes does not yet (it takes -3-4 weeks for thymocytes to migrate through and out into the periphery). Therefore, although proportions within each subpopulation are equal, numbers of thymocytes are building before being released into the periphery.
Following cyclophosphamide treatment of young mice (-2-3 months), total lymphocyte numbers within the spleen of Cx mice, although reduced, were not significantly different from control mice throughout the time-course of analysis (Fig. 9A). However, ShCx mice showed a significant decrease in total splenocyte numbers at 1- and 4-weeks post- treatment (p≤0.05) (Fig. 9A). Within the lymph nodes, a significant decrease in cellularity was observed at 1-week post-treatment for both sham-castrated and castrated mice (p≤O.Ol) (Fig. 9B), possibly reflecting the influence of stress steroids. By 2-weeks post-treatment, lymph node cellularity of castrated mice was comparable to control mice however sham- castrated mice did not restore their lymph node cell numbers until 4-weeks post-treatment,
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with a significant (p≤0.05) reduction in cellularity compared to both control and Cx mice at 2-weeks post-treatment (Fig. 9B). These results indicate that castration may enhance the rate of recovery of total lymphocyte numbers following cyclophosphamide treatment.
Sublethal irradiation (625Rads) induced a profound lymphopenia such that at 1-week post-treatment, both treatment groups (Cx and ShCx), showed a significant reduction in the cellularity of both spleen and lymph nodes (p≤O.OOl) compared to control mice (Figs. 13A and 13B). By 2 weeks post-irradiation, spleen cell numbers were similar to control values for both castrated and sham-castrated mice (Fig. 13 A), whilst lymph node cell numbers were still significantly lower than control values (p≤O.OOl for sham-castrated mice; p≤O.Ol for castrated mice) (Fig. 13B). No significant difference was observed between the Cx and ShCx mice.
Figure 10 illustrates the use of chemical castration compared to surgical castration in enhancement of T cell regeneration. The chemical used in this example, Deslorelin (an LHRH-A), was injected for four weeks, and showed a comparable rate of regeneration post- cyclophosphamide treatment compared to surgical castration (Fig. 10). The enhancing effects were equivalent on thymic expansion and also the recovery of spleen and lymph node (Fig 10). The kinetics of chemical castration are slower than surgical, that is, mice take about 3 weeks longer to decrease their circulating sex steroid levels. However, chemical castration is still as effective as surgical castration and can be considered to have an equivalent effect.
Discussion
The impact of castration on thymic structure and T cell production was investigated in animal models of immunodepletion. Specifically, Example 2 examined the effect of castration on the recovery of the immune system after sublethal irradiation and cyclophosphamide treatment. These forms of immunodepletion act to inhibit DNA synthesis and therefore target rapidly dividing cells. In the thymus these cells are predominantly immature cortical thymocytes, however all subsets are effected (Fredrickson and Basch, (1994) Dev. Comp. Immunol. 18:251). In normal healthy aged animals, the qualitative and quantitative deviations in peripheral T cells seldom lead to pathological states. However, major problems arise following severe depletion of T cells because of the reduced capacity of the thymus for T cell regeneration. Such insults occur in HIV/AIDS, and particularly following chemotherapy and radiotherapy in cancer treatment (Mackall et al, (1995) N. Eng. J. Med. 332:143).
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In both sublethally irradiated and cyclophosphamide treated mice, castration markedly enhanced thymic regeneration. Castration was carried, out on the same day as and seven days prior to immunodepletion in order to appraise the effect of the predominantly corticosteroid induced, stress response to surgical castration on thymic regeneration. Although increases in thymus cellularity and architecture were seen as early as one week after immunodepletion, the major differences were observed two weeks after castration. This was the case whether castration was performed on the same day or one week prior to immunodepletion.
Immunohistology demonstrated that in all instances, two weeks after castration the thymic architecture appeared phenotypically normal, while; that of noncastrated mice was disorganized. Pan epithelial markers demonstrated that immunodepletion caused a collapse in cortical epithelium and a general disruption of thymic architecture in the thymii of noncastrated mice. Medullary markers supported this finding. One of the first features of castration-induced thymic regeneration was a marked upregulation in the extracellular matrix, identified by MTS 16.
Flow cytometry analysis data illustrated a significant increase in the number of cells in all thymocyte subsets in castrated mice. At each time point, there was a synchronous increase in all CD4, CD8 and αβ-TCR - defined subsets following immunodepletion and castration. This is an unusual but consistent result, since T cell development is a progressive process it was expected that there would be an initial increase in precursor cells (contained within the CD4 CD8" gate) and this may have occurred before the first time point. Moreover, since precursors represent a very small proportion of total thymocytes, a shift in their number may not have been, detectable. The effects of castration on other cells, including macrophages and granulocytes were also analyzed. In general there was little alteration in macrophage and granulocyte numbers within the thymus.
In both irradiation and cyclophosphamide models of immunodepletion thymocyte numbers peaked at every two weeks and decreased four weeks after treatment. Almost immediately after irradiation or chemotherapy, thymus weight and cellularity decreased dramatically and approximately 5 days later the first phase of thymic regeneration begun. The first wave of reconstitution (days 5-14) was brought about by the proliferation of radioresistant thymocytes (predominantly double negatives) which gave rise to all thymocyte subsets (Penit and Ezine, (1989) Proc. Natl. Acad. Sci, USA 86:5547). The second decrease,
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observed between days 16 and 22 was due to the limited proliferative ability of the radioresistant cells coupled with a decreased production of thymic precursors by the bone marrow (also effected by irradiation). The second regenerative phase was due to the replenishment of the thymus with bone marrow derived precursors (Huiskamp et al, (1983) Radiat. Res. 95:370).
It is important to note that in adult mice the development from a HSC to a mature T cell takes approximately 28 days (Shortman et al, (1990) Sem. Immunol. 2:3). Therefore, it is not suφrising that little change was seen in peripheral T cells up to four weeks after treatment. The periphery would be supported by some thymic export, but the majority of the T cells found in the periphery up to four weeks after treatment would be expected to be proliferating cyclophosphamide or irradiation resistant clones expanding in the absence of depleted cells. Several long term changes in the periphery would be expected post-castration including, most importantly, a diversification of the TCR repertoire due to an increase in thymic export.
EXAMPLE 3
THYMIC REGENERATION FOLLOWING INHIBITION OF SEX STEROIDS RESULTS IN RESTORATION OF DEFICIENT PERIPHERAL T CELL FUNCTION
Materials and Methods
Materials and methods were as described in Examples 1 and 2. In addition, the following methods were used.
HSV-1 immunization. Aged (>18 months) mice were surgically castrated. 6 weeks after castration (following thymus reactivation). Following anesthetic, mice were injected in the hind leg (foot-hock) with 4xl05 plaque forming units (pfu) of HSV-l(KOS strain) in sterile PBS using a 20-gauge needle. Infected mice were housed in isolated cages and humanely killed on D5 post-immunization at which time the popliteal (draining) lymph nodes were removed for analysis.
Virus was obtained from Assoc. Prof. Frank Carbone (Melbourne University). Virus stocks were grown and titrated on VERO cell monolayers in MEM supplemented with 5% FCS (Gibco-BRL, Australia).
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Analysis of the draining (popliteal) lymph nodes was performed on D5 post-infection. For HSV-1 studies, popliteal lymph node cells were stained for anti-CD25-PE, anti-CD8- APC and anti-VβlO-biotin. For detection of DC, an FcR block was used prior to staining for CD45.1-FITC, I-Ab-PE and CDllc-biotin. All biotinylated antibodies were detected with streptavidin-PerCP. For detection of HSC, BM cells were gated on Lin" cells by collectively staining with anti-CD3, CD4, CD8, Gr-1, B220 and Mac-1 (all conjugated to FITC). HSC were detected by staining with CD117-APC and Sca-l-PE. For TN thymocyte analysis, cells were gated on the Lin" population and detected by staining with CD44-biotin, CD25-PE and c-kit-APC.
Cytotoxicity assay of lymph node cells. Lymph node cells were incubated for three days at 37°C, 6.5% CO2. Specificity was determined using a non-transfected cell line (EL4) pulsed with gB 98.505 peptide (gBp) and EL4 cells alone as a control. A starting effectoπtarget ratio of 30:1 was used. The plates were incubated at 37°C, 6.5% CO2 for four hours and then centrifuged 650gmaχ for 5 minutes. Supernatant (lOOμl) was harvested from each well and transferred into glass fermentation tubes for measurement by a Packard Cobra auto-gamma counter.
Results
To determine the functional consequences of thymus regeneration (e.g., whether castration can enhance the immune response, Heφes Simplex Virus (HSV) immunization was examined as it allows the study of disease progression and role of CTL (cytotoxic) T cells. Castrated mice were found to have a qualitatively and quantitatively improved responsiveness to the virus.
Mice were immunized in the footpad and the popliteal (draining) lymph node analyzed at D5 post-immunization. In addition, the footpad was removed and homogenized to determine the virus titer at particular time-points throughout the experiment. The regional (popliteal) lymph node response to HSV-1 infection (Figs. 14-19) was examined.
A significant decrease in lymph node cellularity was observed with age (Figs. 14A, 14B, and 16). At D5 (i.e., 5 days) post-immunization, the castrated mice have a significantly larger lymph node cellularity than the aged mice (Fig. 16). Although no difference in the proportion of activated (CD8+CD25+) cells was seen with age or post-castration (Fig. 17 A), activated cell numbers within the lymph nodes were significantly increased with castration
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when compared to the aged controls (Fig. 17B). Further, activated cell numbers correlated with that found for the young adult (Fig. 17B), indicating that CTLs were being activated to a greater extent in the castrated mice, but the young adult may have an enlarged lymph node due to B cell activation. This was confirmed with a CTL assay detecting the proportion of specific lysis occurring with age and post-castration (Fig. 18). Aged mice showed a significantly reduced target cell lysis at effectoπtarget ratios of 10:1 and 3: 1 compared to young adult (2-month) mice (Fig. 18). Castration restored the ability of mice to generate specific CTL responses post-HSV infection (Fig. 18).
In addition, while overall expression of VβlO by the activated cells remained constant with age (Fig. 19A), a subgroup of aged (18-month) mice showed a diminution of this clonal response (Figs. 15A-C). By six weeks post-castration, the total number of infiltrating lymph node cells and the number of activated CD25+CD8+ cells had increased to young adult levels (Figs. 16 and 17B). More importantly however, castration significantly enhanced the CTL responsiveness to HSV-infected target cells, which was greatly reduced in the aged mice (Fig. 18) and restored the CD4:CD8 ratio in the lymph nodes (Fig. 19B). Indeed, a decrease in CD4+ T cells in the draining lymph nodes was seen with age compared to both young adult and castrated mice (Fig. 19B), thus illustrating the vital need for increased production of T cells from the thymus throughout life, in order to get maximal immune responsiveness.
EXAMPLE 4
INHIBITION OF SEX STEROIDS ENHANCES UPTAKE OF NEW HAEMOPOIETIC PRECURSOR CELLS INTO THE THYMUS WHICH ENABLES CHIMERIC MIXTURES OF HOST AND DONOR LYMPHOID CELLS (T. B, AND
DENDRITIC CELLS)
Materials and methods were as described in Examples 1-3. In addition, the following techniques were used:
Previous experiments have shown that microchimera formation plays an important role in organ transplant acceptance. DC have also been shown to play an integral role in tolerance to graft antigens. Therefore, the effects of castration on thymic chimera formation and dendritic cell number was studied.
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In order to assess the role of stem cell uptake in thymus regeneration, BM reconstitution was performed as described in Example 2
For the syngeneic experiments, three month old mice (n=4) were used per treatment group. All controls were age matched and untreated.
The total thymus cell numbers of castrated and noncastrated reconstituted mice were compared to untreated age matched controls and are summarized in Fig. 20A. As shown in Fig. 20A, in mice castrated 1 day prior to reconstitution, there was a significant increase (p≤O.Ol) in the rate of thymus regeneration compared to sham-castrated (ShCx) control mice. Thymus cellularity in the sham-castrated mice was below untreated control levels (7.6xl07 ± 5.2xl06) 2 and 4 weeks after congenic BMT, while thymus cellularity of castrated mice had increased above control levels at 4-weeks post-BMT (Fig. 20A). At 6 weeks, cell numbers remained below control levels, however, those of castrated mice were three fold higher than the noncastrated mice (p≤0.05) (Fig. 20A).
There were also significantly more cells (p≤0.05) in the BM of castrated mice 4 weeks after BMT (Fig. 20D). BM cellularity reached untreated control levels (1.5xl07± 1.5xl06) in the sham-castrates by 2 weeks, whereas BM cellularity was increased above control levels in castrated mice at both 2 and 4 weeks after congenic BMT (Fig. 20D). Mesenteric lymph node cell numbers were decreased 2-weeks after irradiation and reconstitution, in both castrated and noncastrated mice; however, by the 4 week time point cell numbers had reached control levels. There was no statistically significant difference in lymph node cell number between castrated and noncastrated treatment groups (Fig. 20C). Spleen cellularity reached untreated control levels (1.5xl07 ± 1.5x10 ) in the sham-castrates and castrates by 2 weeks, but dropped off in the sham group over 4-6 weeks, whereas the castrated mice still had high levels of spleen cells (Fig. 20B). Again, castrated mice showed increased lymphocyte numbers at these time points (i.e., 4 and 6 weeks post-reconstitution) compared to noncastrated mice (p≤0.05) although no difference in total spleen cell number between castrated and noncastrated treatment groups was seen at 2-weeks (Fig. 20B).
Thus, in mice castrated 1 day prior to reconstitution, there was a significant increase
(p≤O.Ol) in the rate of thymus regeneration compared to sham-castrated (ShCx) control mice (Fig. 20A). Thymus cellularity in the sham-castrated mice was below untreated control levels (7.6xl07 ± 5.2xl06) 2 and 4 weeks after congenic BMT, while thymus cellularity of castrated mice had increased above control levels at 4-weeks post-BMT (Fig. 20A).
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Castrated mice had significantly increased congenic (Ly5.2) cells compared to non-castrated animals (data not shown).
In noncastrated mice, there was a profound decrease in thymocyte number over the 4 week time period, with little or no evidence of regeneration (Fig. 21 A). In the castrated group, however, by two weeks there was already extensive thymopoiesis which by four weeks had returned to control levels, being 10 fold higher than in noncastrated mice. Flow cytometeric analysis of the thymii with respect to CD45.2 (donor-derived antigen) demonstrated that no donor derived cells were detectable in the noncastrated group at 4 weeks, but remarkably, virtually all the thymocytes in the castrated mice were donor-derived at this time point (Fig. 21B). Given this extensive enhancement of thymopoiesis from donor- derived haemopoietic precursors, it was important to determine whether T cell differentiation had proceeded normally. CD4, CD8 and TCR defined subsets were analyzed by flow cytometry. There were no proportional differences in thymocytes subset proportions 2 weeks after reconstitution (Fig. 22). This observation was not possible at 4 weeks, because the noncastrated mice were not reconstituted with donor-derived cells. However, at this time point the thymocyte proportions in castrated mice appear normal.
Two weeks after foetal liver reconstitution there were significantly more donor- derived, myeloid dendritic cells (defined as CD45.2+ Macl+ CDI 1C+) in castrated mice than noncastrated mice, the difference was 4-fold (p<0.05). Four weeks after treatment the number of donor-derived myeloid dendritic cells remained above the control in castrated mice (Fig. 23 A). Two weeks after foetal liver reconstitution the number of donor derived lymphoid dendritic cells (defined as CD45.2+Macl-CD11C+) in the thymus of castrated mice was double that found in noncastrated mice. Four weeks after treatment the number of donor-derived lymphoid dendritic cells remained above the control in castrated mice (Fig. 23B).
Immunofluorescent staining for CD11C, epithelium (antikeratin) and CD45.2 (donor- derived marker) localized dendritic cells to the corticomedullary junction and medullary areas of thymii 4 weeks after reconstitution and castration. Using colocalization software, donor- derivation of these cells was confirmed (data not shown). This was supported by flow cytometry data suggesting that 4 weeks after reconstitution approximately 85% of cells in the thymus are donor derived.
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Cell numbers in the bone marrow of castrated and noncastrated reconstituted mice were compared to those of untreated age matched controls and are summarized in Fig. 24A. Bone marrow cell numbers were normal two and four weeks after reconstitution in castrated mice. Those of noncastrated mice were normal at two weeks but dramatically decreased at four weeks (p<0.05). Although, at this time point the noncastrated mice did not reconstitute with donor-derived cells.
Flow cytometeric analysis of the bone marrow with respect to CD45.2 (donor-derived antigen) established that no donor derived cells were detectable in the bone marrow of noncastrated mice 4 weeks after reconstitution, however, almost all the cells in the castrated mice were donor- derived at this time point (Fig. 24B).
Two weeks after reconstitution the donor-derived T cell numbers of both castrated and noncastrated mice were markedly lower than those seen in the control mice (p<0.05). At 4 weeks there were no donor-derived T cells in the bone marrow of noncastrated mice and T cell number remained below control levels in castrated mice (Fig. 25 A).
Donor-derived, myeloid and lymphoid dendritic cells were found at control levels in the bone marrow of noncastrated and castrated mice 2 weeks after reconstitution. Four weeks after treatment numbers decreased further in castrated mice and no donor-derived cells were seen in the noncastrated group (Fig. 25B).
Spleen cell numbers of castrated and noncastrated reconstituted mice were compared to untreated age matched controls and the results are summarized in Fig. 27A. Two weeks after treatment, spleen cell numbers of both castrated and noncastrated mice were approximately 50% that of the control. By four weeks, numbers in castrated mice were approaching normal levels, however, those of noncastrated mice remained decreased. Analysis of CD45.2 (donor-derived) flow cytometry data demonstrated that there was no significant difference in the number of donor derived cells of castrated and noncastrated mice, 2 weeks after reconstitution (Fig. 27B). No donor derived cells were detectable in the spleens of noncastrated mice at 4 weeks, however, almost all the spleen cells in the castrated mice were donor derived.
Two and four weeks after reconstitution there was a marked decrease in T cell number in both castrated and noncastrated mice (p<0.05) (Fig. 28A). Two weeks after foetal liver reconstitution donor-derived myeloid and lymphoid dendritic cells (Figs. 28A and 28B,
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respectively) were found at control levels in noncastrated and castrated mice. At 4 weeks no donor derived dendritic cells were detectable in the spleens of noncastrated mice and numbers remained decreased in castrated mice.
Lymph node cell numbers of castrated and noncastrated, reconstituted mice were compared to those of untreated age matched controls and are summarized in Fig. 26A. Two weeks after reconstitution cell numbers were at control levels in both castrated and noncastrated mice. Four weeks after reconstitution, cell numbers in castrated mice remained at control levels but those of noncastrated mice decreased significantly (Fig. 26B). Flow cytometry analysis with respect to CD45.2 suggested that there was no significant difference in the number of donor-derived cells, in castrated and noncastrated mice, 2 weeks after reconstitution (Fig. 26B). No donor derived cells were detectable in noncastrated mice 4 weeks after reconstitution. However, virtually all lymph node cells in the castrated mice were donor-derived at the same time point.
Two and four weeks after reconstitution donor-derived T cell numbers in both castrated and noncastrated mice were lower than control levels. At 4 weeks the numbers remained low in castrated mice and there were no donor-derived T cells in the lymph nodes of noncastrated mice (Fig. 29). Two weeks after foetal liver reconstitution donor-derived, myeloid and lymphoid dendritic cells were found at control levels in noncastrated and castrated mice (Figs. 29A and 29B, respectively). Four weeks after treatment the number of donor-derived myeloid dendritic cells fell below the control, however, lymphoid dendritic cell number remained unchanged
Thus, castrated mice had significantly increased congenic (Ly5.2) cells compared to non-castrated animals. The observed increase in thymus cellularity of castrated mice was predominantly due to increased numbers of donor-derived thymocytes (Figs. 21 and 23), which correlated with increased numbers of HSC (Lin"c-kit+sca-l+) in the bone marrow of the castrated mice. In addition, castration enhanced generation of B cell precursors and B cells in the marrow following BMT, although this did not correspond with an increase in peripheral B cell numbers at the time-points. Thus, thymic regeneration most likely occurs through synergistic effects on stem cell content in the marrow and their uptake and/or promotion of intrathymic proliferation and differentiation. Importantly, intrathymic analysis demonstrated a significant increase (p≤0.05) in production of donor-derived DC in Cx mice compared to ShCx mice (Fig. 23B) concentrated at the corticomedullary junction as is normal for host DC
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(data not shown). In all cases of thymic reconstitution, thymic structure and cellularity was identical to that of young mice (data not shown).
These HSC transplants (BM or fetal liver) clearly showed the development of host DCs (and T cells) in the regenerating thymus in a manner identical to that which normally occurs in the thymus. There was also a reconstitution of the spleen and lymph node in the transplanted mice which was much more profound in the castrated mice at 4 weeks (see, e.g., Figs. 24, 26, 27, 28, and 29). Since the host HSC clearly enter the patient thymus and create DC which localize in the same regions as host DC in the normal thymus (confirmed by immunohistology; data not shown) it is highly likely that such chimeric thymii will generate T cells tolerant to the donor (by negative selection occurring in donor-reactive T cells after contacting donor DC). This establishes a clear approach to inducing transplantation tolerance because it is long lasting (because the donor HSC are self-renewing) and not requiring prolonged immunosuppression, being due to the actual death of potentially reactive clones.
In a parallel set of experiments, 3 month old, young adults, C57/BL6 mice were castrated or sham-castrated 1 day prior to BMT. For congenic BMT, the mice were subjected to 800RADS TBI and IV injected with 5 x 106 Ly5.1+ BM cells. Mice were killed 2 and 4 weeks later and the BM, thymus and spleen were analyzed for immune reconstitution. Donor/Host origin was determined with anti-CD45.1 antibody, which only reacts with leukocytes of donor origin.
The results from this parallel set of experiments are shown in Figs. 30-39.
Figures 31 and 32 show an increase in the number and proportion of donor derived HSC in the BM of castrated animals. This indicates improved engraftment and suggests faster recovery from BMT.
Figure 33 shows an increase in donor derived B cell precursors and B cells in the BM of castrated mice. However, Figure 35 and 36 show castration does not alter the number or proportion of B cells in the periphery at 2 and 4 weeks post castration.
Figure 37 shows castration increased numbers of donor derived TN, DP, CD4 and
CD8 cells in the thymus. However Figure 34 shows castration does not alter the donor thymocyte proportions of CD4 and CD8 cells. In the periphery, there are very few CD4 or CD8 cells and at the time points considered, there was no increase in these cells with castration.
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Importantly, Figure 39 shows and increased number of donor DC in the thymus by 4 weeks post castration.
Discussion
This example shows the influence of castration on syngeneic and congenic bone marrow transplantation. Starzl et al, (1992) Lancet 339: 1579reported that microchimeras evident in lymphoid and nonlymphoid tissue were a good prognostic indicator for allograft transplantation. It was postulated that they were necessary for the induction of tolerance to the graft (Starzl et al, (1992) Lancet 339:1579). Donor-derived dendritic cells were present in these chimeras and were thought to play an integral role in the avoidance of graft rejection (Thomson and Lu, (1999) Immunol. Today 20:20). Dendritic cells are known to be key players in the negative selection processes of thymus and if donor-derived dendritic cells were present in the recipient thymus, graft reactive T cells may be deleted.
In order to determine if castration would enable increased chimera formation, a study was performed using syngeneic foetal liver transplantation. The results showed an enhanced regeneration of thymii of castrated mice. These trends were again seen when the experiments were repeated using congenic (Ly5) mice. Due to the presence of congenic markers, it was possible to assess the chimeric status of the mice. As early as two weeks after foetal liver reconstitution there were donor-derived dendritic cells detectable in the thymus, the number in castrated mice being four-fold higher than that in noncastrated mice. Four weeks after reconstitution the noncastrated mice did not appear to be reconstituted with donor derived cells, suggesting that castration may in fact increase the probability of chimera formation. Given that castration not only increases thymic regeneration after lethal irradiation and fetal liver reconstitution and that it also increases the number of donor-derived dendritic cells in the thymus, along-side stem cell transplantation this approach increases the probability of graft acceptance.
EXAMPLE 5
IMMUNE CELL DEPLETION
In order to prevent interference with the graft by the existing T cells in the potential graft recipient patient, a human patient underwent T cell depletion (ablation). The patient received anti-T cell antibodies in the form of a daily injection of 15mg/kg of Atgam (xeno anti-T cell globulin, Pharmacia Upjohn) for a period of 10 days in combination with an
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inhibitor of T cell activation, cyclosporin A, 3 mg/kg, as a continuous infusion for 3-4 weeks followed by daily tablets at 9mg/kg as needed. This treatment did not affect early T cell development in the patient's thymus, as the amount of antibody necessary to have such an affect cannot be delivered due to the size and configuration of the human thymus. The treatment was maintained for approximately 4-6 weeks to allow the loss of sex steroids followed by the reconstitution of the thymus.
The prevention of T cell reactivity may also be combined with inhibitors of second level signals such as interleukins, accessory molecules (e.g., antibodies blocking, e.g., CD28), signal transduction molecules or cell adhesion molecules to enhance the T cell ablation and/or other immune cell depletion. The thymic reconstitution phase would be linked to injection of donor HSC (obtained at the same time as the organ or tissue in question either from blood - pre-mobilized from the blood with G-CSF (2 intradermal injections/day for 3 days) or collected directly from the bone marrow of the donor. The enhanced levels of circulating HSC would promote uptake by the thymus (activated by the absence of sex steroids and/or the elevated levels of GnRH). These donor HSC would develop into intrathymic dendritic cells and cause deletion of any newly formed T cells which by chance would be "donor-reactive". This would establish central tolerance to the donor cells and tissues and thereby prevent or greatly minimize any rejection by the host. The development of a new repertoire of T cells would also overcome the immunodeficiency caused by the T cell-depletion regime.
The depletion of peripheral T cells minimizes the risk of graft rejection because it depletes non-specifically all T cells including those potentially reactive against a foreign donor. Simultaneously, however, because of the lack of T cells the procedure induces a state of generalized immunodeficiency which means that the patient is highly susceptible to infection, particularly viral infection.
EXAMPLE 6
SEX STEROID ABLATION THERAPY
The patient was given sex steroid ablation therapy in the form of delivery of an LHRH agonist. This was given in the form of either Leucrin (depot injection; 22.5 mg) or Zoladex (implant; 10.8 mg), either one as a single dose effective for 3 months. This was
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effective in reducing sex steroid levels sufficiently to reactivate the thymus. In some cases it is also necessary to deliver a suppresser of adrenal gland production of sex steroids. Cosudex (5mg/day) may be delivered as one tablet per day for the duration of the sex steroid ablation therapy. Alternatively, the patient is given a GnRH antagonist, e.g., Cetrorelix or Abarelix as a subcutaneous injection
Reduction of sex steroids in the blood to minimal values takes about 1-3 weeks post surgical castration, and about 3-4 weeks following chemical castration. In some cases it is necessary to extend the treatment to a second 3 month injection/implant. The thymic expansion may be increased by simultaneous enhancement of blood HSC either as an allogeneic donor (in the case of grafts of foreign tissue) or autologous HSC (by injecting the host with G-CSF to mobilize these HSC from the bone marrow to the thymus.
EXAMPLE 7
ALTERNATIVE DELIVERY METHOD
In place of the 3 month depot or implant administration of the LHRH agonist, alternative methods can be used. In one example the patient's skin may be irradiated by a laser such as an Er:YAG laser, to ablate or alter the skin so as to reduce the impeding effect of the stratum corneum.
Laser ablation or alteration is described in U.S. Patent Nos. 6,251,100, 6,419,642 and 4,775,361.
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In another example, delivery is by means of laser generated pressure waves. A dose of LHRH agonist is placed on the skin in a suitable container, such as a plastic flexible washer (about 1 inch in diameter and about 1/16 inch thick), at the site where the pressure wave is to be created. The site is then covered with target material such as a black polystyrene sheet about 1 mm thick. A Q-switched solid state ruby laser (20 ns pulse duration, capable of generating up to 2 joules per pulse) is used to generate a single impulse transient, which hits the target material. The black polystyrene target completely absorbs the laser radiation so that the skin is exposed only to the impulse transient, and not laser radiation. The procedure can be repeated daily, or as often as required, to maintain the circulating blood levels of the agonist.
EXAMPLE 8
ADMINISTRATION OF DONOR CELLS
Where practical, the level of hematopoietic stem cells (HSC) in the donor blood is enhanced by injecting into the donor granulocyte-colony stimulating factor (G-CSF) at 10 μg/kg for 2-5 days prior to cell collection (e.g., one or two injections of 10 μg/kg per day for each of 2-5 days). The donor may also be injected with LHRH agonist and/or a cytokine, such as G-CSF or GM-CSF, prior to (e.g., 7-14 days before) collection to enhance the level or quality of stem cells in the blood. CD34+ donor cells are purified from the donor blood or BM, such as by using a flow cytometer or immunomagnetic beading. Antibodies that specifically bind to human CD34 are commercially available (from, e.g., Research Diagnostics Inc., Flanders, NJ; Miltenyi-Biotec, Germany). Donor-derived HSC are identified by flow cytometry as being CD34+. These CD34+ HSC may also be expanded by in vitro culture using feeder cells (e.g., fibroblasts), growth factors such as stem cell factor (SCF), and LIF to prevent differentiation into specific cell types. At approximately 3-4 weeks post LHRH agonist delivery (i.e., just before or at the time the thymus begins to regenerate) the patient is injected with the donor HSC, optimally at a dose of about 2-4 x 106 cells/kg. G-CSF may also be injected into the recipient to assist in expansion of the donor HSC. If this timing schedule is not possible because of the critical nature of clinical condition, the HSC could be administered at the same time as the GnRH. It may be necessary to give a second dose of HSC approximately 2-3 weeks later to assist in the thymic regrowth and the development of donor DC (particularly in the thymus). Once the HSC have engrafted
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(i.e., incoφorated into) and/or migrated to the BM and thymus, the effects should be permanent since HSC are self-renewing.
The reactivating or reactivated thymus takes up the donor HSC and converts them into donor-type T cells and DC, while converting the recipient's HSC into recipient-type T cells and DC. By inducing deletion by cell death, or by inducing tolerance through immunoregulatory cells, the donor and host DC tolerize any new T or NK cells that are potentially reactive with donor or recipient cells.
EXAMPLE 9
TRANSPLANTATION OF GRAFT HSC
While the recipient is still undergoing continuous T cell depletion and/or other immune cell depletion and/or immunosuppressive therapy, the HSC are transplanted from the donor to the recipient patient. The recipient thymus has been activated by GnRH treatment and infiltrated by exogenous HSC.
Within about 3-4 weeks of LHRH therapy the first new T cells will be present in the blood stream of the recipient. However, in order to allow production of a stable chimera of host and donor hematopoietic cells, immunosuppressive therapy may be maintained for about 3-4 months. The new T cells will be purged of potentially donor reactive and host reactive cells, due to the presence of both donor and host DC in the reactivating thymus. Having been positively selected by the host thymic epithelium, the T cells will retain the ability to respond to normal infections by recognizing peptides presented by host APC in the peripheral blood of the recipient. The incoiporation of donor dendritic cells into the recipient's lymphoid organs establishes an immune system situation virtually identical to that of the host alone, other than the tolerance of donor cells, tissue and organs. Hence, normal immunoregulatory mechanisms are present. These may also include the development of regulatory T cells which switch on or off immune responses using cytokines such as IL4, 5, 10, TGF-beta, TNF-alpha.
EXAMPLE 10
IMMUNIZATION AND PREVENTION OF VIRAL INFECTION (INFLUENZA)
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Influenza viruses are segmented RNA viruses that cause highly contagious acute respiratory infections. The major problem associated with vaccine development against influenza is that these viruses have the ability to escape immune surveillance and remain in a host population by altering antigenic sites on the hemagglutinin (HA) and neuraminidase (N) envelope glycoproteins by phenomena termed antigenic drift and antigenic shift. The primary correlate for protection against influenza virus is neutralizing antibody against HA protein that undergoes strong selection for antigenic drift and shift. However, much more conserved antigenic cross-reactivities for different strains of influenza virus occur between internal proteins, such as the nucleoprotein (NP) (Shu et al, (1993) J. Virol. 67:2723). CTL and protection from influenza challenge following immunization with a polynucleotide encoding NP has previously been shown (Ulmer et al. (1993) Science 259: 1745).
Materials and Methods
Surgical Castration. BALB/c mice are anesthetized by intraperitoneal injection of 30-40 μl of a mixture of 5 ml of 100 mg/ml ketamine hydrochloride (Ketalar®; Parke-Davis, Caringbah, NSW, Australia) plus 1 ml of 20 mg/ml xylazine (Rompun®; Bayer Australia Ltd., Botany NSW, Australia) in saline. Surgical castration is performed as described elsewhere herein by a scrotal incision, revealing the testes, which are tied with suture and then removed along with surrounding fatty tissue. The wound is closed using surgical staples. Sham-castrated mice prepared following the above procedure without removal of the testes are used as controls.
Chemical castration. Mice are injected i.m. with 10 mg/kg Lupron® (a GnRH agonist) as a 1 month slow release formulation. Alternatively mice are injected with a GnRH antagonist (e.g., Cetrorelix or Abarelix). Confirmation of loss of sex steroids is performed by standard radioimmunoassay of plasma samples following manufacturer's instructions. Castrate levels (<0.5 ng testosterone or estrogen /ml) should normally be achieved by 3-4 weeks post injection.
Preparation of influenza A/PR/8/34 subunit vaccine. Purified influenza A/PR/8/34
(H1N1) subunit vaccine preparation is prepared following methods known in the art. Briefly, the surface hemagglutinin (HA) and neuraminidase (NA) antigens from influenza A/PR/8/34 particles are extracted using a non-ionic detergent (7.5% N-octyl-β-o-thioglucopyranoside).
After centrifugation, the HA/NA-rich supernatant (55% HA) is used as the subunit vaccine.
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Influenza A/PR/8/34 subunit immunization. Approximately 6 weeks following surgical castration or about 8 weeks following chemical castration, mice are immunized with 100 μl of formalin-inactivated influenza A/PR/8/34 virus (about 7000 HAU) injected subcutaneously.
Booster immunizations can optionally be performed at about 4 weeks (or later) following the primary immunization. Freund's complete adjuvant (CFA) is used for the primary immunization and Freund's incomplete adjuvant is used for the optional booster immunizations.
Alternatively, the influenza A/PR/8/34 subunit vaccine preparation (see above) may be intramuscularly injected directly into, e.g., the quadriceps muscle, at a dose of about 1 μg to about 10 μg dilute in a volume of 40 μl sterile 0.9% saline.
Plasmid DNA. Preparation of plasmid DNA expression vectors are readily known in the art (see, e.g., Current Protocols In Immunology. Unit 2.14, John E. Coligan et al, (eds), Wiley and Sons, New York, NY (1994), and yearly updates including 2002). Briefly, the complete influenza A/PR/8/34 nucleoprotein (NP) gene or hemagglutinin (HA) coding sequence is cloned into an expression vector, such as, pCMV, which is under the transcriptional control of the cytomegalovirus (CMV) immediate early promoter.
Empty plasmid (e.g., pCMV with no insert) is used as a negative control. Plasmids are grown in Escherichia coli DH5α or HB101 cells using standard techniques and purified using Qiagen® Ultra-Pure®- 100 columns (Chatsworth, CA) according to manufacturer's instructions. All plasmids are verified by appropriate restriction enzyme digestion and agarose gel electrophoresis. Purity of DNA preparations is determined by optical density readings at 260 and 280 nm. All plasmids are resuspended in TE buffer and stored at -20°C until use.
DNA immunization. Methods of DNA immunization are well known in the art. For instance, methods of intradermal, intramuscular, and particle-mediated ("gene gun") DNA immunizations are described in detail in, e.g., Current Protocols In Immunology, Unit 2.14, John E. Coligan et al, (eds), Wiley and Sons, New York, NY (1994), and yearly updates including 2002).
Cytokine-encoding DNAs are optionally administered to shift the immune response to a desired Thl- or a Th2-type immune response. Thl-inducing genetic adjuvants include, e.g.,
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IFN-γ and IL-12. Th2-inducing genetic adjuvants include, e.g., IL-4, IL-5, and IL-10. For review of the preparation and use of Thl- and Th2- inducing genetic adjuvants in the induction of immune response (see, e.g., Robinson, et al, (2000) Adv. Virus Res. 55: 1).
Influenza A/PR/8/34 virus challenge In an effort to determine if castrated mice are better protected from influenza virus challenge (with and without vaccination) as compared to their sham-castrated counteφarts, metofane-anesthetized mice are challenged by intranasal inoculation of 50 Dl of influenza A/PR/8/34 (H1N1) influenza virus containing allantoic fluid diluted 10"4 in PBS/2% BSA (50-100 LD50; 0.25 HAU). Mice are weighed daily and sacrificed following >20% loss of pre-challenge weight. At this dose of challenge virus, 100% of na ve mice should succumb to influenza infection by 4-6 days.
Sublethal infections are optionally done to activate memory T cells, but use a 10" dilution of virus. Sublethal infections may also be optionally done to determine if non- immunized, castrated mice have better immune responses than the sham castrated controls, as determined by ELISA, cytokine assays (Th), CTL assays, etc. outlined below. Viral titers for lethal and sublethal infections may be optimized prior to use in these experiments.
Enzyme-linked immunosorbant assays. At various time periods pre- and post- immunization (or pre- and post- infection), mice from each group are bled, and individual mouse serum is tested using standard quantitative enzyme-linked immunosorbant assays (ELISA) to assess anti-HA or -NP specific IgG levels in the serum. IgGl and IgG2a levels may optionally be tested, which are known to correlate with Th2 and Thl -type antibody responses, respectively.
Preparation and stimulation of splenocytes for cytokine production. Spleens are aseptically harvested from the various groups of mice (n=2-3) and pooled in p60 petri dishes containing about 4 ml RPMI-10 media (RPMI-1640, 10% fetal bovine serum, 50 μg/ml gentamycin). Spleens are prepared and RBC lysed using standard procedures. Cells are then counted, and resuspended in RPMI-10 containing 80 U/ml rat IL-2 (Sigma, St. Louis, MO) to a final cell concentration of 2xl07 cells/ml. One hundred microliters of cells are dispensed into wells of a 96-well tissue culture plate for a final concentration of 2x10 cells/well. Stimulations are conducted by adding 100 μl of the appropriate peptide or inactivated influenza virus diluted in RPMI-10. CD8+ T cells are stimulated with either the Kd-restricted HA533.541 peptide (IYSTVASSL; SEQ ID NO: l) (Winter, Fields, and Brownlee, (1981) Nature 292:72) or the Kd-restricted NPι47.ι55 peptide (TYQRTRALV; SEQ ID NO:2)
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(Rotzschke et al, (1990) Nature 348:252). CD4+ T cells are stimulated with inactivated influenza virus (13,000 HAU per well of boiled influenza virus plus 13,000 HAU per well of formalin-inactivated influenza virus) plus anti-CD28 (1 μg/ml) and anti-CD49d (1 μg/ml) (Waldrop et al, (1998) J. Immunol. 161:5284). Negative control stimulations are done with media alone. Cells are then incubated as described below to detect extracellular cytokines by ELISA or intracellular cytokines by FACS staining.
Chromium release assay for CTL. CTL responses to influenza HA and NP are measured using procedures well known to those in the art (see, e.g., Current Protocols In Immunology, John E. Coligan et al, (eds), Unit 3, Wiley and Sons, New York, NY (1994), and yearly updates including 2002). The synthetic peptide HA533-5 ι IYSTVASSL (SEQ ID NO: l) (Winter, Fields, and Brownlee, (1981) Nature 292:72) or ΝP]47-ι55 TYQRTRALV (SEQ ID NO:2) (Rotzschke et al, (1990) Nature 348:252) are used as the peptide in the target preparation step. Responder splenocytes from each animal are washed with RPMI-10 and resuspended to a final concentration of 6.3xl06 cells/ml in RPMI-10 containing 10 U/ml rat IL-2 (Sigma, St. Louis, MO). Stimulator splenocytes are prepared from na ve, syngeneic mice and suspended in RPMI-10 at a concentration of lxlO7 cells/ml. Mitomycin C is added to a final concentration of 25 μg/ml. Cells are incubated at 37°C/5%CO2 for 30 minutes and then washed 3 times with RPMI-10. The stimulator cells are then resuspended to a concentration of 2.4xl06 cells/ml and pulsed with HA peptide at a final concentration of 9xlO"6M or with NP peptide at a final concentration of 2xlO"6M in RPMI-10 and 10 U/ml EL- 2 for 2 hours at 37°C/5% CO2. The peptide-pulsed stimulator cells (2.4xl06) and responder cells (6.3xl06) are then co-incubated in 24-well plates in a volume of 2 ml SM media (RPMI- 10, 1 mM non-essential amino acids, 1 mM sodium pyruvate) for 5 days at 37°C/5%CO2. A chromium-release assay is used to measure the ability of the in vitro stimulated responders (now called effectors) to lyse peptide-pulsed mouse mastocytoma P815 cells (MHC matched, H-2d). P815 cells are labeled with 51Cr by taking 0.1 ml aliquots of p815 in RPMI-10 and adding 25 μl FBS and 0.1 mCi radiolabeled sodium chromate (NEN, Boston, MA) in 0.2 ml normal saline. Target cells are incubated for 2 hours at 37°C/5%CO2, washed 3 times with RPMI-10 and resuspended in 15 ml polypropylene tubes containing RPMI-10 plus HA (9x10" 6M) or NP (lxlO"6) peptide. Targets are incubated for 2 hours at 37°C/5%CO2 . The radiolabeled, peptide-pulsed targets are added to individual wells of a 96-well plate at 5xl04 cells per well in RPMI-10. Stimulated responder cells from individual immunization groups (now effector cells) are collected, washed 3 times with RPMI-10, and added to individual
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wells of the 96-well plate containing the target cells for a final volume of 0.2 ml/well. Effector to target ratios are 50:1, 25:1, 12.5:1 and 6.25:1. Cells are incubated for 5 hours at 37°C/5%CO2 and cell lysis is measured by liquid scintillation counting of 25 μl aliquots of supernatants. Percent specific lysis of labeled target cells for a given effector cell sample is [100 x (Cr release in sample-spontaneous release sample) / (maximum Cr release- spontaneous release sample)]. Spontaneous chromium release is the amount of radioactive released from targets without the addition of effector cells. Maximum chromium release is the amount of radioactivity released following lysis of target cells after the addition of TritonX-100 to a final concentration of 1%. Spontaneous release should not exceed 15%.
Detection of IFNγ or IL-5 in bulk culture supernatants by ELISA. Bulk culture supernatants may be tested for IFNγ and IL-5 cytokine levels, which are known to correlate with Thl and Th2-type response, respectively. Pooled splenocytes are incubated for 2 days at 37°C/ 5% CO2, and then supernatants are harvested and pooled All ELISA antibodies and purified cytokines are purchased from Pharmingen (San Diego, CA). Fifty microliters of purified anti-cytokine monoclonal antibody diluted to 5 μg/ml (rat anti-mouse IFNγ) or 3 μg/ml (rat anti-mouse IL-5) in coating buffer (0.1 M NaHCO3, pH 8.2) is distributed per well of a 96-well ELISA plate (Corning, Corning, NY) and incubated overnight at 4°C. Plates are washed, blocked, and rewashed with PBS-T. Standards (recombinant mouse cytokine) and samples are added to wells at various dilutions in RPMI-10 and incubated overnight at 4°C for maximum sensitivity. Plates are washed 6 times with PBS-T. Biotinylated rat anti-mouse cytokine detecting antibody is diluted in PBS-T to a final concentration of 2 μg/ml and 100 μl was distributed per well. Plates are incubated for 1 hr. at 37°C and then washed 6 times with PBS-T. Streptavidin- AP (Gibco BRL, Grand Island, NY) is diluted 1:2000 according to manufacturer's instructions, and 100 μl is distributed per well. Plates are incubated for 30 min. and washed an additional 6 times with PBS-T. Plates are developed by adding 100 μl/well of AP developing solution (BioRad, Hercules, CA) and incubating at room temperature for 50 minutes. Reactions are stopped by addition of 100 μl 0.4 M NaOH and read at OD405. Data are analyzed using Softmax Pro Version 2.21 computer software (Molecular Devices, Sunnyvale, CA).
Intracellular cytokine staining and FACS analysis. Splenocytes may be tested for intracellular IFNγ and IL-5 cytokine levels, which are known to correlate with Thl and Th2- type response, respectively. Pooled splenocytes are incubated for 5-6 hours at 37°C in a humidified atmosphere containing 5% CO2. A Golgi transport inhibitor, Monensin
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(Pharmingen, San Diego, CA), is added at 0.14 μl/well according to the manufacturer's instructions, and the cells are incubated for an additional 5-6 hours (Waldrop et al, (1998) J. Immunol. 161:5284). Cells are thoroughly resuspended and transferred to a 96-well U- bottom plate. All reagents (GolgiStop kit and antibodies) are purchased from Pharmingen (San Diego, CA) unless otherwise noted, and all FACS staining steps are done on ice with ice-cold reagents. Plates are washed 2 times with FACS buffer (lx PBS, 2% BSA, 0.1% w/v sodium azide). Cells are surface stained with 50 μl of a solution of 1:100 dilutions of rat anti- mouse CD8β-APC, -CD69-PE, and -CD16/CD32 (Fcγlll/RII; 'Fc Block') in FACS buffer. For tetramer staining (see below), cells were similarly stained with CD8β-TriColor, CD69- PE, CD16/CD32, and HA- or NP-tetramer-APC in FACS buffer. Cells are incubated in the dark for 30 min. and washed 3 times with FACS buffer. Cells are permeabilized by thoroughly resuspending in 100 μl of Cytofix/Cytoperm solution per well and incubating in the dark for 20 minutes. Cells are washed 3 times with Permwash solution. Intracellular staining is completed by incubating 50 μl per well of a 1:100 dilution of rat anti-mouse IFNγ- FITC in Permwash solution in the dark for 30 min. Cells are washed 2 times with Permwash solution and 1 time with FACS buffer. Cells are fixed in 200 μl of 1% paraformaldehyde solution and transferred to microtubes arranged in a 96-well format. Tubes are wrapped in foil and stored at 4°C until analysis (less than 2 days). Samples are analyzed on a FACScan® flow cytometer (Becton Dickenson, San Jose, CA). Compensations are done using single- stained control cells stained with rat anti-mouse CD8-FITC, -PE, -Tricolor, or -APC. Results are analyzed using FlowJo Version 2.7 software (Tree Star, San Carlos, CA).
Tetramers. HA and NP tetramers may be used to quantitate HA- and NP-specific CD8+ T cell responses following HA or NP immunization. Tetramers are prepared essentially as described previously (Flynn et al, (1998) Immunity 8:683). The present example utilizes the H-2Kd MHC class I glycoprotein complexed the synthetic influenza A/PR/8/34 virus peptide HA533.54ι (IYSTVASSL; SEQ ID NO:l) (Winter, Fields, and Brownlee, (1981) Nature 292:72) or NP,47.I55 (TYQRTRALV; SEQ ID NO:2) (Rotzschke et al, (1990) Nature 348:252).
It is noted that the methods described in this example are applicable to a wide array agents, with only minor variations, which would be readily determinable by those skilled in the art.
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EXAMPLE 11
IMMUNIZATION AND PREVENTION OF PARASITIC INFECTION (MALARIA)
The circumsporozoite protein (CSP) is a target of this pre-erythocytic immunity (Hoffman et al, (1991) Science 252:520. In the Plasmodium yoelii (P. yoelii) rodent model system, passive transfer P. yoelii CSP-specific monoclonal antibodies (Charoenvit et al, (1991) J. Immunol. 146:1020), as well as adoptive transfer of P. yoelii CSP-specific CD8+ T cells (Rodrigues et al, (1991) Int. Immunol. 3:579, Weiss et al, (1992) J. Immunol. 149:2103) and CD4+ T cells (Renia et al, (1993) J. Immunol. 150:1471) are protective. Numerous vaccines designed to protect mice against sporozoites by inducing immune responses against the P. yoelii CSP have been evaluated.
Any Plasmodium sporozoite proteins known in the art capable of inducing protection against malaria usable in this invention may be used, such as P. falciparum, P. vivax, P. malariae, and P. ovale CSP; SSP2(TRAP); Pfsl6 (Sheba); LSA-1; LSA-2; LSA-3; MSA-1 (PMMSA, PSA, pl85, pl90); MSA-2 (Gymmnsa, gp56, 38-45 kDa antigen); RESA (Pfl55); EBA-175; AMA-1 (Pf83); SERA (pi 13, pl26, SERP, Pfl40); RAP-1; RAP-2; RhopH3; PfHRP-LT; Pf55; Pf35; GBP (96-R); ABRA (plOl); Exp-1 (CRA, Ag5.1); Aldolase; Duffy binding protein of P. vivax; Reticulocyte binding proteins; HSP70-1 (p75); Pfg25; Pfg28; Pfg48/45; and Pfg230.
Materials and Methods
Castration. Surgical and/or chemical castration is performed as above.
Parasites. The 17XNL (nonlethal) strain of P. yoelii is used as described previously (U.S. Patent No. 5,814,617).
Preparation of irradiated P. yoelii sporozoites. Preparation of irradiated P. yoelii sporozoites for immunization has been described previously (see, e.g., Franke et al, (2000) Infect. Immun. 68:3403). Briefly, sporozoites are isolated by the discontinuous gradient technique (Pacheco et al, (1979) J. Parisitol 65:414) from infected Anopheles Stephens mosquitoes that have been irradiated at 10,000 rads (137Ce).
Immunization with irradiated P. yoelii sporozoites. Mice are intravenously immunized with 50,000 sporozoites at approximately 6 weeks following surgical castration or about 8 weeks following chemical castration via the tail vein. Booster immunizations of
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20,000 to 30,000 sporozoites are optionally given at 4 weeks and 6 weeks following the primary immunization (see, e.g., Franke et al, (2000) Infect Immun. 68:3403).
Plasmid DNA and DNA immunization. Plasmid DNA encoding the full length P. yoelii CSP are known in the art. For instance, the pyCSP vector described in detail in Sedegah et al, ((1998) Proc. Natl. Acad. Sci. USA 95:7648) may be used.
Methods of DNA immunization are also well known in the art. For instance, methods of intradermal, intramuscular, and particle-mediated ("gene gun") DNA immunizations are described in detail in, e.g., Current Protocols In Immunology, Unit 2.14, John E. Coligan et al, (eds), Wiley and Sons, New York, NY (1994), and yearly updates including 2002).
Peptide Immunization. Methods of P. yoelii CSP peptide preparation are known in the art (see, e.g., Franke et al, (2000) Infect Immun. 68:3403).
Chromium release assay for CTL. Since CD8+ CTL against the P. yoelii CSP have been shown to adoptively transfer protection (Weiss et al, (1992) J. Immunol. 149:2103), and CD8+ T cells are required for the protection against P. yoelii induced by immunization with irradiated sporozoites (Weiss et al, (1988) Proc. Natl. Acad. Sci USA 85:573), it must be determined if P. yoelii CSP vaccination (e.g., irradiated sporozoite, CSP peptide, or CSP DNA immunizations) elicits a CSP-specific CTL.
CTL responses are measured using procedures well known to those in the art (see, e.g., Current Protocols In Immunology, John E. Coligan et al, (eds), Unit 3, Wiley and Sons, New York, NY (1994_, and yearly updates including 2002). The general procedure described elsewhere herein for influenza HA and NP is used except that the cells are pulsed with the synthetic P. yoelii CSP peptide (281-296; SYVPSAEQILEFVKQI; SEQ ID NO:3).
Inhibition of liver stage development assay. The liver stage development assay and acquisition of mouse hepatocytes from mouse livers by in situ collagenase perfusion have been described previously (Franke et al, (1999) Vaccine 17: 1201 ; Franke et ø/.,(2000) Infect. Immun. 68:3403). Hepatocyte cultures are seeded onto eight-chamber Lab-Tek plastic slides at lxlO5 cells/chamber and incubated with 7.5 x 104 P. yoelii sporozoites for 3 hours. The cultures are then washed and cultured for and additional 24 hours at 37 C/5% CO . Effector cells are obtained as described above for the chromium release assay for CTL and are added and cultured with the infected hepatocytes for about 24-48 hours. The cultures are then washed, and the chamber slides are fixed for 10 min. in ice-cold absolute methanol. The
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chamber slides are then incubated with a monoclonal antibody (NYLS1 or NYLS3, both described previously in U.S. Patent No. 5,814,617) directed against liver stage parasites of P. yoelii before incubating with FITC-labeled goat anti-mouse Ig. The number of liver-stage schizonts in triplicate cultures are then counted using an epifluorescence microscope. Percent inhibition is calculated using the formula [(control-test)/control) xlOO].
Infection and challenge. For a lethal challenge dose, the ID50 of P. yoelii sporozoites must be determined prior to experimental challenge. However, it is also initially possible to inject mice intravenously in the tail vein with a dose of about 50 to 100 P. yoelii sporozoites (nonlethel, strain 17XNL). Forty-two hours after intravenous inoculation, mice are sacrificed and livers are removed. Single cell suspensions of hepatocytes in medium are prepared, and 2xl05 hepatocytes are placed into each of 10 wells of a multi-chamber slide. Slides may be dried and frozen at -70°C until analysis. To count the number of schizonts, slides are dried and incubated with NYLS1 before incubating with FITC-labeled goat anti- mouse Ig, and the numbers of liver-stage schizonts in each chamber are counted using fluorescence microscopy.
Once it is demonstrated that castration and/or immunization reduces the numbers of infected hepatocytes, blood smears are obtained to determine if immunization protect against blood stage infection. Mice are considered protected if no parasites are found in the blood smears at days 5-14 days post-challenge.
To test the preventative efficacy of castration alone (no vaccination) from a P. yoelii sporozoite primary infection, castrated mice are infected and analyzed as described above. Sham-castrated mice are used as controls.
Human studies. After establishing the efficacy in mice, large numbers of humans are immunized in a double blind placebo controlled field trial.
EXAMPLE 12
IMMUNIZATION AND PREVENTION OF BACTERIAL INFECTION (TB Ag85)
Tuberculosis (TB) is a chronic infectious disease of the lung caused by the pathogen
Mycobacterium tuberculosis, and is one of the most clinically significant infections worldwide, (see, e.g., U.S. Patent No. 5,736,524; for review see Bloom and Murray, (1993),
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Science 257, 1055.
M. tuberculosis is an intracellular pathogen that infects macrophages. Immunity to TB involves several types of effector cells. Activation of macrophages by cytokines, such as IFNγ, is an effective means of minimizing intracellular mycobacterial multiplication. Acquisition of protection against TB requires both CD8+ and CD4+ T cells (see, e.g., Orme et al, (1993) J. Infect. Dis. 167: 1481). These cells are known to secrete Thl-type cytokines, such as IFNγ, in response to infection, and possess antigen-specific cytotoxic activity. In fact, it is known in the art that CTL responses are useful for protection against M. tuberculosis (see, e.g., Flynn et al, (1992) Proc. Natl. Acad. Sci. USA 89:12013).
Predominant T cell antigens of TB are those proteins that are secreted by mycobacteria during their residence in macrophages. These T cell antigens include, but are not limited to, the antigen 85 complex of proteins (85A, 85B, 85C) (Wiker and Harboe, ((1992) Microbiol. Rev. 56: 648) and ESAT-6 (Andersen, (1994) Infect. Immunity, 62:2536). Other T cell antigens have also been described in the art (see, e.g., Young and Garbe, (1991) Res. Microbiol. 142:55; Andersen, (1992) J. Infect. Dis. 166:874; Siva and Lowrie, (1994)
Immunol. 82:244; Romain et a.„ (1993) Proc. Natl. Acad. Sci. USA 90:5322; and Faith et al,
(1991) Immunol. 74: 1).
The genes for each of the three antigen 85 proteins (A, B, and C) have been cloned and sequenced (see, e.g., Borremans et al, (1989) Infect. Immunity 57:3123); DeWit et al, (1994) DNA Seq. 4:267), and have been shown to elicit strong T cell responses following both infection and vaccination.
Materials and Methods
Castration. Surgical and/or chemical castration of BALB/c or C57BL/6 is performed as above.
Protein immunization. General methods for Mycobacterium tuberculosis (TB) bacilli purification and immunization are known in the art (see, e.g., Current Protocols In Immunology, Unit 2.4, John E. Coligan et al, (eds), Wiley and Sons, New York, NY (1994), and yearly updates including 2002). The purified TB may be prepare using preparative SDS- PAGE. Approximately 2 mg of the TB protein is loaded across the wells of a standard 1.5 mm slab gel using a large-tooth comb. An edge of the gel may be removed and stained following electrophoresis to identify the TB protein band on the gel. The gel region that contains the TB protein band is then sliced out of the gel, placed in PBS at a final
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concentration 0.5 mg purified TB protein per ml, and stored at 4°C until use. The purified TB protein may then be emulsified with an equal volume of complete Freund's adjuvant (CFA) for immunization.
Approximately 6 weeks following surgical castration or about 8 weeks following chemical castration, 2 ml of the purified TB (0.5 mg/ml in PBS) is emulsified 2 ml CFA and stored at 4°C. The TB/CFA mixture is slowly drawn into and expelled through a 3-ml glass syringe attached to a 19 gauge needle, being certain to avoid excessive air bubbles. Once the emulsion is at a homogenous concentration, the needle is replaced by a 22 gauge needle, and all air bubbles are removed. The castrated and sham-castrated mice are injected intramuscularly with a 50 μl volume of the TB/CFA emulsion (immunization may also be done via the intradermal or subcutaneous routes). M. bovis BCG may also be used in a vaccine preparation.
A booster immunization can optionally be performed 4-8 weeks (or later) following the primary immunization. The TB adjuvant emulsion is prepared in the same manner described above, except that incomplete Freund's adjuvant (IFA) is used in place of CFA for all booster immunizations. Further booster immunizations can be performed at 2-4 week (or later intervals) thereafter.
Plasmid DNA. Suitable Ag85-encoding DNA sequences and vectors have been described previously (see, e.g., U.S. Patent No. 5,736,524). Other suitable expression vectors would be readily ascertainably by hose skilled in the art.
Antigen 85 DNA Immunization. Methods of DNA immunization are well known in the art. For instance, methods of intradermal, intramuscular, and particle-mediated ("gene gun") DNA immunizations are described in detail in, e.g., Current Protocols In Immunology,
Unit 2.14, John E. Coligan et al, (eds), Wiley and Sons, New York, NY (1994), and yearly updates including 2002).
Cytokine-encoding DNAs are optionally administered to shift the immune response to a desired Thl- or a Th2-type immune response. Thl-inducing genetic adjuvants include, e.g.,
IFN-γ and IL-12. Th2-inducing genetic adjuvants include, e.g., IL-4, IL-5, and LL-10. For review of the preparation and use of Thl- and Th2- inducing genetic adjuvants in the induction of immune response, see, e.g., Robinson, et al, (2000) Adv. Virus Res. 55: 1-74.
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Approximately 6 weeks following surgical castration or about 8 weeks following chemical castration, mice are intramuscularly injected with 200 μg of DNA diluted in 100 μl saline.
Booster DNA immunizations are optionally administered at 4 weeks post-prime and 2 weeks post-boost.
Enzyme-linked immunosorbant assays. At various time periods pre- and post- immunization, mice from each group are bled, and individual mouse serum is tested using standard quantitative ELISA to assess anti-Ag85 specific IgG levels in the serum. IgGl and IgG2a levels may optionally be tested, which are known to correlate with Th2 and Th-type antibody responses, respectively.
Serum is collected at various time points pre- and post-prime and post boost, and analyzed for the presence of anti-Ag85 specific antibodies in serum. Basic ELISA methods are described elsewhere herein, except purified Ag85 protein is used.
Cytokine assays. Spleen cells from vaccinated mice are analyzed for cytokine secretion in response to specific Ag85 restimulation, as described, e.g., in Huygen et al,
(1992) Infect. Immunity 60:2880, and in U.S. Patent No. 5,736,524. Briefly, spleen cells are incubated with culture filtrate (CF) proteins from M. bovis BCG purified Ag85A or the
C57BL/6 T cell epitope peptide (amino acids 241-260).
Four weeks post-prime and 2 weeks post boost (or later), cytokines are assayed using standard bio-assays for IL-2,IFNγ and IL-6, and by ELISA for IL-4 and IL-10 using methods well known to those in the art. See, e.g., Current Protocols In Immunology, Unit 6, John E.
Coligan et al, (eds), Wiley and Sons, New York, NY (1994), and yearly updates including
2002.
Mycobacterial infection and challenge. To test the efficacy of the vaccinations, mice are challenged by intravenous injection of live M. bovis BCG (0.5 mg). At various time points post-challenge, BCG multiplication is analyzed in both mouse spleens and lungs.
Positive controls are na ve mice (castrated and/or sham castrated as appropriate) receiving a challenge dose.
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To test the efficacy of sex steroid ablation to prevent primary infection, live M. bovis BCG are injected similarly to that described in the challenge experiment above. Sham castrated mice are used as controls.
The number of colony-forming units (CFU) in the spleen and lungs of the challenged, vaccinated mice, as well as in the lungs of the castrated, primary infected mice is expected to be substantially lower than in negative control animals, which is indicative with protection in the live M. bovis challenge model.
EXAMPLE 13
IMMUNIZATION AND PREVENTION OF CANCER
To determine if sex steroid ablation is effective in preventing cancer and/or in eliciting a protective immune response following vaccination with a cancer antigen, the following studies are performed.
Materials and Methods
Castration of mice. C57BL/6 mice are castrated as described above.
CEA immunization. Approximately 6 weeks following surgical castration or about 8 weeks following chemical castration, mice were inoculated with an adenovirus vector encoding the human carcinoembryonic antigen (CEA) gene (MC38-CEA-2) (Conry et al, 1995), such as AdCMV-hcea described in U.S.P.N. 6,348,450. Alternatively, a plasmid DNA encoding the human CEA gene is injected into the mouse (e.g., intramuscularly into the quadriceps muscle) utilizing one of the various methods of DNA vaccination described elsewhere herein.
Tumor challenge. To asses the efficacy of sex steroid ablation on anti-tumor activity of mice immunized with CEA, mice are subjected to a tumor challenge. At various time points post immunization, syngeneic tumor cells expressing the human CEA gene
(MC38-CEA-2) (Conry et al, 1995) are inoculated into the mice. Mice are observed every other day for development of palpable tumor nodules. Mice are sacrificed when the tumor nodules exceed 1 cm in diameter. The time between inoculation and sacrifice is the survival time.
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To test the efficacy of sex steroid ablation preventing tumors, tumor cells expressing the human CEA gene are inoculated into castrated, non-vaccinated mice as outlined above. Sham castrated mice are used as controls.
EXAMPLE 14
TRANSPLANTATION OF GENETICALLY MODIFIED HSC (GENE THERAPY)
L SCID-hu Mouse Model
Materials and Methods
Mice. SCID-hu mice are prepared essentially as described previously (see, e.g., Namikawa et al, (1990) J. Exp. Med. 172:1055 and Bonyhadi et al, (1997) J. Virol.
71:4707) by surgical transplantation of human fetal liver and thymus fragments into CB-17 scid/scid mice. Methods for the construction of SCID-hu Thy/Liv mice can also be found, e.g., in Coligan et al. Unit 4.8, 1994 and yearly updates including 2002.
Castration of mice. The SCID-hu mice are anesthetized by intraperitoneal injection of 30-40 μl of a mixture of 5 ml of 100 mg/ml ketamine hydrochloride (Ketalar; Parke-Davis, Caringbah, NSW, Australia) plus 1 ml of 20 mg/ml xylazine_(Rompun; Bayer Australia Ltd., Botany NSW, Australia) in saline. Surgical castration is performed as described above by a scrotal incision, revealing the testes, which are tied with suture and then removed along with surrounding fatty tissue. The wound is closed using surgical staples. Sham-castrated mice prepared following the above procedure without removal of the testes are used as controls.
Chemical castration. Chemical castration is performed as above.
Isolation of human CD34 + HSC. Human cord blood (CB) HSC are collected and processed using techniques well known to those skilled in the art (see, e.g., DiGusto et al, (1997) Blood, 87:1261 (1997), Bonyhadi et al, (1997) 7. Virol. 71 :4707). A portion of each CB sample is HLA phonotyped for the MA2.1 surface molecule. CD34+ cells are enriched using immunomagnetic beads using the method described in Bonyhadi et al. ((1997) J. Virol. 71:4707). Briefly, CB cells are incubated with anti-CD34 antibody (QBEND-10, Immunotech) and then washed and resuspended at a final concentration of 2x10 cells/ml. CD34+ cells are then enriched using goat-anti-mouse IgGl magnetic beads (Dynal) following
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manufacturer's instructions. The CD34+ cells are then incubated with 50 μl of glycoprotease (O-sialoglycoprotein endopeptidase), which causes release of the CD34+ cells from the immunomagnetic beads. The beads are removed using a magnet, and the cells are then subjected to flow cytometry using conjugated anti-CD34-PE to determine the total level of CD34+ cells present in the population. Alternatively, the cells are magnetically labeled with anti-CD34 and sorted on an autoMACS™. The autoMACS™ may be used for magnetic presorting of cells before further flow cytometric sorting. For example, anti-FITC- or anti-PE MACS® MicroBeads, may be added to the FITC or PE stained cells. Then the cells are sorted on the autoMACS™ according to their magnetic labeling. The positive and negative fractions may then be collected for sorting by flow cytometry.
Optionally, HSC are expanded ex vivo with IL-3, LL-6, and either SCF or LIF (10 ng/ml each).
RevMlO vectors and preparation of genetically modified (GM) HSC. RevMlO is known in the art, and has been described extensively in studies of GM HSC for the survival of T cells in HIV-infected patients (Woffendin et al, 1996); for review, see Amado et al, (1999). The HIV Rev protein is known to affect viral latency in HIV infected cells and is essential for HIV replication. RevMlO is a derivative of Rev because of mutations within the leucine-rich domain of Rev that interacts with cell factors. RevMlO has a substitution of aspartic acid for leucine at position 78 and of Leucine for glutamic acid at position 79. The result of these mutations is that RevMlO is able to compete effectively with the wild-type HIV Rev for binding to the Rev-responsive element (RRE).
Any of the RevMlO gene transfer vectors known and described in the art may be used. For example, the retroviral RevMlO vector, pLJ-RevMlO is used to transducer the HSC. The pLJ-RevMlO vector has been shown to enhance T cell engraftment after delivery into HIV-infected individuals (Ranga et al, 1998). Other methods of construction and retroviral vectors suitable for the preparation of GM HSC are well known in the art (Bonyhadi et al, 1997).
In another example, the pRSV/TAR RevMlO plasmid is used for non-viral vector delivery using particle-mediated gene transfer into the isolated target HSC essentially as described in Woffendin et al, (1994). The pRSV/TAR RevMlO plasmid contains the Rous sarcoma virus (RSV) promoter and tat-activation response element (TAR) from -18 to +72 of
HIV is used to express the RevMlO open reading frame may also be used (Woffendin et al,
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(1994); Liu et al, (1997). In vitro transfection of this plasmid into human PBL has previously been shown to provide resistance to HIV infection (Woffendin et al, 1994).
A marker gene, such as the Lyt-2α (murine CD8α) gene, may also be incorporated into the RevMlO vector for ease of purification and analysis of GM HSC by FACS analysis in subsequent steps (Bonyhadi et al, 1997).
A ΔRevlO, which contains a deletion of the methionine (Met) initiation codon (ATG), as well as a linker comprising a series of stop codons inserted in-frame into the Bglll site of the RevMlO gene, is constructed and used as a negative control (Bonyhadi et al, 1997).
Injection of GM HSC into mice. SCID-hu mice are analyzed, and the mice determined to be HLA mismatched (MA2.1) with respect to the human donor HSC are given approximately 400 rads of total body irradiation (TBI) about four months following the thymic and liver grafts in an effort to eliminate the cell population. After TBI, mice are reconstituted with the RevMlO GM HSC (see above) as described previously (DiGusto et al, (1997), Bonyhadi et al, (1997)). Control mice are injected with unmodified HSC or with HSC that have been modified with the ΔRevMlO gene or an irrelevant gene.
Analysis of GM HSC by flow cytometry. Approximately 8 to 12 weeks after GM HSC reconstitution, the Thy/Liv grafts are removed, and the thymocytes are obtained and analyzed for the HLA pheonotype (MA2.1) and the distribution of CD4+, CD8+, and Lyt2 (the "marker" murine homolog of CD8α) surface expression using methods of flow cytometry and FACS analysis readily known to those skilled in the art (see, e.g., Bonyhadi et al, J. Virol. 71:4707 (1997)); see also Coligan, et al, Units 4.8 and 5 (1994 and yearly updates including 2002). Thymocytes are also tested for transgenic DNA with primers specific for the RevMlO gene using standard PCR methods.
Analysis of GM HSC resistance to HIV infection. Approximately 8 to 12 weeks (or later) after GM HSC reconstitution, the Thy/Liv grafts are removed and the thymocytes are obtained from the GM HSC reconstituted SCID-hu mice. The thymocytes are stimulated in vitro and infected with the JR-CSF molecular isolate of HIV-1 as described previously (Bonyhadi et al, 1997). Briefly, the thymocytes are stimulated in vitro in the presence of irradiated allogeneic feeder cells (106 peripheral blood mononuclear cells/ml and 10s JY cells/ml) in RPMI medium containing 10% FCS, 50 μg/ml streptomycin, 50 U/G penicillin G, lx MEM vitamin solution, lx insulin transferring-sodium selenite medium supplement
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(Sigma), 40 U human rIL-2/ml, and 2 μg/ml phytohemagglutinin (PHA) (Sigma). About every 10 days, cells are restimulated with feeder cells and PHA as described previously in Vandekerckhove et al, (1992) J. Exp. Med. 1:1033. Approximately 5 days after stimulation, cells were sorted on the basis of donor HLA phenotype (MA2.1) and Lyt2 (the "marker" murine homolog of CD8α). Sorted cells are restimulated and may be expanded to increase the cell composition to greater than about 90% purity. CD4+/Lyt2+ cells are then sorted out and an aliquot of approximately 5xl04 of the sorted cells are place in multiple wells of a 96- well U bottom tissue culture plate. About 200 TCID50 of EW, an HIV-1 primary isolate, or 1000 TCID5o of JR-CSF, an HIV-1 molecular isolate, are added to each well. Methods of virus stock preparation have been described previously (Bonyhadi et al. Nature, 363:728 (1993). Medium is changed every day from days 3 to 12. Aliquots of supernatant are collected every other day and stored at -80° C until use. Tissue culture supernatants are then analyzed using a p24 ELISA following manufacturer's instructions (Coulter).
II. Therapy of HIV Infected Individual
Materials and Methods.
Isolation of human CD34 + HSC. As most HIV infected patients have very low titers of HSC, it is possible to use a donor to supply cells. Where practical, the level of HSC in the donor blood is enhanced by injecting into the donor granulocyte-colony stimulating factor (G-CSF) at lOμg/kg for 2-5 days prior to cell collection.
In this example, human cord blood (CB) HSC are collected and processed using techniques well known to those skilled in the art (DiGusto et al, (1997); Bonyhadi et al, (1997)). A portion of each CB sample is HLA phonotyped, and the CD34+ donor cells are purified from the donor blood (or bone marrow), such as by using a flow cytometer or immunomagnetic beading, essentially as described above. Donor-derived HSC are identified by flow cytometry as being CD34+.
Optionally, HSC are expanded ex vivo with IL-3, IL-6, and either SCF or LIF (10 ng/ml each).
RevMlO vectors and preparation of genetically modified (GM) HSC. Any of the
RevMlO gene transfer vectors known and described in the art, including those described in the mouse studies above, may be used. Methods of gene transduction using GM retroviral
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vectors or gene transfection using particle-mediated delivery are also well known in the art, and are described elsewhere herein.
As described above, a retroviral vector may be constructed to contain the trans- dominant mutant form of HIV-1 rev gene, RevMlO, which has been shown to inhibit HIV replication (Bonyhadi et al. 1997). Amphotropic vector-containing supernatants are generated by infection with filtered supernatants from ecotropic producer cells that were transfected with the vector.
The collected CD34+ cells are optionally pre-stimulated for 24 hours in LCTM media supplemented with IL-3, IL-6 and SCF or LIF (10 ng/ml each) to induce entry of the cells into the cell cycle.
In this example, CD34+-enriched HSC undergo transfection by a linearized RevMlO plasmid utilizing particle-mediated ("gene gun" transfer) essentially as described in Woffendin et al, (1996).
However, if retroviral transduction is done, supernatants containing the vectors are repeatedly added to the cells for 2-3 days to allow transduction of the vectors into the cells.
HA ART Treatment of HIV-infected patients. HA ART therapy is begun before T cell depletion and sex steroid ablation, and therapy is maintained throughout the procedure to reduce the viral titer.
T cell depletion. T cell depletion is performed as given in Example 5 to remove as many HIV infected cells as possible.
Sex steroid ablation therapy. The HIV-infected patient is given sex steroid ablation therapy as described in Example 6
Injection of GM HSC into patients. Prior to injection, the GM HSC are expanded in culture for approximately 10 days in X-Vivo 15 medium comprising 11-2 (Chiron, 300 IU/ml).
At approximately 1-3 weeks post LHRH agonist delivery, just before or at the time the thymus begins to reactivate, the patient is injected with the genetically modified HSC, optimally at a dose of about 2-4 x 106 cells/kg. Optionally G-CSF may also be injected into the recipient to assist in expansion of the GM HSC.
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Immediately prior to patient infusion, the GM HSC are washed four times with Dulbecco's PBS. Cells are resuspended in 100 ml of saline comprising 1.25% human albumin and 4500 U/ml IL-2, and infused into the patient over a course of 30 minutes.
Following sex steroid ablation and injection of the GM HSC in the HIV-infected patient, all new T cells (as well as DC, macrophages, etc.) will be resistant to subsequent infection by this virus. Injection of allogeneic HSC into a patient undergoing thymic reactivation means that the HSC will enter the thymus. The reactivated thymus takes up the genetically modified HSC and converts them into donor-type T cells and dendritic cells, while converting the recipient's HSC into recipient-type T cells and dendritic cells. By inducing deletion by cell death, or by inducing tolerance through immunoregulatory cells, the donor dendritic cells will tolerize any T cells that are potentially reactive with recipient.
When the thymic chimera is established, and the new cohort of mature T cells have begun exiting the thymus, reduction and eventual elimination of immunosuppression occurs.
Post-infusion studies. Following infusion, the persistence and half life of GM HSC in the HIV-infected patient is be tested periodically using limiting dilution PCR of PBL samples obtained from the patient essentially as described in Woffendin et al, (1996). The relative level of GM HSC in the infected patient is compared to the negative control patient that received the ΔRevMlO vector.
Various standard hematologic (e.g., CD4+ T cell counts), immunologic (e.g., neutralizing antibody titers), and virologic (e.g., viral titer) studies will also be performed using methods well known to those skilled in the art.
Termination of immunosuppression. Termination of immunosuppression is performed as given in Example 16.
EXAMPLE 15
ALTERNATIVE PROTOCOLS
In the event of a shortened time available for transplantation of donor cells, tissue or organs, the timeline as used in Examples 1-14 is modified. T cell ablation and/or other immune cell depletion and sex steroid ablation may be begun at the same time. T cell ablation and/or other immune cell depletion is maintained for about 10 days, while sex steroid ablation is maintained for around 3 months. In one embodiment, HSC transplantation
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is performed when the thymus starts to reactivate, at around 10-12 days after start of the combined treatment.
In an even more shortened time table, the two types of ablation and the HSC transplant may be started at the same time. In this event T cell ablation and/or other immune cell depletion may be maintained 3-12 months, and, in one embodiment, for 3-4 months.
EXAMPLE 16
TERMINATION OF IMMUNOSUPPRESSION
When the thymic chimera is established and the new cohort of mature T cells have begun exiting the thymus, blood is taken from the patient and the T cells examined in vitro for their lack of responsiveness to donor cells in a standard mixed lymphocyte reaction (see, e.g., Coligan et al. 1994, and yearly updates including 2002). If there is no response, the immunosuppressive therapy is gradually reduced to allow defense against infection. If there is no sign of rejection, as indicated in part by the presence of activated T cells in the blood, the immunosuppressive therapy is eventually stopped completely. Because the HSC have a strong self -renewal capacity, the hematopoietic chimera so formed will be stable theoretically for the life of the patient (as for normal, non-tolerized and non-grafted people).
EXAMPLE 17
USE OF LHRH AGONIST TO REACTIVATE THE THYMUS IN HUMANS
Materials and Methods:
In order to show that a human thymus can be reactivated by the methods of this invention, these methods were used on patients who had been treated with chemotherapy for prostate cancer.
Patients. Sixteen patients with Stage I-III prostate cancer (assessed by their prostate specific antigen (PSA) score) were chosen for analysis. All subjects were males aged between 60 and 77 who underwent standard combined androgen blockade (CAB) based on monthly injections of GnRH agonist 3.6mg Goserelin (Zoladex) or 7.5 mg Leuprolide (Lupron)
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treatment per month for 4-6 months prior to localized radiation therapy for prostate cancer as necessary.
FACS analysis. The appropriate antibody cocktail (20 Dl) was added to 200 Dl whole blood and incubated in the dark at room temperature (RT) for 30min. RBC, were lysed and remaining cells washed and resuspended in 1%PFA for FACS analysis. Samples were stained with antibodies to CD19-FITC, CD4-FITC, CD8-APC, CD27-FITC, CD45RA-PE, CD45RO- CyChrome, CD62L-FITC and CD56-PE (all from Pharmingen, San Diego, CA).
Statistical analysis. Each patient acted as an internal control by comparing pre- and post-treatment results and were analyzed using paired student t-tests or Wilcoxon signed rank tests.
Results:
Prostate cancer patients were evaluated before and 4 months after sex steroid ablation therapy. The results are summarized in Figs. 30-34. Collectively the data demonstrate qualitative and quantitative improvement of the status of T cells in many patients.
I. The Effect of LHRH Therapy on Total Numbers of Lymphocytes and T cells
Subsets Thereof:
The phenotypic composition of peripheral blood lymphocytes was analyzed in patients (all >60 years) undergoing LHRH agonist treatment for prostate cancer (Fig 40). Patient samples were analyzed before treatment and 4 months after beginning LHRH agonist treatment. Total lymphocyte cell numbers per ml of blood were at the lower end of control values before treatment in all patients. Following treatment, 6/9 patients showed substantial increases in total lymphocyte counts (in some cases a doubling of total cells was observed). Correlating with this was an increase in total T cell numbers in 6/9 patients. Within the CD4+ subset, this increase was even more pronounced with 8/9 patients demonstrating increased levels of CD4+ T cells. A less distinctive trend was seen within the CD8+ subset with 4/9 patients showing increased levels albeit generally to a smaller extent than CD4+ T cells.
II. The Effect Of LHRH Therapy On The Proportion Of T Cells Subsets:
Analysis of patient blood before and after LHRH agonist treatment demonstrated no substantial changes in the overall proportion of T cells, CD4+ or CD8+ T cells and a variable change in the CD4+:CD8+ ratio following treatment (Fig. 41). This indicates that there was
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little effect of treatment on the homeostatic maintenance of T cell subsets despite the substantial increase in overall T cell numbers following treatment. All values were comparative to control values.
III. The Effect Of LHRH Therapy On The Proportion Of B Cells And Myeloid Cells:
Analysis of the proportions of B cells and myeloid cells (NK, NKT and macrophages) within the peripheral blood of patients undergoing LHRH agonist treatment demonstrated a varying degree of change within subsets (Fig 42). While NK, NKT and macrophage proportions remained relatively constant following treatment, the proportion of B cells was decreased in 4/9 patients.
IV. The Effect Of LHRH Agonist Therapy On The Total Number Of B Cells And Myeloid Cells:
Analysis of the total cell numbers of B and myeloid cells within the peripheral blood post-treatment showed clearly increased levels of NK (5/9 patients), NKT (4/9 patients) and macrophage (3/9 patients) cell numbers post-treatment (Fig 43). B cell numbers showed no distinct trend with 2/9 patients showing increased levels; 4/9 patients showing no change and 3/9 patients showing decreased levels.
V. The Effect Of LHRH Therapy On The Level Of Naive Cells Relative To Memory Cells:
The major changes seen post-LHRH agonist treatment were within the T cell population of the peripheral blood. In particular there was a selective increase in the proportion of na ve (CD45RA+) CD4+ cells, with the ratio of na ve (CD45RA+) to memory (CD45RO+) in the CD4+ T cell subset increasing in 6/9 patients (Fig 44).
These results show that LHRH agonist treatment of an animal such as a human having an atrophied thymus can induce regeneration of the thymus. A general improvement has been shown in the status of blood T lymphocytes in these prostate cancer patients who have received sex-steroid ablation therapy.
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EXAMPLE 18
TREATMENT OF A PATIENT WITH PERNICIOUS ANEMIA
A adult (e.g., 35 years old) human female patient is suffering from pernicious anemia, an autoimmune disease. Her CD34+ hematopoietic stem cells (HSC) are recruited from her blood following 3 days of G-CSF treatment (2 injections /day, for 3 days, 10 μg/kg). Her HSC can be purified from her blood using CD34. To collect the CD34+ cells, peripheral blood of the donor (i.e., the person who will be donating his/her organ or skin to the recipient) is collected, and CD34+ cells isolated from the peripheral blood according to standard methods. One non-limiting method is to incubate the peripheral blood with an antibody that specifically binds to human CD34 (e.g., a murine monoclonal anti-human CD34+ antibody commercially available from Abeam Ltd., Cambridge, UK), secondarily stain the cells with a detectably labeled anti-murine antibody (e.g., a FITC-labeled goat anti- mouse antibody), and isolate the FITC-labeled CD34+ cells through fluorescent activated cell sorting (FACS). Because of the low number of CD34+ cells found in circulating peripheral blood, multiple collection and cell sorting may be required from the donor. The CD34+ may be cryopreserved until enough are collected for use.
Because the antigen for pernicious anemia, the patient's collected HSC are transfected by any means to express the antigen (namely, the gastric proton pump). HSC can be transfected by using a variety of techniques including, without limitation, electroporation, viral vectors, laser-based pressure wave technology, lipid-fusion (see, e.g., the methods described in Bonyhadi et al. 1997). In one example, her HSC are transfected with the D chain of the H/K-ATPase proton pump, using the MHC class II promoter for the expression.
To stop the ongoing autoimmune disease, the patient will to undergo T cell depletion and/or other immune cell depletion. She will also undergo thymic regeneration to replace these T cells and hence overcome the immunodeficiency state. To do this, she will receive 4 one monthly injections of Lupron (7.5 mg) to deplete the sex steroids (by 3 weeks) thereby allowing reactivation of her thymus. This will also allow uptake of the HSC and to establish central tolerance to the autoantigen in question. It is not clear why autoimmune disease starts but cross-reaction to a microorganism is a likely possibility; depleting all T cells will thus remove these cross-reactive cells. If the disease was initiated by such cross-reaction if may not be necessary to transfect the HSC with the nominal autoantigen. Simply depleting T cells followed by thymic reactivation by disrupting sex steroid signaling may be sufficient.
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One standard procedure for removing T cells is as follows. The human patient receives anti-T cell antibodies in the form of a daily injection of 15 mg/kg of Atgam (xeno anti-T cell globulin, Pharmacia Upjohn) for a period of 10 days in combination with an inhibitor of T cell activation, cyclosporin A, 3 mg/kg, as a continuous infusion for 3-4 weeks followed by daily tablets at 9mg/kg as needed. This treatment does not affect early T cell development in the patient's thymus, as the amount of antibody necessary to have such an effect cannot be delivered due to the size and configuration of the human thymus. The treatment is maintained for approximately 4-6 weeks to allow the loss of sex steroids followed by the reconstitution of the thymus. The prevention of T cell reactivity may also be combined with inhibitors of second level signals such as interleukins, accessory molecules (blocking, e.g., CD28), signal transduction molecules or cell adhesion molecules to enhance the T cell ablation and/or other immune cell depletion.
by the T cell-depletion regime.
EXAMPLE 19
TREATMENT OF A PATIENT WITH TYPE I DIABETES
A similar approach to that described in Example 18 is undertaken with a patient with Type I diabetes. The T cells will be removed by broad-based depletion methods (see above), thymic rejuvenation instigated by 4 month Lupron treatment and the patient's immune system recovery enhanced by injection of pre-collected autologous HSC transfected with the pro- insulin gene using the MHC class II promoter. The HSC will enter the thymus, differentiate into DC (and all thymocytes), and present pro-insulin to the developing T cells. All those potentially reactive to the pro-insulin will be killed by apoptosis, leaving a repertoire free to attack foreign infectious agents.
In the case that the autoimmune disease arose as a cross-reaction to an infection or simply "bad luck" it would be sufficient to use autologous HSC to help boost the thymic regrowth. If there is a genetic predisposition to the disease (family members can often get autoimmune disease) the thymic recovery would be best performed with allogeneic highly purified HSC to prevent graft versus host reaction through passenger T cells. Umbilical cord blood is also a good source of HSC and there are generally no or very few alloreactive T cells. Although cord blood does not have high levels of CD34+ HSC, they may be sufficient for establishment of a microchimera - even -10% of the blood cells being eventually (after 4-
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6 weeks) could be sufficient to establish tolerance to the autoantigen with sufficient intrathymic dendritic cells.
EXAMPLE 20
TREATMENT OF A PATIENT SUFFERING FROM ALLERGIES
In the case of allergy, a similar principle would be undertaken. The allergic patient would be depleted of T cells as above. In severe cases where there is exacerbation through IgE or IgG producing B cells (plasma cells) it may be necessary to use myeloablation as for chemotherapy. Alternatively, whole body irradiation may be used (e.g., 6 Gy). The entire immune system would be rejuvenated by the use of 3-4 month GnRH and injection intravenously of the HSC (allogenic or autologous as appropriate). Allogeneic would be used in the case of genetic disposition to allergy but otherwise mobilized autologous HSC would be used.
EXAMPLE 21
EFFECTS OF CASTRATION ON NOD AND NZB MICE
Non-obese diabetic mice (NOD mice) are a very well characterized model for type I diabetes. Extensive research has confirmed that the pathology of this disease is due to abnormal T cell infiltration of the pancreas and autoimmune destruction of the insulin- producing islet cells. The structure of the thymus in these animals is abnormal - there is ectopic expression of medullary epithelial cells (identified by mAb MTS 10), the presence of large B cell follicles and thymocyte-rich areas which lack the epithelial cells.
To examine the impact of sex steroids on these mice, 20 three week old female NOD mice were surgically ovariectomised and 20 were sham-operated. This stage was chosen because it is prior to disease onset. Blood glucose was monitored from 10 weeks of age. By 21 weeks of age, over 60% of the sham-castrated mice had developed diabetes but <20% of the castrated group had. There was insulitis (infiltration of the pancreas) but no islet destruction. After surgical castration, there was also a normalization of the thymic defects with well-defined cortex and medulla, loss of the B cell follicles, an increase in CD25+ regulatory cells. The increased in regulatory T cells may be very important because they could alter the pathogenic cytokine profile of the emigrating thymocytes. Hence castration has a dramatic impact on the development and progression of diabetes in NOD mice.
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Sixteen (16) ovariectomised and sixteen (16) sham-operated NOD mice were examined for 21 weeks for the development of diabetes (elevated blood glucose levels; BGL) and insulitis. At autopsy they were also examined for the presence of thymic structural abnormalities. As shown in Fig. 45, whereas 60% of the sham-operated NOD mice had diabetes, fewer than 20% of the castrated group had diabetes. This clearly shows a retarding or even prevention of the diabetes.
As shown in Fig. 45, castrated NOD mice had a marked increase in total thymocyte number but no differences in total spleen cells. In the diabetic castrated mice there was a marked decrease in total thymocyte number, which may have pre-disposed these mice to disease and suggests that the diabetes trigger may have occurred before the castration.
There was a significant increase in all thymocyte subclasses (Fig. 47 A) but there was no change in their proportions (data not shown). Interestingly there no change in B cells compared to sham-castrated mice (Fig. 47C) nor in the total T or B cells in the spleen (Fig. 47B).
In parallel with the increase in total thymocytes post-castration, there was a marked increase in CD25+ regulatory cells (data not shown). There was no such change in the spleen.
The effect of castration was also examined on NZB mice, which are a model for systemic lupus erythematosis (SLE). NZB mice have marked abnormalities in the thymus which are manifest before disease onset and are closely associated with disease. These defects include a poorly-defined cortex-medulla demarcation and abnormal clusters of B cells (see Takeoka et al, (1999) Clin. Immunol. 90:388).
Mice were castrated or sham -castrated at 4-7 weeks of age and examined 4 weeks later.
There was a marked increase in total thymocytes (Fig. 48A) and spleen cells (Fig.
48B). There was also a marked increase in thymic regulatory cells (CD25+ and NKT cells). The cytokines from these mice maybe influencing the effector T cells and modulating their potential pathogenicity. By immunohistology, the castrated mice had a normal thymic architecture and a loss of the B cell follicles (data not shown).
EXAMPLE 22
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EFFECT OF CASTRATION ON IMMUNIZATION WITH TUMOR-SPECIFIC ANTIGEN
Human Papillomavirus (HPV) infection causes genital heφes, which may lead to cervical cancer in some women. In fact, over 90% of all cervical cancers contain HPV DNA. Papillomaviruses are double-stranded DNA viruses that infect skin and mucosal surfaces. More than 80 types of HPV have been identified to date. HPV16 is one of the major types associated with cervical cancers.
E7 is the major oncogenic protein associated with HPV16-induced cervical cancer. Expression of the E7 open reading frame with activated ras has been shown by other groups to be sufficient to transform primary epithelial cells in culture to a malignant phenotype (Lin et α/.(1996) Cancer Res. 56:21). Thus, E7 is an attractive tumor specific antigen for use in immunotherapy and/or vaccination for cervical cancer and precursor lesions. Indeed, other groups have shown that mice immunized with an optimal dose of 50 μg/ml of an E7-GST fusion protein, with Quil A as adjuvant were protected against a subsequent challenge with an HPV16E7-transfected tumor cell line (Fernando et al, (1999) Clin. Exp. Immunol. 115:397.
This experiment was undertaken to determine if castration of mice was able to enhance the efficacy of vaccination (as a prophylactic vaccine) and/or immunotherapy (as an therapeutic vaccine) with a suboptimal dose of HPV16E7.
Materials and Methods
Adult (>9 mon. old) C57BL/6 mice received a subcutaneous injection of E7-positive syngeneic E7+ TCI tumor cells derived from primary epithelial cells of C57BL/6 mice cotransformed with HPV-16 E6 and E7 and c-Ha-ras oncogenes. Five days later, the mice were surgically castrated as described above by a scrotal incision, revealing the testes, which were tied with suture and then removed along with surrounding fatty tissue. The wound was then closed using surgical staples. Sham-castrated mice were prepared following the above procedure without removal of the testes and were used as negative controls. Seven days following tumour challenge, castrated or sham-castrated mice were injected with a suboptimal dose (5 μg/ml) of the E7GST fusion protein. Positive control mice received the optimal 50 μg/ml dose of the E7GST fusion protein. Twenty five days later, mice were analyzed for tumors (visually, and tumor mass), and T cell responsiveness (IFNγ production
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using standard ELIspot methods and CTL lytic activity by 5 lCr release assays using HPV 16E7 -pulsed target cells as described above).
Results
As shown in Figure 49, only 22% (4/18) of castrated mice receiving the suboptimal dose of E7GST had tumors, as compared to 47% (9/19) tumor occurrence in the sham- castrated suboptimal dose controls. Notably, the proportion of protected castrated mice receiving the suboptimal dose E7GST vaccine (78%) was comparable to the proportion of protected positive control mice, which received a 10-fold higher (optimal) dose of the vaccine (3/4, 75%). All of the castrated mice that did not receive the vaccine had tumors.
As shown in Figure 50, castrated mice receiving the suboptimal E7GST dose had a significantly higher number of cells in the spleen as compared to all other groups (Fig. 50A) (p > 0.01). However, the overall number of activated (CD25+) CD4+ (Fig. 50B) and CD8+ (Fig. 50C) T cell in the spleens of the castrated, suboptimal E7GST group remained the same as compared to all the other groups tested.
As shown in Figure 51 A, nonspecific production of IFNγ in the splenocytes of castrated mice receiving the suboptimal GST-E7 dose was comparable to the positive control mice receiving a 10-fold higher (optimal) dose of the vaccine. Sham-castrated mice receiving the suboptimal dose vaccine had a moderate level of IFNγ production, whereas the negative control animals had little to no nonspecific splenocyte production of IFNγ. This data parallels that seen with respect to tumor incidence (see Figure 49), and indicates that the production of IFNγ by splenocytes is directly proportional to tumor incidence in these animals. This is not suiprising given that Thl cell mediated responses are primarily responsible for tumor protection.
As shown in Figure 5 IB, the level of E7-specifc production follows the same trends as discussed above with respect to nonspecific production of IFNγ. While the level of E7- specific IFNγ production in castrated mice receiving the suboptimal GST-E7 dose was slightly lower than that of the positive control mice receiving a 10-fold higher (optimal) dose of the vaccine, it was still higher than the levels observed in the sham-castrated mice receiving the suboptimal dose. Once again, the negative control animals had little to no E7- specific splenocyte production of IFNγ.
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Finally, the mice were analyzed for E7-specific CTL killing of target cells infected with HPV16E7. As shown in Figure 52A, castrated mice receiving the suboptimal E7GST dose had comparable levels of E7-specific CTL as compared to the positive control mice receiving a 10-fold higher (optimal) dose of the vaccine. Sham-castrated mice receiving the suboptimal dose vaccine had a moderate level of E7-specific CTL responses, whereas the negative control animals had little to no E7-specific CTL. This data parallels that seen with respect to tumor incidence (see Figure 49) and IFNγ production (see Figure 51). Figure 52B shows that E7-specifc CTL activity is inversely proportional to tumor mass. That is, mice having the highest levels of CTL activity also had no tumors; whereas the few mice that did have tumors, were shown to have low E7-specifc CTL lytic activity.
Conclusion
These experiments show that castration can potentially increase vaccine efficacy in patients. Equivalent IFNγ production, CTL activity, and protection from tumor challenge is achieved in noncastrated mice receiving a 10-fold higher (optimal) dose of an HPV16-E7 vaccine as compared to castrated mice receiving a suboptimal dose of the vaccine.
A similar experiment could be undertaken to determine the efficacy of castration plus E7 vaccination in the context of an immunotherapeutic vaccination. In this instance, mice would be castrated as indicated above. Mice are injected with TCI cells at 6 weeks post- castration, and immunized with a GSTE7 vaccination 1 week later. Mice are then analyzed for tumors (visually, and tumor mass), and T cell responsiveness (IFNγ production using standard ELIspot methods and CTL lytic activity by 51Cr release assays using HPV16E7- pulsed target cells as described above). It is expected that mice receiving this therapeutic vaccination protocol will have reduced tumor mass as compared to sham castrated controls. Additionally, it is expected that castrated mice will need a lower dose of vaccine than their sham castrated counteφarts.
EXAMPLE 23 ALTERNATIVE VIRAL VACCINATION STRATEGIES
The methods of the present invention can be used to improve the efficacy of a variety of art-recognized viral vaccines by prior, subsequent, or concurrent administration of an inhibitor of sex steroid signaling, such as a GnRH analog (or other method of castration). In
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addition to the examples provided above, non-limiting examples of viral vaccines are as follows.
I. Hepatitis B Viral Vaccine.
An example of a viral vaccine and a recombinant DNA vaccine are those developed for Hepatitis B by Glaxo Smith Kline (Engerix B®), and Merck Shaφe and Dohme (HB VaxIJ®), respectively. The Engerix B® vaccine preparation is a 20 μg per 1 mL dose administered according to manufacturer's instructions. The HB Vax II® vaccine preparation is a 1 mL 10 μg/mL dose administered according to manufacturer's instructions. A number of pediatric formulations are also available for these and other vaccines. These vaccines may or may not contain preservatives such as Thiomersal. (Australian Immunization Handbook, 8th edition). Vaccine doses are typically in the range of 0.5mL to lmL administered by i.m. injection. The usual course of vaccination may vary but usually consists of a single, primary immunization followed by at least one booster immunization at intervals of approximately one or more months.
II. Hepatitis A Viral Vaccine.
Examples of an inactivated viral vaccine are those developed for the treatment of Hepatitis A by Aveniis Pasteur (Avaxim®), Glaxo Smith Kline (Havrix 1440® and Havrix Junior®) and others. In the case of Avaxim®, each 0.5 mL dose contains 160 ELISA units of hepatitis A (GBM strain) viral antigens. In the case of Havrix 1440®, each 0.5mL dose contains 1440 ELISA units of hepatitis A virus (HM 175 strain). Vaccine doses of such monovalent vaccines are in the range of 0.5mL to 1 mL by LM injection. The usual course of vaccination may vary but usually consists of three vaccinations at six month intervals.
III. Hepatitis A and Hepatitis B Multivalent Vaccine.
Additionally, polyvalent formulations may be used, which contain more than one viral antigen. For instance, Glaxo Smith Kline's Twinrix® contains 720 ELISA units of Hepatitis A viral antigens and 20μg of recombinant DNA hepatitis B surface antigen protein, and are administered by i.m. injection at 0, 3, and 6 months. Another polyvalent vaccine is Aventis Pasteur's Vivaxim®. Each 1 mL dose contains 160 ELISA units of inactivated Hepatitis A virus antigens and 25μg purified typhoid capsular polysaccharide. Supplied in a dual chamber syringe this polyvalent vaccine is administered i.m. in two or three doses.
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IV. Cytomegalovirus Vaccine.
Vical, Inc. has developed a immunotheφautic DNA-based vaccine against CMV. The vaccine is administered in three doses of either 1 or 5 mgs, as provided in the manufacturer's instructions. The DNA plasmid encodes CMV phosphoprotein 65 (pp65) and glycoprotein B (gB), and the vaccine is formulated with a poloxamer.
V. EBV Vaccine.
Medlmmune, GlaxoSmithKline, and Henogen have co-developed a soluble recombinant subunit vaccine against EBV. Live recombinant vaccinia vectors have been used to express EBV gp220/350, and have been shown to confer protection in primates and EBV-negative infants. Additionally, clinical trials of an EBNA-3A peptide have been conducted in Australia, (for a review of this virus vaccine and others, see, e.g., THE JORDAN REPORT 2000: ACCELERATED DEVELOPMENT OF VACCINES, published by the Division of Microbiology and Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health (available at http://www.niaid.nih.gov/publications/pdf/iordan.pdf -last visited Apr. 2004))
EXAMPLE 24 ALTERNATIVE CANCER VACCINATION STRATEGIES
The methods of the present invention can be used to improve the efficacy of a variety of art-recognized cancer vaccines by prior, subsequent, or concurrent administration of an inhibitor of sex steroid signaling, such as a GnRH analog (or other method of castration). In addition to the examples provided above, non-limiting examples of cancer vaccines are as follows.
I. Melanoma Vaccine.
The methods of the present invention can be used to improve the efficacy of a melanoma vaccine by prior, subsequent, or concurrent administration of an inhibitor of sex steroid signaling, such as a GnRH analog (or other method of castration). An example of such a melanoma vaccine is the autologous melanoma cellular vaccine developed by AVAX
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coφoration (Philadelphia, PA). Metastatic tumor are excised, maintained at 4 °C, and delivered to the laboratory within 48 h of excision. Tumor cells are extracted by enzymatic dissociation with collagenase and DNAse, aliquotted, frozen in a controlled rate freezer, and stored in liquid nitrogen in a medium containing human albumin and 10% dimethylsulfoxide until needed. On the day that a patient is to be treated, an aliquot of cells are thawed, washed, and irradiated to 2500 cGy. The tumor cells are then washed, incubated for 30 min. with initrofluorobenzene (DNFB), and then washed with saline. (Miller et al. (1976) J. Immunol. 117: 1519. After washing, the cells are counted, suspended in 0.2 ml Hanks solution with human albumin, and maintained at 4°C until administered.
Vaccine doses are in the range of 0.5-25.0 x 106 cells. Just prior to injection, 0.1 ml of BCG (Tice, Organon Teknika, Durham, N.C.) may be added to the vaccine. The dose of BCG may be progressively attenuated to produce a local reaction consisting of an inflammatory papule without ulceration. The mixture is injected intradermally into one or more sites, e.g.,, the upper arm. Multi-dose vaccination is conducted over a variable period (e.g., monthly for 12 months or weekly for 6-12 weeks) and may include the administration of low dose (300 mg/M2) cyclophosphamide, a cytotoxic drug that paradoxically augments cell-mediated immunity when administered at the proper time in relation to immunization.
II. Lung Cancer Vaccine.
The methods of the present invention can also be used to improve the efficacy of a lung cancer vaccine by prior, subsequent, or concurrent administration of an inhibitor of sex steroid signaling, such as a GnRH analog (or other method of castration). An example of a such a lung cancer vaccine is the DNA vaccine for non-small cell lung cancer that by Corixa Coφoration. The vaccine is a recombinant DNA vaccine that is administered with a recombinant adenovirus adjuvant. A Biojector® device (Bioject, Tualatin, OR) is used for vaccine administration and is a reusable needle-free injector powered by a compressed CO2 cartridge.
Other non-limiting lung vaccines that may be used with the methods of the invention include L523S for non-small cell lung cancer (Wang et al. (2003) Br. J. Cancer 88:887) and BEC-2, GM2, Globo H, fucosyl GM1, and polysialic acid for small cell lung cancers (Krug (2004) Sem. Oncol. 31:112).
III. Prostate Cancer Vaccine.
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The methods of the present invention can also be used to improve the efficacy of a prostate cancer vaccine by prior, subsequent, or concurrent administration of an inhibitor of sex steroid signaling, such as a GnRH analog (or other method of castration). One non- limiting example of such a vaccine is Provenge® (Dendreon Coφ.), which is a vaccine against androgen independent prostate cancer. This vaccines results in the generation of a T cell immune response to the prostate cancer associated antigen prostatic acid phosphatase (PAP). DC precursors are obtained from the patient by leukopheresis and isolated from other white blood cells using a cell separation device. These cells are then co-cultured with a recombinant PAP fused delivery cassette for about 36 hours to allow the DC to mature. The mature DC are then used in the vaccine. The Provenge vaccine is delivered as three 30- minute intravenous infusions at two week intervals. Other prostate cancer vaccine candidates for use in the invention are known in the art (see, e.g., Shaffer and Scher (2003) Lancet Oncol. 4:407).
IV. Colorectal Cancer Vaccine.
The methods of the present invention can also be used to improve the efficacy of a colorectal cancer vaccine by prior, subsequent, or concurrent administration of an inhibitor of sex steroid signaling, such as a GnRH analog (or other method of castration). One non- limiting example of such a vaccine is Trovax™ (Oxford BioMedica). This vaccine is a solid tumor associated antigen, 5T4, delivered by modified vaccinia virus Ankara (MVA) via intramuscular injection. It is delivered at weeks 0, 4 and 8, coincident with chemotherapy. Other cytotoxic T-lymphocyte precursor-oriented peptide vaccines for colorectal carcinoma patients are known in the art (see, e.g., Sato et al. (2004) Br. J. Cancer 90:1334.
V. Ovarian Cancer Vaccine.
The methods of the present invention can also be used to improve the efficacy of a ovarian cancer vaccine by prior, subsequent, or concurrent administration of an inhibitor of sex steroid signaling, such as a GnRH analog (or other method of castration). One non- limiting example of such a vaccine is M-FP (CancerVac Pty. Ltd.) for the treatment of metastatic ovarian cancer. It is an autogenic cellular vaccine, whereby DC are injected subcutaneously. A recombinant version of MUC1 is fused to mannan to promote antigen uptake by the patients purified dendritic precursor cells. Activated DC presenting MUC1 peptides are then injected s.c. into the patient.
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All publications mentioned in the above specification are herein incoφorated by reference. Various modifications and variations of the described methods and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are apparent to those skilled in biology or related fields are intended to be within the scope of the following claims.
Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific embodiments described specifically herein. Such equivalents are intended to be encompassed in the scope of the following claims.
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