WO2004103271A2 - Prevention de maladies et vaccination avant reactivation thymique - Google Patents

Prevention de maladies et vaccination avant reactivation thymique Download PDF

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Publication number
WO2004103271A2
WO2004103271A2 PCT/US2004/011921 US2004011921W WO2004103271A2 WO 2004103271 A2 WO2004103271 A2 WO 2004103271A2 US 2004011921 W US2004011921 W US 2004011921W WO 2004103271 A2 WO2004103271 A2 WO 2004103271A2
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WIPO (PCT)
Prior art keywords
patient
cells
sex steroid
thymus
hsc
Prior art date
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PCT/US2004/011921
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English (en)
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WO2004103271A3 (fr
Inventor
Gabrielle Lianne Goldberg
Jayne Suzanne Sutherland
Ann Patricia Chidgey
Richard Boyd
Original Assignee
Norwood Immunology, Ltd.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Publication date
Priority claimed from US10/418,747 external-priority patent/US20040018180A1/en
Priority claimed from US10/419,066 external-priority patent/US20040037817A1/en
Priority claimed from US10/418,727 external-priority patent/US20040013641A1/en
Priority claimed from US10/419,068 external-priority patent/US20050002913A1/en
Priority claimed from US10/748,831 external-priority patent/US20050020524A1/en
Priority claimed from US10/748,450 external-priority patent/US20040241842A1/en
Priority claimed from US10/749,118 external-priority patent/US20040265285A1/en
Priority claimed from US10/749,122 external-priority patent/US20040259803A1/en
Priority to JP2006532426A priority Critical patent/JP2007518699A/ja
Priority to EP04785486A priority patent/EP1620126A4/fr
Priority to US10/553,594 priority patent/US20080279812A1/en
Priority to AU2004241949A priority patent/AU2004241949A1/en
Priority to CA002528521A priority patent/CA2528521A1/fr
Application filed by Norwood Immunology, Ltd. filed Critical Norwood Immunology, Ltd.
Publication of WO2004103271A2 publication Critical patent/WO2004103271A2/fr
Publication of WO2004103271A3 publication Critical patent/WO2004103271A3/fr

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    • A61K35/26—Lymph; Lymph nodes; Thymus; Spleen; Splenocytes; Thymocytes
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Definitions

  • the present invention is in the fields of cellular immunology, disease prevention, vaccination, and gene therapy. More specifically, the invention is directed to enhancing bone marrow (BM) hematopoiesis and functionality, enhancing BM engraftment following hematopoietic stem cell transplant (HSCT), and increasing the functionality of new and preexisting T cells and other cells of the immune system.
  • BM bone marrow
  • HSCT hematopoietic stem cell transplant
  • 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.
  • the immune response has also been described as responding to danger signals.
  • 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.
  • 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 ⁇ -islet cells in diabetes mellitus)
  • 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.
  • MHC major histocompatibility complex
  • Tc or CTL cytotoxic T cells
  • Th helper T cells
  • Th cells are important in virtually all immune responses.
  • HIV/AIDS where their absence through destruction by the virus causes severe immune deficiency, which eventually leads to death due to opportunistic infections.
  • Inappropriate development of Th can also lead to a variety of other conditions such as allergies, cancer, and autoirnmunity.
  • 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 has been found in NZB mice, which develop Lupus-like symptoms (Takeoka et al., (1999) Clin. Immunol.
  • T and B lymphocytes The ability to recognize antigen is encompassed in a plasma membrane receptor in T and B lymphocytes. These receptors are randomly generated 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 elicit varying degrees of responses. Since antigen receptor specificity arises by chance, the problem thus arises as to why the body does not self destruct through lymphocytes reacting against self antigens. Fortunately there are several mechanisms which prevent the T and B cells from doing so. Collectively, these mechanisms create a situation where the immune system is tolerant to self.
  • T regulatory cells such as CD4+CD25+ and NKT cells, provide a means whereby they can suppress potentially autoreactive cells.
  • the thymus essentially consists of developing thymocytes (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.
  • 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.
  • stromal cells predominantly epithelial cell subsets
  • the thymus is an important organ in the immune system because it is the primary site of production of T lymphocytes.
  • the role of the thymus is to attract appropriate BM-derived precursor cells from the blood, as described below, and induce their commitment to the T cell lineage, including the gene rearrangements necessary for the production of the T cell receptor (TCR) for antigen.
  • TCR T cell receptor
  • Each T cell has a single TCR type and is unique in its specificity.
  • 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.
  • 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 or 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.
  • TCR 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.
  • MS multiple sclerosis
  • RA rheumatoid arthritis
  • SLE systemic lupus erythematosus
  • DC dendritic cells
  • thymus 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.
  • 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.
  • thymus 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.
  • conditions sucn 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.
  • T cell dependent immune functions e.g., cytolytic T cell activity and mitogenic responses.
  • T cell dependent immune functions e.g., cytolytic T cell activity and mitogenic responses.
  • 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 HIN 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).
  • 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 na ⁇ ve T cells.TREC levels has also been analysed following hematopoietic stem cell transplantation (Douek et al, (2000) Lancet 355:1875).
  • Thymus and the neuroendocrine 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).
  • 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.
  • thymocytes 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.
  • thymocytes in older aged animals 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) Eur. J. Immunol. 28: 1886).
  • 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 etal, (1997) J. Immunol. 158:3037).
  • 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 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 major cells of the immune system are the B and T lymphocytes (a major class of white blood cells), and the antigen presenting cells (APC). All immune cells are basically derived from hematopoietic stem cells (HSC) and their progeny, the Common Lymphoid Progenitor (CLP) and the Common Myeloid Progenitor (CMP), which are produced in the BM. Some of the precursor cells migrate to the thymus and are converted into T cells and thymic DC. DC play a role in inducing self-tolerance.
  • HSC hematopoietic stem cells
  • CLP Common Lymphoid Progenitor
  • CMP Common Myeloid Progenitor
  • Th and Tc cells Prior to leaving the thymus, Th and Tc cells can effectively distinguish foreign antigen because, as described above, T cells are "selected" in the thymus so that those T cells that leave recognize all of the cells in the body as self and, under normal circumstances, do not respond against them.
  • B cells are also ultimately derived from HSC and develop in the BM before exiting into the peripheral immune system. Following interactions with T cells, and other cells of the immune system, B cells develop into plasma cells that produce and release large amounts of antibodies, which help the body desttoy infective organisms and abnormal cells.
  • HSC produced by the BM are also utilized for the production of all other blood cells, such as NK cells, regulatory cells, common myeloid progenitor derived cells, neutrophils, basophils and eosinophils, dendritic cells, monocytes, macrophages, platelets and red blood cells.
  • Hematopoietic stem cell transplantation also commonly known as bone marrow transplantation (BMT)
  • BMT bone marrow transplantation
  • HSCT Hematopoietic stem cell transplantation
  • BMT bone marrow transplantation
  • HSCT Hematopoietic stem cell transplantation
  • BMT bone marrow transplantation
  • Transplant are used interchangeably and are herein defined as a transplant into a recipitent, 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.
  • CLP common lymphoid progenitors
  • CMLP common myelolympho
  • the transplant may be a peripheral blood stem cell transplant (PBSCT).
  • PBSCT peripheral blood stem cell transplant
  • 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 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.
  • HSCT is given prior to, concurrently with, or after sex steroid inhibion.
  • HSC is a nonlimiting exemplary type of cell, which may be transplanted and/or genetically modified, as used throughout this application.
  • 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.
  • HSC are derived from a fetal liver and/or spleen.
  • HSCT allows, for example, stem cells and their progeny cells that were damaged by, e.g., chemotherapy or radiation treatment to be replaced with healthy stem cells that can ultimately produce the blood cells that the patient needs.
  • HSCT is the basic treatment for a number of hematological cancers, such as leukemias and lymphomas (cancers of the blood and immune system cells), as well as non- malignant immune disorders such as severe combined immunodeficiency, Fanconi's anemia, myelodysplastic syndromes, amyloidosis, aplastic anemia, Diamond Blackfan anemia, hemophagocytic lymphohitiocytosis, Kostmann syndrome, Wiskott-Aldrich syndrome, thrombocytopenias, and hemoglobinopathologies, such as sickle cell disease and thalassemia.
  • leukemias and lymphomas cancers of the blood and immune system cells
  • non- malignant immune disorders such as severe combined immunodeficiency, Fanconi's anemia, myelodysplastic syndromes, amyloidosis, aplastic anemia, Diamond Blackfan anemia, hemophagocytic lymphohitiocytosis, Kostmann syndrome, Wis
  • HSC leukemia and lymphoma
  • HSCT myeloablation or myelodepletion to rid the body of cancerous cells
  • HSC mobilizing agents such as cytokines (e.g., G- CSF or GM-CSF), or drugs (e.g., cyclophosphamide), allow faster and/or better engraftment and may also allow chemotherapy and radiation therapy to be given at higher doses and/or more frequently.
  • HSC HSC mobilization from the BM
  • GM-CSF and G-CSF are presently used for this purpose, but other agents, such as chemotherapy and cytokines have also been shown to be effective.
  • chemotherapy and cytokines have also been shown to be effective.
  • the ability to more effectively mobilize HSC has application beyond hematological repair.
  • Recent studies have shown that HSC are multipotent and may be utilized for repair of damaged tissues, e.g., cardiac muscle, skeletal muscle, liver, bone, connective tissue, epithelial tissue, pancreas, vasculature.
  • the rate of engraftment plays a role, wherein the longer the rate of engraftment, the more likely opportunistic infection will occur.
  • graft versus host disease A second limitation of current HSCT strategies occurs when the grafted cells 'reject' the recipient of the cells. This is known clinically as "graft versus host disease" (GVHD).
  • GVHD graft versus host disease
  • An autologous transplant may avoid GVHD.
  • the overall anti-cancer success rates of autologous transplants are lower as compared to allogeneic transplants.
  • autologous transplants In cancer patients, autologous transplants have the disadvantage that they do not produce a Graft Versus Tumor (GVT) effect (which is similar to the GVH effect) and there is the risk that cancerous cells may be returned to the patient with the transplant.
  • GVT Graft Versus Tumor
  • HSCT treatments A further limitation of HSCT treatments is the lack of donors to treat all the potential candidates.
  • umbilical cord blood (UCB) has been utilized to a fairly limited extent, there are few cells from each donor and as a consequence this has been mainly used in children where the total number of HSC required is lower (HSC number required is linked to patient body weight).
  • HSC number required is linked to patient body weight.
  • donors are in limited supply and there must be an acceptable MHC match or the risk of GVH is high.
  • HSCT could be used more widely, for example to treat autoimmune disease, and sources such as cord blood could be utilized (e.g., 1.5xl0 7 cells/kg for recipient engraftment).
  • T cells are the major component of the immune system, and are produced in the thymus.
  • the most important T cells are Th cells because these are the cells that initiate virtually all immune responses.
  • the absence of these Th cells e.g., caused by HIV infection, chemotherapy, radiation, etc.
  • An important role of a subset of Th cells is to regulate immune responses.
  • the balance between enhancement and suppression of T and B cell function has a major effect on e.g., whether a vaccine is efficacious, whether a cancer or tumor is attacked, or whether a transplant is tolerated or rejected.
  • thymocyte export is directly related to the cellularity in the thymus (Scollay et al, (1980) Eur. J. Immunol. 10:210; Berzins et al, (1998) /. Exp. Med. 187:1839)
  • age-related thymic atrophy results in a gradual decrease in recent thymic emigrants (RTEs) (Steffens et al, (2000) Clin. Immunol 97:95; Sempowski etal, (2002) Mol. Immunol.
  • T cell proliferation in response to non-specific and receptor-mediated (CD3/TCR) stimulation is severely compromised with age (Hertogh- Huijbregts et al, (1990) Mech. Ageing Dev. 53:141-155; Flurkey et al, (1992) J. Gerontol 47:B115; Kirschmann etal, (1992) Cell Immunol 139:426).
  • the most likely defect is encompassed within the T cell compartment because of the dramatic decline in thymus function with age primarily due to the impact of sex steroids. This leads to a loss of new or "na ⁇ ve" T cells exported into the bloodstream, which are needed for responses to new antigens. In addition to the numerical loss of potential responding T cells, the pre-existing T cells may be suppressed to some degree by the presence of sex steroids.
  • chemotherapy and radiotherapy used to treat cancers are often deleterious to the patient's non-cancerous cells, particularly the blood cells.
  • the major limitation to increasing frequency and dose of such treatments is the ability of the patient to survive the treatment and avoid susceptibility to opportunistic infection as a result of the compromised immune system. Thus it would greatly benefit the patient if the immune recovery was more rapid or the damage less severe.
  • the present invention relates to methods for preventing illness or aiding recovery in a patient by enhancing BM haemopoieses and functionality, enhancing BM engraftment following HSCT, and increasing the functionality of pre-existing T cells and other immune cells by disrupting sex steroid and other hormonal signaling.
  • Immune capacity will also be enhanced by increased levels of na ⁇ ve T cells produced through renewed thymic function, and also B lymphocytes and other cells of the immune system produced via activated BM function.
  • the invention provides methods of enhancing engraftment following HSCT.
  • engraftment is enhanced in the BM.
  • engraftment and/or reconstitution is enhanced in the thymus, whereby thymic recovery is ultimately induced.
  • engraftment and/or reconstitution is enhanced in the spleen and/or other lymphoid organs, tissues, and/or blood.
  • the HSC are allogeneic, and in other embodiments, the HSC are autologous.
  • the number of T cell precursors is increased as compared to the number that would have been present in a patient that had HSCT without undergoing interruption of sex steroid signaling.
  • total white blood cells, donor-derived DC, BM precursors, HSC, CLP, MLP, lymphocytes, myeloid cells, granulocytes, neutrophils, macrophage, NK, NKT, platelets, na ⁇ ve T cells, memory T cells, helper T cells, effector T cells, regulatory T cells, RBC, B cells, donor-, and/or host-derived peripheral T cells, APC, and/or donor derived peripheral B cells are increased as compared to the number that would have been present in a patient that had HSCT without undergoing interruption of sex steroid signaling.
  • a patient is also treated with a cytokine (e.g., IL-7, SCF, IL-11, G-CSF, or GM-CSF) or hormone (e.g., growth hormone, or its mediator insulin dependent growth factor (IGF-1) or any member of the fibroblast growth factor family e.g., FGF 7 /Keratinocyte Growth Factor (KGF)) following HSCT to enhance immune recovery and/or engraftment.
  • cytokine e.g., IL-7, SCF, IL-11, G-CSF, or GM-CSF
  • hormone e.g., growth hormone, or its mediator insulin dependent growth factor (IGF-1) or any member of the fibroblast growth factor family e.g., FGF 7 /Keratinocyte Growth Factor (KGF)
  • the present invention either alone, or in combination (concurrently or sequentially) with the administration of hemopoietic agents, such as cytokines (e.g., G-CSF or GM-CSF), allow faster and/or better engraftment and/or homing to the target tissue and/or enhance recovery of immune cells.
  • hemopoietic agents such as cytokines (e.g., G-CSF or GM-CSF)
  • cytokines e.g., G-CSF or GM-CSF
  • the immune cells are T cells.
  • the T cell proliferative responsiveness to T cell receptor (TCR) stimulation is improved.
  • the T cell responsiveness to an antigen e.g., tetanus toxoid (TT) or pokeweed mitogen (PWM), or Keyhole Limpet Hemocyanin (KLH)
  • TT tetanus toxoid
  • PWM pokeweed mitogen
  • KLH Keyhole Limpet Hemocyanin
  • the T cell responsiveness is improved to a recall antigen (i.e., an improved T cell memory response).
  • the T cell proliferative responsiveness in respect of co-stimulatory or secondary signaling is improved.
  • the kinetics of T cell responsiveness is improved.
  • the T cell response to antigen presented by APC is improved.
  • immune cell responsiveness is improved within about five-, four-, three- or two months post transplant. In certain embodiments of the invention, immune cell responsiveness is improved within about one month post-transplant. In other embodiments of the invention, immune cell responsiveness is improved within two weeks post-transplant. In another embodiment of the invention, immune cell responsiveness is improved within one week post-transplant. In yet other embodiments of the invention, immune cell responsiveness is improved within three days post transplant. In other embodiments the immune rpsonse is improved after 3 or more months post treatment involving at this time input from newly thymic derived T cells in addition to pre-existing T cells.
  • the invention provides methods of enhancing pre-existing immune cell functionality, including, but not limited to, immune cells in the periphery.
  • the cells are T cells.
  • the cells are DC or other APC.
  • the cells are NK cells or regulatory cells, such as CD4+CD25+ T cells and natural killer T (NKT) cells.
  • T cell proliferative responsiveness to TCR stimulation is improved in the patient.
  • the T cell responsiveness to antigen (e.g., TT, PWM, or KLH) stimulation is improved.
  • the T cell proliferative responsiveness to secondary or co-stimulatory signaling is improved.
  • the T cell response to antigen presented by APC is improved.
  • a LHRH GnRH analog has a direct effect or indirect effect on the responsiveness of pre-existing immune cells.
  • sex steroid analogs agonist and antagonists thereto, such as GnRH/LHRH analogs, are used in the methods of the invention to disrupt sex steroid-mediated signaling, immune cells or BM.
  • sex steroid analogs directly stimulate (i.e., directly increase the functional activity of) the thymus, BM, and/or pre-existing cells of the immune system, such as T cells, B cells, and DC.
  • the invention provides methods of enhancing HSC engraftment and mobilization in a patient, or in a blood, HSC, or BM donor.
  • disrupting sex steroid signaling increases the number and/or functionality of peripheral immune progenitor cells such as HSC, CD34+ cells, CLP, or CMP.
  • One embodiment provides a method for enhancing HSC mobilization comprising disrupting sex steroid signaling, either alone or in combination with administration of an HSC mobilizing agent, for example, cytokines, GM-CSF, G-CSF, CSF, chemotherapeutics, cyclophosphamide, flt-3 ligand, KGF/FGF 7 or other members of the FGF familyor IL-7.
  • an HSC mobilizing agent for example, cytokines, GM-CSF, G-CSF, CSF, chemotherapeutics, cyclophosphamide, flt-3 ligand, KGF/FGF 7 or other members of the FGF familyor IL-7.
  • the present invention provides methods to allow chemotherapy and radiation therapy to be given at higher doses and/or more frequently and/or allow faster recovery of or less damage to the immune system after chemotherapy and radiation therapy.
  • the invention provides methods to prevent or treat illness in a patient.
  • One embodiment provides a method for preventing or diminishing the risk of an infection, illness, or disease in a patient, the method comprising disrupting sex steroid mediated signaling in the patient.
  • signaling is disrupted to the BM.
  • signaling is disrupted to the thymus.
  • signaling is disrupted to the spleen.
  • signaling is disrupted to the peripheral immune cells.
  • the methods of the invention are used to prevent or treat viral infections, such as HTN, herpes, influenza, and hepatitis.
  • the methods of the invention are used to prevent or treat bacterial infections, such as pneumonia and tuberculosis (TB).
  • 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.
  • the patient receives non-genetically modified HSC transplantation.
  • BM or HSC are transplanted into the patient to provide a reservoir of precursor cells, which may ultimately be used for the renewed thymic growth.
  • Some of these HSC have the ability to turn into DC or other APC, which may have the effect of providing better antigen presentation to the T cells and therefore a better immune response (e.g., increased Ab production and effector T cells number and/or function).
  • the atrophic thymus in an aged (post-pubertal) patient is in the process of being reactivated by disruption of sex-steroid signaling at the time of HSCT. 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.
  • the invention provides methods to improve the immune responsiveness of a patient to a vaccine.
  • gene therapy utilizing genetically modified HSC, lymphoid progenitor, myeloid progenitor or epithelial stem cells, or combinations thereof (the group and each member herein referred to as "GM cells"), are delivered to the patient to create , particular immunities useful in treating or preventing an illness.
  • GM cells myeloid progenitor or epithelial stem cells, or combinations thereof
  • the illness is one that has a defined genetic basis, such as that caused by a genetic defect.
  • 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
  • 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 functionally, either directly or indirectly, or causes T cells to function in a manner which is harmful to the individual.
  • viral infections such as human immunodeficiency virus (HIV)
  • T cell functional disorders such as T cell functional disorders, and any other disease or condition that reduces T cells numerically or functionally, either directly or indirectly, or causes T cells to function in a manner which is harmful to the individual.
  • the present invention provides methods for treating or preventing infection by an infectious agent, such as HIV, by transplanting GM cells that have been genetically modified to resist or prevent infection, activity, replication, and the like, and combinations thereof, of the infectious agent may be injected into a patient prior to, or concurrently with, thymic reactivation.
  • the HSC are modified to include a gene whose product interferes with HIV infection, function, and/or replication in the T cells (and/or other HSC-derived cells) of the patient.
  • HSC are genetically modified with viral resistance gene, such as the RevMlO gene (see, e.g., Bonyhadi et al, (1997) /. 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.
  • the invention provides for disruption of sex steroid mediated signaling to, and subsequent reactivation of, the thymus.
  • castration is used to disrupt the sex steroid mediated signaling.
  • chemical castration is used.
  • surgical castration e.g., by removal of the testes or by ovariectomy
  • complete inhibition of sex steroid . signaling occurs.
  • partial disruption of sex steroid signaling occurs.
  • castration reverses the state of the thymus towards its pre-pubertal state, thereby reactivating it.
  • castration modifies the level of other molecules, which enhance immune cell responsiveness and/or proliferation and/or activation state by having, e.g., a direct effect on pre-existing immune cells.
  • 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").
  • 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, GnR
  • one or more blocker is used.
  • 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 incorporated herein by reference.
  • Figs. 1A-B Castration rapidly regenerates thymus cellularity.
  • Figs. 1 A-B 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. 1A) or by the number of cells per thymus (Fig. IB).
  • aged (i.e., 2-year old) male mice were surgically castrated.
  • Thymus weight in relation to body weight (Fig. 1A) and thymus cellularity (Figs. IB) were analyzed in aged (1 and 2 years) and at 2-4 weeks post-castration (post-cx) male mice.
  • Figs. 2A-D Castration restores the CD4:CD8 T cell ratio in the periphery.
  • 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).
  • Fig. 2B 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).
  • Fig.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.
  • FACS Fluorescence Activated Cell Sorter
  • 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.
  • Figs. 4A-B Regeneration of thymocyte proliferation by castration. Mice were injected with a pulse of BrdU and analyzed for proliferating (BrdU + ) thymocytes. 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+ cells within the thymus with age and post-castration are shown (Fig. 4A). No difference was observed in the total proportion of proliferation within the thymus, as this proportion remains constant with age and following castration (Fig. 4A and left-graph in Fig. 4B).
  • Figs. 5A-H Castration enhances proliferation within all thymocyte subsets.
  • aged (2-year old) mice were castrated and injected with a pulse of bromodeoxyuridine (BrdU) to determine levels of proliferation.
  • BrdU bromodeoxyuridine
  • Fig. 5A shows that the proportion of each thymocyte subset within the BrdU+ population did not change with age or post-castration.
  • Fig. 5B a significant decrease in the proportion of DN (CD4-CD8-) thymocytes proliferating was seen with age.
  • Fig. 5A shows that the proportion of each thymocyte subset within the BrdU+ population did not change with age or post-castration.
  • Fig. 5B a significant decrease in the proportion of DN (CD4-CD8-) thymocytes proliferating was seen with age.
  • Figs. 6A-C Castration increases T cell export from the aged thymus.
  • aged (2-year old) mice were castrated and were injected intrathymically with FJ C to determine thymic export rates.
  • the number of FITC+ cells in the periphery was calculated 24 hours later.
  • 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.
  • the rate of emigration export/total thymus cellularity
  • Fig. 6C shows that a significant increase in the ratio of CD4 + to CD8 RTE was seen; this was normalized by 1-week post-cx (Fig. 6C).
  • Figs. 7A-B Castration enhances thymocyte regeneration following T cell depletion.
  • 3-month old mice were either treated with cyclophosphamide (intraperitoneal injection with 200 mg/kg body weight cyclophosphamide, twice over 2 days) (Fig. 7A) or exposed to sublethal irradiation (625 Rads) (Fig. 7B).
  • cyclophosphamide intraperitoneal injection with 200 mg/kg body weight cyclophosphamide, twice over 2 days
  • Fig. 7B sublethal irradiation (625 Rads) mice
  • Cx mice castrated mice showed a significant increase in the rate of thymus regeneration compared to their sham-castrated (ShCx) counterparts.
  • Figs. 8A-B Total lymphocyte numbers within the spleen and lymph nodes post- cyclophosphamide treatment.
  • mice 3 month old mice were depleted of lymphocytes using cyclophosphamide (intraperitoneal injection with 200 mg/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 were depleted of lymphocytes using cyclophosphamide (intraperitoneal injection with 200 mg/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
  • Figs. 10A-C Changes in thymus (Fig. 10A), spleen (Fig. 10B) and lymph node (Fig. 1 IC) cell numbers following irradiation (625 Rads) one week after surgical castration.
  • Fig. 10A 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. 10A).
  • spleen Fig. 11B
  • lymph node Fig. 1 IC cell numbers following irradiation and castration on the same day.
  • 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 counterparts (Fig. 11 A). Note the rapid expansion of the thymus in castrated animals when compared to the non-castrate group at 2 weeks post-treatment. No difference in spleen (Fig. 1 IB) or lymph node (Fig.
  • Figs. 13A-B Castration restores responsiveness to HSV-1 immunization. Mice were immunized in the hind foot-hock with 4xl0 5 pfu of HSV. On Day 5 post-infection, the draining lymph nodes (popliteal) were analyzed for responding cells.
  • Fig. 13A shows the lymph node cellularity following foot-pad immunization with Herpes Simplex Virus- 1 (HSV- 1). Note the increased cellularity in the aged post-castration as compared to the aged non- castrated group.
  • Figs. 14A-C VD 10 expression (HSV-specific) on CTL (cytotoxic T lymphocytes) in activated LN (lymph nodes) following HSV-1 inoculation.
  • CTL cytotoxic T lymphocytes
  • LN lymph nodes
  • Figs. 15A-B Castration enhances activation following HSV-1 infection.
  • Fig. 16 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.Ol, **) 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.
  • Figs. 17A-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 TCR VD markers (Fig. 17A) and CD4/CD8 T cells (Fig. 17B). The percentage of activated (CD25+) CD8 + T cells expressing either VD10 or VD8.1 is shown as mean ⁇ ISD for 8 mice per group in Fig. 17A. 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. 17B). This decrease was restored post-castration.
  • Figs. 18A-D Castration enhances regeneration of the thymus (Fig. 18 A), spleen (Fig. 18B) and BM (Fig. 18D), but not lymph node (Fig. 18C) following BM transplantation (BMT) of Ly5 congenic mice. 3 month old, young adults, C57/BL6 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 BM cells (10 6 cells).
  • thymus Fig. 18A
  • spleen Fig. 18B
  • lymph node Fig. 18C
  • BM Fig. 18D
  • Donor/Host origin was determined with anti- CD45.2 (Ly5.2), which only reacts with leukocytes of donor origin.
  • Ly5.2 anti- CD45.2
  • Fig. 18A There were significantly more donor cells in the thymus of castrated mice 2 and 4 weeks after BMT compared to sham-castrated mice (Fig. 18A). Note the rapid expansion of the thymus in castrated animals when compared to the non-castrate group at all time points post-treatment.
  • Figs. 19A-C Castration increases BM and thymic cellularity following congenic BMT. As shown in Fig. 19A, there are significantly more cells in the BM of castrated mice 2 and 4 weeks after BMT. BM cellularity reached untreated control levels (1.5xl0 7 ⁇ 1.5xl0 6 ) in the sham-castrates by 2 weeks. BM cellularity is above control levels in castrated mice 2 and 4 weeks after congenic BMT. Fig. 19b shows that there are 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 (7.6xl0 7 ⁇ 5.2xl0 6 ) 2 and 4 weeks after congenics BMT. 4 weeks after congenic BMT and castration thymic cellularity is increased above control levels.
  • Fig. 19C shows that there is no significant difference in splenic cellularity 2 and 4 weeks after BMT. Spleen cellularity has reached control levels (8.5xl0 7 ⁇ 1.1x10 ) in sham-castrated and castrated mice by 2 weeks. Each group contains 4 to 5 animals. Open bars indicate sham-castration; closed bars indicate castration.
  • Fig.20 Castration increases the proportion of HSC following congenic BMT.
  • FIG. 21A-B Castration increases the proportion and number of HSC following congenic BMT. As shown in Fig. 21 A, there was a significant increase in the proportion of HSCs following castration, 2 and 4 weeks after BMT (* ⁇ 0.05). Fig.
  • 21B 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. Open bars indicate sham-castration; closed bars indicate castration.
  • Figs.22A-B There are significantly more donor-derived B cell precursors and B cells in the BM of castrated mice following BMT. As shown in Fig. 22A, there were significantly more donor-derived CD45.1 + B220 + IgM " B cell precursors in the BM of castrated mice compared to the sham-castrated controls (* p ⁇ 0.05). Fig. 22B shows that there were significantly more donor-derived B220 + IgM + B cells in the BM of castrated mice compared to the sham-castrated controls (* p ⁇ 0.05). Each group contains 4 to 5 animals. Open bars indicate sham-castration; closed bars indicate castration.
  • Fig. 23 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.
  • Fig.25 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 10 7 + 4.5xl0 6 ). Each group contains 4 to 5 animals. Open bars indicate sham-castration; closed bars indicate castration.
  • Fig. 26 Donor-derived triple negative, double positive and CD4 and CD8 single positive thymocyte numbers are increased in castrated mice following BMT.
  • Fig. 26A 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. 26B 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).
  • Fig. 26A 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. 27 There are very few donor-derived, peripheral T cells 2 and 4 weeks after congenic BMT. As shown in Fig. 27 A, 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. 27B shows that there was no significant difference in donor- derived T cell numbers 2 and 4 weeks after BMT.
  • Fig. 28 Castration increases the number of donor-derived DC in the thymus 4 weeks after congenics BMT.
  • donor-derived DC were CD45.1 + CDllc + MHC ⁇ + .
  • Fig. 28B shows there were significantly more donor-derived thymic DC 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 (1.4xl0 5 ⁇ 2.8xl0 4 ). 4 weeks after congenic BMT dendritic cell numbers are above control levels in castrated mice. Each group contains 4 to 5 animals. Open bars indicate sham-castration; closed bars indicate castration.
  • Figs.29A-C Castration enhances immune cell reconstitution in allogeneic HSCT recipients.
  • Recipients were either castrated or sham- castrated one day before transplant. Animals were humanely killed on days 14, 28 and 42 and BM (Fig. 29 A), thymus (Fig. 29B), and spleen (Fig. 29C) organ cellularity was assessed. * ( p ⁇ 0.05). Each group contained 4 to 5 animals.
  • Figs. 30A-C Castration enhances donor-derived HSC and B cells in allogeneic HSCT recipients. Castrated and sham-castrated recipients were transplanted as in described in Fig. 29. As shown in Fig. 30A, 14 days after HSCT there were very few donor-derived HSC and B cells.
  • HSCs (Ly9.1"Lin " Sca-l + c-kit + ) in both sham-castrated and castrated mice; however, by day 28, donor HSC numbers were 4-fold higher in the castrated group. Additionally, as shown in Figs. 30B-C, there are significantly more donor-derived B cells in the BM (Fig. 30B) and spleen (Fig. 30C) of castrated mice. Central and peripheral B cell populations were analyzed using total BM or splenic cell counts and multicolor flow cytometry. B cells were separated into developmental stages based on CD45R, IgM and CD43 expression. Total B cells
  • CD45R+ pro-B cells
  • CD43 + CD45R+IgM- pro-B cells
  • pre-B cells CD43"CD45R + IgM
  • B cells (CD43"CD45R + IgM + ). Donor/host origin was determined with anti-Ly9.1, which only reacts with leukocytes of host origin. Each group contained 4 to 5 animals. Open bars indicate sham-castrated animals, and closed bars represent castrated animals. * ( p ⁇ 0.05) represents a significant increase in cell number in the castrated group compared to the sham- castrated control.
  • Figs. 31A-E Castration enhances thymocyte and peripheral T cell reconstitution as well as the number of host and donor-derived DC in allogeneic HSCT recipients. Castrated and sham-castrated recipients were transplanted as described in Fig. 29. Animals were humanely killed on days 14, 28 and 42, and thymocyte and T cell populations were analyzed using total thymic (Fig. 31A-F) or splenic (Fig. 31G) cell counts and multicolor flow cytometry. DC were defined as CD1 lc hi Ia-k hi .
  • Fig. 31 A depicts numbers of TN (CD3 " CD4-CD8") thymocytes.
  • Fig. 31A-E Castration enhances thymocyte and peripheral T cell reconstitution as well as the number of host and donor-derived DC in allogeneic HSCT recipients. Castrated and sham-castrated recipients were transplanted as described in Fig. 29. Animals were humanely killed on days 14,
  • FIG. 3 IB depicts numbers of DP (CD4+CD8+) thymocytes.
  • Fig. 31C depicts numbers of CD4+ SP (CD3+CD4+CD8") thymocytes.
  • Fig. 3 ID depicts numbers of CD8+ SP (CD3+CD4-CD8+) thymocytes.
  • Fig. 33 IE there are significantly more host-derived DC in castrated recipients at both 14 and 28 days after allogeneic HSCT as compared to sham-castrated control recipients.
  • Fig. 3 IF there are significantly more donor-derived DC in castrated recipients 28 days following allogeneic HSCT, as compared to sham-castrated controls.
  • Fig. 33 IE there are significantly more host-derived DC in castrated recipients at both 14 and 28 days after allogeneic HSCT as compared to sham-castrated control recipients.
  • Fig. 3 IF there are significantly more donor-derived DC in castrated recipients 28 days following allogene
  • 31G depicts numbers of peripheral T cells, which were identified using anti-CD3, anti-CD4 and anti-CD8.
  • Donor/host origin was determined with anti-Ly9.1, which only reacts with leukocytes of host origin.
  • Donor CD4 T cells were Ly9.1-CD3+CD4+CD8- and donor CD8 T cells were Ly9.1 " CD3 + CD4"CD8 + .
  • Each group contained 4 to 5 animals. Open bars indicate sham-castrated animals, and closed bars represent castrated animals. * ( p ⁇ 0.05) and ** (p ⁇ 0.01) represent a significant increase in cell number in the castrated group compared to the sham-castrated control. Figs.
  • FIG. 32A-G Castration does not alter the function of donor-derived T cells following allogeneic HSCT. Castrated and sham-castrated recipients were transplanted as in Figs. 29. T cell functionality was assessed 42 days after transplantation.
  • Fig. 32A shows the number of donor-derived T cells (CD3 + CD4 + and CD3 + CD8 + ) six weeks after allo-HSCT.
  • Fig. 32B shows that castration has no effect on the proliferative capability of T cells after allogeneic HSCT.
  • Fig. 32C shows no difference in alloreactive T-cell proliferation in an MLR.
  • Fig. 32D shows no difference in cytolytic activity of donor-derived T cells.
  • Fig.32E shows intracellular IFN ⁇ expression of alloreactive T cells.
  • Splenic B6 T cells were harvested on day 42 from sham- castrated or castrated recipients as described above and incubated with irradiated (20 Gy) (BALB/C - third party) splenic stimulator cells in 24-well plates for 5 days.
  • Fig. 32E Representative plots are shown in Fig. 32E and graphically represented as the percentage of donor-derived CD8 + T cells that express IFN- ⁇ in Fig. 32F.
  • Fig. 32G shows that T cell functionality was significantly enhanced 48 hrs. after challenge when mice were castrated at the time of allo-HSCT.
  • the DTH assay was performed at week 6 following allogeneic HSCT in sham-castrated and castrated mice, and the swelling was measured by subtracting left hind footpad swell from right hind one. Open bars indicate sham-castrated animals, and closed bars represent castrated animals.
  • Figs.33A-B Castration does not aggravate GVHD or decrease GVL activity in allogeneic HSCT recipients.
  • Figs. 34A-I Castration and IL-7 treatment have an additive effect in the thymus following allogeneic HSCT. Castrated and sham-castrated recipients were transplanted described in Fig. 1. Recipients killed on day 14 (Fig. 34 A) received, in addition, 10 g/day IL- 7 or PBS (control) by intraperitoneal injection from day 0 to day 13. Recipients killed on day 28 (Fig. 34B) received 10 g/day E -7 or PBS (control) from day 21 to day 28. Thymic cellularity was calculated from total cell counts. * ( p ⁇ 0.05) represents a significant increase in cell number in the castrated group compared to the sham-castrated control.
  • Fig. 34G-I shows the results from the thymus (Fig. 34G), BM (Fig. 34H), and spleen (Fig. 341) of IL7-/- mice.
  • Fig. 35 Castration enhances engraftment in the BM, thymus, and spleen following HSCT. Mice were castrated 1 day before congenic HSCT. 5xl0 6 Ly5.1+ BM cells were injected intravenously into irradiated (800 rads) C57/BL6 mice. The BM, spleen and thymus were analyzed by flow cytometry at various time points (2-6 weeks) post-transplant. As shown in Fig. 35B, two weeks after castration and HSCT, there are significantly more cells in the BM of castrated mice as compared to sham-castrated controls. Similarly, as shown in Fig.
  • Fig. 36A-B Castration enhances engraftment of HSC in the BM following congenic
  • HSCT HSCT. Mice were castrated 1 day before congenic HSCT. 5x10 Ly5.1 + BM cells were injected intravenously into irradiated (800 rads) C57/BL6 mice. The BM was analyzed for lin-c-kit4-sca-l+ HSC by flow cytometry at two weeks post-transplant (Fig. 36A). Two weeks after BMT transplantation and castration there are significantly more donor-derived HSCs in the BM of castrated mice compared to sham castrated controls (Fig. 36B).
  • Figs. 37A-B Castration enhances engraftment of HSC in the BM following congenic HSCT (2.5xl0 6 cells and 5xl0 6 cells). Mice were castrated 1 day before congenic HSCT. 2.5x10 (Fig. 37A-B) or 5xl0 6 (Fig. 37C-D) Ly5.1+ BM cells were injected intravenously into irradiated (800 rads) C57 BL6 mice. The BM was analyzed for lin-c-kit+sca-l+ HSC by flow cytometry at two weeks post-transplant. Figs. 37A-D depict percent of common lymphoid precursors in the BM. Two weeks after BMT transplantation and castration there is a significantly increased proportion of donor-derived HSCs in the BM of castrated mice compared to sham castrated controls.
  • Figs.38A-B Castration enhances the rate of engraftment of donor-derived DC in the thymus following congenic HSCT (2.5xl0 6 cells and 5xl0 6 cells).
  • 5x10 Ly5.1+ BM cells were injected intravenously into irradiated (800 rads) C57/BL6 mice.
  • Thymocytes were analyzed by flow cytometry at two weeks post-transplant (Fig. 38A).
  • Donor-derived DC were defined as CD45.1+CDllc+MHC class H+ CDllb + ° r -Donor-derived CD 11 b+ and
  • CD lib DC are significantly increased in the thymii of castrated mice compared to sham- castrated controls 2 weeks after BMT (Fig. 38B).
  • Figs. 39A-D Castration enhances the rate of engraftment of donor-derived B cells in
  • Fig. 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, six out of nine 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 six out of nine patients. Within the CD4 + subset, this increase was even more pronounced with eight out of nine patients demonstrating increased levels of CD4 T cells. A less distinctive trend was seen within the CD8 T subset with four out of nine patients showing increased levels, albeit generally to a smaller extent than CD4 + T cells.
  • Fig. 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.
  • Fig. 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 four out of nine patients.
  • Fig.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
  • B cell numbers showed no distinct trend with two out of nine patients showing increased levels; four out of nine patients showing no change and three out of nine patients showing decreased levels.
  • Figs.44A-B Chemical castration in humans enhances na ⁇ ve and memory T cells.
  • a significant increase in na ⁇ ve (CD62L + CD45RA + CD45RO ⁇ ) CD4 + T cells was observed following LHRH- A treatment.
  • both na ⁇ ve and memory (CD62L " CD45RA " CD45RO + ) CD8 + T cells numbers were enhanced following the LHRH agonist treatment.
  • Fig.45A-B Chemical castration in humans enhances peripheral blood lymphocyte numbers.
  • the phenotypic composition of peripheral blood was analyzed in human patients (all >60years of age) undergoing chemical castration with a LHRH-A as part of their routine treatment for prostate cancer. Patients were analysed prior to treatment and at 4-months of treatment.
  • Fig. 45A total lymphocyte number per ⁇ l peripheral blood was significantly increased following LHRH-A treatment. T his was reflected by a significant increase in total T cells, CD4 + and CD8 + T cells (Fig 45B).
  • Fig. 46A-B LHRH-A treatment effectively depletes serum testosterone, and increases thymic function and T cell export.
  • Fig.46A prostate cancer patients were treated with LHRH-A for 4 months. Blood was analyzed by FACS and serum was analyzed by RIA both prior to treatment and following 4-months of LHRH-A treatment. As shown in Fig. 83 A, no testosterone was detected in patient sera at 4-months of LHRH-A treatment. The bar represents the mean of 13 patients analyzed.
  • Fig. 83 A no testosterone was detected in patient sera at 4-months of LHRH-A treatment. The bar represents the mean of 13 patients analyzed.
  • Fig 46B experiment direct evidence for an increase in thymic function and T cell export was found following analysis of TREC levels in 10 patients. Within both the CD4 + and CD8 + T cell population, five out of ten patients showed an increase (>25% above initial presentation values) in absolute TREC levels (per ml of blood) by 4 months of LHRH-A treatment. This was also reflected in a proportional increase (per l lO 5 cells; data not shown). This correlated with six out of ten
  • Figs. 48A-B Chemical castration in humans does not increase proliferation of T cells.
  • Figs. 48A-B depict analyses of cellular proliferation performed using Ki-67 antigen detection. In all patients, levels of proliferation within na ⁇ ve, activated and memory cell subsets for both CD4 + (Fig. 48A) and CD8 + T cells (Fig.48B), was not altered with LHRH-A treatment.
  • Fig. 50 FACS analysis of NKT cell reconstitution at various time points (day 14, 21, 28 and 35) following HSCT in control patients. An early recovery was observed in allogeneic patients, and was seen predominantly within the CD8+ population early post- transplant, which indicated extrathymic routes of regeneration. Also, CD4+NKT cells were evident from 1 month post-transplant.
  • Figs. 51A-B B cell reconstitution following HSCT at various time points (2-12 months) following HSCT in control patients. As shown in Fig. 51B, B cell regeneration occurs occurring relatively faster in autologous transplant patients as compared to that of allogeneic patients (Fig. 51 A). However, a return to control values (shaded) was not evident until at least 6 months post-transplant in both groups.
  • Figs. 52A-B CD44- reconstitution following HSCT at various time points (2-12 months) following HSCT in control patients. While B cell numbers were returning to control values by 6 months post-transplant (see Figs. 48A-B), CD4+ T cell numbers were severely reduced, even at 12 months post-transplant, in both autologous (Fig. 52B) and allogeneic (Fig. 52A) recipients.
  • Figs. 53A-C CD8+ regeneration following HSCT at various time points (2-12 months) following HSCT in control patients.
  • CD8+ T cell numbers regenerated quite rapidly post-transplant in both allogeneic and autologous recipients, respectively.
  • the CD8+ T cells are mainly of extrathymic origin as indicated by the increase in TCR ⁇ + T CD8+T cells, CD8 ⁇ T cells, and CD28 " CD8 + T cells.
  • Figs. 54A-B FACS analysis of proliferation in various populations of CD4+ and CD8+ T cells before (Fig. 54A) and 28 days after (Fig. 54B) HSCT in control patients using the marker Ki-67.
  • Cells were analyzed on the basis of na ⁇ ve, memory and activated phenotypes using the markers CD45RO and CD27. The majority of proliferation occurred in CD8+ T cell subset, which further indicated that these cells were extrathymically derived and that the predominance of proliferation occurred within peripheral T cell subsets.
  • Figs. 55A-D Na ⁇ ve CD4+ T cell regeneration at various time points (2-12 months) following HSCT in control patients and LHRH-A treated patients.
  • Fig. 55A depicts FACS analysis of na ⁇ ve CD4+ T cells (CD45RA+CD45RO-CD62L+) in control (no LHRH-A treatment) patients, and shows a severe loss of these cells throughout the study.
  • Figs. 55B-C na ⁇ ve CD4+ T cell began to regenerate in the control patients by 12 months post-HSCT in autologous transplant patients (Fig. 55C) but were still considerably lower than the control values in allogeneic control patients (Fig. 55B).
  • Figs. 56A-D TREC levels at various time points (1-12 months) following HSCT in control patients and LHRH-A treated patients.
  • Analysis of TREC levels which are only seen in recent thymic emigrants (RTE), emphasized the inability of the thymus to restore levels following transplant in both allogeneic (Fig. 52A) and autologous (Fig. 52B) patients. Again, this was due to the age of the patients, as well as the lack of thymic function due to thymic atrophy, which has considerable implications in the morbidity and mortality of these patients.
  • patients undergoing allogeneic peripheral blood stem cell transplantation demonstrated a significant increase in CD4+TREC+ cells/ml blood when treated with an
  • Fig. 57A-C LHRH-A administration enhances responsiveness to TCR specific stimulation following allogeneic (Fig. 57A-B) and autologous (Fig. 57C) HSCT. Three weeks prior to HSCT, patients were given LHRH-A. Patients who did not receive the agonist were used as control patients. Analysis of TCR specific stimulation was performed using 5 ⁇ g anti-CD3 and 10 ⁇ g anti-CD28 cross-linking at various time points (1-12 months) post- transplant. As shown in Figs.
  • LHRH-A treated patients showed enhanced proliferative responses (assessed by 3 H-Thymidine incorporation) compared to control patients at all time-points except 6 and 9 months (due to low patient numbers analyzed at this time). At 6 and 9 months post-transplant control patients had similar responsiveness to pre- treatment values. However at all other time-points, they were considerably lower. In contrast, LHRH-A treated patients had equivalent responsiveness at all time-points except 6 months compared to pre-treatment. LHRH-A treated patients showed enhanced proliferative responses (assessed by 3 H-Thymidine incorporation) compared to control patients at 1, 3 and 4 months post-transplant.
  • Fig. 58A-B LHRH-A administration enhances responsiveness to PWM and TT rnitogenic stimulation following allogeneic HSCT.
  • Three weeks prior to HSCT patients were treated with LHRH-A. Patients who did not receive the agonist were used as control patients. Analysis of rnitogenic responsiveness was performed using pokeweed mitogen (PWM) or tetanus toxoid (TT) at various time points (1-12 months) post-transplant.
  • PWM pokeweed mitogen
  • TT tetanus toxoid
  • Fig.59A-B LHRH-A administration enhances responsiveness to PWM and TT rnitogenic stimulation following autologous HSCT.
  • Three weeks prior to HSCT patients were treated with LHRH-A. Patients who did not receive the agonist were used as control patients. Analysis of rnitogenic responsiveness was performed using PWM or TT at various time point (1-12 months) post-transplant. Patients treated with LHRH-A prior to HSCT showed an enhanced responsiveness to PWM (Fig. 59A) and TT (Fig. 59B) stimulation at the majority of time-points studied compared to control patients (p ⁇ O.OOl at 3 months). By 12- months post-transplant, LHRH-A treated patients had restored responsiveness.
  • Figs. 60A-D LHRH-A treatment enhances the rate of engraftment in autologous HSCT patients.
  • Three weeks prior to HSCT patients were treated with LHRH-A (Figs. 60A, C and D). Patients who did not receive the agonist were used as control patients (Figs. 60B).
  • Total white blood cell (WBC) counts and granulocyte (G) counts per ⁇ l of blood were determined at days 14, 28, and 35 post transplant.
  • WBC white blood cell
  • G granulocyte
  • Figs. 61A-D LHRH-A treatment enhances the rate of engraftment in allogeneic HSCT patients.
  • LHRH-A Three weeks prior to HSCT, patients were treated with LHRH-A (Figs. 61A, C and D). Patients who did not receive the agonist were used as control patients (Fig. 61B).
  • Total white blood cell (WBC) counts and granulocyte (G) counts per ⁇ l of blood were determined at day 14, 28, and 35 post transplant.
  • WBC white blood cell
  • G granulocyte
  • Figs.62A-F TCR specific peripheral T cell proliferative responses are enhanced within one week of castration. Eight week-old mice were castrated and analyzed for anti- CD3/anti-CD28 stimulated T cell proliferative response 3 days (Figs. 62A, C, and E) and 7 days (Figs. 62B, D, and F) after surgery. Peripheral (cervical, axillary, brachial and inguinal) lymph node (Figs. 62A and B), mesenteric lymph node (Figs. 62C and D), and spleen cells (Figs.
  • 62E and F were stimulated with varying concentrations of anti-CD3 and co-stimulated with anti-CD28 at a constant concentration of 10 ⁇ g/ml for 48 hours.
  • Cells were then pulsed with tritiated thymidine for 18 hours and proliferation was measured as 3 H-T incorporation.
  • LHRH-A administration enhances responsiveness to TCR specific stimulation following treatment for chronic cancer sufferers.
  • Patients with chronic malignancies were treated with LHRH-A.
  • Analysis of TCR specific stimulation was performed using anti-CD3 and anti-CD28 cross-linking from at various time points (day 7 - 12 months) following LHRH-A administration.
  • LHRH-A treated patients showed enhanced proliferative responses (assessed by 3 H-Thymidine incorporation) compared to pre-treatment levels in a cyclical fashion. This reflected the administration of the agonist with monthly depot injections.
  • Figure 64 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 65 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 66A-C are bar graphs showing that there was a significant increase in all thymocyte subclasses (Fig. 66A) in castrated NOD mice. There no change in B cells compared to sham-castrated NOD mice (Fig. 66C) nor in the total T or B cells in the spleen (Fig. 66B).
  • Figures 67A and 67B show a marked in total thymocytes (Fig. 67A) and spleen cells (Fig. 68B) in castrated NZB mice.
  • Figure 68 is a graph showing decreased tumor incidence in mice that have been castrated and immunized as compared to controls.
  • Figures 69A-C are bar graphs showing that castrated and immunized mice have increased splenic cellularity as compared to controls.
  • Figures 70A-B are graphs showing increased ⁇ lFN production in mice that have castrated and immunized as compared to controls.
  • Figures 71A-B are graphs showing that castrated and immunized mice exhibit enhanced antigen-specific CTL responses as compared to controls.
  • Figures 72A-E are graphs showing that thymectomy does not impact the effect of sex steroid inhibition/BMT on common lymphoid progenitors in the BM (Fig. 72A), total BM B cells (Fig. 72B), immature B cells in the BM (Fig. 64C), total cell numbers in the spleen (Fig. 72D) or on total B cells in the spleen (Fig. 72E).
  • the present invention comprises methods for increasing the BM functionality following sex steroid ablation and/or interruption of sex steroid signalling, either without, prior to, or in combination with, thymus regeneration.
  • "Increasing the function of BM” and "enhancing BM functionality” is herein defined as an improvement in the production and/or output of immune cells, including precursors, for example HSC (and consequent increases in blood cells) from the BM, including improvement in haemopoiesis and/or enhancement of engraftment following HSCT.
  • An improvement in output may include, but is not limited to, an improved ability to mobilize immune cells, including HSC into the periphery or to a target tissue, in particular, immune or damaged tissue).
  • HSC haemopoiesis is improved.
  • HSC output is improved.
  • blood and/or immune cell numbers are increased.
  • HSC engraftment is improved following HSCT.
  • HSC mobilization into the periphery or homing to target tissue is improved.
  • proliferative ability, and/or the ability to differentiate into haematopoietic or non-haemopoitetic progeny is improved.
  • the present invention also comprises methods for increasing the function of T cells and other immune cells following sex steroid ablation and/or interruption of sex steroid signaling, either without, prior to, or in combination with, thymus regeneration.
  • immune cells and “cells of the immune system” are used interchangeably and are herein defined as HSC, T cells, B cells, DC, and/or other blood cells, including, but not limited to HSC progeny, CLP, MLP, lymphocytes, myeloid cells, neutrophils, granulocytes, basophils, eosinophils, NK, NKT, platelets, red blood cells, monocytes, macrophage, na ⁇ ve T cells, and precursors of the aforementioned.
  • the cells may or may not be peripheral, and the cells may be found in any one or more of the BM, blood, spleen, lymph nodes, thymus. mucosal membranes, skin, or other tissues.
  • the immune cells are more able to provide an adequate required immune response, when compared to the immune response normally expected without sex steroid ablation.
  • the immune cells are T cells.
  • the immune cells are B cells, DC, and/or HSC.
  • “Increased functionality” includes, but is not limited to, improved killing of target cells; increased lymphocyte proliferative response; improved signaling ability; improved homing ability; improved APC activation, increased levels or activity of receptors, cell adhesion molecules, or co-stimulatory molecules; decreased apoptosis; increased release of cytokines, interleukins, and other growth factors; increased levels of antibody (Ab) in the plasma; and increased levels of innate immunity (e.g., natural killer (NK) cells, DC, neutrophils, macrophages, etc.) in the blood and throughout the body.
  • NK natural killer
  • NK natural killer
  • the present invention further comprises methods for preventing, diminishing the risk, or treating illness or disease in a patient.
  • the disease is a T cell disorder.
  • the disease is an autoimmune disease or allergy.
  • 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.
  • a vaccine antigen e.g., that of an agent
  • the functional status of the peripheral T cells may be improved and may be accomplished by quantitatively and qualitatively restoring the peripheral T cell pool, particularly at the level of na ⁇ ve T cells. These na ⁇ ve T cells are then able to respond to a greater degree to presented foreign antigen.
  • 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.
  • Praubertal 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.
  • vaccinating 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.
  • infection by, e.g., a virus or other agent is also a method of vaccinating an individual.
  • “Ulness” and “disease” are used interchangeably and are herein defined as any disease, infection or medical condition (symptomatic or asymptomatic) in which an immune response, defence or modified immune system would be beneficial to the patient.
  • the illness 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.
  • 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 have improved response to, respond better to, or to overcome, a new (by prevention) or existing (by treatment) illness involves improving the immune system of the body, which includes increasing the number and/or functionality of the BM cells and/or thymic-derived factors, and/or increasing the number and/or functionality of immune cells.
  • Activation of the immune system also increases the number of lymphocytes capable of responding to the antigen of the agent in question, which leads to the elimination (complete or partial) of the antigen and/or foreign agent creating a situation where the host is treated for or resistant to the infection or disease.
  • the individual 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).
  • a vaccination e.g., increased levels of antibody (Ab) specific to that vaccine or antigen, and development of effector T cells.
  • 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.
  • 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)).
  • HPV human papilloma virus
  • influenza peptide e.g., hemagglutinin (HA), nucleoprotein (NP), or neuraminidase (N)
  • Retroviridae e.g., human immunodeficiency viruses, such as HIV-1 (also referred to as HTLV-DI, LAV or HTLV- r ⁇ /LAV, or H ⁇ - ⁇ I) and other isolates, such as HIV-LP; Picomaviridae (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, rab
  • infectious bacteria include: Helicobacter pylons, Borelia burgdorferi, Legionella pneumopliilia, Mycobacteria sporozoites (sp.) (e.g., M. tuberculosis, M. avium, M. intracellulare, M. kansaii, M. gordonae), Staphylococcus aureus, Neisse ⁇ a gonorrhoeae, Neisseria meningitidis, Listeria monocyto genes, Streptococcus pyogenes
  • sp. e.g., M. tuberculosis, M. avium, M. intracellulare, M. kansaii, M. gordonae
  • Staphylococcus aureus e.g., Neisse ⁇ a gonorrhoeae, Neisseria meningitidis, Listeria monocyto genes, Streptococcus pyogenes
  • Streptococcus pneumoniae pathogenic Campylobacter sp., Enterococcus sp., Haemophilus influenzae, Bacillus antracis, Corynebacterium diphtheriae, Corynebacterium sp., Erysipelothrix rhusiopat iae, Clostridium perfringens, Clostridium tetani, Enterobacter aerogenes, Klebsiella pneumoniae, Pasturella multocida, Bacteroides sp., Fusobacterium nucleatum, Streptobacillus moniliformis, Treponema
  • Non-limiting examples of infectious fungi include: Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Chlamydia trachomatis, Candida albicans.
  • infectious organisms include, but are not limited to, Plasmodium falciparum and Toxoplasma gondii.
  • 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.
  • the agent is a cancer or tumor.
  • the cancer or tumor may be malignant or non-malignant.
  • a tumor or cancer includes, e.g., tumors of 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 hematological system (e.g., blood, myelodysplastic syndromes, myelop), and/or the
  • This invention 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 examples, the invention is used with large mammals, such as humans.
  • 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 disrapted, 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).
  • 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.
  • 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 present invention further provides methods of disruption of sex steroid-mediated signaling in a patient, wherein the patient's thymus may or may not be subsequently reactivated. Additionally, the present invention provides methods of improving the functional status of immune cells (e.g., T cells) of the patient.
  • T cells the thymus begins to increase the rate of proliferation of the early precursor cells (CD3 ⁇ CD4 ⁇ CD8 " cells) and converts them into CD4 + CD8 + , and subsequently new mature
  • CD3 hi CD4 + CD8 " T helper (Th) lymphocytes
  • CD3 hi CD4 " CD8 + cytotoxic T lymphocytes (CTL)
  • the rejuvenating thymus also increases its uptake of 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 DC.
  • the increased activity in the thymus resembles in many ways that found in a normal younger thymus (e.g., a prepubertal patient).
  • the result of this renewed thymic output is increased levels of na ⁇ ve T cells (those T cells which have not yet encountered antigen) in the blood.
  • peripheral T cells There is also an increase in the ability of the peripheral T cells to respond to stimulation, 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) and this increased T cell responsiveness can occur before thymic regeneration, such as within 2, 3, 4, 5 6, 7, 14 or 21 days.
  • mitogens such as pokeweed mitogen (PWM)
  • PWM pokeweed mitogen
  • This combination of events results in the body becoming better able to defend against infection and other immune system challenges (e.g., cancers), or recover from immune system challenges (e.g., becoming better able to recover from chemotherapy and radiotherapy).
  • the methods of the invention may be used to prevent or treat an illness or infection, increase a patient's immune responsiveness to a vaccine, and for optional gene therapy.
  • sex steroid ablation 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.
  • sex steroid signaling to the thymus is interrupted.
  • 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.
  • 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 signalling is through castration.
  • Methods for castration include, but are not limited to, chemical castration and surgical 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.
  • “chemical castration” is the administration of a chemical for a period of time, which results 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.
  • the patient's hormone production may be turned off or reduced.
  • the castration may be reversed upon termination of chemical delivery or by delivery of the relevant sex hormones.
  • ex steroid analog sex steroid ablating agent
  • sex steroid inhibitor sex steroid inhibitor
  • inhibitor of sex steroid signalling modifier of sex steroid signalling
  • GnRH also called LHRH or GnRH/LHRH herein
  • analogs thereof are nonlimiting exemplary inhibitors of sex steroid signalling used throughout this application.
  • GnRH/LHRH 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.
  • one nonlimiting method of, inhibiting sex steroid signaling, reactivating the thymus and/or enhancing the functionality of BM and immune cells 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.
  • 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).
  • LH leutinizing hormone
  • FSH follicle-stimulating hormone
  • GnRH agonists e.g., in the form of Synarel ® or Lupron®
  • LH leutinizing hormone
  • FSH follicle-stimulating hormone
  • the sex steroid mediated signaling is disrupted by administration of a sex steroid analog, such as an analog of leutinizing hormone-releasing hormone (LHRH).
  • a sex steroid analog such as an analog of leutinizing hormone-releasing hormone (LHRH).
  • LHRH leutinizing hormone-releasing hormone
  • Such analogs include, but are not limited to, the following agonists ofthe 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 ⁇ g per nostril, every 8 hrs.), Hoechst, also described in U.S. Patent Nos.
  • 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 ⁇ g per nostril, every 8 hrs.)
  • Suprefact® e.g., 0.5-02 mg s.c./day
  • Suprefact Depot® e.g., 0.5-02 mg s.c./day
  • Cystorelin® e.g., gonadorelin diacetate tetrahydrate, Hoechst
  • deslorelin e.g., desorelin acetate, Deslorell®, Balance Pharmaceuticals
  • gonadorelin e.g., gonadorelin hydrocholoride, trade name Factrel® (100 ⁇ g i.v. or s.c), Ayerst Laboratories
  • goserelin goserelin acetate, trade name Zoladex®, AstraZeneca, Aukland, NZ, also described in U.S. Patent Nos.
  • histrelin e.g., histerelin acetate, Supprelin®, (s.c, 10 ⁇ g/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.
  • leuprorelin e.g., leuprorelin 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.
  • 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 ⁇ g/day), Syntex, also described in U.S. Patent No.
  • 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 PlenaxisTM (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 CetrotideTM (e.g., 0.25 or 3 mg s.c), Zentaris, Frankfurt, Germany).
  • PlenaxisTM e.g., 100 mg i.m. on days 1, 15 and 29, then every 4 weeks thereafter
  • Praecis Pharmaceuticals, Inc., Cambridge, MA Praecis Pharmaceuticals, Inc., Cambridge, MA
  • cetrorelix e.g., cetrorelix acetate, trade name CetrotideTM (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.
  • 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 10089
  • One non-limiting analog of the invention is deslorelin (described in U.S. Patent No. 4,218,439).
  • Deslorelin described in U.S. Patent No. 4,218,439.
  • LHRH AND ITS ANALOGS CONTRACEPTIVE & THERAPEUTIC APPLICATIONS (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.
  • 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).
  • GnRH agonist e.g., one, three, or four month Lupron® injections
  • 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.
  • Lupron® e.g., Lupron Depot® '(leuprolide acetate for depot suspension) TAP Pharmaceuticals Products, Inc., Lake Forest, IL.
  • Lupron® injection is sufficient for four months of sex steroid ablation to allow the thymus to rejuvenate and export new naive T cells into the blood stream.
  • the thymus of the patient is ultimately reactivated by sex steroid ablation and/or interruption or disruption of sex steroid-mediated signalling.
  • disruption reverses the hormonal status of the patient.
  • the hormonal status of the recipient is reversed such that the hormones of the recipient approach pre-pubertal levels.
  • 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.
  • 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.
  • a treatment of a disease may be anti-viral, immunosuppression, chemotherapy, and/or radiation treatment.
  • the patient is menopausal or has had sex steroid (or other hormonal levels) decreased by another means, e.g., trauma, drugs, etc.
  • Sex steroid ablation or interruption of sex steroid mediated signaling has one or more direct effect on the BM and/or cells of the immune system, wherein functionality is improved. The effects may occur prior to, or concurrently with, thymic reactivation.
  • 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 QID, AstraZeneca, Aukland, NZ), either alone or in combination with an LHRH analog or any other method of castration.
  • an anti-androgen such as an androgen blocker (e.g., bicalutamide, trade names Cosudex® or Casodex®, 5-500 mg, e.g., 50 mg po QID, AstraZeneca, Aukland, NZ), either alone or in combination with an LHRH analog or any other method of castration.
  • an anti-androgen such as an androgen blocker (e.g., bicalutamide, trade names Cosudex® or Casodex®
  • 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 EVI 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.
  • cyproterone acetate trade name, Androcor®, Shering AG, Germany; e.g., 10-1000 mg, 100 mg bd or tds, or 300 mg EVI weekly
  • a 17- hydroxyprogesterone acetate acts as a progestin, either alone or in combination with an LHRH analog or any other method of castration.
  • 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 Corp, N.J.; 50-500 mg e.g., 250 or 750 mg po QID), megestrol acetate
  • imidazole class such as liarozole (Liazol® e.g., 150 mg/day, an aromatase inhibitor) and ketoconazole, flutamide (trade names Euflex® and Eulexin®, Shering Plough Corp, N.J.; 50-500 mg e.g., 250 or 750 mg po QID), megestrol acetate
  • 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.
  • SARMS selective androgen receptor modulators
  • quinoline derivatives e.g., bicalutamide (trade name Cosudex® or Casodex®, as above), and flutamide (trade name Eulexin®, e.g., orally, 250 mg/day)
  • flutamide trade name Eulexin®, e.g., orally, 250 mg/day
  • 5 alpha reductase inhibitors e.g., dutasteride,(e.j ., po 0.5 mg/day)
  • 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
  • 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 IM monthly) act by binding the estrogen receptor (ER) with high affinity similar to estradiol and consequently inhibiting estrogen from binding. Faslodex® binding also triggers conformational change to the receptor and down-regulation of estrogen receptors, without significant change in FSH or LH levels.
  • ER estrogen receptor
  • 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.
  • tamoxifen trade name Nolvadex®
  • Clomiphene trade name Clomid®
  • SERMS selective estrogen receptor modulators
  • toremifene trade name Fareston®, 5-1000 mg, e.g., 60 mg po QID
  • raloxofene trade name Evista®
  • tamoxifen trade name Nolvadex®, 1-1000 mg, e.g., 20 mg po bd
  • Estrogen receptor , downregulators ELDs
  • tamoxifen trade name, Nolvadex®
  • 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-
  • adrenal gland blockers e.g., Aminoglutethimide, formestane, vorazole, exemestane
  • 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)).
  • SPRM selective progesterone response modulators
  • anti-GnRH vaccines see, e.g., Hsu et al, (2000) Cancer Res. 60:3701; Talwar, (1999) Immunol. Rev. 171:173-92
  • 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.
  • 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.
  • 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.
  • prolactin 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 hyperprolactine ia 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.
  • Antidopaminergic agents include haloperidol, fluphenazine, sulphide, 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.
  • 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.
  • an LHRH-R antagonist is delivered to the patient, followed by an LHRH-R agonist.
  • 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).
  • 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.
  • 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.
  • 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, 17 ⁇ -hydroxy progesterone, 20 ⁇ -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 17 ⁇ -hydroxy androstenedione.
  • Non-limiting members of the estrogen family of C17 steroids include estrone, estradiol- 17 ⁇ , and estradiol- 17 ⁇ .
  • sex steroids 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.
  • 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.
  • the direct methods include methods of influencing sex steroid biosynthesis and metabolism, 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.
  • FSH follicle stimulating hormone
  • LH luteinizing hormone
  • IGF-1 insulin-like growth factor- 1
  • 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.
  • 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.
  • P450scc P450 cholesterol side chain cleavage
  • inhibition of enzymes later in the pathway such as P450 aromatase (P450arom) that converts androgens to estrogens, or 5 ⁇ -reductase that converts testosterone to DHT, will only effect the production of estrogens or DHT, respectively.
  • oxidoreductase enzymes that catalyze the interconversion of inactive to bioactive steroids, for example, androstenedione to testosterone or estrone to estradiol-17Dby 1-7-hydroxysteroid dehydrogenase (17-HSD).
  • These enzymes are tissue and cell specific and generally catalyze either the reduction or oxidation reaction e.g., 17 ⁇ HSD type 3 is found exclusively in the Leydig cells of the testes, whereas 17 ⁇ 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.
  • 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.
  • 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.
  • 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 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, PR A and PRB, and three forms of the estrogen receptor, ERoc, ER ⁇ l and ER ⁇ 2. 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 ofthe genes encoding the regulators. Additionally, c-AMP
  • antiandrogens that interact with the specific steroid receptors
  • antiprogestins that interact with the specific steroid receptors
  • 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.
  • SARMS selective receptor modulators
  • SERMS selective receptor modulators
  • 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.
  • 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.
  • FSH and LH pituitary gonadotrophins
  • 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.
  • GnRH/LHRH hypothalamic hormone
  • 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.
  • 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 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.
  • BM lymphopoiesis While the stimulus for the direct and indirect effects on BM functionality, BM lymphopoiesis, and immune cell functionality 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 (IL-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 (TL-11; 1-1000 ⁇ g/kg) members ofthe 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 ⁇ g/kg/day TV or SC), granulocyte macrophage stimulating factor (GM-CSF; 50-1000 ⁇ g/sq meter/day SC or IV), insulin dependent growth factor (IGF-1), and keratinocyte growth
  • a non-exclusive 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, E -l, JL-4, JL-5, IL-6, IL-8, IL-9, IL-10, IL-12, IL-13, LIF, flf3/flk2, human growth hormone, B-cell growth factor, B-cell differentiation factor and eosinophil differentiation factor, or combinations thereof.
  • Meg-CSF Meg-CSF
  • MIF Macrophage Inhibitory Fact
  • 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 (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 functionality of the BM and other immune cells.
  • 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.
  • mobilizing agent is herein defined as agents such as SDF-1 (e.g., AMD3100), growth hormone, GM-CSF, G-CSF and chemotherapeutics (e.g., cyclophosphamide), which enhance mobilisation of stem cells from the BM.
  • SDF-1 e.g., AMD3100
  • growth hormone e.g., GM-CSF
  • G-CSF e.g., chemotherapeutics
  • chemotherapeutics e.g., cyclophosphamide
  • G-CSF and GM-CSF are known to mobilize the production of granulocytes (primarily neutrophils) and macrophages, respectively, and also result in increased production of DC from the BM, which help provide a non-specific immune response, in the patient to antigenic challenge (Janeway et al. , (2001) Immunobiology 5 th Ed., p. 325).
  • G-CSF and GM-CSF are used, for example, to decrease the incidence of infection (as manifested by febrile neutropenia) in patients with non-myeloid malignancies receiving myelosuppressive anti-cancer drugs, which are typically associated with a significant incidence of severe neutropenia and fever.
  • both of these drugs are approved clinically to prevent infections in patients receiving HSCT.
  • G-CSF and GM-CSF are currently used in patients undergoing peripheral blood progenitor cell collection or therapy.
  • Colony stimulating factors (CSFs) which stimulate the differentiation and/or proliferation of BM stem cells, have generated much interest because of their therapeutic potential for restoring depressed levels of hematopoietic stem cell-derived cells. CSFs in both human and murine systems have been identified and distinguished according to their activities.
  • G-CSF granulocyte-CSF
  • M-CSF macrophage-CSF
  • G-CSF granulocyte-CSF
  • IL-3 interleukin-3
  • IL-3 also stimulates the formation of mast, megakaryocyte and pure and mixed erythroid colonies (when erythropoietin is added).
  • GM-CSF accelerates recovery of neutrophils and maintains functional capacity, yet has little demonstrable effect on platelet recovery.
  • G-CSF and/or GM-CSF are used in some of the methods of the invention. Sex steroid ablation together (sequentially or concurrently) with G-CSF and/or GM-CSF therapy results in an increase in the output from the BM of both lymphoid and myeloid cell, which in turn significantly improves both the short and long term outcomes for patients suffering, or likely to suffer from, infections.
  • the CSFs are administered 3-4 days after chemotherapy or radiation therapy. Clinical outcomes already associated with the use of the CSFs are also greatly enhanced by an interruption to sex steroid signaling.
  • the concurrent use of two separate classes of drags may allow for the same immune system regeneration but may require a reduced dosage of the G-CSF or GM- CSF.
  • the concurrent use of these two separate classes of drugs may allow for a greater, or more prolonged rejuvenation of immune system cells, while utilizing the same dosage of G-CSF or GM-CSF.
  • the concurrent use of two separate classes of drags may allows for the same rejuvenation of immune system cells, while utilizing a reduced dosage (i.e., a reduction compared to the "normally" used dosages used for the treatment of prostate cancer, endometriosis, or breast cancer) of the drug, or combination of drags, used to ablate or interrapt sex steroid signaling.
  • a reduced dosage i.e., a reduction compared to the "normally" used dosages used for the treatment of prostate cancer, endometriosis, or breast cancer
  • the concurrent use of these two separate classes of drugs allows for a greater, or prolonged rejuvenation of immune system cells, while utilizing a reduced dosage of the drag, or combination of drugs, used to ablate or interrupt sex steroid signaling.
  • drugs known to cause sex steroid ablation, or which interrapt sex steroid signaling, either alone or in combination, with or without the aforementioned growth factors and cytokines may be used for the following: reduction of infections associated with a number of treatment regimens; rejuvenation of the BM following ablative therapy (see, e.g., Example 19); as an adjunct in enabling HSC engraftment (see, e.g., Example 22); as an adjunct in the effective management of allogeneic or autologous organ or cell transplants (see, e.g., Examples 21 and 22, and co-pending, co-owned U.S. Serial Nos.
  • the use of these drugs in these diseases will either result in more effective treatment outcomes or will result in the overall treatment protocols being more efficient.
  • the doses or administration ofthe various chemotherapy drugs may be altered such that they now produce less side effects and/or result in better quality of life outcomes for the patients.
  • the coadministration of the various cytokines and growth factors may allow for a reduced number of HSC that need to be transplanted. For example, using the method of the invention, it may now be possible to use human cord blood for adult HSCT, since a reduced number of cells is required to obtain engraftment.
  • compositions can be prepared according to standard methods (see, e.g., Remington. The Science and Practice of Pharmacy, Gennaro A.R., ed., 20 th edition, Williams & Wilkins PA, USA (2000)).
  • Non-limiting examples of pharmaceutically acceptable carriers include physiologically compatible coatings, solvents and diluents.
  • the compositions may be protected such as by encapsulation.
  • the compositions may be provided with carriers that protect the active ingredient(s), while allowing a slow release of those ingredients.
  • 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., 20 th edition, Williams & Wilkins PA, USA (2000)).
  • LHRH analogs i.e., compounds that do not block the ability of an LHRH analog to disrupt sex steroid hormone signalling
  • examples are various growth factors and other cytokines as described herein.
  • 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 drags and/or radiation; and various patient-related issues as identified by the attending physician such as factors which modify the action of drags, e.g., the condition, body weight, sex and diet of the patient, the severity of any illness, time of administration and other clinical factors.
  • 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).
  • 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.
  • T cell depleting reagents such as chemotherapy or radiotherapy
  • 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. Hence, TRECs are only found in new (na ⁇ ve) T cells. TREC levels are an indicator of thymic function in humans.
  • Dose varies with the sex steroid inhibitor or, e.g. anti-sex steroid vaccine or other blocker used.
  • a dose may be prepared to last as long as a periodic epidemic lasts.
  • "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.
  • the formulation can be prepared to specifically deter infection by e.g., influenza (flu) virases 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).
  • 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 vims over the length of the flu season.
  • 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.
  • Administration of sex steroid ablating agents may be by any method which delivers the agent into the body.
  • 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.
  • 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 steroid mediated signalling utilizes a single dose of an LHRH agonist that is effective for three months.
  • 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.
  • 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.
  • 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.
  • 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.
  • HSC hematopoietic progenitor cells
  • CD34 + hematopoietic cells hematopoietic progenitor cells
  • 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.
  • CD34 + HSC and/or epithelial stem 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 + or CD34 10 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.
  • G-CSF Neurogen, Amgen
  • 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 eg embryonic stem cells and adult stem cells now found in may tissues, e.g., BM, pancreas, brain, and the olfactory system.
  • nonautologous (donor) cells tolerance to these cells is created during or after thymus reactivation.
  • GM genetically modified
  • non-genetically modified donor cells are transplanted into the recipient.
  • These cells ideally stem or progenitor cells, are incorporated 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 eactive 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/IBOl/02740).
  • the administration of stem or precursor donor cells 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 incorporation 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 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 CD344- 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 stem cells may also be injected intrathymically in combination with activation of thymic regrowth through use of sex steroid inhibitors, e.g., LHRH/GnRH analogues. Even non-HSC are likely to be induced to form into DC within the thymic microenvironment and its content of appropriate growth factors for such cells.
  • sex steroid inhibitors e.g., LHRH/GnRH analogues.
  • 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 incorporation 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.
  • 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.
  • the invention further comprises methods of gene therapy through enhancing the functionality of BM and/or immune cells in conjunction with a regenerating thymus, or alternatively, prior to, or without reactivation of the thymus.
  • the genetically modified cells may be HSC, epithelial stem cells, embryonic or adult stem cells, or myeloid or lymphoid progenitor cells.
  • the genetically modified cells are CD34+ or CD341o HSC, lymphoid progenitor cells, or myeloid progenitor cells.
  • 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 tiiat 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.
  • the first new T cells are present in the blood stream.
  • Full development of the T cell pool may take 3-4 (or more) months.
  • 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.
  • the modified stem and progenitor cells are taken up by the thymus and converted 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.
  • the methods ofthe 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.
  • GM cells genetically modified HSC, lymphoid progenitor cells, myeloid progenitor cells, epithelial stem cells or combinations thereof
  • An appropriate gene or polynucleotide i.e., the nucleic acid sequence defining a specific protein
  • the cell differentiates into, e.g., an APC, it expresses 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.
  • antineoplastic or cytotoxic anticancer drugs used in the clinic today cause moderate to severe bone maixow toxicity (e.g., vinblastine, cisplatin, methotrexate, alkylating agents, anti-folate, a vinca alkaloid and anthracyclines).
  • drug resistance genes can be introduced into HSC to confer resistance to anticancer drags. 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 etal, (1992) Proc. Natl. Acad. Sci. USA 89:9676; Banerjee etal, (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, a patient has already been in contact with an agent, or is at a high risk of doing so.
  • the person may be given a sex steroid analog to activate their thymus, and/or to 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.
  • the HSC Following injection into the patient, the HSC enter the bone marrow 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.
  • 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.
  • a patient is infected with HIV.
  • 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 a sex steroid analog, such as an
  • hematopoietic stem or progenitor cells, or epithelial stem cells from the donor may be transplanted into the recipient patient. 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, in the case of nonautologus transplants) as self. Tolerance for a graft from the donor may also be created in the recipient. The graft may be cells, tissues or organs of the donor, or combinations thereof.
  • thymic grafts can be used in the methods of the invention to improve engraftment of the donor cells or tolerance to the donor graft.
  • thymic grafts are when the patient is athymic, when the patient's thymus is resistant to regeneration, or to hasten regeneration.
  • a thymic xenograft to induce tolerance is used (see .e.g., U.S. Patent No. 5,658,564).
  • an allogenic thymic graft is used.
  • the phrase "creating tolerance” or “inducing tolerance” in a patient 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.
  • a patient receives a HSCT during or after castration.
  • the patient is injected with their own HSC.
  • the patient is injected with HSC from an appropriate donor.
  • the patient or donor may or may not be pretreated with G- CSF (e.g., 2 s.c. injections per day for three days, followed by collection of HSC from the blood on days 4 and 5).
  • G- CSF e.g., 2 s.c. injections per day for three days, followed by collection of HSC from the blood on days 4 and 5.
  • hematopoietic cells are supplied to the patient before or concurrently with thymic reactivation, which increases the immune capabilities of the patient's body.
  • the transplanted cells may or may not be genetically modified.
  • the transplanted cells may be HSC, epithelial stem cells, or hematopoietic progenitor cells.
  • the transplanted cells may be CD34 + HSC, lymphoid progenitor cells, or myeloid progenitor cells. In certain cases, the transplanted cells are CD34+ or CD341o HSC.
  • the HSC may or may not be genetically modified.
  • the HSC are transfected or transduced with a gene (e.g., encoding the protein, peptide, or antigen from the agent or other gene of interest) to produce a protein or antigen of interest.
  • a gene e.g., encoding the protein, peptide, or antigen from the agent or other gene of interest
  • 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 (see, e.g., Example 14). This method is described in more detail in co-owned U.S. Serial Nos. 09/758,910, 10/419,068, and 10/399,213.
  • the uptake into the thymus of HSC is substantially increased in the absence of sex steroids. These cells become integrated into the thymus and produce DC 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 a reactivating thymus, which are capable of rapid, specific responses to antigen.
  • the first new T cells may be preset in the blood stream. Full development of the T cell pool may take three to four months.
  • the HSC Following injection into the patient, the HSC enter the bone and bone marrow from the the blood and then some exit back to the blood to be eventually converted into T cells, DC, APC throughout the body.
  • Antigens are expressed in the context of MHC class I and/or MHC class II molecules on the surface of these APC.
  • 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.
  • 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 of autoimmune diseases.
  • B cells are also increased in number in the BM, blood and peripheral lymphoid organs, such as the spleen and lymph nodes, within e.g., two weeks of castration.
  • 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
  • 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 block either the further transcription or translation. In this manner, mutant phenotypes may be generated.
  • 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.
  • 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 etal, (1992) Gene 113:157) and the hairpin ribozyme (Shippy et al, (1999) Mol. Biotechnol ⁇ 2:lll).
  • Double stranded RNA is particularly useful for specifically inhibiting the production of a particular protein.
  • dsRNA Double stranded RNA
  • 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.
  • 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; Waterhouse etal, (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.
  • Useful genes and gene fragments (polynucleotides) for use in the methods of the invention involving GM HSCTs include those that code for resistance to infection of T cells by a particular infectious agent or agents.
  • infectious agents include, but are not limited to, HIV, T cell leukemia virus, and other virases that cause lymphoproliferative diseases.
  • 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 el 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.
  • HIV- 1 rev-responsive element (RRE) (Kohn et al, ( ⁇ 999) 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.
  • RRE rev-responsive element
  • genes or gene fragments may be used in a stably expressible form.
  • 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 also codes for translation signals.
  • 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 well-known to those skilled in the art.
  • Expression vectors useful for expressing the proteins of the present invention 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.
  • the DNA vector construct comprises a promoter, enhancer, and a polyadenylation signal.
  • the promoter may be selected from the non-limiting group consisting of HIV, such as the Long Terminal Repeat (LTR), Simian Virus 40 (SV40), Epstein Barr virus (EBV), cytomegaloviras (CMV), Rous sarcoma virus (RSV), Moloney virus, mouse mammary tumor virus (MMTV), human actin, human myosin, human hemoglobin, human muscle creatine, human metalothionein.
  • 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, for example, from the group consisting of: LTR polyadenylation signal and SV40 polyadenylation signal, particularly the SV40 minor polyadenylation signal among others.
  • the expression vectors of the present invention 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.
  • 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 virases. An inducible promoter may be used so that the amount and timing of expression of the inserted gene or polynucleotide can be controlled.
  • DNA constructs which are functional in cells can be produced by one having ordinary skill in the art.
  • 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.
  • Standard recombinant methods can be used to introduce genetic modifications into the cells being used for gene therapy.
  • retro viral 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)
  • Additional vectors include, but are not limited to, those that are adenovirus derived or lentivirus derived, and Moloney murine leukemia virus-derived vectors.
  • 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.
  • 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.
  • the stem cells can be transfected with specific gene(s) which eventually become expressed in the dendritic cells in the thymus (and elsewhere in the body).
  • genes can 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 direct effects of sex steroid inhibition on the BM functionality and immune cell functionality, and on the eventual 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. As the thymus is reactivating, a new or modified immune system is created, one that no longer recognizes and/or responds to a self antigen.
  • a patient diagnosed with an autoimmune disease e.g., type I diabetes
  • an immunosuppressant e.g., cyclosporine or rapamycin
  • 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.
  • sex steroid analogs or other methods of castration
  • His own T cells may then be mobilized with GCSF.
  • the autoimmune patient is reconstituted with allogeneic stem cells.
  • these allogeneic stem cells are umbilical cord blood cells, which do not include mature T cells.
  • the transplanted HSC may follow full myeloablation or myelodepletion, and thus result in a full HSC transplant (e.g., 5xl0 6 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 xlO 5 cells/kg body weight.
  • T cell depletion (TCD), and/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.
  • the donor HSC are from umbilical cord blood (e.g., 1.5x10 cells/kg for recipient engraftment).
  • 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).
  • 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).
  • the transplanted HSC may follow lymphoablation.
  • T cells and/or B cells may be selectively ablated, to remove cells, as needed (e.g., those cells involved in autoirnmunity 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, TGF ⁇ .
  • 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, TGF ⁇ .
  • IL2-R B chain cytokine receptors
  • Other methods of selecting and sorting cells are well known and include magnetic and fluorescent cell separation, centrifugation, and more specifically, hemaphere
  • HSCT is performed without myeloablation, myelodepletion, lymphodepletion, T cell ablation, and/or other selective immune cell ablation.
  • 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 methoftexate) (see, e.g., U.S. Patent No. 5,876,708).
  • an immunosuppressing agent e.g., cyclosporin, prednisone, ozothioprine, FK506, Imunran, and/or methoftexate
  • immunosuppression is performed in the absence of HSCT.
  • immunosuppression is performed in conjunction with (e.g., prior to, concurrently with, or after) HSCT.
  • immunosuppresion is performed in the absence of myeloablation, lymphoablation, T cell ablation and/or other selective immune cell ablation, deletion, or depletion.
  • immunosuppression is performed in conjunction with (e.g., prior to, concurrently with, or after) myeloablation, lymphoablation, T cell ablation, and/or other selective immune cell ablation, deletion, or depletion.
  • 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).
  • myeloablation, myelodepletion, lymphoablation, T cell ablation, and/or other selective immune cell ablation e.g., B cell or NK cell depletion.
  • NK cells are depleted.
  • NK populations are known in the art.
  • 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 methodsm, as well as non-myeloablative therapy and formation of a chimeric lymphohematopoietic population, all of which may be used in the methods ofthe invention.
  • the methods of the invention further comprise, e.g., prior to
  • the present invention further includes a T cell help-reducing treatment, such as increasing the level of the activity of a cytokine which directly or indirectly (e.g., by the stimulation or inhibition ofthe level of activity of a second cytokine) promotes tolerance to a graft (e.g., IL-10, IL-4, or TGF-.beta.), or which decreased the level of the activity of a cytokine which promotes rejection of a graft (i.e., a cytokine which is antagonistic to or inhibits tolerance (e.g., IFN.beta., IL-1, IL-2, or IL-12)).
  • a cytokine which directly or indirectly e.g., by the stimulation or inhibition ofthe level of activity of a second cytokine
  • a graft e.g., IL-10, IL-4, or TGF-.beta.
  • a cytokine which is antagonistic to or inhibits tolerance e.g.,
  • a cytokine is administered to promote tolerance.
  • the cytokine may be derived from the donor species or from the recipient species (see, e.g., U.S. Patent No. 5,624,823, which describes DNA encoding porcine interleukin-10 for such use).
  • the duration of the help-reducing treatment may be approximately equal to, or is less than, the period required for mature T cells of the recipient species to initiate rejection of an antigen after first being stimulated by the antigen (in humans this is usually 8-12 days). In other embodiments, the duration is approximately equal to or is less than two-, three-, four-, five-, or ten times the period required for mature T cells of the recipient to initiate rejection of an antigen after first being stimulated by the antigen.
  • the short course of help-reducing treatment may be administered in the presence or absence of a treatment which may stimulate the release of a cytokine by mature T cells in the recipient, e.g., in the absence of Prednisone (17,21- dihydroxypregna-l,4-diene-3,ll,20-trione).
  • the help-reducing treatment may be begun before or about the time the graft is introduced.
  • the short course of help-reducing treatment may be pre-operative or post-operative.
  • the donor and recipient are class I matched.
  • the antigen is not an auto-antigen but, rather, an external antigen (e.g., pollen or seafood).
  • 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.
  • an external antigen e.g., pollen or seafood
  • 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.
  • an organ or cell transplant to repair or replace damaged cells, tissues or organs.
  • 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.
  • genetic modification of the HSC may be employed if the antigen involved in the autoimmune disease or allergy is known.
  • the antigen may be myelinglycoprotein (MOG) myelin oligodendroglial protein, myelin basic protein or proteolipid protein.
  • MOG myelinglycoprotein
  • the antigen may be the gastric proton pump.
  • the antigen may be pro- insulin ( J Clin Invest. (2003) 111: 1365., GAD or an islet cell antigen. T cell epitopes of type jT collagen have been described with rheumatoid arthritis in (Ohnishi et al. (2003) Int. J. Mol. Med. 1:331).
  • an antigen is thyroid peroxidase
  • an antigen is the thyroid-stimulating hormone receptor
  • Systemic lupus erythematosus antigens include DNA, histones, ribosomes, snRNP, scRNP e.g., HI histone protein.
  • SS-A systemic lupus erythematosus
  • SS-B La 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) Cril. Rev. Immunol. 21:1.
  • certain allergic reactions are in response to known antigens (e.g., allergy to feline saliva antigen in cat allergies).
  • 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.
  • 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.
  • 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.
  • immune cell depletion or suppression is also used.
  • thymic epithelial stem cells e.g., autologous epithelial stem cells
  • 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 counterpart (see Gill et al, (2002) Nat. Immunol 3:635).
  • 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.
  • the autoimmune disease is diagnosed, and a determination is made as to whether or not there is a familial (genetic) predisposition.
  • 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 activation. In practice it may not be possible to determine the cause of the disease.
  • T cell depletion is performed and, as appropriate, B cell depletion is performed (or other immune cell depletion), combined with chemotherapy, radiation therapy and/or anti-B cell reagents (e.g., CD 19, CD20, and CD21) or antibodies to specific Ig subclasses (anti IgE).
  • anti IgE antibodies to specific Ig subclasses
  • the bone marrow and immune cell functionality is improved by administering GnRH to the patient. Simultaneous with this 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 will also produce the antigen in the bone maixow, 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 would overcome any untoward activation of pre-existing potentially autoreactive T and B cells.
  • GnRH may be combined with G-CSF injection to increase blood levels of autologous HSC to enhance the thymic regrowth.
  • hematopoietic or lymphoid stem and/or progenitor cells from a donor are transplanted into the recipient to increase the speed of ultimate regeneration of the thymus.
  • these cells are transplanted from a healthy donor, without autoimmune disease or allergies, to replace aberrant stem and/or progenitor cells in the patient.
  • 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 abeixant T cells that failed tolerance induction to self remain eliminated from the T cell population.??
  • a patient's immune system cells causing allergies are eliminated by the same lymphocyte ablation treatements 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. In other cases, allergies and autoimmune diseases are alleviated following sex steroid signaling disruption due to increased functionality of the BM and other immune cells.
  • the invention further provides methods for preventing, increasing resistance to, or treating an infection of a patient through enhancing the functionality of BM and/or immune cells in conjunction with a regenerating thymus, or alternatively, prior to, or without reactivation of the thymus.
  • 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 Figs. 13-17, which show the effects of thymic reactivation on the mouse immune system, as demonstrated with viral (HSV) challenge.
  • HSV viral
  • 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.
  • 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 show human responses. This is reinforced by the data showing the effects of thymic reactivation in humans.
  • the ability to increase the functionality of immune cells is exemplified in Example 23, in which TCR responsiveness and proliferation is increased in castrated mice as early as 3-7 days following castration, and before thymus regeneration.
  • 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.
  • the methods of the invention may be used with virtually any method of vaccination in combination with sex steroid inhibition without undue experimentation.
  • the vaccination is given prior to or concurrently with, thymic reactivation. Multiple doses (e.g., booster immunizations) may also be given as needed.
  • the vaccine is a killed or inactivated vaccine (e.g., by heat or other chemicals).
  • the vaccine is an attenuated vaccine (e.g., polioviras and smallpox vaccines).
  • the vaccine is a subunit vaccine (e.g., hepatitis B vaccine, in which hepatitis B surface antigen (HBsAg) is the agent-specific protein).
  • the vaccine is a recombinant vaccine.
  • recombinant vaccine is an attenuated vaccine in which the agent (e.g., a virus) has specific virulence-causing genes deleted, which renders the viras non- virulent.
  • agent e.g., a virus
  • recombinant vaccine employs the use of infective, but non- virulent, vectors which are genetically modified to insert a gene encoding target antigens. Examples of a recombinant vaccines is a vaccinia virus vaccines.
  • 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.
  • T cells are the most vulnerable because of the marked sex steroid-induced shutdown in thymic export that becomes profound from the unset of puberty and the global suppresson 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).
  • type 1 responses have been associated with the raising of cytotoxic T cells and type 2 responses with the raising of antibody.
  • 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).
  • Thl- or Th2- type cytokines e.g., delivery of recombinant cytokines or DNA encoding cytokines
  • Immunostimulatory CpG oligonucletides have also been utilized to shift immune response to various vaccine formulations to a more Thl- type response.
  • sex steroid inhibition results in BM and immune cell functionality is increased without, prior to, or concurrently with thymic regrowth, which allows for improved immune responsiveness to vaccines.
  • cytokine therapies 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 IFN ⁇ to skew the response to Thl (cell mediated, inflammatory responses), EL 12 to promote Thl and ILIO to promote Th2 cells.
  • IL-2 interleukin 2
  • IL-15 IL-4 to skew the response to Th2 (humoral immunity)
  • IFN ⁇ to skew the response to Thl (cell mediated, inflammatory responses)
  • EL 12 to promote Thl
  • ILIO 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.
  • 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 polynucleotides of the invention, but may also be viruses or retro viruses.
  • the vectors used in the polynucleotide vaccines may be "naked" (i.e., not associated with a delivery vehicle such as liposomes, colloidal particles, etc.).
  • 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.
  • Retroviral vectors may be derivatives of a murine or avian retroviras.
  • retroviral include, but are not limited to: Moloney murine leukemia viras (MoMuLV), Harvey murine sarcoma viras (HaMuS-V), murine mammary tumor viras (MuMTV), and Rous Sarcoma Virus (RSV).
  • MoMuLV Moloney murine leukemia viras
  • HaMuS-V Harvey murine sarcoma viras
  • MuMTV murine mammary tumor viras
  • RSV Rous Sarcoma Virus
  • Other plasmids and viral vectors useful in the vaccination methods of the invention are well known in the vaccine art.
  • the present invention provides methods for increasing the function of BM in a patient, including increasing production of HSC and enhancing haemopoiesis. These methods are useful in a number of applications.
  • one of the difficult side effects of chemotherapy or radiotherapy can be its negative impact on the patient's BM.
  • the BM may be damaged or ablated and production of blood cells may be impeded.
  • Administration of a dose 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.
  • administration of the LHRH analog in the weeks prior to delivery of chemotherapy increases the population of HSC and other blood cells so that some of the deleterious effects of chemotherapy will be decreased.
  • the improvement in BM function may be applicable to, for example, patients with blood disorders.
  • blood disorders is herein defined as any disorder or malady that involves the cells of the blood system in a patient, either directly or indirectly, including, but not limited to, disorders associated with hematopoiesis, e.g., leukemia.
  • the methods of the present invention are useful to replace the cancerous blood system cells with new cells from a donor (matched or unmatched) in an allogenic HSCT, or following autologous HSCT with the patient's own cells.
  • Increased HSC production by the BM causes consequential increase in red blood cells, which are, in turn, useful for management of RBC production. This can be easily determined by looking for, e.g., increased hematocrit.
  • the increased HSC are CD344- or CD341o HSC.
  • Mobilized HSCs are CD344- or CD341o HSC.
  • HSC have the potential to generate non-hematopoietic tissue. While much of the work has been carried out in vitro, a study at the Mayo Clinic, Rochester has shown that after BMT a small number of cardiomyocytes are donor derived. Similarly, the Beuamont Hospital, Michigan have used HSC to repair damaged heart muscle, although it is unclear whether the HSC become myocytes or vasculature. Mice experiments have also shown the potential of HSC to become insulin producing ⁇ - cells. Other work has shown HSC are capable of becoming skeletal muscle (myocytes), liver (hepatocytes), bone, connective tissue, epithelial tissue (e.g. of lung, gut and skin), vasculature, neurons, and islet D cells.
  • 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 drags, radiation therapy ) or diseases (e.g., HIV, chronic renal failure).
  • therapies e.g., cancer chemotherapy drags, radiation therapy
  • diseases e.g., HIV, chronic renal failure
  • ablation of the BM is a desired effect.
  • the methods of the invention may be used immediately after ablation occurs to stimulate the BM and increase the production of HSC and their progeny blood cells, so as to decrease the patient's recovery time.
  • 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 BM or hematopoietic stem or progenitor cells, as well as other factors such as colony stimulating factors (CSFs) and stem cell factor (SCF).
  • CSFs colony stimulating factors
  • SCF stem cell factor
  • 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.
  • 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.
  • Sex steroids in males and females can be inhibited temporarily by taking disraptors of sex steroid mediated signaling (e.g., GnRH agonists). It has been shown that loss of steroids causes a reactivation of thymus function and enhanced production of na ⁇ ve T cells. Furthermore, even before those new T cells have had a chance to leave the thymus, the pre- existing T cells are much more sensitive to stimulation, which results in a much more effective immune response. This increased responsiveness is evident within days of the loss of sex steroids (see, e.g., Example 23). This may be because there are no inhibitory effects of sex steroids.
  • GnRH can be given as a single treatment simultaneously with the delivery of, for example, a vaccine.
  • a one month formulation is useful which has the beneficial effects of stimulating immune responses but without the side effects of longer loss of sex steroids.
  • a subsequent "booster" injection of the antigen can also be administered.
  • Sex steroid inhibitors e.g., GnRH analogs
  • GnRH analogs are useful to boost all forms of immunotherapy in cancer patient, particularly for the removal of cancer cells with have escaped chemotherapy or surgery, but also for the defense against opportunistic infections.
  • These analogs may also be used prophylacticly to improve immune response to vaccination programs designed to prevent, e.g., infections or cancer.
  • the methods of the invention utilize inhibion of sex steroid signaling.
  • Sex steroids suppress the function of the thymus, BM, and also T and B lymphocytes throughout the body, which are concentrated in the major lymphoid areas of the body including, but not limited to, the blood, lymph nodes, mucosal tissue (e.g., respiratory, gastrointestinal, genital).
  • ablation of sex steroids and/or interruption of sex steroid- mediated signaling may be used not only to regenerate the thymus (and thus the number and 'quality' of T cells), but also to improve the functionality of pre-existing and newly produce T cells (and other cells of the immune system) either without, prior to, or concurrently with, thymus regeneration.
  • a poor immune response can have immediate and clinically important consequences. It can mean an increased susceptibility to common infections (e.g., influenza), increased susceptibility to cancers and tumors, and/or poor responsiveness to vaccinations.
  • common infections e.g., influenza
  • An increase in the number and/or proportion of na ⁇ ve T cells in the total T cell pool has a positive immediate therapeutic effect on a number of clinical (or potentially clinical) conditions and diseases, including, but not limited to, cancer, immunodeficiency (particularly viral infections, e.g., Acquired Immune Deficiency Syndrome (AIDS) and Severe Acute Respiratory Syndrome (SARS) or iniiuenz), autoirnmunity, transplantation, allergies, as well as improving the general efficacy of vaccination programs.
  • AIDS Acquired Immune Deficiency Syndrome
  • SARS Severe Acute Respiratory Syndrome
  • inhibion of sex steroid signaling is used to boost the responsiveness of T and B lymphocytes to stimulation with antigen.
  • This stimulation may be a microorganism (e.g., bacteria, viras, fungi, parasites, etc.) entering the body.
  • inhibion of sex steroid signaling is used to enhance the immune response to a vaccine antigen.
  • inhibion of sex steroid signaling occurs prior to immune system challenge. This allows time for the loss or sex steroids to occur.
  • the inhibion of sex steroid signaling may also be accomplished simultaneously or sequentially with the administration of the stimulant to act as an "adjuvant" for enhancing the immune response directly (which could be mediated via direct signaling at the cell surface, increase in cytokines, decrease in inhibitors, etc.).
  • the inhibion of sex steroid signaling (e.g., using LHRH/GnRH analogs) may also be given on multiple occasions.
  • the immediate effects are due to enhancing the functionality of pre-existing lymphoid (and non-lymphoid) cells.
  • the reduction of sex steroids increases the production and functionality of T cells, B cells and APC, which additively, synergistically, or complementarily continue to enhance the response.
  • Increases of new T cells, B cells, and other immune cells, such as APC, together with increased sensitivity of pre-existing T cells, B cells, and APC to stimulation, may then be used for the generation of more efficacious immune responses to primary infection, secondary infection, vaccination, etc.
  • disrupters of sex steroid signaling are used according to the methods of the instant invention, to cause a clinically positive effect by initiating an increased activation or functionality of these immune cells even before these drugs have been able to cause significant thymic regrowth.
  • Drags also may be used to assist in the replacement of blood cells that may have been injured or destroyed by various other therapies or diseases, including but not limited to cancer chemotherapy drugs and/or cancer radiation therapy, as wells as diseases, such as chronic renal failure.
  • cancer chemotherapy drugs and/or cancer radiation therapy including but not limited to cancer chemotherapy drugs and/or cancer radiation therapy, as wells as diseases, such as chronic renal failure.
  • diseases such as chronic renal failure.
  • the use of sex steroid inhibition drags in combination with G-CSF, GM-CSF, erythropoietin (EPO), SCF, or other hormones or cytokines may also be used to further improve the enhancement of the production of blood cells by those compounds.
  • modifying the T-cell population makeup is herein defined as altering the nature and/or ratio of T cell subsets defined functionally and by expression of characteristic molecules.
  • characteristic molecules include, but are not limited to, the T cell receptor, CD4, CD8, CD3, CD25, CD28, CD44, CD45, CD62L and CD69.
  • Increasing the number of cells e.g., T-cells
  • T-cells is herein defined as 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 BM 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.
  • the HSC are genetically modified such that they and their progeny, in particular T cells, macrophages and DC, 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.
  • the HSC may be genetically modified to normalize the defect.
  • the modification may include the introduction of nucleic acid constructs or genes which normalise the HSC and inhibit or reduce its likelihood of becoming a cancer cell.
  • the methods of the present invention are also useful for the treatment of AIDS, where the treatment involves reduction of viral load, increases in T cell number and functionality, reactivation of thymic function through inhibition of sex steroids signalling.
  • the patients may receive HSC which have been genetically modified such that all progeny (e.g., T cells and 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 HIV will be able to remove any remnant viral infected cells.
  • a similar strategy could be applied to gene therapy in HSC for any T cell defect or any viral infection which targets T cells.
  • B cell function is also diminished with age, which is in part due to the decline in T cell production and consequent lack of T cell help.
  • age-associated changes in B cell function Hu et al, (1993) Int., Immunol. 5:1035-1039.
  • B cell numbers remaining relatively constant, throughout life due to tightly regulated homeostatic mechanisms, there is a decrease in export from the BM, clonal expansion of peripheral B cells, and a narrowing of the antibody response (LeMaoult et al., (1999) /. Immunol 162:6384-6391).
  • Castration of aged mice results in an increase in IL-7 responsive B cell progenitors, including late pro-B cells, pre- B cells, and immature B cells (Ellis et ⁇ /.,(2001) Int. Immunol. 13:553-558).
  • the absolute number of B cells in the periphery is also increased (Ellis et al, (20001) Int. Immunol. 13:553-558).
  • This increase in circulating B cells is largely due to an increase in the number of recent BM emigrants (CD45R lo CD24 hl ) and these cells remain at an elevated level for up to 54 days after castration (Ellis et al, (2001) Int Immunol. 13:553-558).
  • the methods of the invention may be used to increase the number and functionality of B cells without, prior to. or concurrently with thymic regeneration. This may be useful for, e.g., increased control (by prevention or treatment) of bacterial infections in normal patients, in patients with compromised immune systems, such as those patients receiving a disease treatment regimen. Increased B cell number and functionality may also be useful following surgery and/or in burn victims, and in other instances wherein the patient's immune system is compromised.
  • the present invention also provides methods for increasing DC functionality and/or
  • DC number Following sex steroid ablation (e.g., following delivery of an LHRH analog) DC are increased in the thymus, and in the periphery, which may also assist the T cell stimulation. DC are important antigen presenting cells and increased numbers and/or function may be useful in improving responsiveness to agents, e.g., cancers. Enhanced DC functionality may also be useful in achieving resistance to agents such as allergens or self- antigens (in the case of autoimmune disorders).
  • the present invention also provides methods for increasing platelet cell number and/or functionality.
  • Thrombocytopenia is common and has a variety of causes, including, but not limited to, poor BM and splenomegaly. The condition generally results in bleeding disorders that are very difficult to treat.
  • the most common diseases associated with thrombocytopenia include leukemia, aplastic anemia, cirrhosis, and Gauchers disease. Massive blood replacement is often needed because platelets have a short half life in stored blood which used for transfusions.
  • the present invention also provides methods for increasing NK and NKT cell number and/or functionality (see, e.g., Example 17 and Figures 43 and 49). This may be useful in the treatment of diseases displaying NK deficiencies, e.g., Crohn's disease, Chediak-Highashi syndrome. Impaired NK cell function has been reported in patients with connective tissue diseases including lupus and rheumatoid arthritis. NK cells are important in defending against cancer and infectious agents.
  • NK cell deficiency diseases associated with NK cell deficiency (numerical and/or functional), which may benefit from the methods of the invention include herpes vims infections (e.g., varicella-zoster viras (VZV, chicken pox, shingles), HSV CMV, and EBV), other vitral infections (e.g., measles, mumps, influenza and HIV, which are now thought to be controlled, at least in part, by NK cells), mycobacterial infections (e.g., M.
  • herpes vims infections e.g., varicella-zoster viras (VZV, chicken pox, shingles), HSV CMV, and EBV
  • other vitral infections e.g., measles, mumps, influenza and HIV, which are now thought to be controlled, at least in part, by NK cells
  • mycobacterial infections e.g., M.
  • NKT cells are important regulators of the immune response because they are very highproducers of cytokines without the need for prior activation. They have a role in preventing autoimmune disease but also promote anti-cancer effects. Tregs (commonly defined as being CD4+CD25+) are also major regulators of the immune response primarily through their cytokine production.
  • the present invention also provides methods for increasing macrophage number and functionality (see, e.g., Example 17 and Figure 43). which would have a primary role in helping remove infectious agents particularly bacteria.
  • mice 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, Vicotoria) 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 hydiOchloride (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 100 ⁇ l 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 Inc., Indiana), snap frozen in liquid nitrogen, and stored at -70°C until use.
  • BrdU Sigma Chemical Co., St. Louis, MO
  • 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 500 ⁇ l 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.
  • PFA paraformaldehyde
  • 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, San Diego, CA) and CD44-B (Pharmingen, San Diego, CA) 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.
  • 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- C ⁇ 3 (Amersham, Uppsala, Sweden). 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 HCI, neutralized by washing in Borate Buffer (Sigma), followed by two washes in PBS. BrdU was detected using anti-BrdU-FITC (Becton-Dickinson).
  • Sections were analyzed using a Leica fluorescent and Nikon confocal microscopes.
  • FITC labeling of thymocytes technique are similar to those described elsewhere (Scollay et al, (1980) Proc. Natl. Acad. Sci, USA 86:5547; Berzins et al, (1998) /. Exp. Med. 187:1839). Briefly, thymic lobes were exposed and each lobe was injected with approximately 10 ⁇ m of 350 ⁇ g/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 CO 2 asphyxiation approximately 24 hours after injection and lymphoid organs were removed for analysis.
  • Migrant cells were identified as live-gated FITC + cells expressing either CD4 or CD8 (to omit autofluorescing cells and doublets). The percentages 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) /. Exp. Med. 187: 1839).
  • thymic weight Fig. 1A
  • total thymocyte number Figs. IB
  • Relative thymic weight mg thymus/g body
  • 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 about 6.7 x 10 7 thymocytes, decreasing to about 4.5 x cells by 24 months.
  • 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. 1 A) and cellularity (Fig. IB).
  • Fig. 1 A weight
  • Fig. IB cellularity
  • thymocytes were labeled with defining markers in order to analyze the separate subpopulations. hi 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 ⁇ TCR revealed no change in the proportions of these populations with age.
  • 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.
  • telomeres As shown in Figs. 4A-4B, 15-20% of thymocytes were proliferating at 2-4 months of age. The majority (about 80%) of these are double positive (DP) i.e., CD44-, CD8+) with the triple negative (TN) (i.e., CD3 CD4GD8 " ) subset making up the second largest population at about 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.
  • DP double positive
  • TN triple negative
  • the DN subpopulation in addition to the thymocyte precursors, contains 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.
  • Figs. 5E-H illustrate the extent of proliferation within each subset of TN cells in young, old and castrated mice.
  • T cells migrate from the thymus daily in the young mouse
  • 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) /. 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.
  • the aged thymus shows a significant increase in both thymic size and cell number, surpassing that of the young adult thymus presumably due to the actions of sex steroids already exerting themselves in the 2 month old mouse.
  • thymocyte differentiation has been shown 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 analysed to determine if this was a contributing factor in thymus atrophy.
  • thymocytes Proliferation of thymocytes was not affected by age-induced thymic atrophy or by removal of sex-steroid influences post-castration with about 14% of all thymocytes proliferating.
  • the localisation 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 disorganisation with age, localisation of proliferation was difficult to distinguish but appeared to be less uniform in pattern than the young and relegated to the outer cortex.
  • Thymocyte migration was shown to occur at a constant proportion of thymocytes with age conflicting with previous data by ScoUay et al, ( (1980) Proc. Natl. Acad. Sci, USA.
  • J. Med. 332: 1483 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 immunodeficiency in the aged.
  • Castration allows the thymus to repopulate the periphery through significantly increasing the production of naive T cells.
  • the aged thymus is capable of functioning in a nature equivalent to the pre-pubertal thymus.
  • 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.
  • two important findings were noted. Firstly, there appears to be an adverse affect on the TN cells in their ability to proliferate, correlating with findings by Aspinall ((1997) /. Immunol. 158:3037). This defect could be attributed to an inherent defect in the thymocytes themselves.
  • mice were injected intravenously with 5xl0 6 donor BM cells.
  • BM cells were obtained by passing RPMI-1640 media through the tibias and femurs of donor (2-month old congenic
  • mice 3-4 month old mice were subjected to 625Rads of whole body D- irradiation.
  • mice e.g., 2 years old were injected with cyclophosphamide (200 mg/kg body wt over two days) and castrated.
  • Castration enhanced regeneration following severe T cell depletion For both models of T cell depletion studied (chemotherapy using cyclophosphamide or sublethal irradiation using 625Rads), castrated (Cx) mice showed a significant increase in the rate of thymus regeneration compared to their sham-castrated (ShCx) counterparts (Figs. 7 A and 7B). By 1 week post-treatment castrated mice showed significant thymic regeneration even at this early stage (Figs. 7 and 9-11). In comparison, non-castrated animals, showed severe loss of DN and DP thymocytes (rapidly-dividing cells) and subsequent increase in proportion of CD4 and CD8 cells (radio-resistant).
  • thymocyte numbers with castrated animals showing at least a 4-fold increase in thymus size even at 1 week post-treatment.
  • the non-castrated animals showed relative thymocyte normality with regeneration of both DN and DP thymocytes.
  • 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.OOl) 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).
  • both groups of Cx mice had significantly (5-6 fold) greater thymocyte numbers (p ⁇ O.OOl) than the ShCx mice (Fig. 7A).
  • 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 (PIrr) compared to ShCx mice (p ⁇ O.OOl) (Figs. 7B, 10A, and 11 A). 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.
  • PIrr thymus cellularity post-irradiation
  • thymus size appears to 'overshoot' the baseline of the control thymus.
  • 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, with a significant (p ⁇ 0.05) reduction in cellularity compared to both control and Cx mice at 2-weeks post-treatment (Fig. 8B). These results indicate that castration may enhance the rate of recovery of total lymphocyte numbers following cyclophosphamide treatment.
  • Fig. 9 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.
  • the enhancing effects were equivalent on thymic expansion and also the recovery of spleen and lymph node.
  • the kinetics of chemical castration are slower than surgical, that is, mice take about 3 weeks longer to decrease their circulating sex steroid levels.
  • chemical castration is still as effective as surgical castration and can be considered to have an equivalent effect.
  • 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, but 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.
  • 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 disorganised.
  • 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.
  • the second decrease, 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 BM (also effected by irradiation).
  • the second regenerative phase was due to the replenishment of the thymus with BM derived precursors (Huiskamp et al, (1983) Radiat. Res. 95:370).
  • mice 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 4xl0 5 plaque forming units (pfu) of HSV-1 (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.
  • pfu plaque forming units
  • Virus was obtained from Assoc. Prof. Frank Carbone (Melbourne University). Virus stocks were grown and titrated on VERO cell r ⁇ onolayers in MEM supplemented with 5% FCS (Gibco-BRL, Australia).
  • HSC HSC were detected by staining with CD117-APC and Sca-l-PE.
  • TN thymocyte analysis cells were gated on the Lin " population and detected by staining with CD44-biotin, CD25-PE and c-kit-APC.
  • Lymph node cells were incubated for three days at 37°C, 6.5% C0 2 . Specificity was determined using a non-transfected cell line (EL4) pulsed with gB 49 s-505 peptide (gBp) and EL4 cells alone as a control. A starting effector.target ratio of 30:1 was used. The plates were incubated at 37°C, 6.5% CO 2 for four hours and then centrifuged 650 g max for 5 minutes. Supernatant (lOO ⁇ l) was harvested from each well and transferred into glass fermentation tubes for measurement by a Packard Cobra auto-gamma counter.
  • EL4 non-transfected cell line
  • gBp gB 49 s-505 peptide
  • HSV herpes simplex viras
  • mice were immunized in the footpad and the popliteal (draining) lymph node analyzed at D5 post-immunization.
  • 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. 13-17) was examined.
  • 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 (7.6xl0 7 ⁇ 5.2xl0 6 ) 2 and 4 weeks after congenic BMT, while thymus cellularity of castrated mice had increased above control levels at 4- weeks post-BMT (Fig. 18A). At 6 weeks, cell numbers remained below control levels. However, those of castrated mice were three fold higher than the noncastrated mice ( ⁇ 0.05) (Fig. 18A).
  • BM cellularity reached untreated control levels (1.5xl0 7 ⁇ 1.5xl0 6 ) 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. 18D).
  • 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. 18C).
  • 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. 18A).
  • Thymus cellularity in the sham-castrated mice was below untreated control levels (7.6xl0 7 ⁇ 5.2xl0 6 ) 2 and 4 weeks after congenic BMT, while thymus cellularity of castrated mice had increased above control levels at 4- weeks post-BMT (Fig. 18 A).
  • Castrated mice had significantly increased congenic (Ly5.2) cells compared to non-castrated animals.
  • mice 3 month old, young adults, C57/BL6 mice were castrated or sham-castrated 1 day prior to BMT.
  • the mice were subjected to 800RADS TBI and IV injected with 5 x 10 6 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.
  • Figures 20 and 21 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 22 shows an increase in donor derived B cell precursors and B cells in the BM of castrated mice.
  • Figure 24 and 25 show castration does not alter the number or proportion of B cells in the periphery at 2 and 4 weeks post castration.
  • Figure 26 shows castration increased numbers of donor derived TN, DP, CD4 and
  • Figure 28 shows and increased number of donor DC in the thymus by 4 weeks post castration.
  • Example 4 shows the influence of castration on syngeneic and congenic BM transplantation.
  • Starzl et al, (1992) Lancet 339:1579 reported that microchimeras evident in lymphoid and nonlymphoid tissue were a good prognostic indicator for allograft transplantation. That is it was postulated that they were necessary for the induction of tolerance to the graft (Starzl et al, (1992) Lancet 339:1579).
  • Donor-derived DC 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).
  • DC are known to be key players in the negative selection processes of thymus and if donor-derived DC were present in the recipient thymus, graft reactive T cells may be deleted.
  • the patient underwent T cell depletion (ablation).
  • T cell depletion One standard procedure for this step is as follows.
  • the human patient received anti-T cell antibodies in the form of a daily injection of 15 mg/kg of Atga (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 9 mg/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 or other immune cell depletion.
  • 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 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 BM 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).
  • donor HSC would develop into intrathymic DC 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.
  • peripheral T cells minimize the risk of graft rejection because it depletes non-specifically all T cells including those potentially reactive against a foreign donor.
  • the procedure induces a state of generalized immunodeficiency which means that the patient is highly susceptible to infection, particularly viral infection.
  • 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 effective in reducing sex steroid levels sufficiently to reactivate the thymus. hi some cases it is also necessary to deliver a suppresser of adrenal gland production of sex steroids.
  • Cosudex® (5 mg/day or 50 mg/day) may also be given as one tablet per day for the duration of the sex steroid ablation therapy.
  • the patient is given a GnRH antagonist, e.g., Cetrorelix or Abarelix as a subcutaneous injection
  • sex steroids in the blood 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 BM to the thymus.
  • 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.
  • a laser such as an Er:YAG laser
  • 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.
  • 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.
  • 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 ⁇ g/kg for 2-5 days prior to cell collection (e.g., one or two injections of 10 ⁇ g/kg per day for each of 2-5 days).
  • G-CSF granulocyte-colony stimulating factor
  • 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 LIE to prevent differentiation into specific cell types.
  • feeder cells e.g., fibroblasts
  • SCF stem cell factor
  • LIE stem cell factor
  • 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 (i.e., incorporated 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.
  • the donor and host DC tolerize any new T or NK cells that are potentially reactive with donor or recipient cells.
  • the 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.
  • immunosuppressive therapy may be maintained for about 3-4 weeks
  • the new T cells are 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 retain the ability to respond to normal infections by recognizing peptides presented by host APC in the peripheral blood of the recipient.
  • the incorporation of donor DC 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.
  • 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.
  • NP nucleoprotein
  • CTL and protection from influenza challenge following immunization with a polynucleotide encoding NP has previously been shown (Ulmer et al. (1993) Science 259:1745).
  • mice are anesthetized by intraperitoneal injection of
  • mice are injected i.m. with 10 mg kg Lupron® (a GnRH agonist) as a 1 month slow release formulation.
  • mice are injected with a GnRH antagonist (e.g., Cetrorelix or Abarelix).
  • 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.
  • influenza A PR/8/34 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- ⁇ -o-thioglucopyranoside).
  • HA hemagglutinin
  • NA neuraminidase
  • the HA/NA-rich supernatant (55% HA) is used as the subunit vaccine.
  • 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 ⁇ l 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.
  • influenza A/PR/8/34 subunit vaccine preparation may be intramuscularly injected directly into, e.g., the quadriceps muscle, at a dose of about 1 ⁇ g to about 10 ⁇ g dilute in a volume of 40 ⁇ l 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 cytomegaloviras (CMV) immediate early promoter.
  • NP nucleoprotein
  • HA hemagglutinin
  • Plasmids are grown in Escherichia coli DH5 ⁇ or HBIOI cells using standard techniques and purified using Qiagen® Ultra-Pure®-! 00 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,
  • 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.,
  • Th2-inducing genetic adjuvants include, e.g., IL-4, E -5, and IL-10.
  • Thl- and Th2- inducing genetic adjuvants See, e.g., Robinson, et al, (2000) Adv. Virus Res. 55:1).
  • Influenza A/PR/8/34 virus challenge hi an effort to determine if castrated mice are better protected from influenza virus challenge (with and without vaccination) as compared to their sham-castrated counterparts, metofane-anesthetized mice are challenged by intranasal inoculation of 50 DI of influenza A/PR/8/34 (HlNl) influenza virus containing allantoic fluid diluted 10 '4 in PBS/2% BSA (50-100 LD 50 ; 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 "7 dilution of viras. 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-hnked 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.
  • ELISA quantitative enzyme-hnked immunosorbant assays
  • Stimulations are conducted by adding 100 ⁇ l of the appropriate peptide or inactivated influenza virus diluted in RPMI-10.
  • CD8 + T cells are stimulated with either the K d -restricted
  • HA 533-5 4i peptide (IYSTVASSL; SEQ ID NO:l) (Winter, Fields, and Brownlee, (1981)
  • 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 viras) plus anti-CD28 (1 ⁇ g/ml) and anti-CD49d (1 ⁇ g/ml)
  • 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.
  • 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 HA 533-54 ⁇ IYSTVASSL (SEQ ID NO:l) (Winter, Fields, and Brownlee, (1981) Nature 292:72) or NP ⁇ 7- ⁇ 55 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 6.3xl0 6 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 lxlO 7 cells/ml. Mitomycin C is added to a final concentration of 25 ⁇ g/ml. Cells are incubated at 37°C/5%CO 2 for 30 minutes and then washed 3 times with RPMI-10.
  • the stimulator cells are then resuspended to a concentration of 2.4x10 6 cells/ml and pulsed with HA peptide at a final concentration of 9xl0 "6 M or with NP peptide at a final concentration of 2xl0 "6 M in RPMI-10 and 10 U/ml IL- 2 for 2 hours at 37°C/5% CO 2 .
  • the peptide-pulsed stimulator cells (2.4xl0 6 ) and responder cells (6.3xl0 6 ) 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 pyravate) for 5 days at 37°C/5%CO 2 .
  • a chromium-release assay is used to measure the ability of the in vitro sthnulated responders (now called effectors) to lyse peptide-pulsed mouse mastocytoma P815 cells (MHC matched, H-2d).
  • P815 cells are labeled with 51 Cr by taking 0.1 ml aliquots of ⁇ 815 in RPMI-10 and adding 25 ⁇ l 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%CO , washed 3 times with RPMI-10 and resuspended in 15 ml polypropylene tubes containing RPMI-10 plus HA (9x10 " 6 M) or NP (lxlO "6 ) peptide. Targets are incubated for 2 hours at 37°C/5%CO 2 .
  • the radiolabeled, peptide-pulsed targets are added to individual wells of a 96-well plate at 5xl0 4 cells per well in RPMI-10.
  • Stimulated responder cells from individual immunization groups are collected, washed 3 times with RPMI-10, and added to individual 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%CO 2 and cell lysis is measured by liquid scintillation counting of 25 ⁇ l aliquots of supematants. 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 IFN ⁇ or IL-5 in bulk culture supematants by ELISA Bulk culture supematants may be tested for IFN ⁇ 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% CO 2 , and then supematants are harvested and pooled All ELISA antibodies and purified cytokines are purchased from Pharmingen (San Diego, CA).
  • Biotinylated rat anti-mouse cytokine detecting antibody is diluted in PBS-T to a final concentration of 2 ⁇ g/ml and 100 ⁇ l 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 ⁇ l 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 ⁇ l/well of AP developing solution (BioRad, Hercules, CA) and incubating at room temperature for 50 minutes. Reactions are stopped by addition of 100 ⁇ l 0.4 M NaOH and read at. OD u5 . Data are analyzed using Softmax Pro Version 2.21 computer software (Molecular Devices, Sunnyvale, CA).
  • Splenocytes may be tested for intracellular IFN ⁇ 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% CO 2 .
  • a Golgi transport inhibitor, Monensin (Pharmingen, San Diego, CA) is added at 0.14 ⁇ l/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.
  • tetramer staining For tetramer staining (see below), cells were similarly stained with CD8 ⁇ -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 ⁇ l 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 ⁇ l per well of a 1:100 dilution of rat anti-mouse IFN ⁇ - 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 ⁇ l 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-FTTC, -PE, -Tricolor, or -APC. Results are analyzed using Flow Jo Version 2.7 software (Tree Star, San Carlos, CA).
  • 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-2K d MHC class I glycoprotein complexed the synthetic influenza A/PR/8/34 virus peptide HA 533 - 5 4i (IYSTVASSL; SEQ ID NO:l) (Winter, Fields, and Brownlee, (1981) Nature 292:72) or NP 147 - 155 (TYQRTRALV; SEQ ID NO:2) (Rotzschke et al, (1990) Nature 348:252).
  • CSP circumsporozoite protein
  • 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- ⁇ ; Pf55; Pf35; GBP (96-R); ABRA (plOl); Exp-1 (CRA, Ag5.1); Aldolase; Duffy binding protein of P. vivax; Reticulocyte binding proteins; HSP70-1 ( ⁇ 75);
  • 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 ( 137 Ce).
  • 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 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 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 etal, (2000) Infect Immun. 68:3403).
  • 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; SYVPSAEQJLEFVKQI; SEQ ID NO:3).
  • liver stage development assay Inhibition of liver stage development assay.
  • Hepatocyte cultures are seeded onto eight-chamber Lab-Tek plastic slides at lxlO 5 cells/chamber and incubated with 7.5 x 10 4 P. yoelii sporozoites for 3 hours. The cultures are then washed and cultured for and additional 24 hours at 37 C/5% CO 2 .
  • 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 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 triphcate cultures are then counted using an epifluorescence microscope.
  • 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.
  • Percent inhibition is calculated using the formula [(control-test)/control) xlOO]. Infection and challenge. For a lethal challenge dose, the ED 50 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 2xl0 5 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.
  • castrated mice are infected and analyzed as described above. Sham-castrated mice are used as controls.
  • mice Human studies. After establishing the efficacy in mice, large numbers of humans are immunized in a double blind placebo controlled field trial.
  • Tuberculosis 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), Science 257, 1055.
  • M. tuberculosis is an intracellular pathogen that infects macrophages. Immunity to
  • cytokines such as IL-4, 5-FU, 5-FU, 5-FU, 5-FU, 5-FU, 5-FU, 5-FU, 5-FU, 5-FU, 5-FU, 5-FU, 5-FU, 5-FU, 5-FU, 5-FU, 5-FU, 5-FU, 5-FU, 5-FU, 5-FU, 5-FU, 5-FU, 5-FU, 5-FU, 5-FU, 5-FU, 5-FU, 5-FU, 5-FU, 5-FU, 5-FU, 5-FU, 5-FU, 5-FU, and others.
  • IFN ⁇ is an effective means of minimizing intracellular mycobacterial multiplication.
  • CD8 + and CD4 + T cells are known to secrete Thl-type cytokines, such as IFN ⁇ , in response to infection, and possess antigen-specific cytotoxic activity.
  • Thl-type cytokines such as IFN ⁇
  • CTL responses are useful for protection against M. tuberculosis (see, e.g., Flynn etal, (1992) Proc. Natl. Acad. Sci. USA 89:12013).
  • 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 (85 A, 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) /. Infect. Dis. 166:874; Siva and Lowrie, (1994) Immunol. 82:244; Romain et a. dislike (1993) Proc. Natl. Acad. Sci. USA 90:5322; and Faith et al, (1991) Immunol 74:1).
  • mice Castration of mice. Surgical and/or chemical castration of BALB/c or C57BL/6 mice is performed as above.
  • TB Mycobacterium tuberculosis
  • 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 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.
  • CFA complete Freund's adjuvant
  • 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.
  • 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 ⁇ l 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.
  • IFA incomplete Freund's adjuvant
  • Plasmid DNA 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.
  • 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.
  • 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.,
  • Th2-inducing genetic adjuvants include, e.g., B -4, IL-5. and IL-10.
  • Thl- and Th2- inducing genetic adjuvants See, e.g., Robinson, et al, (2000) Adv. Virus Res. 55:1-74.
  • mice are intramuscularly injected with 200 ⁇ g of DNA diluted in 100 ⁇ l saline.
  • Booster DNA immunizations are optionally administered at 4 weeks post-prime and 2 weeks post-boost.
  • 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,
  • 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).
  • CF culture filtrate
  • cytokines are assayed using standard bio-assays for IL-2,IFN ⁇ and IL-6, and by ELISA for IL-4 and E -10 using methods well known to those in the art. See, e.g., Current Protocols hi Immunology, Unit 6, John E.
  • mice are challenged by intravenous injection of live M. bovis BCG (0.5 mg).
  • 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.
  • CFU colony-forming units
  • mice are inoculated with an adenoviras vector encoding the human carcinoembryonic antigen (CEA) gene (MC38-CEA-2) (Conry et al, (1995) Cancer Gene Then 2:33), such as AdCMV-hcea described in U.S. Patent No. 6,348,450.
  • 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.
  • mice are subjected to a tumor challenge.
  • syngeneic tumor cells expressing the human CEA gene (MC38-CEA-2) (Conry et al, (1995) Cancer Gene Ther. 2:33) 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.
  • tumor cells expressing the human CEA gene are inoculated into castrated, non- vaccinated mice as outlined above. Sham castrated mice are used as controls.
  • SCID-hu mice are prepared essentially as described previously (see, e.g., Namikawa et al, (1990) /. Exp. Med. 172:1055 and Bonyhadi et al, (1997) /. 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 Current Protocols In Immunology, Unit 4.8, John E. Coligan et al, (eds), Wiley and Sons, New York, NY (1994), and yearly updates including 2002.
  • mice Surgical castration of mice.
  • the SCID-hu mice are anesthetized by intraperitoneal injection of 30-40 ⁇ l of a mixture of 5 ml of 100 mg/ml ketamine hydrochioride (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 is performed as above.
  • 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) /. Virol
  • CB cells are incubated with anti-CD34 antibody (QBEND-10, Immunotech) and then washed and resuspended at a final concentration of 2xl0 7 cells/ml.
  • CD34 + cells are then enriched using goat-anti-mouse IgGl magnetic beads (Dynal) following manufacturer's instructions.
  • the CD34 + cells are then incubated with 50 ⁇ l of glycoprotease
  • CD34 + cells present in the population are magnetically labeled with anti-CD34 and sorted on an autoMACSTM.
  • the autoMACSTM may be used for magnetic presorting of cells before further flow cytometric sorting.
  • anti-FITC- or anti-PE MACS® MicroBeads may be added to the FITC or PE stained cells. Then the cells are sorted on the autoMACSTM according to their magnetic labeling. The positive and negative fractions may then be collected for sorting by flow cytometry.
  • 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 are known in the art, and has been described extensively in studies of GM HSC for the survival of T cells in HIV-infected patients (see, e.g., Woffendin et al, (1996) Proc. Natl. Acad. Sci. USA, 93:2889; for review, see Amado et al, (1999) Front. Biosci. 4:d468).
  • 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).
  • RevMlO gene transfer vectors any of the RevMlO gene transfer vectors known and described in the art may be used.
  • 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) Proc. Natl. Acad. Sci. USA 95:1201).
  • Other methods of construction and retroviral vectors suitable for the preparation of GM HSC are well known in the art (see, e.g., Bonyhadi et al, (1997) J. Virol. 71:4707).
  • 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) Proc. Natl. Acad. Sci. USA, 91:11581.
  • the pRSV/TAR RevMlO plasmid contains the Rous sarcoma viras (RSV) promoter and tat-activation response element (TAR) from -18 to +72 of HJN is used to express the RevMlO open reading frame may also be used (Woffendin et al, (1994) Proc. Natl. Acad. Sci.
  • a marker gene such as the Lyt-2 ⁇ (murine CD8 ⁇ ) gene, may also be incorporated into the RevMlO vector for ease of purification and analysis of GM HSC by FACS analysis in subsequent steps (see, e g., Bonyhadi et al, (1997) /. Virol 71:4707).
  • a ⁇ RevlO 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 (see, e g., Bonyhadi et al, (1997) J. Virol. 71:4707).
  • mice 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 give 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.
  • TBI total body irradiation
  • mice are reconstituted with the RevMlO GM HSC (see above) as described previously (see, e.g., DiGusto et al, (1997) Blood, 87:1261, Bonyhadi et al, (1997) /. Virol. 71:4707).
  • Control mice are injected with unmodified HSC or with HSC that have been modified with the ⁇ RevMlO gene or an irrelevant gene.
  • 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 CD8 ⁇ ) surface expression using methods of flow cytometry and FACS analysis readily known to those skilled in the art (see, e.g., Bonyhadi et al, (1997) J. Virol. 71:4707; see also Cuixent Protocols In Immunology, Units 4.8 and 5, John E. Coligan et al, (eds), Wiley and Sons, New York, NY (1994), and yearly updates including 2002). Thymocytes are also tested for transgenic DNA with primers specific for the RevMlO gene using standard PCR methods.
  • 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) J. Virol. 71:4707).
  • the thymocytes are stimulated in vitro in the presence of irradiated allogeneic feeder cells (10 6 peripheral blood mononuclear cells/ml and 10 5 JY cells/ml) in RPMI medium containing 10% FCS, 50 ⁇ g/ml streptomycin, 50 U/G penicillin G, lx MEM vitamin solution, lx insulin transferring-sodium selenite medium supplement (Sigma), 40 U human rIL-2/ml, and 2 ⁇ g/ml phytohemagglutinin (PHA) (Sigma). About every 10 days, cells are restimulated with feeder cells and PHA as described previously in Vandekerckhove et al, (1992) /. Exp. Med.
  • CB HSC human cord blood
  • CB HSC human cord blood
  • a portion of each CB sample is HLA phonotyped, and the CD34 + donor cells are purified from the donor blood (or BM), 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 + .
  • 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 vectors or gene transfection using particle-mediated delivery are also well known in the art, and are described elsewhere herein.
  • 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 HJV replication (Bonyhadi et al, (1997) J. Virol. 71:4707).
  • Amphotropic vector-containing supematants are generated by infection with filtered supematants 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, JL-6 and SCF or LIF (10 ng/ml each) to induce entry of the cells into the cell cycle.
  • CD34 + -enriched HSC undergo transfection by a linearized RevMlO plasmid utilizing particle-mediated ("gene gun" transfer) essentially as described in
  • HAART Treatment of HIV-infected patients HAART 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 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.
  • GM HSC Injection of GM HSC into patients.
  • the GM HSC Prior to injection, the GM HSC are expanded in culture for approximately 10 days in X-Vivo 15 medium comprising H-2 (Chiron, 300 IU/ml).
  • H-2 Choiron, 300 IU/ml
  • the patient is injected with the genetically modified HSC, optimally at a dose of about 2-4 x 10 6 cells/kg.
  • G-CSF may also be injected into the recipient to assist in expansion of the GM HSC.
  • 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.
  • all new T cells are 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 reactivating or reactivated thymus takes up the genetically modified HSC and converts them into donor-type T cells and DC, while converting the HSC of the recipient into recipient-type T cells and DC.
  • the donor DC will tolerize any T cells that are potentially reactive with recipient.
  • the relative level of GM HSC in the infected patient is compared to the negative control patient that received the ⁇ RevMlO vector.
  • hematologic e.g., CD4+ T cell counts
  • immunologic e.g., neutralizing antibody titers
  • virologic e.g., viral titer
  • Termination of immunosuppression is performed as given in Example 16.
  • T cell ablation or other immune cell depletion and sex steroid ablation are begun at the same time.
  • T cell ablation or other immune cell depletion is maintained for about 10 days, while sex steroid ablation is maintained for around 3 months.
  • HSC transplantation is performed when the thymus starts to reactivate, at around 10-12 days after start of the combined treatment.
  • T cell ablation or other immune cell depletion is maintained 3-12 months, for example, for 3-4 months.
  • the thymic chimera 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., Current Protocols In Immunology, John E. Coligan et al, (eds), Wiley and Sons, New York, NY (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 (that is a normal, non-tolerized and non-grafted person).
  • peripheral blood lymphocytes 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.
  • NK, NKT and macrophages 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 four out of nine.
  • NK five out of nine patients
  • NKT four out of nine patients
  • macrophage three out of nine patients
  • 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. It is likely that such cells are derived from the thymus as no other source of mainstream (TCR ⁇ +CD8 ⁇ chain) T cells has been described. Gastrointestinal tract T cells are predominantly TCR ⁇ or CD8 ⁇ chain.
  • This example relates to clinical trials undertaken with HSCT patients.
  • prostate cancer patients >60 years
  • sex-steroid ablation therapy based on LHRH- agonist (chemical castration) treatment have been analyzed.
  • Patients were examined at the time of presentation and after 4-months of treatment by which time seram testosterone concentration was at castrate levels for all patients.
  • Test patients were given 3.6 mg (effective for 4 weeks) Zoladex (LHRH-A) 3-weeks prior to autologous or allogeneic stem cell transplantation and then monthly injections for 4-months. All patients were analyzed pre-treatment, weekly for 5- weeks after transplantation and then monthly up to 12 months. Ethics approval was obtained from The Alfred Committee for Ethical Research on Humans (Trial Number 01/006).
  • CD27-FITC CD45RO-CyChrome
  • CD4- or CD8-APC CD4- or CD8-APC
  • samples were incubated for 20 min, RT, in the dark in 500 ⁇ l of IX FACS permeabilizing solution (Becton-Dickinson, USA; IX solution was made from 10X stock in R.O.H 2 O). Washed samples (2 ml FACS buffer, 5 min., 600g ma ⁇ , RT) were incubated with either anti-Ki67-PE or anti-Ki67-FITC (or the appropriate isotype controls) for 30 min. at RT, in the dark. Samples were then washed and resuspended in 1% PFA for analysis.
  • IX FACS permeabilizing solution Becton-Dickinson, USA; IX solution was made from 10X stock in R.O.H 2 O. Washed samples (2 ml FACS buffer, 5 min., 600g ma ⁇ , RT) were incubated with either anti-Ki67-PE or anti-Ki67-FITC (or the appropriate isotype controls) for 30 min. at RT, in the
  • PBMC Preparation of PBMC.
  • Purified lymphocytes were prepared for T-cell stimulation assays and TREC analysis, by ficoll-hypaque separation and following centrifugation, the plasma layer was removed and stored at -20°C prior to analysis of sex steroid levels.
  • Cells not used for T lymphocyte stimulation assays were resuspended in freezing media and stored in liquid nitrogen prior to TREC analysis.
  • T Lymphocyte Stimulation Assay For mitogen stimulation, purified lymphocytes were plated out in 96-well round-bottom plates at a concentration of 1 x 10 5 cells/well in 100 ⁇ l of RPMI-FCS. Cells were incubated at 37°C, 5% CO 2 with PHA in doses from 1-10 ⁇ g/ml. For TCR-specific stimulation, cells were incubated for 48 hours on plates previously coated with purified anti-CD3 (1-10 ⁇ g/ml) and anti-CD28 (10 ⁇ g/ml). Following plaque formation (48-72 hours), 1 ⁇ Ci of 3 H-thymidine was added to each well and plates incubated for a further 16-24 hours. Plates were harvested onto filter mats and incorporation of 3 H- Thymidine was determined using liquid scintillation on a ⁇ -counter (Packard-Coulter, USA).
  • Real-Time PCR using Molecular Beacons Real-Time PCR using Molecular Beacons. Real-time PCR for analysis of TREC content in sorted cells was performed as described previously (Zhang et al, (1999) /. Exp.
  • Fig. 50 depicts FACS analysis of NKT cell reconstitution at various time points (day 14, 21, 28 and 35) following HSCT in control patients. An early recovery was observed in allogeneic patients, and was seen predominantly within the CD8+ population early post- transplant, which indicated extrathymic routes of regeneration. Also, CD4+NKT cells were evident from 1 month post-transplant.
  • Fig. 51 depicts B cell reconstitution following HSCT at various time points (2-12 months) following HSCT in control patients.
  • B cell regeneration occurs relatively faster in autologous transplant patients as compared to that of allogeneic patients (Fig. 51 A).
  • a return to control values (shaded) was not evident until at least 6 months post-transplant in both groups.
  • Fig. 52 depicts CD4+ reconstitution following HSCT at various time points (2-12 months) following HSCT in control patients. While B cell numbers were returning to control values by 6 months post-transplant (see Figs. 48A-B), CD4+ T cell numbers were severely reduced, even at 12 months post-transplant, in both autologous (Fig. 52B) and allogeneic (Fig. 52A) recipients.
  • Fig. 53 depicts CD8+ regeneration following HSCT at various time points (2-12 months) following HSCT in control patients. As shown in Fig. 53A-B, CD8+ T cell numbers regenerated quite rapidly post-transplant in both allogeneic and autologous recipients, respectively. However, as shown in Fig. 53C, the CD8+ T cells are mainly of extrathymic origin as indicated by the increase in TCR ⁇ + T CD8+ T cells, CD8 ⁇ T cells, and CD28 " CD8 + T cells.
  • Fig. 54 depicts FACS analysis of proliferation in various populations of CD4+ and CD8+ T cells before (Fig. 54A) and 28 days after (Fig. 54B) HSCT in control patients using the marker Ki-67.
  • Cells were analyzed on the basis of na ⁇ ve, memory and activated phenotypes using the markers CD45RO and CD27. The majority of proliferation occurred in CD8+ T cell subset, which further indicated that these cells were extxathymically derived and that the predominance of proliferation occurred within peripheral T cell subsets.
  • Fig. 55 depicts na ⁇ ve CD4+ T cell regeneration at various time points (2-12 months) following HSCT in control patients and LHRH-A treated patients.
  • Fig. 55A depicts FACS analysis of na ⁇ ve CD4+ T cells (CD45RA+CD45RO-CD62L+), and shows a severe loss of these cells throughout the study.
  • Figs. 55B-C na ⁇ ve CD4+ T cell began to regenerate by 12 months post-HSCT in autologous transplant patients (Fig. 55C) but were still considerably lower than the control values in allogeneic patients (Fig. 55B). These results indicated that the thymus was unable to restore adequate numbers of na ⁇ ve T cells post-transplant due to the age of the patients.
  • Fig. 56 depicts TREC levels at various time points (1-12 months) following HSCT in control patients.
  • Analysis of TREC levels which are only seen in recent thymic emigrants (RTE), emphasized the inability of the thymus to restore levels following transplant in both allogeneic (Fig. 52A) and autologous (Fig. 52B) patients. Again, this was due to the age of the patients, as well as the lack of thymic function due to thymic atrophy, which has considerable implications in the morbidity and mortality of these patients.
  • LHRH-A administration significantly increases NK but not B cell numbers in the peripheral blood. Overall, no significant change in B cell numbers was observed with LHRH-A treatment (Fig. 47). However, a significant increase in NK cell numbers was observed with treatment (p ⁇ O.Ol) (Fig. 47). Therefore, removal of sex steroid results in significantly increased numbers of T cells and NK cells.
  • TRECs are diluted out with mitosis (Zhang et al, (1999) J. Exp. Med. 190:725-732), which could occur intrathymically as part of normal T cell development or following export (Hazenberg et al, (2001) J. Mol. Med. 79:631-40), the absolute TREC levels would represent very much an underestimate of T cell export.
  • GVHD hematopoietic recovery following allogeneic HSCT without increasing GVHD and maintaining GVT.
  • Anlimurine CD16/CD32 FcR block (2.4G2) and all following fluorochrome-labeled antibodies against murine antigens were obtained from Pharmingen (San Diego, CA): Ly-9.1(30C7), CD3(145-2C11), CD4 (RM4-5), CD8 ⁇ .2(53-5.8), T-cell receptor- ⁇ (TCR- ⁇ ; H57-597), CD45R/B220 (RA3-6B2), CD43 (S7), IgM-FITC (R6-60.2), CDllb (Ml/70), Ly-6G(Gr-l) (RB6-8C5), c-kit (2B8), Sca-1 (D7), CDllc (HL3) I-A k (11- 5.2), isotypic controls: rat IgG2a-k (R35-95), rat IgG2a-l (B39-4), rat IgG2b-(A95-l), rat IgGl-k (R3
  • Streptavidin-FITC, PercP -phycoerythrin (PE) also were obtained from Pharmingen (San Diego, CA).
  • Recombinant human IL-7 was provided by Dr Michel Morre (Cytheris, Vanves, France).
  • Tissue culture medium consisted of RPMI 1640 supplemented with 10% heat inactivated fetal calf serum, 100 U/mL penicillin, 100 mg/mL streptomycin, and 2 mM L-glutamine (as well as 50 mM 2-mercaptoethanol for the culture of 32Dp210 cells and proliferation assays).
  • mice and HSCT Male C57BL/6J (B6, H-2b), C3FeB6Fl/J([B6 3 C3HJF1; H-2b/k), B10.BR (H-2k), B6D2F1/J (H-2b/d), CBA J (H-2k), Balb/c (H2-d), 1L7-/- and KGF-/- mice were obtained from the Jackson Laboratory (Bar Harbor, ME) and used in experiments when they were between 8 and 12 weeks of age. KGF-/- and IL7-/- were used between 4 and 7 months of age. HSCT protocols were approved by the Memorial Sloan-Kettering Cancer Center Institutional Animal Care and Use Committee.
  • the BM cells were removed aseptically from femurs and tibias.
  • Donor BM was depleted of T cells by incubation with anti-Thy-1.2 for 30 minutes at 4°C followed by incubation with Low-TOX-M rabbit complement (Cedarlane Laboratories, Hornby, ON, Canada) for 1 hour at 37°C.
  • Splenic T cells (for GVHD analysis) were obtained by purification over a nylon wool column followed by red cell removal with ammonium chloride red cell lysis buffer.
  • Surgical Castration Mice were anaesthetized and a small scrotal incision was made to reveal the testes. These were sutured and removed along with surrounding fatty tissue. The wound was closed using surgical staples. Sham-castration required the same surgical procedure, except for the removal of the testes. Castration was performed one day prior to
  • IL-7 IL-7 were either given from days 0 to 13 or 21 to 27 intraperitoneally at 10 ⁇ g/day for immune reconstitution studies. PBS was injected into control mice at the same time points.
  • BM cells, splenocytes or thymocytes were washed in
  • FACS buffer phosphate buffered saline (PBS)/2% bovine serum albumin (BSA)/0.1% azide
  • 1-3 x 10 6 cells were incubated for 30 minutes at 4°C with CD16/CD32 FcR block. Cells were then incubated for 30 minutes at 4°C with primary antibodies and washed twice with FACS buffer. Where necessary, cells were incubated with conjugated Streptavidin for a further 30 minutes at 4°C. The stained cells were resuspended in FACS buffer and analyzed on a FACSCaliburTM flow cytometer (Becton Dickinson, San Jose, CA) with CellQuestTM software.
  • FACSCaliburTM flow cytometer Becton Dickinson, San Jose, CA
  • SI Stimulation indices
  • Target cells were labelled with 100 mCi 51Cr at 2 xlO 6 cells/mL for 2 hours at 37°C and 5% CO 2 . After 3 washes, labelled targets were plated at 2.5xl0 3 cells/well in U-bottomed plates (Costar, Cambridge, MA). Splenocytes cultured with irradiated BALB/C splenocytes (1:2 ratio) for 5 days were added at various effector-to- target ratios in a final volume of 200 mL to 4 to 6 wells and incubated for 4 to 6 hours at 37°C and 5% CO 2 .
  • Detection of alloreactive T-cell clones with intracellular IFN- ⁇ staining Briefly, cells were incubated for 12 to 15 hours (for secondary allogeneic stimulation with T cell- depleted [TCD], irradiated stimulator cells) with Brefeldin A (10 mg/mL), harvested, washed, stained with primary (surface) fluorochrome (FITC, PerCP, and APC)-conjugated antibodies, fixed, and permeabilized with the Cytofix/Cytoperm kit (Pharmingen), and subsequently stained with odFN ⁇ - PE. FACS analysis was conducted by gating for the designated populations. Flow cytometer and software were used as mentioned below.
  • GVHD The severity of GVHD was assessed with a clinical GVHD scoring system as first described by Cooke et al. ((1996) Blood 88:3230-9). Briefly, ear- tagged animals in coded cages were individually scored every week for 5 clinical parameters (weight loss, posture, activity, fur, and skin) on a scale from 0 to 2. A clinical GVHD index was generated by summation of the 5 criteria scores (0-10). Survival was monitored daily. Animals with scores of 5 or more were considered moribund and were humanely killed.
  • death from leukemia was characterized by hepatosplenomegaly and the presence of mastocytoma cells in liver and spleen on microscopic examination, whereas death from GVHD was defined as the absence of hepatosplenomegaly and leukemic cells in liver and spleen, and the presence of clinical symptoms of GVHD as assessed by the clinical GVHD scoring system at the time of death.
  • RNA from whole BM was reverse-transcribed using Superscript II reverse transcriptase (Life Technologies, Rockville, USA).
  • cDNA was PCR-amplified for 35 cycles (94°C for 30 sees; 56°C for 30 sees; 72°C for 60 sees) with PCR Master Mix (Promega, Madison, USA .
  • HPRT 5'CACAggACTAgAACACCT gC 3' and 5' gCTggTgAAAAggACCTCT 3' TGF ⁇ ,: 5'CTACTgCTTCAgCTC CACAg 3' and 5' TgCACTTgCAggAgCgCAC 3' and KGF: 5'gCCTTgTCACg ACCTgTTTC 3' and 5' AgTTCACACTCgTAgCCgTTTg 3'.
  • CD45- thymic stromal cells and each thymus was subjected to enzymic digestion in 0.125% (w/v) collagenase/dispase (Roche Applied Sciences, Indianapolis, USA) with 0.1% (w/v) DNase, releasing most of the stromal and haematopoietic cells from the thymi allowing for the accurate calculation of thymic cellularity.
  • Anti-CD45 was used to identify CD45- stromal cells.
  • the number of donor-derived HSC was very low in both sham-castrated and castrated mice 14 days after allogeneic HSCT (2.98x10 2 ⁇ 1.25xl0 2 and 2.66xl0 2 ⁇ 8.8xl0 1 respectively) (Fig. 30A). However, by day +28 there are significantly more Ly9.1 + Lin " Sca-1 + c-kit + donor-derived HSCs in the castrated mice (4.8xl0 3 ⁇ l.lxl0 3 ), compared to the sham-castrated controls (I.lxl0 3 ⁇ 4.1xl0 2 ) (Fig. 30A).
  • T cell reconstitution following allogeneic HSCT is enhanced by castration.
  • Thymocytes and peripheral cells were divided into developmental stages on the basis of expression of CD3, CD4 and CD8: Triple Negatives (TN) (CD3 " CD4 " CD8 " ), double positive (DP) (CD4 + CD8 + ), single positive CD4 (SP CD4) CD3 + CD4 + CD8 " and single positive CD8 (SP CD8) CD3 + CD4 " CD8 + (Figs. 31A-D).
  • Fig. 32A The proliferative capacity of the splenic T cells was tested in 2 ways: CD3/ ⁇ CD28 cross-linking (Fig. 32B) and in a 3 rd party MLR (using irradiated BALB/C splenocytes as stimulators) (Fig. 32C).
  • Fig. 32E shows IFN ⁇ production by donor- derived CD8 + splenic T cells following BALB/C primary stimulation and either BALB/C or B10.BR secondary stimulation (control). This is represented graphically in Fig. 32F. There is no significant difference in the proportion IFN- ⁇ producing donor-derived CD8 + when comparing sham-castrated and castrated mice.
  • a DTH assay was used whereby, 42 days after castration and allo-BMT mice were sensitised with sRBCs. On day 46 days they were challenged and 24 and 48 hr later footpad swelling was determined. The DTH response is significantly enhanced 48 hrs after challenge when mice are castrated at the time of allo- HSCT compared to sham-castrated controls (Fig. 32G).
  • T cells in castrated recipients are comparable on a per cell basis with T cells from sham-castrated recipients and are capable to respond to novel antigens with intact proliferation, cytoxicity and cytokine production.
  • mastocytoma cell line P815 H-2d was injected into B6D2F1/J recipients at the time of transplant. Animals that died during the experiment were autopsied and the cause of mortality (tumor vs. GVHD) was determined. Mortality due to mastocytoma remained unchanged following castration (six out of nine mice) when compared to sham-castrated controls (five out of eight mice). This suggests that castration does not diminish GVT response following HSCT (Fig. 33B).
  • IL-7 and Castration have and additive effect following allogeneic HSCT. It has previously been shown that IL-7 treatment can increase the number of T and B cells in otherwise untreated animals and can also enhance lymphoid recovery following cyclophosphamide treatment, irradiation, syngeneic and allogeneic HSCT (Alpdogan et al, (2001) Blood 98:2256; Bolotin et al, (1996) Blood 88:1887; Faltynek et al, (1992) /. Immunol 149:1276; Morrissey et al, (1991) /. Immunol. 146:1547). IL-7 is known to increase T cell numbers through increased thymic activity as well as peripheral expansion.
  • TGF- ⁇ i and KGF Semi-Quantitative RT-PCR for IL-7, TGF- ⁇ i and KGF reveals an increase in KGF and a decrease in TGF- ⁇ i following allo-HSCT and castration.
  • RT-PCR analysis of whole bone marrow cells revealed undetectable levels of IL-7 transcript in both sham- castrated and castrated mice as late as 6 weeks after allo-HSCT. When template from control, untransplanted mice was used IL-7 was detected (data not shown).
  • TGF ⁇ i and KGF are known to be key mediators of hematopoiesis. Using HPRT equibrated template there appears to be a decrease in TGF ⁇ iand an increase in KGF 2 weeks after castration and allo-BMT (Fig. 34C).
  • mice (4-6 months old) were castrated and 2 weeks later thymus, spleen and BM were analysed (TGF ⁇ 1 "/” mice die prepubertally, Shull et al. (1992) Nature 359:693). Thymic cellularity is significantly increased when comparing sham-castrated and castrated KGF _ " mice.
  • peripheral T cell number that occurs following castration may decrease the incidence of these infections leading to enhanced overall survival of transplant patients.
  • G-CSF for example, is used to mobilise donor stem cells (Dreger et al, (1993) Blood 81:1404). Noach et al, showed that pre-treatment with SCF and IL-11 or
  • TGF- ⁇ has also been shown to down-regulate stromal IL-7 production and subsequently inhibit the proliferation of B cell progenitors (Tang et al, (1997) J. Immunol 159:117).
  • TGF- ⁇ The proliferation of hematopoietic stem cells is also regulated by TGF- ⁇ . Batard et al, have demonstrated that physiological concentrations of TGF- ⁇ i inhibit the proliferation and differentiation of HSCs in vitro ((2000) /. Cell Sci. 113:383-90). Furthermore, disruption of TGF- ⁇ signaling in HSCs (via the transient expression of a mutant type II receptor) enhances survival and proliferation of these cells (Fan et al, (2002) /. Immunol. 168:755-62). It is therefore possible that the increased number of HSCs seen 28 days after allogeneic HSCT and castration may be due do a decrease in the production of TGF- ⁇ by BM stromal cells.
  • Estrogen directly inhibits the proliferation and differentiation of HSC, as well as some lymphoid precursor subsets (Medina et ⁇ /.,(2001) Nat. Immunol. 2:718; Kouro et al. (2001) Blood 97:2708).
  • HSCs express functional estrogen receptors (ERs) and estrogen administration decreases the number of Lin " c-kit + Sca-l + HSCs (Thurmond et al, (2000) Endocrinol.
  • Olsen et al. have shown that it is the presence of a functional androgen receptor on the thymic epithelium but not the thymocytes that is essential for age-related thymic involution and the subsequent regeneration via sex steroid ablation Olsen et al, (2001) Endocrinol. 142:1278).
  • Thymic IL-7 levels decline with age (Aspinall, et al, (2000) Vaccine 18:1629; Andrew et al, (2002) Exp. Gerontol. 37:455; Ortman et ⁇ /.,(2002) Int. Immunol. 14:813). It remains unclear as to whether this is due to a decrease in the number of cells that produce JL-7 or a decrease in the ability of the existing cells to produce the cytokine.
  • DC are the key mediators of negative selection in the thymus (Jenkinson et al, (1985)
  • Test patients were given Zoladex (LHRH-A) 3 -weeks prior to stem cell transplantation and then monthly injections for 4-months. All patients were analyzed pre- treatment, weekly for 5-weeks after transplantation and monthly up to 12 months. Ethics approval was obtained from The Alfred Committee for Ethical Research on Humans (Trial Number 01/006).
  • PBMC Preparation of PBMC. Purified lymphocytes were used for T-cell stimulation assays and TREC analysis nd were prepared as above.
  • T Lymphocyte Stimulation Assay Analysis of TCR specific stimulation was performed using anti-CD3 and anti-CD28 cross-linking from 1-12 months post-transplant, unless otherwise indicated.
  • TCR-specific stimulation cells were incubated for 48 hours on plates previously coated with purified anti-CD3 (1-10 ⁇ g/ml) and anti-CD28 (10 ⁇ g/ml). Following plaque formation (48-72 hours), I ⁇ Ci of 3 H-Thymidine was added to each well and plates incubated for a further 16-24 hours. Plates were harvested onto filter mats and incorporation of 3 H-thymidine was determined using liquid scintillation on a ⁇ -counter (Packard-Coulter, USA).
  • LHRH-A administration enhances responsiveness to TCR specific stimulation following allogeneic stem cell transplantation.
  • LHRH-A treated patients showed enhanced proliferative responses (assessed by 3 H-thymidine incorporation) compared to control patients at all time-points except 6 and 9 months due to low patient numbers analyzed at this time; (Figs. 57A-B).
  • Figs. 57A-B In allogeneic transplant patients treated with a LHRH-A, a significant increase in responsiveness to anti-CD3/CD28 stimulation was observed at 4 and 5-months post-transplant compared to control patients. While control patients showed an enhanced response at both 6 and 9 months post-transplant, LHRH-A treated patients showed a greater responsiveness at 12-months post-transplant.
  • LHRH-A treated patients had equivalent responsiveness at all time-points except 6 months compared to pre-treatment.
  • LHRH-A treated patients showed enhanced proliferative responses (assessed by 3 H-Thyrnidine incorporation) compared to control patients at 1, 3 and 4 months post-transplant. This indicates a contribution of direct peripheral T cell effects, as new CD4+ T cells are not evident until at least 1-2 months post-transplant (Fig. 57A-B).
  • LHRH-A administration enhances responsiveness to TCR specific stimulation following autologous stem cell transplantation. A similar response as that seen in allograft recipients was also observed with autograft recipients (Fig. 57C). Those patients treated with a LHRH-A demonstrating an enhanced proliferative response to TCR stimulation at both 4 and 9 months post-transplant. LHRH-A treated patients showed enhanced proliferative responses (a * ssessed by 3 H-thymidine incorporation) compared to control patients at all time- points except 5 months. Restoration to pre-treatment values was observed by 12 months post- transplant in both control and LHRH-A treated patients.
  • LHRH-A administration enhances responsiveness to TCR specific stimulation following treatment for chronic cancer sufferers.
  • dO Zoladex administered (malesl0.8mg; females 3.6mg) d+28: females-Zoladex 3.6mg administered d+56: females-Zoladex 3.6mg administered d+84: females and males-Zoladex 3.6mg administered
  • SEX STEROID ABLATION ENHANCES MITOGENIC STIMULATION FOLLOWING HEMATOPOIETIC STEM CELL TRANSPLANTATION
  • Patients were those enrolled in Clinical Trial protocol No. 01/006 as above. Prior to stem cell transplant, patients were given LHRH-A (3-weeks prior). Patients who did not receive the agonist were used as control patients.
  • PBMC Preparation of PBMC. Purified lymphocytes were used for T-cell stimulation assays and TREC analysis and were prepared as above.
  • Mitogen Stimulation Assay Analysis of rnitogenic responsiveness was performed using pokeweed mitogen (PWM) and tetanus toxoid (TT) from 1-12 months post-transplant.
  • PWM pokeweed mitogen
  • TT tetanus toxoid
  • PBMC peripheral blood mononuclear cells
  • PBMC peripheral blood mononuclear cells
  • LHRH-A administration enhances responsiveness to rnitogenic stimulation following allogeneic stem cell transplantation.
  • LHRH-A administration enhances responsiveness to rnitogenic stimulation following autologous stem cell transplantation.
  • Patients treated with LHRH-A prior to stem cell transplantation showed an enhanced responsiveness to PWM stimulation at the majority of time-points studied compared to control patients (p ⁇ O.001 at 3 months) (Fig. 59A).
  • Fig. 59A By 12-months post-transplantation, LHRH-A treated patients had restored responsiveness to pre-treatment levels while control patients were still considerably reduced.
  • Allogeneic and autologous patients were analyzed for total WBC and total granulocyte or neutrophil numbers following HSCT. Three weeks prior to HSCT, patients were treated with LHRH-A. Patients who did not receive the agonist were used as control patients.
  • Total white blood cell (WBC) counts, granulocyte (G) and neutrophil counts per ⁇ l of blood were determined up to 35 days post transplant.
  • a sample of whole peripheral blood was analyzed either using a Cell-Dyn 1200 automated cell counter (Abbott) or hemocytometer counts done in duplicate. This allows calculation of total white blood cells, lymphocytes and granulocyte numbers following transplant. Analysis of engraftment was performed from D14-D35 post-transplant.
  • WBC and granulocyte numbers at D14 post-transplant was observed with LHRH-A treated patients compared to controls (Figs. 60 and 61).
  • This enhanced rate of engraftment is crucial for the overall patient morbidity with neutropenia ( ⁇ 200 neutrophils/ml blood) indicative of increased infection rates.
  • neutropenia ⁇ 200 neutrophils/ml blood
  • an early recovery of WBC and granulocyte numbers demonstrates a better survival rate for LHRH-A treated patients
  • inhibition of sex steroids enhances engraftment and reconstitution prior to full thymic regeneration or the release of new T cells as a result of full thymic regeneration.
  • mice Eight week-old mice were castrated and analyzed for anti-CD3/anti-CD28 stimulated T cell proliferative response 3 days (Figs. 62A, C, and E) and 7 days (Figs. 62B, D, and F) after surgery.
  • Peripheral (cervical, axillary, brachial and inguinal) lymph node (Figs. 62 A and B), mesenteric lymph node (Figs. 62C and D), and spleen cells (Figs. 62E and F) were stimulated with varying concentrations of anti-CD3 and co-stimulated with anti-CD28 at a constant concentration of 10 ⁇ g/ml for 48 hours.
  • Sex steroid ablated mice show enhanced CD28/CD3-stimulated T cell proliferation at 3 days (Figs. 62A,C, and E) and 7 days (Figs. 62 B,D, and F) post-castration.
  • T cells isolated from the peripheral LN showed a significant increase in proliferative responses at 3 days (10 ⁇ g/ml anti CD-3) and 7 days (2.5 ⁇ g/ml and 1.25 ⁇ g/ml anti-CD3) post-castration.
  • T cells isolated from the mesenteric lymph nodes (Figs. 62C and D) and spleen (Figs. 62E and F) also showed a significant increase in anti-CD3-stimulated proliferation over sham-castrated mice at 3 days post-castration.
  • HSCT HSCT. Mice were castrated 1 day before congenic HSCT. 5x10 Ly5.1 + BM cells were injected intravenously into irradiated (800 rads) C57/BL6 mice. The BM, spleen and thymus were analyzed by flow cytometry at various time points (2-6 weeks) post-transplant (Fig. 35). As shown in Fig. 35B, two weeks after castration and HSCT, there are significantly more cells in the BM of castrated mice as compared to sham-castrated controls. Similarly, as shown in Fig. 35C, there is a significant increase in thymic cell number 2, 4 and 6 weeks post-transplant as compared to sham castrated controls. As shown in Fig. 35C, in the periphery, splenic cell numbers are also significantly higher than controls 4 and 6 weeks post- transplant in the castrated recipients.
  • Castration enhances engraftment of HSC in the BM following congenic HSCT.
  • mice were castrated 1 day before congenic HSCT. 5x10 Ly5.1 + BM cells were injected intravenously into irradiated (800 rads) C57/BL6 mice. The BM was analyzed for lin-c- kit+sca-l+ HSC by flow cytometry at two weeks post-transplant (Fig. 36). Two weeks after BMT transplantation and castration there are significantly more donor-derived HSCs in the BM of castrated mice compared to sham castrated controls.
  • Fig. 37A-B depicts percent of common lymphoid precursors in the BM.
  • Fig. 37B depicts the number of common lymphoid precursors in the BM. Two weeks after BMT transplantation and castration there is a significantly increased proportion of donor-derived HSCs in the BM of castrated mice compared to sham castrated controls.
  • CDl lb • Donor-derived CDl lb + and CDl lb" DC are significantly increased in the thymii of castrated mice compared to sham-castrated controls 2 weeks after BMT (Fig. 38).
  • This example illustrates the use of G-CSF and/or GM-CSF for the increase in neutrophil levels and decrease of incidence of infection in patients receiving chemotherapy.
  • a randomized double blind, placebo controlled study is conducted in 100 patients with small cell lung cancer.
  • Neupogen® Amgen, Thousand Oaks, CA
  • Neupogen® is administered at a dosage of 4-8 ⁇ g/kg/day s.c. from days 4-17 following chemotherapy according to manufacturer's instructions.
  • GM-CSF administration hi a first study, patients receive a single SC dose of 6 mg of Neulasta ® (Amgen, Thousand Oaks, CA) on day 2 of each chemotherapy cycle or Filgrastim® (Amgen, Thousand Oaks, CA) at 5 ⁇ g/kg/day SC beginning on day 2 of each cycle according to manufacturer's instructions.
  • subjects were randomized to receive a single SC injection of Neulasta ® at 100 ⁇ g/kg on day 2 or Filgrastim® at 5 ⁇ g/kg/day SC beginning on day 2 of each cycle of chemotherapy.
  • All patients had been diagnosed with small cell lung cancer and were treated with standard cycles of cyclophosphamide, doxombicin and etoposide.
  • GM-CSF patients are diagnosed with metastatic breast cancer.
  • G-CSF Neuronal Com
  • Treatment with G-CSF resultsed in a clinically and statistically significant decrease in the incidence of infection as measured by febrile neutropenia, infection rates, in-patient hospitalization and antibiotic use.
  • Numerous other Phase VU trials reported by this company (www.neupogen.com) report that the use of G-CSF resulted in measurable increases in neutrophils, thereby supporting the clinical use of G-CSF to treat cancer patients receiving immunosuppressive chemotherapy.

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Abstract

L'invention concerne des méthodes de prévention ou de traitement de maladies, permettant d'améliorer la faculté de réponse aux vaccinations et d'améliorer l'efficacité de la thérapie génique chez des patients, par interruption de la signalisation de stéroïdes sexuels chez un patient. Selon l'invention, la fonctionnalité de la moelle osseuse et celle d'autres cellules immunitaires sont améliorées, sans régénération thymique préalable ou simultanée. Dans certains modes de réalisation, la signalisation des stéroïdes sexuels est interrompue ou supprimée chez le patient concerné, par administration d'agonistes de la LHRH, d'antagonistes de la LHRH, d'anticorps du récepteur anti-LHRH, de vaccins anti-LHRH, d'anti-androgènes, d'anti-oestrogènes, de modulateurs sélectifs du récepteur d'oestrogènes (SERM), de modulateurs sélectifs du récepteur d'androgènes (SARM), de modulateurs sélectifs de réponse à la progestérone (SPRM), d'ERD, d'inhibiteurs d'aromatase ou de différentes combinaisons des constituants précités.
PCT/US2004/011921 2003-04-18 2004-04-19 Prevention de maladies et vaccination avant reactivation thymique WO2004103271A2 (fr)

Priority Applications (5)

Application Number Priority Date Filing Date Title
CA002528521A CA2528521A1 (fr) 2003-04-18 2004-04-19 Prevention de maladies et vaccination avant reactivation thymique
AU2004241949A AU2004241949A1 (en) 2003-04-18 2004-04-19 Disease prevention and vaccination prior to thymic reactivations
US10/553,594 US20080279812A1 (en) 2003-12-05 2004-04-19 Disease Prevention and Vaccination Prior to Thymic Reactivation
EP04785486A EP1620126A4 (fr) 2003-04-18 2004-04-19 Prevention de maladies et vaccination avant reactivation thymique
JP2006532426A JP2007518699A (ja) 2003-04-18 2004-04-19 胸腺再生前の病気の予防およびワクチン接種

Applications Claiming Priority (18)

Application Number Priority Date Filing Date Title
US10/418,747 US20040018180A1 (en) 1999-04-15 2003-04-18 Stimulation of thymus for vaccination development
US10/418,747 2003-04-18
US10/418,727 2003-04-18
US10/419,068 US20050002913A1 (en) 1999-04-15 2003-04-18 Hematopoietic stem cell gene therapy
US10/419,066 2003-04-18
US10/418,727 US20040013641A1 (en) 1999-04-15 2003-04-18 Disease prevention by reactivation of the thymus
US10/419,066 US20040037817A1 (en) 1999-04-15 2003-04-18 Normalization of defective T cell responsiveness through manipulation of thymic regeneration
US10/419,068 2003-04-18
US52700103P 2003-12-05 2003-12-05
US60/527,001 2003-12-05
US10/749,122 2003-12-30
US10/748,831 2003-12-30
US10/748,450 2003-12-30
US10/749,118 2003-12-30
US10/748,831 US20050020524A1 (en) 1999-04-15 2003-12-30 Hematopoietic stem cell gene therapy
US10/748,450 US20040241842A1 (en) 1999-04-15 2003-12-30 Stimulation of thymus for vaccination development
US10/749,118 US20040265285A1 (en) 1999-04-15 2003-12-30 Normalization of defective T cell responsiveness through manipulation of thymic regeneration
US10/749,122 US20040259803A1 (en) 1999-04-15 2003-12-30 Disease prevention by reactivation of the thymus

Publications (2)

Publication Number Publication Date
WO2004103271A2 true WO2004103271A2 (fr) 2004-12-02
WO2004103271A3 WO2004103271A3 (fr) 2005-11-24

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PCT/US2004/011913 WO2004094599A2 (fr) 2003-04-18 2004-04-19 Prvention contre une maladie et vaccination apres reactivation thymique
PCT/US2004/011921 WO2004103271A2 (fr) 2003-04-18 2004-04-19 Prevention de maladies et vaccination avant reactivation thymique

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PCT/US2004/011913 WO2004094599A2 (fr) 2003-04-18 2004-04-19 Prvention contre une maladie et vaccination apres reactivation thymique

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EP (2) EP1620545A4 (fr)
JP (1) JP2007518699A (fr)
KR (1) KR20060022232A (fr)
AU (1) AU2004241949A1 (fr)
CA (1) CA2528521A1 (fr)
WO (2) WO2004094599A2 (fr)

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EP1620545A2 (fr) * 2003-04-18 2006-02-01 Norwood Immunology, Ltd. Prvention contre une maladie et vaccination apres reactivation thymique
CN100425288C (zh) * 2005-01-28 2008-10-15 北京金迪克生物技术研究所 鼻腔喷雾型流感病毒灭活疫苗及其制备方法
JP2009508923A (ja) * 2005-09-21 2009-03-05 オックスフォード バイオメディカ（ユーケー）リミテッド 化学免疫療法の方法
WO2010015036A1 (fr) * 2008-08-08 2010-02-11 Christopher Hovens Applications biologiques de domaines de liaison à un stéroïde
US20110086051A1 (en) * 2009-10-08 2011-04-14 Dartmouth-Hitchcock Clinic System and method for monitoring and optimizing immune status in transplant recipients
US11564969B2 (en) 2017-01-20 2023-01-31 ISR Immune System Regulation Holding AB (publ) Immunorhelin compounds for intracellular infections
US11672842B2 (en) 2017-02-22 2023-06-13 ISR Immune System Regulation Holding AB (publ) Gonadotropin-releasing hormones for use as adjuvant immunotherapeutics

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BRPI0910686A2 (pt) * 2008-04-21 2015-09-29 Tissue Regeneration Therapeutics Inc células perivasculares do cordão umbilical humano geneticamente modificadas para a profilaxia contra agentes biológicos e químicos ou para o tratamento dos mesmos.
CA2888166A1 (fr) * 2012-10-15 2014-04-24 Chamaeleo Pharma Bvba Fosfestrol pour une utilisation dans le traitement curatif ou palliatif du cancer chez les mammiferes femelles
JP2020505379A (ja) 2017-01-20 2020-02-20 イミューン システム レギュレェイション ホールディング エービー 新規化合物（イムノレリン）

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US20020086000A1 (en) * 1999-04-15 2002-07-04 Richard Boyd Stimulation of thymus for vaccination development
US20040258672A1 (en) * 1999-04-15 2004-12-23 Monash University Graft acceptance through manipulation of thymic regeneration
AUPP977899A0 (en) * 1999-04-15 1999-05-13 Monash University Improvement of t cell mediated immunity
CA2462073A1 (fr) * 2000-10-13 2002-04-18 Monash University Therapie genique aux cellules souches hematopoietiques
AP2003002796A0 (en) * 2000-10-13 2003-06-30 Richard Boyd Disease prevention by reactivation of the thymus
KR20060022232A (ko) * 2003-04-18 2006-03-09 노르우드 이뮤놀러지 리미티드 흉선 재활성화 이전에 수행되는 질병 예방법 및 면역방법

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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1620545A2 (fr) * 2003-04-18 2006-02-01 Norwood Immunology, Ltd. Prvention contre une maladie et vaccination apres reactivation thymique
EP1620545A4 (fr) * 2003-04-18 2007-07-04 Norwood Immunology Ltd Prvention contre une maladie et vaccination apres reactivation thymique
CN100425288C (zh) * 2005-01-28 2008-10-15 北京金迪克生物技术研究所 鼻腔喷雾型流感病毒灭活疫苗及其制备方法
JP2009508923A (ja) * 2005-09-21 2009-03-05 オックスフォード バイオメディカ（ユーケー）リミテッド 化学免疫療法の方法
JP2012144563A (ja) * 2005-09-21 2012-08-02 Oxford Biomedica (Uk) Ltd 化学免疫療法の方法
WO2010015036A1 (fr) * 2008-08-08 2010-02-11 Christopher Hovens Applications biologiques de domaines de liaison à un stéroïde
US20110086051A1 (en) * 2009-10-08 2011-04-14 Dartmouth-Hitchcock Clinic System and method for monitoring and optimizing immune status in transplant recipients
US11564969B2 (en) 2017-01-20 2023-01-31 ISR Immune System Regulation Holding AB (publ) Immunorhelin compounds for intracellular infections
US11672842B2 (en) 2017-02-22 2023-06-13 ISR Immune System Regulation Holding AB (publ) Gonadotropin-releasing hormones for use as adjuvant immunotherapeutics

Also Published As

Publication number Publication date
CA2528521A1 (fr) 2004-12-02
EP1620545A2 (fr) 2006-02-01
EP1620126A4 (fr) 2007-07-04
JP2007518699A (ja) 2007-07-12
EP1620545A4 (fr) 2007-07-04
EP1620126A2 (fr) 2006-02-01
KR20060022232A (ko) 2006-03-09
AU2004241949A1 (en) 2004-12-02
WO2004103271A3 (fr) 2005-11-24
WO2004094599A2 (fr) 2004-11-04
WO2004094599A3 (fr) 2005-12-29

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