WO2003105874A1

WO2003105874A1 - Thymic epithelial cells with progenitor capacity

Info

Publication number: WO2003105874A1
Application number: PCT/AU2003/000749
Authority: WO
Inventors: Jason William Gill; Richard Lennox Boyd
Original assignee: Monash University
Priority date: 2002-06-14
Filing date: 2003-06-16
Publication date: 2003-12-24
Also published as: AU2003233271A1

Abstract

The present invention provides thymic progenitor cells which give rise to the complete thymus microenvironment including both cortical and medullary epithelial lineages. These progenitor cells are identified by cell surface expression of the glycoprotein MTS24. These cells can be utilized in a variety of methods to prevent or treat diseases which can be alleviated by increasing the number of T cells and/or altering the T cell population of a subject. The new thymic tissue derived from MTS24+ thymic epithelial progenitor cells is able to uptake appropriate T cell precursors such as hematopoietic stem cells (HSC) or common lymphoid progenitor cells and/or other bone marrow cells from the blood and convert them in the new thymic tissue to both new T cells and dendritic cells (DC).

Description

THYMIC EPITHELIAL CELLS WITH PROGENITOR CAPACITY

FIELD OF THE INVENTION

The present invention relates to thymic epithelial progenitor cells, and their use in methods for reconstituting a thymic epithelial microenvironment which supports T cell development, and which can be used to manipulate the thymic microenvironment.

BACKGROUND OF THE INVENTION

The thymus is responsible for the generation of a T cell repertoire that is concurrently restricted to self-MHC molecules and tolerant to self-antigens. The generation of these T cells is under strict control of non-lymphocytic components of the thymic microenvironment which are mainly comprised of epithelial cells and cells of mesenchymal origin. These stromal cells help to impart the specificity of the thymus and regulate the programs controlling thymocyte survival, lineage commitment and selection (reviewed in ¹). The central role of epithelial cells for the development and selection of T cells in the thymus is widely acknowledged ², but little is known about the molecular mechanisms involved in the commitment to, and development of, the different thymic epithelial cell lineages.

The thymic epithelial primordium is formed from an outpocketing of the pharyngeal endoderm of the third pharyngeal pouch with inductive contributions by neural crest-derived mesoderm (reviewed in reference ⁸). Several transcription factors have been described as having roles in thymic organogenesis by their expression patterns and by analysis of mice with spontaneous and targeted gene mutations. Mice with a deficiency for either transcription factor Hoxa3 or the down-stream positioned Paxl fail to initiate the formation and development of a regular thymic primordium ⁹'¹⁰. Similarly, loss-of-function mutation for either the Pax3 or Pax9 genes results in moderate to severe defects in early thymus organogenesis ^u"13. The homozygous lack of Foxnl (also known as Whn) causes an early arrest in thymic epithelial cell (TEC) expansion and the capacity to attract hematopoietic precursor cells, a complex phenotype known as "nude" ¹⁴'¹⁵. The absence of fibroblast growth factor (Fgf)-10 or of its specific receptor, FgfR2IIIb, on thymic epithelial cells results in a severe thymic hypoplasia ^16"18. demonstrating that epithelial-mesenchymal interactions constitute an essential role in thymus organogenesis. Together these studies reflect an essential function for these molecules and have started to define the molecular mechanisms that control the earliest events in thymic organogenesis. Morphological and functional studies have demonstrated a broad heterogeneity among thymic epithelial cells ^19"23, but conflicting views as to the lineage relationship among these different subpopulations have remained. It has been suggested that the cortex develops from a stem cell of ectodermal origin, and the medulla derives from the endodermal lining of the 3^rd pharyngeal pouch. Other studies propose that both TEC lineages are derived exclusively from a common precursor within the endodermal epithelium, independent of a contribution by surface ectoderm. In support of the latter contention are studies that describe rare cells bearing phenotypic markers typical for both cortical and medullary epithelia ^24"26. While recently a clonal relationship has been demonstrated for individual medullary compartments ²⁷, characterization of the putative thymic epithelial stem cell population and the possibility for repopulation studies have so far been prevented due to the lack of known cell surface markers typical for these cells. Accordingly, there is a need for the identification and isolation of thymic epithelial progenitor cells which can be used in methods for reconstituting a thymic epithelial microenvironment which supports T cell development.

SUMMARY OF THE INVENTION

Thus, in one aspect the present invention provides a method of modifying T cell population makeup or increasing the number of T cells in a subject, the method comprising administering MTS24+ thymic epithelial progenitor cells, wherein the MTS24+ thymic epithelial progenitor cells provide a competent microenvironment for T cell development.

Preferably, the T cells are CD4+CD8- or CD4-CD8+ T cells or regulatory T cells such as CD25+CD4 T cells, CD4-CD8- αβTCR T cells, NKT cells and γδTCR T cells. In a further embodiment, it is preferred that the MTS24+ thymic epithelial progenitor cells are CD45-MHCII+. Other characteristics of the MTS24+ thymic epithelial progenitor cells of the present invention are that they lack of expression of at least some mature epithelial cell markers. Furthermore, the MTS24+ thymic epithelial progenitor cells of the present invention also express at least some cortex markers (eg 4F1, CDR 1, LY51, or 6C3) and/or medullary markers (eg UEA-1).

The present disclosure also concerns methods of modifying the responsiveness of host T-cell populations to accept grafts from a non-identical, or mismatched, donor. New thymic tissue derived from the administered MTS24+ thymic epithelial progenitor cells thus becomes capable of taking up hematopoietic precursor cells from the blood and converting them in the new thymic tissue to both new T cells and dendritic cells (DC). The latter DC then induce tolerance in subsequent T cells to grafts of the same histocompatibility as that of the precursor cell donor. This vastly improves allogeneic graft acceptance. This process may be facilitated by administering hematopoietic stem cells (HSC) and/or bone marrow cells from the donor. The HSC could be also incorporated with MTS 24+ thymic epithelial progenitor cells prior to in vivo transfer, in a multicellular reaggregate formed by incubating the component cells overnight in eg hanging drops. This process brings the component cells into close contact and facilitates a 3D architecture which T cell development requires. Accordingly, the present invention provides a method for inducing tolerance in a subject to a graft from a mismatched donor comprising the steps of; host T cell ablation to remove pre-existing donor reactive cells, administering MTS24+ thymic epithelial progenitor cells, and transplanting an organ, tissue or cells from a donor to the subject.

Preferably, the method further comprises the step of transplanting bone marrow, or haemopoietic stem cells or lymphoid stem cells or any other stem cell which enter the thymus, from the donor to the subject.

Furthermore, hematopoietic and/or lymphoid stem cells from the donor can also transplanted into the recipient, further creating tolerance to a graft from the donor. In some embodiments, the HSC donor cells are CD34⁺ precursor cells. The present disclosure also concerns methods for destroying a patient's T cells to reduce clinical disease, where the disease is related to the presence of an abnormal set of T cells. This step is followed by the induction of new thymic tissue in the subject. The degree and kinetics of thymic regrowth can be enhanced by injection of CD34+ hematopoietic stem cells (HSC), such as autologous HSC. The patient, having been depleted of T cells, will no longer have the disease and in the presence of a new thymic tissue will soon produce a new cohort of T cells in the blood and lymphoid organs.

Thus, in a further aspect the present invention provides a method of preventing or treating an autoimmune disease in a subject, the method comprising the steps of T cell ablation and administering MTS24+ thymic epithelial progenitor cells.

In yet a further aspect, the present invention provides a method for preventing or treating an allergy in a subject, the method comprising the steps of T cell ablation and administering MTS24+ thymic epithelial progenitor cells.

In some embodiments, hematopoietic or lymphoid stem and/or progenitor cells from a donor (e.g., an MHC-matched donor) are transplanted into the recipient to increase the speed of regeneration of thymic tissue. In another embodiment these cells are transplanted from a healthy donor, without autoimmune disease or allergies, to replace aberrant stem and/or progenitor cells in the patient.

In a further embodiment, the cells of the invention can be genetically modified with a gene or protein the expression of which could prevent or minimise further development of autoimmune disease. Examples include pro-insulin to prevent/minimise Type I diabetes, MOG or equivalent myelin protein to prevent/minimise multiple sclerosis, gastic proton pump to prevent/minimise autoimmune gastritis.

The present disclosure also concerns methods for improving a patient's immune response to a vaccine. This is accomplished by quantitatively and qualitatively restoring the peripheral T cell pool, particularly at the level of naive T cells. These naive T cells are then able to respond, and improve memory cell response, to a greater degree to presented foreign antigen.

A patient's immune response to a vaccine may be improved by causing the patient's to produce new thymic tissue, and the functional status of the peripheral T cells to be improved. In this instance, the thymus will begin to increase the rate of proliferation of the early precursor cells (CD3^"CD4^"CD8^" cells) and convert them into CD4⁺CD8⁺, and subsequently new mature CD3^hiCD4⁺CD8^_ (T helper (Th) lymphocytes) or CD3^ωCD4^"CD8⁺ (T cytotoxic lymphocytes (CTL)). The new thymic tissue will also take up new haemopoietic stem cells (HSC) from the blood stream and convert them into new T cells and intrathymic dendritic cells. The increased activity in the thymic tissue resembles that found in a normal younger thymus (prior to puberty). The result of this renewed thymic output is increased levels of naive T cells (those T cells which have not yet encountered antigen) in the blood. There is also an increase in the ability of the blood T cells to respond to stimulation, e.g., by using anti-CD28 Abs, cross-linking the TCR with, e.g., anti-CD3 antibodies, or stimulation with mitogens. such as pokeweed mitogen (PWM). This combination of events results in the body becoming better able to respond to vaccine antigens, thereby ultimately being able to better defend against infection and other immune system challenges (e.g., cancers), or becoming better able to recover from chemotherapy and radiotherapy. Thus, the present invention also provides a method of enhancing an immune response to an antigen in a subject, the method comprising administering MTS24+ thymic epithelial progenitor cells.

Preferably, the method further comprises administering the antigen.

Preferably, the subject has an infection. In one embodiment, the infection is a HIN infection. In this instance it is preferred that the cells are administered following anti-retroviral treatment such as HAART.

In a further embodiment, bone marrow or HSC are also transplanted into the patient to provide a reservoir of precursor cells for the renewed thymic growth. These HSC have the capability of turning into DC, which may have the effect of providing better antigen presentation to the T cells and therefore a better immune response (e.g., increased antibody (Ab) production and effector T cells number and/or function). In one embodiment, the bone marrow or HSC are transplanted just before, at the time of, or after the generation of new thymic tissue, thereby creating a new population of T cells.

In yet another aspect, the present invention provides a method of preventing or treating cancer in a subject, the method comprising administering MTS24+ thymic epithelial progenitor cells.

Preferably, the cells are administered following chemotherapy, radiation therapy or bone marrow transplantation.

In a further embodiment of the invention, the MTS24+ thymic epithelial progenitor cells are genetically modified.

Such genetically modified MTS24+ thymic epithelial progenitor cells can be used to resist or prevent infection, activity, replication, and the like, and combinations thereof, of the infectious agent are administered to the subject.

Preferably, the genetic modification is selected from the group consisting of: expressing a transgene, and the deletion of at least one endogenous gene of the cell.

Preferably, the transgene encodes a molecule selected from the group consisting of: a polypeptide, dsRΝA, a catalytic nucleic acid, and an antisense oligonucleotide. The thymic progenitor cells of the invention can be used to induce lymphoid commitment in appropriate stem or progenitor cells. Thus, in a further preferred embodiment, the method further comprises administering hematopoietic stem cells (HSC) and/or bone marrow cells. These hematopoietic stem cells (HSC) and/or bone marrow cells may also be genetically modified.

In one embodiment, HSC are genetically modified to create resistance to HIN in the T cells formed during and after the generation of new thymic tissue. For example, the HSC are modified to include a gene whose product will interfere with HIN infection, function and/or replication in the T cell. HSC that have been genetically modified to resist or prevent infection, activity, replication, and the like, and combinations thereof, of the infectious agent are injected into a patient concurrently with MTS24+ thymic epithelial progenitor cells. In another embodiment, HSC are genetically modified to create resistance (complete or partial) to HIN in the T cells formed administered to the subject. For example, the HSC are modified to include a gene whose product will interfere with HIN infection, function and/or replication in the T cell. In one embodiment, HSC are genetically modified with the RevMlO gene (see, e.g., reference ⁴) or the CXCR4 or PolyTAR genes ⁴⁸. This confers a degree of resistance to the virus, thereby preventing disease caused by the virus.

The MTS24+ thymic epithelial progenitor cells can be administered by any means known in the art. In one embodiment, the MTS24+ thymic epithelial progenitor cells are administered by a subcutaneous injection. In another embodiment, the MTS24+ thymic epithelial progenitor cells are administered to the thymus. In a further embodiment, the MTS24+ thymic epithelial progenitor cells are transplanted directly into the kidney capsule.

In a further embodiment, the MTS24+ thymic epithelial progenitor cells are administered as an aggregate. In an alternate embodiment, the MTS24+ thymic epithelial progenitor cells are seeded on a matrix prior to administration.

It is envisaged that the co-administration of mesenchymal cells, or extracts thereof, with the cells of the invention may assist in the generation of new thymic tissue. Thus, it is preferred that the MTS24+ thymic epithelial progenitor cells are administered in a composition comprising mesenchymal cells, or an extract thereof. Further, it is preferred that the MTS24+ thymic epithelial progenitor cells are administered in a composition comprising at least one epithelial cell growth factor. Preferably, the growth factor is selected from the group consisting of: IL-7, a fibroblast growth factor (FGF) - including FGF1, FGF2, FGF3, FGF8, FGF10, a keratinocyte growth factor, insulin-like growth factor 1, epidermal growth factor, hydrocortisone, transferrin, high density lipoprotein, growth hormones and bone morphogenetic proteins. The composition may also include inhibitors of, for example, transforming growth factor beta. The composition may also comprise mimics of the above- mentioned growth factors.

In a further aspect, the present invention provides for the use of MTS24+ thymic epithelial progenitor cells for the manufacture of a medicament for modifying T cell population makeup or increasing the number of T cells in a subject, wherein the

MTS24+ thymic epithelial progenitor cells provide a competent microenvironment for

T cell development.

In another aspect, the present invention provides for the use of MTS24+ thymic epithelial progenitor cells for the manufacture of a medicament for inducing tolerance in a subject to a graft from a mismatched donor, wherein the subjects T cells have been ablated.

In yet another aspect, the present invention provides for the use of MTS24+ thymic epithelial progenitor cells for the manufacture of a medicament for preventing or treating an autoimmune disease in a subject. In another aspect, the present invention provides for the use of MTS24+ thymic epithelial progenitor cells for the manufacture of a medicament for preventing or treating an allergy in a subject.

In yet another aspect, the present invention provides for the use of MTS24+ thymic epithelial progenitor cells for the manufacture of a medicament for enhancing an immune response to an antigen in a subject.

In a further aspect, the present invention provides for the use of MTS24+ thymic epithelial progenitor cells for the manufacture of a medicament for preventing or treating cancer in a subject.

In another aspect, the present invention provides a method of generating thymic tissue, the method comprising culturing, in vitro or in vivo, MTS24+ thymic epithelial progenitor cells under suitable conditions. In particular, culturing MTS24+ cthymic epithelial progenitor cells in vitro in combination with HSC and/or lymphoid progenitor cells can be used as a mechanisms of producing a population of T cells. Preferably, the co-culturing occurs in the presence of mesenchymal cells, extracts thereof, and/or suitable growth factors.

Furthermore, the present invention provides an isolated thymic epithelial progenitor cell, wherein the cell produces a protein designated MTS24.

In a further aspect, the present invention provides a composition comprising an thymic epithelial progenitor cell according to the invention, and a carrier or diluent. In another aspect, the present invention provides a method of purifying a cell according to the invention, the method comprising; i) contacting a population of cells with an agent that binds MTS24, and ii) separating agent/MTS24 complexes.

Preferably, the agent is an antibody. More preferably, the antibody is a monoclonal antibody. The present disclosure also provides a diagnostic method for determining the susceptibility of a patient to generate thymic tissue upon the administration of thymic progenitor cells of the invention, within a week, within 4 to 5 days, within 2-3 days, or within 24 hours after administration of the MTS24+ thymic epithelial progenitor cells. As used herein, "determining the susceptibility of a thymus to regeneration" means to assess whether or not a thymic tissue is being generated following administration the MTS24+ thymic epithelial progenitor cells.

In one embodiment, the diagnosis is accomplished by measuring the amount of thymic induced factors in a blood sample of the patient before and after administration of the MTS24+ thymic epithelial progenitor cells. In yet another embodiment, the invention is used to identify previously unidentified thymic factors.

In another embodiment, the diagnosis is accomplished by measuring thymic activity. In addition to the above, this will be achieved by determining levels of newly produced T cells identified by the presence in these cells of small circles of DNA termed T cell receptor excision circles (TREC's). These TREC's are produced as a normal part of T cell development in the thymus, in particular as a result of gene rearrangements in the formation of the T cell receptor for antigen. Basic increases in total T cell number (as measured by flow cytometry staining for CD3, CD4 and CD8) and shifts in their in vitro responsiveness to stimulation with anti-CD3 cross-linking can also be used to monitor thymic function but they are expected to take several days to weeks before any changes may be detectable.

The invention may be used with any animal species (including humans) having MTS24+ thymic epithelial progenitor cells.

As will be apparent, preferred features and characteristics of one aspect of the invention are applicable to many other aspects of the invention.

Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps. Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application. The invention is hereinafter described by way of the following non-limiting

Examples and with reference to the accompanying figures.

DESCRIPTION OF THE ACCOMPANYING FIGURES Figure 1. The MTS24 antigen is a high molecular weight membrane glycoprotein. (a) Immunoblot of membrane lysates from different tissues analyzed using either mAb MTS24 or an IgG2a isotype control, (b) MTS24 Immunoblot of salivary gland extracts treated with the deglycosylating enzyme Neuraminidase (NA) at different concentrations or exposed to proteinase K.

Figure 2. The MTS24 antigen is highly expressed on primordial pharyngeal endoderm Immunohistochemistry of MTS24 antigen expression in early thymic organogenesis. (a) E7.5 gastrulation stage embryo. (Closed arrowhead: presumptive endoderm; open arrowhead: caudal mesoderm; *: parietal endoderm) (b) Developing pharyngeal region at E10.5. pp = pharyngeal pouch, I-IN branchial arches.

Figure 3. Spatial and temporal expression of the MTS24 antigen during thymic development.

IHC at El 1.5, E12.5, E15.5 and 4 week old adult of thymic MTS24 antigen expression relative to cytokeratins-5 and 8 (a) and CD45⁺ hematopoietic cells relative to cytokeratins-5 and 8 distribution (b). Arrow in (a) denotes rare "triple positive" TEC in 4 week old thymus.

Figure 4. Flow-cytometric analysis of thymic epithelial cells for MTS24 antigen expression. (a) Analysis of non-hematopoietic (CD45^") stromal cells at E12.5, E15.5 and 4 weeks of age for the expression MTS24 antigen and MHC class II molecules, (b) Analysis of CD45 TS24⁺ and CD45MTS24^" cells at E14.5, E15.5 and 4-weeks of age for Ly51 expression and binding of UEA1 lectin.

Figure 5. Antibody binding to MTS24 antigen inhibits thymocyte differentiation in fetal thymic organ cultures. Analysis of thymocytes from anti-MTS24 mAb and isotype IgG2a treated fetal thymic organ cultures for CD4 and CD8 cell surface expression and forward scatter characteristics.

Figure 6. MTS24⁺ TEC generate a functional epithelial microenvironment.

(a) CD45^"MHCII MTS24^" (Rl) and CD45^"MHCII⁺MTS24⁺ (R2) thymic epithelial cells were separated by FACS from E15.5 C57B/6 (CD45.2) donor mice, (b) Gross anatomy of kidneys engrafted 8 weeks earlier with CD45^"MHCII⁴MTS24^" (Rl) and CD45^" MHCπ⁺MTS24⁺ (R2) cell aggregates. Arrow denotes site of engraftment. Analysis of thymocyte subset distribution as defined by CD4 and CD8 and by CD44 and CD25 expression, respectively, on thymocytes derived from unmanipulated age matched control thymic lobes (c) compared to thymocytes derived from CD45 MHCII⁺MTS24 (R2) cell aggregates engrafted in an identical fashion (d) Arithmetic means (± standard deviation) of TCRNβ⁺ cells as a percentage of all CD3⁺ thymocytes on thymocytes derived from; unmanipulated age matched control thymic lobes, CD45 MHCII⁺MTS24 (R2) cell aggregates engrafted in an identical fashion and the endogenous thymus. Total number of host derived thymocytes from the CD45 MHClrtViTS24⁺ (R2) cell grafts were 2.52 x 10⁷ (±0.76) (n=6) cells compared to 2.75 η x 10 (±0.53) cells (n=6) from E15.5 fetal thymic lobe transplants.

Figure 7. Thymic tissue derived from MTS24⁺ cell aggregates display a phenotypically normal thymic microenvironment.

IHC analysis of engrafted aged matched control thymic lobes (a, c, e, g) and engrafted CD45^"MHCπ^"tMTS24⁺ cell aggregates (b, d, f, h).

DETAILED DESCRIPTION OF THE INVENTION

General Techniques and Definitions

Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular genetics, immunology, nucleic acid chemistry, hybridisation techniques and biochemistry).

Unless otherwise indicated, the recombinant DNA, cell culture, and immunological techniques utilized in the present invention are standard procedures, well known to those skilled in the art. Such techniques are described and explained throughout the literature in sources such as, J. Perbal, A Practical Guide to Molecular

Cloning, John Wiley and Sons (1984), J. Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989), T.A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D.M. Glover and B.D. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and F.M. Ausubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley- Interscience (1988, including all updates until present), Ed Harlow and David Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory, (1988), and J.E. Coligan et al. (editors) Current Protocols in Immunology, John Wiley & Sons (including all updates until present), and are incorporated herein by reference. As used herein, "prevention" and "preventing" refer to complete as well as partial protection (reduced severity of clinical symptoms) of disease using the methods of the invention. For instance, with an improved immune system the individual will have a reduced likelihood of succumbing to a tumor or cancer, a prevailing infection (e.g., viral, bacterial, fungal, or parasitic), and will show better responses to a vaccination (e.g., increased levels of Ab specific to that vaccine or antigen, and development of effector T cells). For example, the methods of this invention would be applicable to prevention of viral infections, such as influenza and hepatitis, and prevention of bacterial infections, such as pneumonia and tuberculosis (TB).

"Recipient," "patient" "subject" and "host" are used interchangeably herein to indicate the individual that is receiving the epithelial progenitor cells.

"Donor" refers to the source of the transplant, which may be syngeneic, allogeneic or xenogeneic. Allogeneic grafts may be used, and such allogeneic grafts are those that occur between unmatched members of the same species, while in xenogeneic grafts the donor and recipient are of different species. Syngeneic grafts, between matched animals, may also be used in one embodiment. The terms "matched," "unmatched," "mismatched," and "non-identical" with reference to grafts are used to indicate that 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.

Induction of Tolerance

The production of new thymic tissue in a patient who requires an allograft transplantation will facilitate the acceptance by the patient of that allograft. In some embodiments, the patient also receives a transfer of, for example, hematopoietic stem cells (HSC) from the donor. Once new thymic tissue is generated, a new immune system is created, one that no longer recognizes and/or responds to antigens on the allograft. In other words, the allograft is seen as "self, and not as foreign.

The T cell population of an individual can be altered through the methods of this invention. In particular, modifications can be induced that will create tolerance of non- identical (i.e., allogenic) grafts. The establishment of tolerance to exogenous antigens, particularly non-self donor antigens in clinical graft situations, can be best achieved if dendritic cells of donor origin are incorporated into the recipient's thymus. This form of tolerance may also be made more effective through the use of inhibitory immunoregulatory cells. Given that a major mechanism underlying the prevention of T cells reacting against self antigens is due to the negative selection (by clonal deletion) of such cells by thymic dendritic cells, the ability to create new thymic tissue which has dendritic cells from a potential organ or tissue donor has major importance in the prevention of graft rejection. This is because the T cells which could potentially reject the graft will have encountered the donor dendritic cells in the thymus and be deleted before they have the opportunity to enter the blood stream. The blood precursor cells which give rise to the dendritic cells are the same as those which give rise to T cells themselves.

Potentially any cell or foreign protein incorporated into the new thymic tissue will assist in tolerance induction. Most efficient at this process will be methods for incorporation of foreign dendritic cells into a patient's thymus. This is accomplished by the administration of donor cells to a recipient to create tolerance in the recipient. The donor cells may be hematopoietic stem cells (HSC), epithelial stem cells, or hematopoietic progenitor cells or any other type of donor cells or stem cells. In some embodiments, the donor cells are CD34⁺ HSC, lymphoid progenitor cells, or myeloid progenitor cells. In some embodiments, the donor cells are CD34⁺ HSC. The donor HSC can develop into dendritic cells in the recipient. The donor cells are administered to the recipient and migrate through the peripheral blood system to the newly generated thymic tissue. These cells become integrated into the new thymic tissue and produce dendritic cells and T cells in the same manner as do the recipient's cells. The result is a chimera of T cells that are tolerant to both the host and donor and circulate in the peripheral blood of the recipient, and the accompanying increase in the population of cells, tissues and organs that are recognized by the recipient's immune system as self.

In some embodiments involving co-administration of HSC, the transplanted HSC may be following full myeloablation, and thus result in a full HSC transplant (e.g., 5X10⁶ 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 X10⁵ cells/kg body weight. In other words, it may be that as little as 10% chimerism may be sufficient to establish tolerance to a donor's graft. In some embodiments, the donor HSC is from umbilical cord blood.

Moreover, the ability to enhance the uptake into the new thymic tissue of hematopoietic stem cells means that the nature and type of dendritic cells can be manipulated. For example, the stem cells could be transfected with specific gene(s) which eventually become expressed in the dendritic cells in the thymus (and elsewhere in the body). In one non-limiting example of the invention, where the donor is related to the recipient but expresses an additional MHC molecule or a molecule expressed by the Y chromosome (e.g., where the recipient is female and the donor is male), the genes encoding that molecule could be transfected and expressed in either the donor's HSC before reconstitution of the recipient with the donor's HSC, or could be transfected and expressed in the recipient's own HSC. Some of the HSC, whether donor or recipient, would then develop into dendritic cells, and so educate the newly formed T cells that the additional molecule is "self. T cells thus educated, when encountering such a molecule expressed by the donor graft tissue, will recognize the tissue as self and not attempt to reject it. Indeed, thymocyte selection can involve multiple cell types: the cortical epithelium provides the specific differentiation molecules for positive selection, dendritic cells mediate negative selection and third party cells provide the MHC/peptide ligands.

Skewing of Developing TCR Repertoire Towards, or Away From. Specific Antigens

The present invention also stems from the discovery that new thymic tissue of an autoimmune patient will facilitate in overcoming an autoimmune disease suffered by that patient. This same principle also applies to patients suffering from allergies. Once new thymic tissue is generated, a new immune system can be created, one that no longer recognizes and/or responds to a self antigen.

New thymic tissue is generated by the administration of MTS24+ thymic epithelial progenitor cells of the invention. During or after the administration step, hematopoietic stem and/or progenitor cells, from a donor can be transplanted into the recipient. These cells are accepted by the thymus as belonging to the recipient and become part of the production of new T cells and DC by the thymic tissue. The resulting population of T cells recognize both the recipient and donor as self, thereby creating tolerance for a graft from the donor. The ability to generate new thymic tissue means that the nature and type of dendritic cells can be manipulated. For example, hematopoietic 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). Such genes could include those which encode specific antigens for which an immune response would be detrimental, as in autoimmune diseases and allergies. The present disclosure also provides methods for incorporation of foreign dendritic cells into a patient's thymus. This is accomplished by the administration of donor cells to a recipient to create tolerance in the recipient. The donor cells may be hematopoietic stem cells (HSC) or hematopoietic progenitor cells. In some embodiments, the donor cells are CD34⁺ HSC, lymphoid progenitor cells, or myeloid progenitor cells. In some embodiments, the donor cells are CD34⁺ HSC. The donor HSC can develop into dendritic cells in the recipient. The donor cells are administered to the recipient and migrate through the peripheral blood system to the newly generated thymic tissue. The uptake of the hematopoietic precursor cells is substantially increased upon the generation of new thymic tissue using the cells of the invention. These hematopoietic precursor cells become integrated into the new thymic tissue and produce dendritic cells and T cells in the same manner as do the recipient's cells. The result is a chimera of T cells that circulate in the peripheral blood of the recipient, and the accompanying increase in the population of cells, tissues and organs that are recognized by the recipient's immune system as self. In accordance with the invention, the following protocol may be applied. A patient diagnosed with an autoimmune disease (e.g., type I diabetes) is first immunosuppressed to stop disease progression. This may be done by administering an immunosuppressant (e.g., cyclosporine or rapamycin) alone or together with anti-T and B cell antibodies, such as anti-CD3 or anti-T cell gamma globulin to get rid of T cells and anti-CD 19, CD20, or CD21 to get rid of B cells. After which, new thymic tissue can be generated by administering MTS24+ thymic epithelial progenitor cells. The patients own T cells may then be mobilized with GCSF. If the autoimmunity arose as a result of a cross-reaction of the subjects T cells with a pathogen the patient had previously encountered, the ablation of the T cells will remove the auto-reactive T cells, and the newly developed T cells will not continue to recognize his cells (e.g., β- islet cells) as foreign. In this manner, his autoimmune disease is alleviated.

In another non-limiting example of the invention, the autoimmune patient is reconstituted with allogeneic stem cells. In some embodiments, these allogeneic stem cells are umbilical cord blood cells, which do not include mature T cells. In some embodiments when HSC are co-administered, the transplanted HSC may be following full myeloablation, and thus result in a full HSC transplant (e.g., 5X10⁶ 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 XI 0⁵ cells/kg body weight. In other words, it may be that as little as 10% chimerism may be sufficient to alleviate the symptoms of the patient's autoimmune disease.

In yet further embodiments, where the antigen is not an auto-antigen but, rather, an external antigen (e.g., pollen or seafood), similar strategies can be employed. If the allergy arose from some chance activation of an aberrant T or B cell clone, immunosuppression to remove T cells and B cells, followed by the generation of new thymic tissue 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 generated thymic tissue 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 no longer present. In further embodiments of the invention, genetic modification of the HSC may be employed if the antigen involved in the autoimmune disease or allergy is known. For example, in multiple sclerosis, the antigen may be myelin glycoprotein (MOG). In pernicious anaemia, the antigen may be the gastric proton pump. In type I diabetes, the antigen may be pro-insulin. Likewise, certain allergic reactions are in response to known antigens (e.g., allergy to feline saliva antigen in cat allergies). In these situations, MTS24+ thymic epithelial progenitor cells (or cells co-administered with MTS24+ thymic epithelial progenitor cells) may first be genetically modified to express the antigen prior to being administered to the recipient. Accordingly, genetically-modified hematopoietic progenitor cells result in the development of dendritic cells, and so tolerize the newly formed T cells, but they also enter the bone marrow as dendritic cells and delete new, autoreactive or allergic B cells. Thus, central tolerance to the auto-antigen or allergen is achieved in both the thymus and the bone marrow, thereby alleviating the patient's autoimmune disease or allergic symptoms.

In another example for the depletion of hyperreactive T cells, for which the target antigen is known, the MTS24+ thymic epithelial progenitor cells (e.g., autologous epithelial stem cells) can be transfected with the gene encoding the specific antigen for which tolerance is desired.

Thus, in accordance with the invention, the basic principle is to stop ongoing autoimmune disease or prevent one developing in highly predictive cases (e.g., in familial distribution) with T and B as appropriate cell depletion followed by building new thymic tissue. First, the autoimmune disease is diagnosed, and a determination is made as to whether or not there is a familial (genetic) predisposition. Next, a determination is made as to whether or not there had been a recent prolonged infection in the patient which may have lead to the autoimmune disease through antigen mimicry or inadvertent clonal activation. In practice it may be impossible to determine the cause of the disease. Next, T cell depletion is performed and, as appropriate, B cell depletion is performed, combined with chemotherapy, radiation therapy or anti-B cell reagents (e.g., CD 19, CD20, and CD21) or antibodies to specific Ig subclasses (anti IgE). New thymic tissue is then produced by administering MTS24+ thymic epithelial progenitor cells. These cells can be co-administered with HSC which have been in vitro transfected with a gene encoding the autoantigen to enter the new thymic tissue 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 marrow, and present the antigen to developing immature B cells, thereby causing their deletion, similar to that occurring to T cells in the thymus. Use of the immunosuppressive regimes (anti-T, -B therapy) would overcome any untoward activation of pre-existing potentially autoreactive T and B cells. Moreover, in the case of no-obvious genetic predisposition, the thymic and marrow the generation of new thymic tissue may be combined with G-CSF injection to increase blood levels of autologous HSC to enhance the thymic regrowth.

Agents - Infectious and Non-infectious

As used herein, "infectious agents," "foreign agents," and "agents" are used interchangeably and include any cause of disease in an individual. Agents include, but are not limited to viruses, bacteria, fungi, parasites, prions, cancers, allergens, asthma- inducing agents, "self proteins and antigens which cause autoimmune disease, etc.

In one embodiment, 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). Examples of infectious viruses include: Retroviridae (e.g., human immunodeficiency viruses, such as HJN-l (also referred to as HTLN-III, LAN or HTLN-1II/LAN, or HJN- III; and other isolates, such as HIN-LP; Picornaviridae (e.g., polio viruses, hepatitis A virus; enteroviruses, human coxsackie viruses, rhinoviruses, echoviruses); Calciviridae (e.g., strains that cause gastroenteritis); Togaviridae (e.g., equine encephalitis viruses, rubella viruses); Flaviridae (e.g., dengue viruses, encephalitis viruses, yellow fever viruses); Coronaviridae (e.g., coronaviruses, severe acute respiratory syndrome (SARS) virus); Rhabdoviridae (e.g., vesicular stomatitis viruses, rabies viruses); Filoviridae (e.g., ebola viruses); Paramyxoviridae (e.g., parainfluenza viruses, mumps virus, measles virus, respiratory syncytial virus); Orthomyxoviridae (e.g., influenza viruses); Bungaviridae (e.g., Hantaan viruses, bunga viruses, phleboviruses and Nairo viruses); Arena viridae (hemorrhagic fever viruses); Reoviridae (e.g., reoviruses, orbiviurses and rotaviruses); Birnaviridae; Hepadnaviridae (e.g, Hepatitis B virus); Parvoviridae (parvoviruses); Papovaviridae (papilloma viruses, polyoma viruses); Adenoviridae (most adeno viruses); Herpesviridae (e.g., herpes simplex virus (HSV) 1 and 2, varicella zoster virus, cytomegalovirus (CMV), herpes viruses); Poxviridae (e.g., variola viruses, vaccinia viruses, pox viruses); and Iridoviridae (e.g., African swine fever virus); and unclassified viruses (e.g., the etiological agents of Spongiform encephalopathies, the agent of delta hepatities (thought to be a defective satellite of hepatitis B virus), the agents of non-A, non-B hepatitis (class l=internally transmitted; class 2=parenterally transmitted (i.e., Hepatitis C); Norwalk and related viruses, and astroviruses).

Examples of infectious bacteria include: Helicobacter pyloris, Borelia burgdorferi, Legionella pneumophilia, Mycobacteria sporozoites (sp.) (e.g. M. tuberculosis, M. avium, M. intracellulare, M. kansaii, M. gordonae), Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitidis, Listeria monocytogenes, Streptococcus pyogenes (Group A Streptococcus), Streptococcus agalactiae (Group B Streptococcus), Sti-eptococcus (viridans group), Streptococcus faecalis, Streptococcus bovis, Streptococcus (anaerobic sps.), Streptococcus pneumoniae, pathogenic Campylobacter sp., Enterococcus sp., Haemophilus influenzae, Bacillus antracis, corynebacterium diphtheriae, corynebacterium sp., Erysipelothrix rhusiopathiae, Clostridium perfringers, Clostridium tetani, Enterobacter aerogenes, Klebsiella pneumoniae, Pasturella multocida, Bacteroides sp., Fusobacterium nucleatum, Streptobacillus moniliformis, Treponema pallidium, Treponema pertenue, Leptospira, and Actinomyces israelli.

Examples of infectious fungi include: Cryptococcus neqformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Chlamydia trachomatis, Candida albicans. Other infectious organisms (i.e., protists) include: Plasmodium falciparuin and

Toxoplasma gondii.

In another embodiment, the agent is an allergen. Allergic conditions include eczema, allergic rhinitis or coryza, hay fever, bronchial asthma, urticaria (hives) and food allergies, and other atopic conditions. In another embodiment, the agent is a cancer or tumor. As used herein, a tumor or cancer includes, e.g., tumors of the brain, lung (e.g. small cell and non-small cell), ovary, breast, prostate, colon, as well as other carcinomas, melanomas, and sarcomas. The generation of new thymic tissue will increase the number of lymphocytes capable of responding to the antigen of the agent in question, which will lead to the elimination (complete or partial) of the antigen creating a situation where the host is resistant to the infection.

In one embodiment, a patient is infected with HJN. In one embodiment, the method for treating the subject 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) administration of epithelial progenitor cells that have been genetically modified to contain a gene that expresses a protein that will prevent HIN infection, prevent HIN replication, disable the HIN virus, or other action that will stop the infection of T cells by HIN;

4) if the genetically modified epithelial progenitor cells are not autologous, administration of the donor cells will prime the immune system to recognize the donor cells as self; and

5) when the thymic chimera is established and the new cohort of mature T cells have begun exiting the thymus, reduction and eventual elimination of immunosuppression. In an embodiment, the epithelial progenitor cells are unmodified, but administered with HSC that have been genetically modified to contain a gene that expresses a protein that will prevent HIN infection, prevent HIN replication, disable the HIN virus, or other action that will stop the infection of T cells by HIN.

Vaccine Response

The present disclosure also provides methods for improving vaccine response in a patient. This is accomplished by quantitatively and qualitatively restoring the peripheral T cell pool, particularly at the level of naϊve T cells. Νaϊve T cells are those that have not yet contacted antigen and therefore have broad based specificity, i.e., are able to respond to any one of a wide variety of antigens. As a result of the procedures of this invention a large pool of naϊve T cells becomes available to respond to antigen administered in a vaccine.

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). The present disclosure uses the generation of new thymic tissue to improve immune system function, as exemplified by increased functionality of T lymphocytes (e.g.,Th and CTL) including, but not limited to, better killing of target cells; increased release of cytokines, interleukins and other growth factors; increased levels of Ab in the plasma; and increased levels of innate immunity (e.g., natural killer (NK) cells, DC, neutrophils, macrophages, etc.) in the blood, all of which can be beneficial in increasing the response to vaccine antigens.

In one embodiment, administering epithelial progenitor cells of the invention to the thymus creates this pool of naϊve T cells by generating new thymic tissue which has a competent microenvironment for T cell development. As used herein, the terms "vaccinating," "vaccination," "vaccine, "

"immunizing," "immunization," are related to the process of preparing a patient to respond to an antigen of an agent. Vaccination may include both prophylactic and therapeutic vaccines.

The generation of new thymic tissue can be supplemented by the addition of CD34⁺ hematopoietic stem cells (HSC) slightly before or at the time the new thymic tissue is generated in vivo. Ideally these cells are autologous or syngeneic and have been obtained from the patient or twin prior to thymic tissue generation. The HSC can be obtained by sorting CD34⁺ cells from the patient's blood and/or bone marrow. The number of HSC can be enhanced in several ways, including (but not limited to) by administering G-CSF (Neupogen, Amgen) to the patient prior to collecting cells, culturing the collected cells in Stem Cell Growth Factor, and/or administering G-CSF to the patient after CD34⁺ cell supplementation. Alternatively, the CD34⁺ cells need not be sorted from the blood or BM if their population is enhanced by prior injection of G-CSF into the patient. In one embodiment, hematopoietic cells are supplied to the patient, which increases the immune capabilities of the patient's body.

The HSC are administered to the patient and migrate through the peripheral blood system to the new thymic tissue. These cells become integrated into the new thymic tissue and produce dendritic cells and T cells. The results are a population of T cells and other immune cells that circulate in the peripheral blood of the recipient, and the accompanying increase in the population of cells, tissues and organs, which are capable improved responses to the vaccine antigen.

This procedure can be combined with any other form of immune system stimulation, including adjuvant, accessory molecules, and cytokine therapies. For example, useful cytokines include but are not limited to interleukin 2 (JL-2) as a general immune growth factor, JL-4 to skew the response to Th2 (humoral immunity), and JFNgamma to skew the response to Thl (cell mediated, inflammatory responses). 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.

Genetic Modification

Useful genes and gene fragments (polynucleotides) for this invention include those that affect genetically based diseases and conditions of T cells. Such diseases and conditions include, but are not limited to, HIN infection/AIDS, T cell leukemia virus infection, and other viruses that cause lymphoproliferative diseases.

With respect to HIN/AIDS, a number of genes and gene fragments may be used, including, but not limited to, the nef transcription factor; a gene that codes for a ribozyme that specifically cuts HIN genes, such as tat and rev ³; the trans-dominant mutant form of HIN-l rev gene, RevMlO, which has been shown to inhibit HIN replication ⁴; an overexpression construct of the HJN-1 7'ev-responsive element (RRE) ⁵; any gene that codes for an RΝA or protein whose expression is inhibitory to HIN infection of the cell or replication; and fragments and combinations thereof.

These genes or gene fragments are used in a stably expressible form. The term "stably expressible form" as used herein means that the product (RΝA 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 signalling sequences for transcription of the DΝA to RΝA. Additionally, when a protein coded for by the gene or gene fragment is the active molecule that affects the patient's condition, the DΝA will also code for translation signals.

In most cases the genes or gene fragments will be contained in vectors. Those of ordinary skill in the art are aware of expression vectors that may be used to express the desired RΝA or protein. Expression vectors are vectors that are capable of directing transcription of DNA sequences contained therein and translation of the resulting RNA. Expression vectors are capable of replication in the cells to be genetically modified, and include plasmids, bacteriophage, viruses, and minichromosomes. Alternatively the gene or gene fragment may become an integral part of the cell's chromosomal DNA. Recombinant vectors and methodology are in general well-known.

Expression vectors useful for expressing the proteins of the present disclosure contain an origin of replication. Suitably constructed expression vectors contain an origin of replication for autonomous replication in the cells, or are capable of integrating into the host cell chromosomes. Such vectors may also contain selective markers, a limited number of useful restriction enzyme sites, a high copy number, and strong promoters. Promoters are DNA sequences that direct RNA polymerase to bind to DNA and initiate RNA synthesis; strong promoters cause such initiation at high frequency. In one embodiment, the DNA vector construct comprises a promoter, enhancer, and a polyadenylation signal. The promoter may be selected from the group consisting of HIV, such as the Long Terminal Repeat (LTR), Simian Virus 40 (SV40), Epstein Barr virus, cytomegalovirus (CMV), Rous sarcoma virus (RSN), Moloney virus, mouse mammary tumor virus (MMTN), human actin, human myosin, human hemoglobin, human muscle creatine, human metalothionein. In one embodiment, an inducible promoter is used so that the amount and timing of expression of the inserted gene or polynucleotide can be controlled.

The enhancer may be selected from the group including, but not limited to, human actin, human myosin, human hemoglobin, human muscle creatine and viral enhancers such as those from CMN, RSN and EBN. The promoter and enhancer may be from the same or different gene.

The polyadenylation signal may be selected from the group consisting of: LTR polyadenylation signal and SN40 polyadenylation signal, particularly the SN40 minor polyadenylation signal among others. The expression vectors of the present disclosure are operably linked to DΝA coding for an RΝA or protein to be used in this invention, i.e., the vectors are capable of directing both replication of the attached DΝA molecule and expression of the RΝA or protein encoded by the DΝA molecule. Thus, for proteins, the expression vector must have an appropriate transcription start signal upstream of the attached DΝA molecule, maintaining the correct reading frame to permit expression of the DΝA molecule under the control of the control sequences and production of the desired protein encoded by the DNA molecule. Expression vectors may include, but are not limited to, cloning vectors, modified cloning vectors and specifically designed plasmids or viruses. In one embodiment, an inducible promoter may be used so that the amount and timing of expression of the inserted gene or polynucleotide can be controlled. One having ordinary skill in the art can produce DNA constructs which are functional in cells. In order to test expression, genetic constructs can be tested for expression levels in vitro using tissue culture of cells of the same type of those to be genetically modified.

Gene Therapy

The present disclosure provides methods for gene therapy. This is accomplished by the administration of genetically modified cells to a recipient. Preferably, the genetically modified cell is an MTS24+ thymic epithelial progenitor cells, however, these MTS24+ thymic epithelial progenitor cells may be administered inconjunction with other cells that have been genetically modified such as HSC. The HSC can be obtained by sorting CD34⁺ cells from the patient's blood and/or bone marrow. The number of HSC can be enhanced in several ways, including (but not limited to) by administering G-CSF (Neupogen, Amgen) to the patient prior to collecting cells, culturing the collected cells in Stem Cell Growth Factor, and/or administering G-CSF to the patient after CD34⁺ cell supplementation. Alternatively, the CD34⁺ cells need not be sorted from the blood or BM if their population is enhanced by prior injection of G-CSF into the patient.

The genetically modified cells may be, for example, HSC, epithelial stem cells, or myeloid or lymphoid progenitor cells. In one embodiment, the genetically modified cells are CD34⁺ HSC, lymphoid progenitor cells, or myeloid progenitor cells. In another embodiment, the genetically modified cells are CD34⁺ HSC. The genetically modified cells are administered to the patient and migrate through the peripheral blood system to the newly generated thymic tissue. These cells become integrated into the thymic tissue and produce dendritic cells and T cells carrying the genetic modification from the altered cells. The results are a population of T cells with the desired genetic change that circulate in the peripheral blood of the recipient, and the accompanying increase in the population of cells, tissues and organs caused by the new thymic tissue.

Standard recombinant methods can be used to introduce genetic modifications into the cells being used for gene therapy. For example, retroviral vector transduction of cultured epithelial progenitor cells is one successful method ⁶'⁷. Additional vectors include, but are not limited to, those that are adenovirus derived or lentivirus derived, and Moloney murine leukemia virus-derived vectors.

Also useful are the following methods: particle-mediated gene transfer such as with the gene gun ²⁹, liposome-mediated gene transfer ³⁰, coprecipitation of genetically modified vectors with calcium phosphate ³¹, electroporation ⁴³, and microinjection ⁴⁴, as well as any other method that can stably transfer a gene or oligonucleotide, which may be in a vector, into the epithelial progenitor cell such that the gene will be expressed at least part of the time.

Diagnostic Indicators of Thymic Function

Given the broad range of patient age, diseases and treatments, it is anticipated that many patients will respond differently to the treatment of the present invention, including some very poorly. Hence, a diagnostic early indicator of this responsiveness of the generation of new thymic tissue is provided to formulate rational clinical management of T cell based disorders.

Since the thymus is an endocrine organ, the generation of new thymic function involves release of not only new T cells into the blood stream after 2-4 weeks, but prior to this the new thymic will also release increased levels of cytokines. These will be detectable in the blood or plasma. The present disclosure utilizes these released cells and molecules to detect the degree of response of a patient to the treatments of the present invention. Provided here is a set of diagnostic techniques for making this determination.

Known Markers Certain markers are associated with the activation of the thymus. By following the concentration of any one, or any combination, of these markers, one can monitor the level of thymic function. Changes in the levels of these marker molecules pre-and post- generation of new thymic tissue can be examined using bioinformatics. For example, two-dimensional gel electrophoresis of plasma (i.e., blood depleted of all cells by centrifugation) is performed on patients' samples pre- and post-administration of MTS24+ thymic epithelial progenitor cells. The differentially expressed "dots" on the gels are recorded and analyzed by computer. 1. Interleukin-7 (11-7) Immune recovery in mice after T cell-depleted bone marrow transplantation has been documented to be enhanced following administration of IL-7, suggesting the production of IL-7 may be one of the mechanisms regulating de novo production of T cells after bone marrow transplantation. Analysis of IL-7 serum levels in patients before and after bone marrow transplantation by ELISA revealed an inverse relationship to absolute lymphocyte count. Studies measuring EL-7 levels in HIN- infected pediatric and adult patients also indicate a strong inverse correlation between IL-7 and absolute CD4 counts and lesser but significant correlations with CD3 and CD8 counts.

The mechanism underlying the increase in circulating JL-7 are unclear but it has been suggested that decreased T cell numbers result in diminished IL-7 receptor availability leading to increased levels of free IL-7 with no change in IL-7 production. That is, binding to lymphocytes that express IL-7 receptors homeostatically regulates circulating IL-7 levels. An alternative mechanism is the direct upregulation of IL-7 in response to lymphopoenia through the interaction of T cells and JL-7-producing cells via a soluble mediator or through direct contact within the lymphoid microenvironment. In children aged 6-months to 5.5 years, the normal mean concentration of IL-7 is

10.7 ± 3.9 pg/ml. In adults aged 22.2 to 53.5 years the mean is appreciably lower, at 3.1 ± 2.5 pg/ml. It has thus been suggested that IL-7 levels may be determined by age since IL-7 levels are highest in infants less than one year of age and lower in children and adults. This would support previous studies which demonstrated an age-dependent decline in thymopoietic capacity in chemotherapy and bone marrow transplant patients beginning in adolescence. Moreover studies of bone marrow stroma from aged mice have shown decreased secretion of IL-7 with age.

According to an embodiment of the present disclosure, concentration of IL-7 in a patient's blood or serum is monitored before and after administration of the MTS24+ thymic epithelial progenitor cells. Rise in the concentration of EL-7 within 2-3 days, within 24 hours, or within 2-3 hours, of administration of the cells signifies that new thymic tissue is being generated. Concentration of IL-7 is periodically monitored to determine the level of tissue production and function over time. 2. Facteur Thymique Serique (FTS) FTS or thymulin is a nonapeptide hormone secreted exclusively by the thymic subcapsular and medullary cells. Found in both early and late stages of T cell differentiation as well as T cell function, FTS also induces expression of several T cell markers, and promotes T cell functions such as allogeneic cytotoxicity, suppressor functions and IL-2 production. FTS titers in children gradually increase with increasing age from 2.69 ± 1.10 at a few days of age to 4.77 ± 0.44 at a few years of age, then gradually decrease to 0.66 + 0.26 at 36 years of age to old age. As the thymus is physiologically under neuroendocrine control, peptide hormones and neuropeptides influence age-related fluctuations in FTS levels. As noted above, impaired hormonal activity has been shown to be associated with age-related thymic atrophy. In another embodiment of the present disclosure, the concentration of FTS in a patient's blood or serum is monitored before and after administration of the MTS24+ thymic epithelial progenitor cells. Rise in the concentration of FTS within 2-3 days, within 24 hours, or within 2-3 hours, of administration of the cells signifies that new thymic tissue is being generated. Concentration of FTS is periodically monitored to determine the level of tissue production and function over time. 3. Thymosin And Thymopoietin

In contrast to FTS which begins to decline after 20 years of age in humans, thymosin-alpha 1 and thymopoietin serum levels seem to decline as early as 10 years of age. In a further embodiment of the invention, the concentration of thymopoietin, thymosin-alpha 1, thymosin-beta 4, or combinations thereof are measured before and after administration of the MTS24+ thymic epithelial progenitor cells. Rise in the concentration of any of these compounds or combinations within 2-3 days, within 24 hours, or within 2-3 hours of administration of the cells signifies the generation of new thymic tissue. Concentration of any of these compounds or combinations is periodically monitored to determine the level of tissue production and function over time.

Newly Identified Markers In addition to the known markers for thymic activity, several additional markers have been identified and used, based on the methods of the present disclosure.

Procedures for obtaining these markers can mimic those for following the already identified markers. For example, 2D gel electrophoresis can be used and the intensity of the various spots monitored over time. The spots will usually correspond to individual proteins, although occasionally there may be overlap or concurrence of spots from two or more different proteins. The identity of the molecules is revealed by solid phase amino acid sequencing. A new molecule(s) so identified as being altered in expression (increase or decrease) as a result of thymic tissue generation will form the basis of a new diagnostic test for thymic responsiveness. T Cell Analysis

Monitoring of T cell production is another method that may be used to determine generation of new thymic tissue. Techniques such as flow cytometric analysis of whole peripheral blood, detection of proliferating cells by monitoring the marker Ki67, and TREC analysis are among the methods known to those of skill in the field for such monitoring. In an embodiment of the invention, numbers of T cells, as well as proliferating T cells, are determined before and after administration of the MTS24+ thymic epithelial progenitor cells. Rise in the number of any of these T cells or combinations within 2-3 days, within 24 hours, or within 2-3 hours of administration of the cells signifies the generation of new thymic tissue. Concentration of any of these T cells or combinations is periodically monitored to determine the level of tissue production and function over time.

Cell Isolation For the preparation of substantially pure MTS24+ thymic epithelial progenitor cells, a subset of progenitor cells is separated from other cells on the basis of MTS24 binding. Progenitor cells may be further separated by binding to other surface markers such as MHCII+. Other characteristics of the MTS24+ thymic epithelial progenitor cells of the present invention are that they lack of expression of at least some mature epithelial cell markers. Furthermore, the MTS24+ thymic epithelial progenitor cells of the present invention also express at least some cortex markers (eg 4F1, CDR 1, LY51, or 6C3) and/or medullary markers (eg UEA-1).

Procedures for separation may include magnetic separation, using antibody- coated magnetic beads, affinity chromatography and "panning" with antibody attached to a solid matrix, e.g. plate, or other convenient technique. Techniques providing accurate separation include fluorescence activated cell sorters, which can have varying degrees of sophistication, such as multiple color channels, low angle and obtuse light scattering detecting channels, impedance channels, etc. Dead cells may be eliminated by selection with dyes associated with dead cells (propidium iodide [PI], LDS). Any technique may be employed which is not unduly detrimental to the viability of the selected cells.

Conveniently, the antibodies are conjugated with labels to allow for ease of separation of the particular cell type, e.g. magnetic beads; biotin, which binds with high affinity to avidin or streptavidin; fluorochromes, which can be used with a fluorescence activated cell sorter; haptens; and the like. Multi-color analyses may be employed with the FACS or in a combination of immunomagnetic separation and flow cytometry. Multi-color analysis is of interest for the separation of cells based on multiple surface antigens. Fluorochromes which find use in a multi-color analysis include phycobiliproteins, e.g. phycoerythrin and allophycocyanins; fluorescein and Texas red. A negative designation indicates that the level of staining is at or below the brightness of an isotype matched negative control. A dim designation indicates that the level of staining may be near the level of a negative stain, but may also be brighter than an isotype matched control.

In one embodiment, the MTS24 antibody is directly or indirectly conjugated to a magnetic reagent, such as a superparamagnetic microparticle (microparticle). Direct conjugation to a magnetic particle is achieved by use of various chemical linking groups, as known in the art. Antibody can be coupled to the microparticles through side chain amino or sufhydryl groups and heterofunctional cross-linking reagents. A large number of heterofunctional compounds are available for linking to entities. A preferred linking group is 3-(2-pyridyidithio)propionic acid N-hydroxysuccinimide ester (SPDP) or 4-(N-maleimidomethyl)-cyclohexane-l -carboxylic acid N-hydroxysuccinimide ester (SMCC) with a reactive sulfhydryl group on the antibody and a reactive amino group on the magnetic particle.

Alternatively, MTS24 antibody is indirectly coupled to the magnetic particles. The antibody is directly conjugated to a hapten, and hapten-specific, second stage antibodies are conjugated to the particles. Suitable haptens include digoxin, digoxigenin, FITC, dinitrophenyl, nitrophenyl, avidin, biotin, etc. Methods for conjugation of the hapten to a protein, i.e. are known in the art, and kits for such conjugations are commercially available.

To practice the method, the MTS24 antibody is added to a cell sample. The amount of Ab necessary to bind a particular cell subset is empirically determined by performing a test separation and analysis. The cells and MTS24 antibody are incubated for a period of time sufficient for complexes to form, usually at least about 5 min, more usually at least about 10 min, and usually not more than one hr, more usually not more than about 30 min. The cells may additionally be incubated with antibodies or binding molecules specific for cell surface markers known to be present or absent on thymic progenitor cells.

The labeled cells are separated in accordance with the specific antibody preparation. Fluorochrome labeled antibodies are useful for FACS separation, magnetic particles for immunomagnetic selection, particularly high gradient magnetic selection (HGMS), etc. Exemplary magnetic separation devices are described in WO 90/07380, PCT/US96/00953, and EP 438,520.

The purified cell population may be collected in any appropriate medium. Various media are commercially available and may be used, including Dulbecco's Modified Eagle Medium (dMEM), Hank's Basic Salt Solution (HBSS), Dulbecco's phosphate buffered saline (dPBS), RPMI, Iscove's modified Dulbecco's medium (IMDM), phosphate buffered saline (PBS) with 5 mM EDTA, etc., frequently supplemented with fetal calf serum (FCS), bovine serum albumin (BSA), human serum albumin (HSA), as well as media described herein etc. Populations highly enriched for progenitor cells are achieved in this manner.

The desired cells will be 30% or more of the cell composition, preferably 50% or more of the cell population, more preferably 90% or more of the cell population, and most preferably 95% or more (substantially pure) of the cell population.

An isolated/substantially pure MTS24+ thymic epithelial progenitor cells obtained in accordance with the present invention may be allowed or caused to differentiate into a mature thymic epithelial cell-type, e.g. into a cortical or medullary thymic epithelial cell. Such differentiation may occur in vitro or in vivo. Such progeny of the cells of the present invention may also be used in the methods of disease treatment or prevention described herein. For use in the methods herein, MTS24+ thymic epithelial progenitor cells may be obtained from the thymus of the patient to be treated and expanded in vitro before re-administration. Alternatively, an MTS24+ thymic epithelial progenitor cell line can be established using known techniques and these cells used. In a further embodiment, MTS24+ thymic epithelial progenitor cells within a patient may be expanded in vivo by the administration of suitable growth factors etc.

Other cells for use in the present invention, for instance administering hematopoietic stem cells (HSC) and/or bone marrow cells, can be isolated using techniques known in the art.

MTS24+ Thymic Epithelial Progenitor Cell Compositions and Methods for the

Administration Thereof

The thymic epithelial progenitor cells according to this invention can be supplied in the form of a pharmaceutical composition, comprising an isotonic excipient/carrier (such as water, phosphate buffered saline, or saline) prepared under sufficiently sterile conditions for human administration. For general principles in medicinal formulation, the reader is referred to Cell Therapy: Stem Cell Transplantation, Gene Therapy, and Cellular Immunotherapy, by G. Morstyn & W. Sheridan eds, Cambridge University Press, 1996; and Hematopoietic Stem Cell Therapy, E. D. Ball, J. Lister & P. Law, Churchill Livingstone, 2000.

A "therapeutically beneficial amount" of the cells of the invention is a quantity sufficient to enhance thymic function in a subject.

The cells of the invention can be, for example, transplanted or placed at any suitable site in an animal. In one embodiment, the MTS24+ thymic epithelial progenitor cells are administered by a subcutaneous injection. In another embodiment, the MTS24+ thymic epithelial progenitor cells are administered to the thymus. In a further embodiment, the MTS24+ thymic epithelial progenitor cells are transplanted directly into the kidney capsule.

In addition, routes of administration of the cells of the invention, or when cells of the invention are admixed with pharmaceutical carriers, encompassed by the present invention include, for example, intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous routes.

The cells of the invention can be administered alone or as admixtures with conventional excipients, for example, pharmaceutically, or physiologically, acceptable organic, or inorganic carrier substances suitable for enteral or parenteral application which do not deleteriously react with the cells of the invention. Suitable pharmaceutically acceptable carriers include water, salt solutions (such as Ringer's solution), alcohols, oils, gelatins and carbohydrates such as lactose, amylose or starch, fatty acid esters, hydroxymethycellulose, and polyvinyl pyrolidine. Such preparations can be sterilized and, if desired, mixed with auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, and/or aromatic substances and the like which do not deleteriously react with the cells of the invention.

When parenteral application is needed or desired, particularly suitable admixtures for the cells are injectable, sterile solutions, preferably oily or aqueous solutions, as well as suspensions, emulsions, or implants, including suppositories. In particular, carriers for parenteral administration include aqueous solutions of dextrose, saline, pure water, ethanol, glycerol, propylene glycol, peanut oil, sesame oil and polyoxyethylene-block polymers. Pharmaceutical admixtures suitable for use in the present invention are well-known to those of skill in the art and are described, for example, in Pharmaceutical Sciences (17th Ed., Mack Pub. Co., Easton, Pa.) and WO 96/05309 the teachings of both of which are hereby incorporated by reference. The dosage and frequency (single or multiple doses) of the administration or transplantation of the cells to a human, including the actual number of cells transplanted into the human, can vary depending upon a variety of factors, including the particular condition being treated, size, age, sex, health, body weight, body mass index, diet of the human, nature and extent of symptoms of the subject being treated or other health-related problems. In one embodiment, approximately 10⁶ to 10⁷ MTS24+ thymic epithelial progenitor cells are administered.

MTS24+ thymic epithelial progenitor cells can be cultured in vitro using any technique known in the art, such as those described herein as well as that described by Gray et al¹⁹. MTS24+ thymic epithelial progenitor cells may be maintained in vitro by seeding cells into: (i) wells coated with extra cellular matrix gel; (ii) uncoated wells; (iii) wells coated with defined extracellular matrix components (e.g. laminin, collagen); (iv) wells coated with gelatin; or wells coated with irradiated feeder cells, e.g. irradiated mesenchymal cells. In vitro culturing may be aided by immortalizing the cells by genetically modifying the cells to produce a suitable oncogene.

It is preferred that the MTS24+ thymic epithelial progenitor cells are re- aggregated prior to administration into a subject. In one embodiment, the cells are re- aggregated in a "hanging drop" suspension as is known in the art. In another embodiment, the cells are adhered to a suitable matrix. Such matrices are known in the art and can be of biological or synthetic origin.

EXAMPLES

MATERIALS AND METHODS Animals and tissues Four-week-old CBA X C57BL/6 mice were used as a source of thymic stromal cells. C57BL/6 Ly5.1 mice at 4 weeks of age were used as graft recipients. Embryonic material was obtained from CBA X C57BL/6 FI, or C57BL/6 Ly5.2 mouse embryos at indicated gestational ages. Timed mating was conducted and the day of gestation was calculated by the morning of the plug date (= d 0.5). Animals were bred and housed at Monash University Central Animal Services according to institute guidelines.

Antibodies and immunoconjugates

The mAbs used for flow cytometric and immunohistologic analysis were commercially obtained or purified and conjugated in our laboratory; rat mAb anti- CD31 (clone MTS12), rat mAb MTS16 (recognizing ECM), rat mAb MTS24

(described by Blackburn et al.¹⁵), purified rat IgG2a (Pharmingen, USA), biotinylated UEAl lectin (Vector, USA), polyclonal rabbit anti-mouse keratin (DAKO, USA), biotinylated mouse mAb anti-cytokeratin 8 (Progen, Australia), rabbit polyclonal anti- cytokeratin 5 (Covance, USA), FITC-conjugated rat anti-Ly51 (clone 6C3, Pharmingen, USA), FITC-conjugated rat mAb anti-CD45.1 (Pharmingen, USA), PE- conjugated rat mAb anti-CD45 (Pharmingen, USA), PE- conjugated mAb anti-MHCII I-A^b/I-A^e (Pharmingen, USA), APC-conjugated rat mAb anti-CD8 (Pharmingen, USA), APC-conjugated rat mAb anti-CD45 (Pharmingen, USA), PE-conjugated rat mAb anti- CD4 (Pharmingen, USA), PE-conjugated rat mAb anti-CD25 (Pharmingen, USA), biotinylated rat mAb anti-CD44 (Pharmingen, USA), PE-conjugated rat mAb anti- TCRVβ2 (Pharmingen, USA), PE-conjugated rat mAb anti-TCRVβ5 (Pharmingen, USA), PE-conjugated rat mAb anti-TCRVβ6 (Pharmingen, USA), PE-conjugated rat mAb anti-TCRVβ8 (Pharmingen, USA), PE-conjugated rat mAb anti-TCRVβlO (Pharmingen, USA) . Secondary reagents used were PE-conjugated goat polyclonal anti-rat IgG (Southern Biotechnologies, USA), biotinylated rabbit anti-rat IgG (Vector, USA), APC-conjugated goat-anti rat IgG (Caltag, USA), Alexa-568-conjugated goat anti-rabbit IgG (Molecular Probes, USA), Alexa-488-conjugated goat anti-rat IgG (Molecular Probes, USA), PerCP-conjugated Streptavidin (Pharmingen, USA) CyChrome-conjugated Streptavidin (Pharmingen, USA), Cy5-conjugated Streptavidin, (Amersham, USA), Cy5-conjugated goat anti-rabbit Ig (Amersham, USA).

Western blotting

Mouse tissues was homogenised in buffer A (140mMNaCl, 10 mM TRIS-HCl pH 7.3) at 4°C, and the resultant crude suspension was lysed in buffer A supplemented with 2.5% Tween 20 and protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 5 μg/ml aprotinin) for 30 minutes at 4°C. Non-disrupted cells were pelleted by centrifugation of 500g for 5 minutes and the supernatant was subsequently used to remove the nuclei by centrifugation at 10,000g. The remaining supernatant was further centrifuged at 90,000g for 1 hour. The pellet containing the crude membrane fraction was extracted with 0.5% Triton X-100 in buffer A. Solubilized samples were boiled in reducing or non-reducing sample buffer and resolved by 10% SDS-PAGE. Subsequently, proteins were immunoblotted from gels to PDVF membrane (Millipore, USA). The membranes were blocked using 5% skim milk powder prior to exposure to primary antibodies, and subsequently to secondary antibodies coupled to horseradish peroxidase. Peroxidase activity was developed using an enhanced chemiluminescence kit (ECL⁺, Amersham, USA). Neuraminidase digestion

For digestion of MTS24 with neuraminidase, membrane containing pellets of salivary gland extract were solubilized in 50 mM acetate buffer pH 5.5 and treated with 1, 10 or 100 mM neuraminidase from C.perfringens (Boehringer Mannheim, Germany) in for 2 hours at 37°C prior to MW determination as described above.

Immunohistology

Embryos at the indicated ages were embedded in OCT (Miles Laboratories, Australia) and frozen in liquid nitrogen. Sections of 5-20μm were mounted on glass slides and fixed by brief immersion in cold acetone (100%). For immmunohistochemical staining, sections were exposed to PBS/FCS for 10 minutes prior to incubation with primary antibody for 20 minutes at room temperature (RT). Washing in PBS and incubation with the appropriate secondary antibody (20 min, RT) followed this step. Isotype controls were used in all experiments and, where necessary, 10% normal rat sera was employed to block non-specific binding prior to incubation with biotinylated mAbs. Sections were mounted with fluorescent mounting medium (DAKO, USA). Images were captured on a BIO-RAD MRC 1024 confocal microscope using the BIO-Rad LaserSharp v3.2 acquisition software.

Thymic Stromal cell enrichment and analysis

The thymic stromal cell isolation and staining procedures used have been described elsewhere in detail ¹⁹. Briefly, isolated mouse thymi were stripped of any connective tissue and fat and 5-10 small incisions were made in each thymic lobe. The tissue was then slowly stirred for 1 hr at 4 °C in serum free RPM 640. After which remaining tissue aggregates were digested to a single cell suspension by a series of enzymatic digestions for 20 minutes at 37°C with 0.01% (w/v) DNase I and 0.15% (w/v) collagenase D (both Boehringer Mannheim, Germany). Cells from the resultant suspension were washed in cold FACS buffer (5mM EDTA in PBS supplemented with 1%FCS and 0.02% (w/v) NaN3), and subsequently stained as outlined above. Flow cytometric analysis on a FACScalibur used the Cell Quest software (both Becton- Dickinson, USA). Non-viable cells and macrophages, dendritic cells and excess lymphocytes were excluded from the analysis based on their PI incorporation, expression of CD45 and their forward and side scatter characteristics. Data shown represents at least 1 X 10⁴ CD45^" cells within the live gate. For cell separation, cells were prepared as outlined above and sorted using a FACS Star (Becton Dickinson, USA). The cell purity was routinely greater than 98%. Thymic epithelial cell reaggregate cultures and engraftment

Reaggregate cultures were prepared according to a modified protocol previously published ³⁷. Briefly, 2500 thymic epithelial cells were resuspended in a small volume of complete RPMI and placed as a drop on a small fragment of gel foam, (Upjohn, USA) supported on 0.8μm Isopore membrane filter (Gelman Sciences, Ann Arbor, MI) resting on Gelfoam gelatin sponges soaked in fetal thymic organ culture (FTOC) medium; (RPMI supplemented with 10% v/v FCS, 2 mM glutamine, 10 mM HEPES, 0.5 mg/ml folic acid, and 0.2 mg/ml glucose (Flow Laboratories, Australia)). After 24 hours in culture, the solidified reaggregate was grafted under the kidney capsule of congenic mice ⁴¹. Eight weeks post implantation; the transplants were removed and analyzed by flow cytometry or immunohistochemistry.

Fetal thymic organ culture Thymic lobes were removed from embryos at E15.5 of gestation and cultured for 6 days in complete medium supplemented with 1.35 mM 2-deoxyguanosine ⁴⁷. Subsequently, thymus lobes were washed twice by immersion in 50 ml of culture medium for at least 2 h at 37°C, and single lobes placed into individual wells of a Terasaki plate containing 5 x 10⁴ viable El 4.5 fetal liver cells in a final volume of 30 μl of FTOC medium (see above). Thymic lobes were first incubated as hanging drop cultures for 24 h at 37°C and 5% CO₂. Subsequently thymic lobes were placed on polysulfone filters suspended by gelatin sponges, and cultured in FTOC media supplemented either with purified mAb anti-MTS24 or an IgG2a isotype control mAb (both at 200 μg/ml). After 18 days of culture, a single cell suspension was prepared; cells were counted and analyzed by flow cytometry.

RN4 isolation andRT-PCR analysis

Total RNA was isolated from sorted cells using TRI reagent (TVJRC. USA). For the generation of cDNA, RNA was treated with RNAse-free DNasel (Roche Biochemicals, Switzerland) and then reverse transcribed using Superscript II reverse transcriptase with random hexamers as primers (Life Technologies, Scotland). For the PCR reaction, various amounts of cDNA were used with 5 μl 1 X Taq PCR buffer (2mM Mg⁺⁺, 50mMKCL, lOmMTris-HCL, 0.1% gelatin, all from Sigma, Switzerland), 5U/μL Taq (Roche Biochemicals, Switzerland), 250μM dNTP (Roche, Switzerland) and 2μl each primer (Life Technologies, Scotland). The following oligonucleotide pairs were designed from publicly available data bases: Gapdh 5 -ACC ACA GTC CAT GCC ATC AC (SEQ ID NO:l) and 5'-TCC ACC ACC CTG TTG CTG TA (SEQ ID NO:2); KGFR 5'-CAC TCG GGG ATA AAT AGC TC (SEQ ID NO:3) and 5'-GTC CTT CTC TGT GGC ATC AT (SEQ ID NO:4); SCF5'-ATG ATA ACC CTC AAC TAT GTC GCC (SEQ ID NO:5) and 5'-CAC TGA CTC TGG AAT CTT TCT CGG (SEQ ID NO:6); Paxl 5'- AGG CCA CGG ATG CAC TCG GTA G (SEQ ID NO:7) and 5'-AGA TTG GGT CCT TGA AGA ATG C (SEQ ID NO:8); Pax3 5'- TTC GTC TCG CCT TCA CCT GGA TA (SEQ ID NO:9) and 5'- GGT TTG CTG CCG CCG ATG (SEQ ED NO: 10); Pax9 5'- TTG GGC TGG TGT AGG GTA AGG AG (SEQ ID NO: 11) and 5'- GGA TGG CGT GTG CGA CAA GT (SEQ ID NO: 12); Foxnl 5'- TCT ACA ATT TCA TGA CGG AGC ACT (SEQ ID NO: 13) and 5'- TCC ACC TTC TCA AAG CAC TTG TT (SEQ ID NO: 14); Hoxa3 5'- TTC CCT TTT CTC CTC TGC ACC (SEQ ID NO:15) and 5'- GAC AGC CTT TCC AGC AAC CA (SEQ ID NO: 16). The PCR amplification used 30 and 40 cycles for Foxnl and Hoxa3. PCR was performed using the following amplification protocol: 94°C for 5 minutes initially followed by 25- 40 cycles (as indicated) of 30 s at 94°C, 30 s at 60°C and 2 min at 72°C, 1 cycle at 30 s at 72°C for 10 minutes). PCR products were separated on a 1.5% agarose gel, visualized by staining with Sybr Gold (Molecular Probes, USA) and images were analyzed using the Quantity One gel-doc system (BioRad, USA). For data shown in Table 1, the relative amount of the first strand cDNAs produced from each sorted stromal subpopulation was estimated after amplification of a reference (Gapdh) cDNA fragment.

EXAMPLE 1

The MTS24 antigen is an integral membrane mucin-like glycoprotein The mAb MTS24 recognizes an antigen expressed on a rare subset of epithelial cells in the adult thymus as well as the thymic rudiment of the nude mice ¹⁵'²⁸. To characterize the biochemical features of MTS24 antigen, cell membrane fractions isolated from thymus, kidney, uterus, liver and salivary glands were analyzed by SDS- PAGE followed by immunoblotting. Under these conditions, the MTS24 antigen represented a molecule of 80-200 kDa (Figure la). Salivary gland cell membrane extracts digested with neuraminidase (Na), in various concentrations, resulted in a shift in mobility from 80-200 kDa to 80-90 kDa (Figure lb), indicating a large contribution of sialic acid residues to the overall molecular weight. Furthermore, exposure of membrane fractions to proteinase K resulted in an ablation of MTS24 antigen detection. Taken together, these results demonstrate MTS24 antigen to be a glycoprotein with mucin-like characteristics, and an apparent peptide backbone of approximately 80 kDa. EXAMPLE 2

Expression and localisation of the MTS24 antigen during early embryogenesis

To examine the temporal and spatial expression of MTS24 antigen during mouse development, embryos at different stages were investigated by immunohistochemistry (IHC). At day 7.5 of gestation (E7.5), embryonic MTS24 antigen expression was restricted to the presumptive endoderm (foregut) and the caudal mesoderm, but was not detected in cells of ectodermal origin. Strong expression of MTS24 antigen was also observed in the degenerating parietal endoderm (Figure 2a). At E9.5-10.5, expression of MTS24 antigen occurred throughout the pharyngeal endoderm, including the emerging pouches (Figure 2b). Expression was also detected in the intermediate mesoderm of the caudal part of the trunk region, which represents the earliest site of urogenital differentiation (data not shown). Thus, MTS24 antigen was highly expressed during early embryogenesis in a restricted set of anterior endodermal epithelium, pharyngeal endoderm, and a well-defined portion of the intermediate mesoderm destined to develop into urogenital epithelium.

EXAMPLE 3

Relationship between MTS24 antigen expression and thymic epithelium development during thymic organogenesis

To investigate the relationship between MTS24 expression and the phenotypic differentiation of thymic epithelial cell subsets, tissue sections of embryos at distinct developmental stages were analyzed by IHC. MTS24 was co-localized with markers delineating cortical (cytokeratin-8, K8) and medullary (cytokeratin-5, K5) epithelium, respectively ²⁵. At El 0.5, MTS24 expression was restricted to the endodermal cell layers of the pharyngeal pouches and the emerging thymic anlage. The latter structure stained positive for K8, but not for K5 (Figure 2b). Concomitant with thymic colonization by hematopoietic precursors at El 1.5 (¹⁴ and Figure 3b), the K5 marker was now detected on a rare population of MTS24⁺K8⁺ cells within the developing anlage. A day later, the epithelial cells had formed a meshwork structure interspersed by the expanding population of CD45⁺ hematopoietic cells. The epithelium remained positive for both MTS24 and K8 but a substantial number of these cells also co- expressed K5 (Figures 3a). In contrast, MTS24 antigen expression had became spatially restricted at E15.5 to a rare subset of TECs that co-expressed both K8 and K5 and that were located in the emerging medulla (Figure 3). The relative frequency of the MTS24⁺ did not diminish upon further development (Figure 3). In 4 week and older mice, TECs expressing concomitantly MTS24 and markers of both cortical and medullary epithelium resided in the medullary compartment of the thymus

To further characterize expression of MTS24 during thymus development, epithelial cells were isolated at different maturational stages and analyzed by flow cytometry. At E12.5, approximately half of the non-hematopoietic (i.e. CD45-) cells expressed MTS24 antigen (Figure 4a). Of these, approximately 7% co-expressed intermediate to high cell surface concentrations of MHC class II (MHCII) molecules. Concurrent with an expansion of thymocytes at E15.5, the frequency of CD45^' cells diminished to 24% and MHCH⁺ TEC now accounted for 53% of non-hematopoietic stromal cells. At this point TEC expressing the MTS24 antigen had decreased proportionally, comprising only 25% of the CD45^" cells. Notwithstanding a decreased MHCII cell surface expression in 4 week-old mice when compared to embryonic tissue at E15.5 (MFI of 750 versus 1420, Figure 4a), MHCII^VITS24⁺ cells remained detectable at a similar frequency (27%) through all time points examined beyond E15.5.

The cell surface expression of the cortical epithelial marker Ly51 and binding of the lectin UEAl as a medullary marker were next used to further characterize MTS24⁺ epithelial cells ¹⁹'²². At E14.5 and E15.5 of gestation, all MTS24⁺ thymic epithelium co- expressed Ly51, but only 1% and 5% of these cells, respectively, bound to UEAl lectin. The relative frequency of these "double positive" cells remained at 5% in adult thymus (Figure 4b). Thus, MTS24⁺, but not MTS24^" epithelial cells contain in both embryonic and 4-week-old thymus, a rare population that concomitantly express markers of both cortical and medullary mature thymic epithelium.

EXAMPLE 4

The role oftheMTS24 antigen in thymopoiesis

To test for the functional role of the MTS24 antigen in thymus development purified anti-MTS24 mAbs or isotype control mAbs were added to E15.5 fetal thymic organ cultures (FTOC) depleted of lymphoid cells and subsequently reconstituted with hematopoietic precursors. Analysis by flow cytometry at the end of an 18-day culture period revealed 8-fold fewer thymocytes in cultures treated with anti-MTS24 mAbs when compared to control cultures (35.7+8.3 and 4.3±1.8 xlθ4 thymocytes/lobe respectively; n=6). Thymocytes from anti-MTS24-treated cultures were mostly of a CD4 CD8- phenotype while the CD4⁺CD8⁺, CD4⁺CD8^' and CD4^"CD8⁺ cells were severely reduced or absent indicative of a block in early T cell development. Moreover the total number of CD4^"CD8^" cells in cultures exposed to anti-MTS24 mAb was less than 40% of that of isotype mAbs treated cultures (Figure 5).

EXAMPLE 5 MTS24⁺ thymic epithelial cells transcribed gene products necessary for normal thymic organogenesis

To characterize MTS24⁺ TECs at a molecular level, epithelial cells from El 5.5 thymi were fractionated by flow cytometry according to their MTS24 cell surface phenotype and analyzed by RT-PCR for gene transcripts known to be of primary importance in thymus organogenesis. Transcripts for Foxnl, Hoxa3, Paxl, SCF, FgfR2πib and KGFR were present in both MTS24⁺ and MTS24^" TEC, but the expression of Pax3 and Pax9 could not be detected (data not shown). MTS24⁺ cells thus harboured gene transcripts previously shown to be associated with regular thymus organogenesis. There were, however, no major differences in their expression profiles between MTS24⁺ and MTS24^" epithelial cells at this age.

EXAMPLE 6

MTS24⁺ thymic epithelial cells contain epithelial precursors competent to generate a functional microenvironment Next the in vivo capacity of the population of MTS24⁺ cells was tested as a source of precursor cells competent to reconstitute the thymic epithelial compartment. To this end, El 5.5 TECs were purified and separated by flow cytometry into two populations, i.e. CD457vIHCrf TS24^" and CD45^"MHCJΪMTS24⁺ (Figure 6a). Sorted populations were reaggregated, for 24 hours in vitro in the absence of other cell types and subsequently transplanted under the kidney capsule of congenic recipient mice. Eight weeks later, an ectopic thymus could only be detected in recipients of MTS24⁺ epithelial cell aggregates (Figure 6b). Compared either to the endogenous thymus or to a transplanted, age-matched lobe used as control, thymocytes derived from the MTS24⁺ grafts displayed a normal subset distribution as defined by CD4 and CD8 and by CD44 and CD25 expression, respectively, when compared to normal thymic tissue (Figure 6 c and d). Furthermore, TCR Nβ repertoire usage (Nβ 2, 5, 6, 8 and 10) on among mature T cells was identical between all thymi (Figure 6e and data not shown). Thus, CD45^" MHCπ^'1MTS24⁺, but not CD45TVIHCπ MTS24^" cell aggregates formed an epithelial stromal compartment able to support normal thymopoiesis. To assess the architectural organization and cellular composition of the thymic epithelial compartment formed by the transplanted MTS24⁺ cells, the engrafted tissue was phenotypically analyzed by immunohistology using a broad panel of antibodies. This clearly demonstrated an outer cortex densely populated by thymocytes and a distinct inner medulla. The normal differentiation of epithelial cells and their compartmentalization into cortical and medullary subpopulations was determined by staining for cortical (Ly51) and medullary markers (K5, MTS10), respectively. The cellular meshwork of the separate cortical and medullary compartments was identical for the MTS24⁺ cell-derived grafts and the transplanted, age-matched lobes used as controls (Figure 7). A restricted expression of the MTS24 antigen and a typical distribution of extracellular matrix (MTS16) and vasculature (CD31) complemented this regular architectural organization of epithelial cells (Figure 7e-h). Moreover, the expression of MHCII and the distribution of vascular-related fibroblasts were also the same between the different thymic samples (data not shown). Taken together, these grafting experiments revealed that CD45 HCLI⁺MTS24⁺, but not CD45^" MHCII⁺MTS24^" TECs encompass a precursor potential to establish and maintain a complete thymic epithelial microenvironment. This cell aggregate attracted exogenous hematopoietic precursors and supported subsequent T cell development indistinguishable from that of age-matched controls.

EXAMPLE 7 T Cell Depletion

In order to prevent interference with a graft by the existing T cells in the potential graft recipient patient, the patient is subjected to T cell depletion. One standard procedure for this step is as follows. The human patient receives anti-T cell antibodies in the form of a daily injection of 15mg/kg of Atgam (xeno anti-T cell globulin, Pharmacia Upjohn) for a period of 10 days in combination with an inhibitor of T cell activation, cyclosporin A, 3mg/kg, as a continuous infusion for 3-4 weeks followed by daily tablets at 9mg/kg as needed. This treatment does not affect early T cell development in the patient's thymus, as the amount of antibody necessary to have such an affect cannot be delivered due to the size and configuration of the human thymus. The treatment is maintained for approximately 4-6 weeks.

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. The generation of new thymic tissue following administration of MTS+ thymic epithelial progenitor cells to the thymus would be linked to injection of donor HSC (obtained at the same time as the organ or tissue in question either from blood - pre-mobilized from the blood with G-CSF (2 intradermal injections/day for 3 days)) or collected directly from the bone marrow of the donor. The enhanced levels of circulating HSC would promote uptake by the new thymic tissue. These donor HSC would develop into intrathymic dendritic cells and cause deletion of any newly formed T cells which by chance would be "donor-reactive". This would establish central tolerance to the donor cells and tissues and thereby prevent or greatly minimize any rejection by the host. The development of a new repertoire of T cells would also overcome the immunodeficiency caused by the T cell-depletion regime.

The depletion of peripheral T cells minimizes the risk of graft rejection because it depletes non-specifically all T cells including those potentially reactive against a foreign donor. Simultaneously, however, because of the lack of T cells the procedure induces a state of generalized immunodeficiency which means that the patient is highly susceptible to infection, particularly viral infection. Even B cell responses will not function normally in the absence of appropriate T cell help.

EXAMPLE 8

Administration of Donor Cells to Create Tolerance

Where practical, the level of hematopoietic stem cells (HSC) in the donor blood is enhanced by injecting into the donor granulocyte-colony stimulating factor (G-CSF) at lOμg/kg for 2-5 days prior to cell collection (e.g., one or two injections of lOμg/kg per day for each of 2-5 days). CD34⁺ donor cells are purified from the donor blood or bone marrow, for example, 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). 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 LJF to prevent differentiation into specific cell types. The patient is injected with the donor HSC and MTS24+ thymic epithelial progenitor cells, optimally at a dose of about 2-4 x 10⁶ cells/kg. G-CSF may also be injected into the recipient to assist in expansion of the donor HSC. It may be necessary to give a second dose of HSC 2-3 weeks later to assist in the thymic tissue development and the development of donor DC (particularly in the thymus). Once the HSC have engraftment (incorporated into the bone marrow (and new thymic tissue), the effects should be permanent since the HSC are self-renewing in the new thymic tissue. The new thymic tissue takes up the purified HSC and converts them into donor- type T cells and dendritic cells, while converting the recipient's HSC into recipient- type T cells and dendritic cells. By inducing deletion by cell death, or by inducing tolerance through immunoregulatory cells, the donor and host dendritic cells will tolerize any new T cells that are potentially reactive with donor or recipient.

EXAMPLE 9

Transplantation of Graft

In one embodiment of the invention, while the recipient is still undergoing continuous T cell depletion immunosuppressive therapy, an organ, tissue, or group of cells that has been at least partly depleted of donor T cells is transplanted from the donor to the recipient patient. New thymic tissue can be produced using the methods of the invention and infiltrated by exogenous HSC.

In order to allow production of a stable chimera of host and donor hematopoietic cells, immunosuppressive therapy may be maintained for about 3-4 months. The new T cells will be purged of potentially donor reactive and host reactive cells, due to the presence of both donor and host DC in the new thymic tissue. Having been positively selected by the host thymic epithelium, the T cells will retain the ability to respond to normal infections by recognizing peptides presented by host APC in the peripheral blood of the recipient. The incorporation of donor dendritic cells into the recipient's lymphoid organs establishes an immune system situation virtually identical to that of the host alone, other than the tolerance of donor cells, tissue and organs. Hence, normal immunoregulatory mechanisms are present. These may also include the development of regulatory T cells which switch on or off immune responses using cytokines such as IL4, 5, 10, TGF-beta, TNFalpha.

EXAMPLE 10

Induction of Tolerance in Humans

A human patient requiring a skin or organ transplant is administered with MTS24+ thymic epithelial progenitor cells. The patient is given an intravenous injection of CD34+ cells collected from the peripheral blood of an allogeneic donor. To collect the CD34+ cells, peripheral blood of the donor (i.e., the person who will be donating his/her organ or skin to the recipient) is collected, and CD34+ cells isolated from the peripheral blood according to standard methods. One non-limiting method is to incubate the peripheral blood with an antibody that specifically binds to human CD34 (e.g., a murine monoclonal anti-human CD34+ antibody commercially available from Abeam Ltd., Cambridge, UK), secondarily stain the cells with a detectably labeled anti-murine antibody (e.g., a FITC-labeled goat anti-mouse antibody), and isolate the FITC-labeled CD34+ cells through fluorescent activated cell sorting (FACS). Because of the low number of CD34+ cells found in circulating peripheral blood, multiple collection and cell sorting may be required from the donor. The CD34+ may be cryopreserved until used to reconstitute the recipient patient. In one example, at least 5xl0⁵ HSC per kg body weight are administered to the recipient patient.

The recipient patient will be monitored to detect the presence of donor blood and dendritic cells in his/her peripheral blood. When such donor cells are detected, the transplantation of the donor tissue (t.e., skin and/or organ) is made. The donor tissue is accepted by the recipient to a greater degree (t.e., survives longer in the recipient) than in a recipient who had not had new thymic tissue generated by MTS24+ thymic epithelial progenitor cells and had not been reconstituted with donor CD34+ cells.

EXAMPLE 11 Treatment of a Patient with Pernicious Anemia

An adult (e.g., 35 years old) human female patient suffering from pernicious anaemia, an autoimmune disease has her CD34+ hematopoietic stem cells (HSC) recruited from her blood following 3 days of G-CSF treatment (2 injections /day, for 3 days, lOμg/kg). Her HSC can be purified from her blood using CD34. To collect the CD34+ cells, peripheral blood of the donor (i.e., the person who will be donating his/her organ or skin to the recipient) is collected, and CD34+ cells isolated from the peripheral blood according to standard methods. One non-limiting method is to incubate the peripheral blood with an antibody that specifically binds to human CD34 (e.g., a murine monoclonal anti-human CD34+ antibody commercially available from Abeam Ltd., Cambridge, UK), secondarily stain the cells with a detectably labeled anti- murine antibody (e.g., a FITC-labeled goat anti-mouse antibody), and isolate the FITC- labeled CD34+ cells through fluorescent activated cell sorting (FACS). Because of the low number of CD34+ cells found in circulating peripheral blood, multiple collection and cell sorting may be required from the donor. The CD34+ may be cryopreserved until enough are collected for use.

Because the antigen for pernicious anaemia, the patient's collected HSC are transfected by any means to express the antigen (namely, the gastric proton pump). HSC can be transfected by using a variety of techniques including, without limitation, electroporation, viral vectors, laser-based pressure wave technology, lipid-fusion (see, e.g., the methods described in reference ⁴). In one example, her HSC are transfected with the β chain of the H/K-ATPase proton pump, using the MHC class II promoter for the expression. To stop the ongoing autoimmune disease, the patient will to undergo T cell depletion. She will also undergo the generation of new thymic tissue to provide a suitable microenvironment to replace these T cells and hence overcome the immunodeficiency state. To do this, she will receive MTS24+ thymic epithelial progenitor cells which will produce new thymic tissue. This will also allow uptake of the HSC and to establish central tolerance to the autoantigen in question. It is not clear why autoimmune disease starts but cross-reaction to a microorganism is a likely possibility; depleting all T cells will thus remove these cross-reactive cells. If the disease was initiated by such cross-reaction if may not be necessary to transfect the HSC with the nominal autoantigen.

EXAMPLE 12

Treatment of a Patient with Type I Diabetes

A similar approach to that described in Example 11 is undertaken with a patient with Type I diabetes. The T cells will be removed by broad-based depletion methods (see above), new thymic tissue is generated by the administration of MTS24+ thymic epithelial progenitor cells, and the patient's immune system recovery enhanced by injection of pre-collected autologous HSC transfected with the pro-insulin gene using the MHC class II promoter. The HSC will enter the new thymic tissue, differentiate into DC (and all thymocytes), and present pro-insulin to the developing T cells. All those potentially reactive to the pro-insulin will be killed by apoptosis, leaving a repertoire free to attack foreign infections agents.

In the case that the autoimmune disease arose as a cross-reaction to an infection or simply "bad luck" it would be sufficient to use autologous HSC to help boost the thymic regrowth. If there is a genetic predisposition to the disease (family members can often get autoimmune disease) the thymic tissue recovery would be best performed with allogeneic highly purified HSC to prevent graft versus host reaction through passenger T cells. Umbilical cord blood is also a good source of HSC and there are generally no or very few alloreactive T cells. Although cord blood does not have high levels of CD34+ HSC, they may be sufficient for establishment of a microchimera - even ~10% of the blood cells being eventually (after 4-6 weeks) could be sufficient to establish tolerance to the autoantigen with sufficient intrathymic dendritic cells.

EXAMPLE 13

Treatment of a Patient Suffering from Allergies

In the case of allergy, a similar principle would be undertaken as for as autoimmune disease. The allergic patient would be depleted of T cells as above. In severe cases where there is exaccerbaton through IgE or IgG producing B cells (plasma cells) it may be necessary to use myeloablation as for chemotherapy. Alternatively, whole body irradiation may be used (eg 6 Gy). The entire immune system would be rejuvenated by the use MTS24+ thymic epithelial progenitor cells and injection intravenously of the HSC (allogeneic or auologous as appropriate). Allogeneic would be used in the case of genetic disposition to allergy but otherwise mobilized autologous HSC would be used.

EXAMPLE 14 General Discussion

The broad heterogeneity amongst thymic epithelial cells has been established for many years ^19"23; despite this, the precise developmental origin and the lineage relationships between precursor cells and the distinct thymic epithelial cell subsets have remained controversial. In particular there has been no means of identifying thymic epithelial progenitor cells, which is crucial for addressing these issues so critical for thymic organogenesis. Here we demonstrate that the differential expression of the glycoprotein MTS24, exclusively defines a subpopulation of embryonic thymic epithelial cells. This population possesses the progenitor capacity for forming an entire epithelial microenvironment capable of supporting thymocyte development. Detectable at different stages of development, the MTS24 antigen was expressed as early as E10.5 on the endodermal epithelium lining the 3^rd pharyngeal pouch, and by El 5.5 was restricted to a rare subpopulation of TEC concurrently expressing markers for both cortical and medullary epithelium. Importantly, engraftment experiments showed that the MTS24⁺, but not the MTS24^" TEC population, was both necessary and sufficient to generate structurally and functionally normal cortical and medullary thymic epithelial compartments. These microenvironments are proficient to fully support normal thymopoiesis with all defined T cell subsets present in the predicted proportions including a normal repertoire of TCRβ chain usage on mature CD4⁺ and CD8⁺ cells.

Tissue resident epithelial stem cells are characterized by their capacity for self- renewal and their potential to differentiate into morphologically and functionally different cell lineages. While epithelial stem cells have been isolated in a few selected organs, such as skin and gastrointestinal tract (reviewed in reference ³²), their counterpart in the thymus has not been identified, even though a clonal precursor- progeny relationship has recently been demonstrated for individual medullary compartments ²⁷. The experimental evidence provided now demonstrates that the presumptive thymic epithelial stem cell is included within the population of MTS24⁺ TEC. Importantly, the MTS24⁺ cells taken from El 5.5 thymi for grafting contain rare cells, which also co-express cytokeratins5 and 8, i.e. markers exclusively associated in the adult thymus with medullary and cortical epithelium, respectively. Thus, these "triple positive" cells at stages beyond E12.5 could include TEC that represent an intermediate cellular stage between earlier precursors and defined compartment- specific epithelium, and represent, by analogy to other organ systems transit amplifying cells. Clearly the MTS24⁺ despite exclusively containing the progenitor population and being uniformly MHCH ^", are still heterogenous to some degree, with approximately 5% being UEA-1⁺ and Ly51⁺. Given the fact that these two determinants are often used to define medullary and cortical epithelium respectively, this could imply a "triple positive progenitor epithelial cell". Although as we have recently shown UEA-1 also binds to a subpopulation of thymic epithelial cells expressing the putative transcription factor Aire ³³ as well as thymic vascular endothelium¹⁹. Thymic epithelial cells expressing markers of both the cortex and medulla have previously been proposed to be putative precursors ²⁴ but in the absence of any functional data this remains speculative. The rare subset of MTS24⁺UEA-1⁺/Ly51⁺ identified herein may, however, constitute such a cell.

While numerous phenotypic studies have clearly identified heterogeneity within the thymic stromal compartments, there has been no prior description of progenitor- type epithelial cells, primarily due to the lack of known cell surface markers typical for these cells. These phenotypic studies have assisted in the assignment of function to thymic stromal cell subsets. Mesenchymal cells are essential for early stages of thymic organogenesis ³⁴ and in concert with epithelium are important for early stage thymocyte development ⁷'^35,36. Purified epithelial cells, however, have not been able to form a complete thymic microenvironment in the absence of mesenchymal cells . In our study, the presumption is that fibroblasts from either the kidney capsule or from the renal interstitium provide the necessary inductive signals for the progression of progenitor cells within the MTS24⁺ population of TECs to phenotypically and functionally mature epithelium. Cortical epithelium supports the positive-selection mediated transition from immature CD4⁺CD8⁺ thymocytes ³⁸'³⁹-⁴⁰' and medullary epithelium, presumably at a later stage thymocyte maturation, some forms of tolerance induction ⁴¹'⁴².

It is noteworthy that the genetic profiling of MTS24⁺ TEC revealed that these cells expressed gene products that have previously been shown to be critical for thymus organogenesis (reviewed in ^1,s). The continued expression of the selected transcription factors may argue that at least some of these molecules play a significant role in the capacity of the MTS24⁺ epithelial progenitor cells to differentiate sufficiently to regenerate an intact thymic epithelial compartment. Interestingly, MTS24^" TEC displayed a similar expression profile for the genes analyzed, consequently none of these gene products account for the unique capacity of MT24⁺ cells to generate a normal thymic microenvironment. Thus the gene products specifically rendering MTS24⁺ cells competent to affect the observed precursor function remain to be defined. While Pax3 and Pax9 have previously been described as essential for growth and differentiation during early thymic epithelial development ^u'¹³, our findings imply, by their absence within the E15.5 MTS24⁺ TEC compartment, expression is not needed for later stages of thymus organogenesis. An important functional role for the MTS24 antigen in thymopoiesis was demonstrated in fetal thymic organ cultures to which anti-MTS24 mAb were added. Under these experimental conditions, T cell development was markedly affected in as much as the antibody inhibited the progression of CD4^"CD8^" thymocytes to more mature stages of development. Two distinct, yet mutually exclusive reasons may account for this observed effect. First, cross-linking of the glycoprotein MTS24 by mAb may directly affect TEC function so that thymocyte development is not further assisted beyond the stage of CD4^"CD8^" cells. Accordingly, the mucin-like glycoprotein CD 164 supports cell-cell and cell-ECM interactions; its cross-linking suppresses cell proliferation in a wide range of tissues ⁴⁵. Alternatively, binding of anti-MTS24 mAb to its specific epitope may block thymocyte interaction with a thymic epithelial cell surface molecule necessary for the maturational progression of double negative (CD4^" CD8^") cells to a more advanced stage in development. A comparable block in early thymocyte differentiation is a consequence of several loss-of-function mutants of surface and signal transduction molecules expressed by thymocytes (reviewed in reference ^4δ). MTS24, however, constitutes the only identified cell surface molecule on thymic epithelial cells, which upon specific binding by an antibody causes an almost complete arrest in early thymopoiesis. This result directly suggests a critical role for MTS24 in the provision of environmental signals necessary for regular thymopoiesis.

It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

All publications discussed above are incorporated herein in their entirety. Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application.

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Claims

1. A method of modifying T cell population makeup or increasing the number of T cells in a subject, the method comprising administering MTS24+ thymic epithelial progenitor cells, wherein the MTS24+ thymic epithelial progenitor cells provide a competent microenvironment for T cell development.

2. The method of claim 1, wherein the T cells are CD4+CD8-, CD4-CD8+ T cells, CD25+CD4 T cells, CD4-CD8- αβTCR T cells, NKT cells or γδTCR T cells.

3. A method for inducing tolerance in a subject to a graft from a mismatched donor comprising the steps of; host T cell ablation to remove pre-existing donor reactive cells, administering MTS24+ thymic epithelial progenitor cells, and transplanting an organ, tissue or cells from a donor to the subject.

4. The method of claim 3, further comprising the step of transplanting bone marrow, or haemopoietic stem cells or lymphoid stem cells or any other stem cell which enter the thymus, from the donor to the subject.

5. A method of preventing or treating an autoimmune disease in a subject, the method comprising the steps of T cell ablation and administering MTS24+ thymic epithelial progenitor cells.

6. A method for preventing or treating an allergy in a subject, the method comprising the steps of T cell ablation and administering MTS24+ thymic epithelial progenitor cells.

7. A method of enhancing an immune response to an antigen in a subject, the method comprising administering MTS24+ thymic epithelial progenitor cells.

8. The method of claim 7, further comprising administering the antigen.

9. The method of claim 7 or claim 8, wherein the subject has an infection.

10. The method of claim 9, wherein the infection is a HIN infection.

11. The method of claim 10, wherein the cells are administered following HAART treatment.

12. A method of preventing or treating cancer in a subject, the method comprising administering MTS24+ thymic epithelial progenitor cells.

13. The method of claim 12, wherein the cells are administered following chemotherapy, radiation therapy or bone marrow transplantation.

14. The method according to any one of claims 1 to 13, wherein the MTS24+ thymic epithelial progenitor cells are genetically modified.

15. The method of claim 14, wherein the genetic modification is selected from the group consisting of: expressing a transgene, and the deletion of at least one endogenous gene of the cell.

16. The method of claim 15, wherein the transgene encodes a molecule selected from the group consisting of: a polypeptide, dsRNA, a catalytic nucleic acid, and an antisense oligonucleotide.

17. The method according to any one of claims 1 to 16, wherein the method further comprises administering a hematopoietic stem cells (HSC) and/or bone marrow cells.

18. The method of claim 17, wherein the hematopoietic stem cells (HSC) and/or bone marrow cells are genetically modified.

19. The method according to any one of claims 1 to 18, wherein the cells are administered by a subcutaneous injection.

20. The method according to any one of claims 1 to 18, wherein the cells are administered to the thymus.

21. The method according to any one of claims 1 to 18, wherein the cells are transplanted directly into the kidney capsule.

22. The method according to any one of claims 1 to 21, wherein the cells are administered as an aggregate.

23. The method according to any one of claims 1 to 21, wherein the cells are seeded on an extracellular prior to administration.

24. The method according to any one of claims 1 to 23, wherein the cells are administered in a composition comprising mesenchymal cells, or an extract thereof.

25. The method according to any one of claims 1 to 24, wherein the cells are administered in a composition comprising at least one epithelial cell growth factor.

26. The method of claim 25, wherein the growth factor is selected from the group consisting of: IL-7, a fibroblast growth factor, and a keratinocyte growth factor.

27. Use of MTS24+ thymic epithelial progenitor cells for the manufacture of a medicament for modifying T cell population makeup or increasing the number of T cells in a subject, wherein the MTS24+ cells provide a competent microenvironment for T cell development.

28. Use of MTS24+ thymic epithelial progenitor cells for the manufacture of a medicament for inducing tolerance in a subject to a graft from a mismatched donor, wherein the subjects T cells have been ablated.

29. Use of MTS24+ thymic epithelial progenitor cells for the manufacture of a medicament for preventing or treating an autoimmune disease in a subject.

30. Use of MTS24+ thymic epithelial progenitor cells for the manufacture of a medicament for preventing or treating an allergy in a subject.

31. Use of MTS24+ thymic epithelial progenitor cells for the manufacture of a medicament for enhancing an immune response to an antigen in a subject.

32. Use of MTS24+ thymic epithelial progenitor cells for the manufacture of a medicament for preventing or treating cancer in a subject.

33. A method of generating thymic tissue, the method comprising culturing, in vitro or in vivo, MTS24+ thymic epithelial progenitor cells under suitable conditions.

34. An isolated thymic epithelial progenitor cell, wherein the cell produces a protein designated MTS24.

35. A composition comprising an thymic epithelial progenitor cell according to claim 34, and a carrier or diluent.

36. A method of purifying a cell according to claim 34, the method comprising; i) contacting a population of cells with an agent that binds MTS24, and ii) separating agent/MTS24 complexes.

37. The method of claim 36, wherein the agent is an antibody.

38. The method of claim 36, wherein the antibody is a monoclonal antibody.