WO2008070861A2 - Methods and compositions for treating systemic lupus erythematosus - Google Patents

Abstract

The present disclosure relates to compositions and method for treating a patient suffering from systemic lupus erythematosus and related conditions, comprising subjecting said patient to myeloablation and administering to said patient hematopoietic stem cells.

Description

METHODS AND COMPOSITIONS FOR TREATING SYSTEMIC LUPUS ERYTHEMATOSUS

1. CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit under 35 U.S.C. § 119(e) of U. S.S.N. 60/869,085, filed December 7, 2006, which is hereby incorporated by reference in its entirety.

2. TECHNICAL FIELD

[0002] The present disclosure relates to compositions and methods for treating conditions associated with autoimmune diseases that are characterized by abnormal production of B cells.

3. INTRODUCTION

[0003] Hematopoietic stem cell transplantation (HSCT) is a standard therapy for various hematological malignancies, and in some cases, is the only viable treatment option, particularly when the disease is refractory to chemotherapy. Bone marrow, peripheral blood, or cord blood serve as typical sources of hematopoietic stem cells (HSCs), but cells from peripheral blood display more rapid engraftment characteristics and may be mobilized by treatment of the donor with cytokines G-CSF, GM-CSF, or with cytoreductive drugs. Cord blood is readily available, and shows lower incidences of graft versus host disease but is characterized by delayed engraftment. Prior to transplantation, the recipient is given myeloablative doses of chemotherapy and/or radiation to treat the underlying disease and make the recipient suitable for engraftment of the donor HSCs.

[0004] Generally, there are two types of HSCT: autologous and allogeneic. Autologous transplantation involves infusion of a recipient's own cells following myeloablative treatment. Autologous cell transplants minimize the risk of graft versus host disease (GVHD) and result in reduced complications. However, since chemotherapy with myeloablative agents is used to eliminate malignant cells in the HSC preparation, autologous transplantation is problematic where the disease is unresponsive to chemotherapy. Allogeneic transplantation involves infusion of donor stem cells, typically using a donor that matches the recipient's major histocompatibility complex (MHC). An advantage of allogeneic transplants is the accompanying cell-mediated graft versus host reaction that may develop against malignant cells. However, matched unrelated donor (MUD) transplants are also associated with a stronger graft versus host reaction, and thus can result in higher mortality rates.

[0005] Systemic lupus erythematosus (SLE) is a multifactorial, polygenic autoimmune disorder, that produces auto-antibodies to nuclear antigens and that causes inflammation of various body tissues. Among the parts of the body affected are the skin, kidneys, heart, lungs, joints, brain and blood vessels. SLE induces symptoms such as fatigue, severe joint and muscle pain, chest pain, fever, rashes, exhaustion and photosensitivity. In a number of cases, lupus can lead to death. In the United States, it is estimated that some form of lupus afflicts at least 1.5 million people, disproportionately affecting women and non-Caucasians. The cause of lupus is unknown, but it is believed that genetic and environmental factors are involved. Susceptibility to lupus is thought to be governed by a combination of genetic factors and environmental stimuli leading to a loss of self tolerance (Harley, J. B., and Kelly, J.A. 2002. Genetic basis of systemic lupus erythematosus: a review of the unique genetic contributions in African Americans. J Natl Med Assoc 94:670-677; Kelly, J.A., Moser, K.L., and Harley, J. B. 2002. The genetics of systemic lupus erythematosus: putting the pieces together. Genes lmmun 3 Suppl 1:S71-85; Tsao, B.P., Cantor, R.M., Grossman, J. M., Kim, S.K., Strong, N., Lau, C. S., Chen, CJ. , Shen, N., Ginzler, E.M., Goldstein, R., et al. 2002. Linkage and interaction of loci on 1q23 and 16q12 may contribute to susceptibility to systemic lupus erythematosus. Arthritis Rheum 46:2928-2936; James, J.A., Neas, B.R., Moser, K.L., Hall, T., Bruner, G.R., Sestak, A.L., and Harley, J. B. 2001. Systemic lupus erythematosus in adults is associated with previous Epstein-Barr virus exposure. Arthritis Rheum 44:1122-1126; James, J.A., Harley, J. B., and Scofield, R.H. 2001. Role of viruses in systemic lupus erythematosus and Sjogren syndrome. Curr Opin Rheumatol 13:370-376).

[0006] SLE is associated with several intrinsic defects of the immune system; auto-reactive B and T cells, loss of suppressor function, impaired clearance of apoptotic bodies by phagocytes, and deficiencies in complement (Mok, CC, and Lau, CS. 2003. Pathogenesis of systemic lupus erythematosus. J Clin Pathol 56:481-490; Gaipl, U.S., Kuhn, A., Sheriff, A., Munoz, LE., Franz, S., VoII, R. E., Kalden, J. R., and Herrmann, M. 2006. Clearance of apoptotic cells in human SLE. Curr Dir Autoimmun 9:173-187; Agnello, V. 1978. Association of systemic lupus erythematosus and SLE-like syndromes with hereditary and acquired complement deficiency states. Arthritis Rheum 21:S146- 152). Recent research suggests that B cell dysfunction and pathogenic autoantibody formation are critical in the pathogenesis of SLE. B cells produce antibodies that normally target foreign pathogens that invade the body. Occasionally, such antibodies fail to distinguish foreign molecules from self- molecules, resulting in a destructive autoimmune disease that can be fatal. Patients suffering from SLE exhibit enhanced autoantibody production that is thought to be due to cytokine levels that are different from those found in non-SLE subjects. Specifically, SLE patients tend to have higher levels of the proinflammatory cytokines TNFα, interleukin-6 (IL-6), interferon-γ (IFN-γ) and IL-12. SLE patients also produce elevated levels of IL-10, which reduces the production of IL-12, activates B cells and inhibits T cell function. The imbalance between IL-10 and IL-12 in SLE patients is believed to contribute to increased autoantibody production. Furthermore, pathogenic T cells in SLE patients can activate B cells, further leading to higher levels of autoantibodies.

[0007] Currently, there is no cure for lupus. Certain treatments are available, however, to prevent or slow the process of the disease as well as to control its symptoms. Treatments include the administration of corticosteroids and immunosuppressive drugs such as azathioprine and cyclophosphamide. SLE is most often controlled with aggressive immunosuppressive therapy, frequently as a course of intravenous cyclophosphamide followed by corticosteroids. These broad spectrum treatments do not offer a cure for the disease, functioning to suppress the immune system, and have significant side effects (Zonana-Nacach, A., Barr, S. G., Magder, L.S., and Petri, M. 2000. Damage in systemic lupus erythematosus and its association with corticosteroids. Arthritis Rheum 43:1801-1808).

[0008] Because of the involvement of B cells in the disease, one line of research has sought the use of monoclonal antibodies such as rituximab to deplete SLE patients of CD20+ B cells, thus reducing the production of autoantibodies. Transplantation of MHC-matched purified HSCs has been found to prevent T cell disorders such as diabetes in nonobese diabetic mice. Beilhack, G. F. et al., Diabetes 54:1770-1779 (2005).

[0009] Patients with severe lupus failing to respond to conventional therapies are considered for nearly ablative doses of cyclophosphamide alone or in combination with autologous hematopoietic cell transplant (HCT) in an effort to eliminate autoreactive lymphocytes and reset the immunological clock (Brodsky, R.A., Petri, M., Smith, B.D., Seifter, E.J., Spivak, J. L., Styler, M., Dang, C.V., Brodsky, I., and Jones, R.J. 1998. Immunoablative high-dose cyclophosphamide without stem-cell rescue for refractory, severe autoimmune disease. Ann Intern Med 129:1031-1035; Burt, R. K., Traynor, A.E., Pope, R., Schroeder, J., Cohen, B., Karlin, K.H., Lobeck, L., Goolsby, C, Rowlings, P., Davis, F. A., et al. 1998. Treatment of autoimmune disease by intense immunosuppressive conditioning and autologous hematopoietic stem cell transplantation. Blood 92:3505-3514; Petri, M., Jones, R. J., and Brodsky, R.A. 2003. High-dose cyclophosphamide without stem cell transplantation in systemic lupus erythematosus. Arthritis Rheum 48:166-173). Transplantation is considered a salvage therapy, that is, often clinical transplantation is used as a salvage regimen when patients have failed or become refractory to other therapies. Lupus patients that have been immunocompromised for an extended period of time, often have advanced end-organ dysfunction and active or refractory disease at the time of transplant. In these patients, treatment related mortality is significant, approaching 11 % (Tyndall, A., and Daikeler, T. 2005. Autologous hematopoietic stem cell transplantation for autoimmune diseases. Acta Haematol 114:239-247).

[0010] To improve safety, less toxic nonmyeloablative conditioning regimens that employ lower doses of chemoablation combined with polyclonal antibodies that eliminate lymphocytes are currently being investigated. These protocols target self-reactive lymphocytes with limited myeloablation. Initial trials for autologous nonmyeloablative HCT, however, still report a treatment related mortality (TRM) of 4% (Burt, R.K., Traynor, A., Statkute, L., Barr, W.G., Rosa, R., Schroeder, J., Verda, L., Krosnjar, N., Quigley, K., Yaung, K., et al. 2006. Nonmyeloablative hematopoietic stem cell transplantation for systemic lupus erythematosus. Jama 295:527-535). Currently, several trials in the US and Europe are in progress for treatment of severe autoimmune disease by autologous HCT. Over 1000 patients worldwide have been treated; with over 30% maintaining sustained disease-free durable remission without the need of immunosuppressive drugs (Tyndall, A. 2006. Allogeneic bone marrow transplantation for autoimmune disease-the jury is still out. J Rheumatol 33:644-646; Tyndall, A., and Saccardi, R. 2005. Haematopoietic stem cell transplantation in the treatment of severe autoimmune disease: results from phase l/ll studies, prospective randomized trials and future directions. Clin Exp Immunol 141 :1-9). The most current results for treatment of SLE are encouraging, with approximately 50% disease-free survival at 5 years (Burt, R.K., Traynor, A., Statkute, L., Barr, W.G., Rosa, R., Schroeder, J., Verda, L., Krosnjar, N., Quigley, K., Yaung, K., et al. 2006. Nonmyeloablative hematopoietic stem cell transplantation for systemic lupus erythematosus. Jama 295:527-535; Jayne, D., and Tyndall, A. 2004. Autologous stem cell transplantation for systemic lupus erythematosus. Lupus 13:359-365). Additional trials investigating the use of high dose cyclophosphamide without stem cell rescue to treat severe SLE have reported 40% of patients achieving long-lasting remission (Petri, M., Jones, R.J., and Brodsky, R.A. 2003. High-dose cyclophosphamide without stem cell transplantation in systemic lupus erythematosus. Arthritis Rheum 48:166-173). Nonmyeloablative conditioning regimens are being investigated in order to reduce the risk of the transplantation procedure to reduce TRM. However it is clear that not all patients respond to intense lymphoablation or autologous transplantation.

[0011] Accordingly, there remains a need in the art for improvements regarding the treatment of SLE and related autoimmune conditions. For example, approaches using non-myeloblative conditioning; approaches using transplantation before the disease progressives significantly; and approaches using allogenic HSC transplantation. The present invention meets this and other needs.

4. SUMMARY

[0012] The present disclosure describes a method of treating a patient suffering from systemic lupus erythematosus and related conditions comprising subjecting said patient to myeloablation and administering to said patient allogeneic hematopoietic stem cells. In a preferred embodiment, nonmyeloablative conditioning is employed in conjunction with allogeneic stem transplantation to create a chimeric individual, and more preferably to establish durable mixed chimerism in said patient. As demonstrated herein, the systemic lupus erythematosus may be at an early stage of disease progression or at a later stage of disease progression, and a beneficial therapeutic effect obtained.

[0013] Hematopoietic stem cells may be derived from peripheral blood, mobilized peripheral blood, umbilical cord blood, bone marrow, and/or other organs known to harbor hematopoietic stem cells, such as fetal liver. Cell populations may be mixtures of cells as obtained from a source or cells isolated, particularly as an enriched or substantially pure population, based on a desired cell marker phenotype (e.g., CD34+ and/or CD90+ and/or AC133+ and/or ALDH+ cells). Preferably, the starting cell population is enriched for HSC based on the presence of the cell marker CD34+ or CD90+; and still more preferably, the starting cell population is purified HSC that are both CD34+ and CD90+. In a further embodiment, the cells may also have the cell marker phenotype |_in^ne9"^ow.

[0014] In a preferred embodiment, the hemtopoeitc stem cells are from an allogeneic donor or donors. In a particularly preferred embodiment, the cells are from a haplo-identical allogeneic donor.

[0015] Some embodiments also include adjunctive treatments, for example, where one or more antifungal agents anti-bacterial agents and/or anti-viral agents are used

[0016] In another aspect, the invention provides therapeutic compositions for treatment of SLE including allogeneic hematopoietic stem cells for use in the contemplated methods. In one embodiment, the therapeutic composition comprises or consists essentially of allogeneic hematopoietic stem cells in a pharmaceutically acceptable carrier.

5. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Fig. 1 shows the percentage of donor chimerism. Fig. 1A shows donor chimerism in the peripheral blood at 32 weeks post transplantation and Fig. 1 B shows donor chimerism of hematopoietic tissues in mice transplanted with purified allogeneic HSC.

[0018] Fig. 2 shows the survival of NZBW mice following transplantation.

[0019] Fig. 3 shows the serology of NZBW mice following transplantation. Fig. 3A shows circulating immune complexes (CIC), Fig. 3B shows anti-dsDNA, Fig. 3C shows anti-nuclear antigen (ANA), and Fig. 3D shows anti-histone.

[0020] Fig. 4 shows donor chimerism in the peripheral blood at 32 weeks post transplantation.

[0021] Fig. 5 shows survival of aged NZBW mice following transplantation or conditioning.

[0022] Fig. 6 shows the reversal of lupus-like disease in 8 month old NZBW mice treated by nonmyeloablative allogeneic HSC transplantation. Fig. 6A shows how eight month old NZBW mice transplanted with haplo-identical allogeneic HSC had a reversal of their disease as measured by a decrease in the frequency of mice with anti-histone auto-antibody titers post transplant. Fig. 6B shows the reversal and stabilization of proteinuria in transplanted mice with mild to moderate disease.

6. DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

[0023] The instant invention is directed to methods and compositions for treating autoimmune diseases, such as systemic lupus erythematosus (SLE). The treatment methods and compositions find use with respect to other conditions related to SLE, including other autoimmune diseases such as, rheumatoid arthritis, juvenile idiopathic (chronic) arthritis, type 1 diabetes mellitus, Grave's disease, multiple sclerosis, pernicious anemia, scleroderma, systemic sclerosis, Crohn's disease, and the like. [0024] The instant disclosure provides data demonstrating that active autoimmunity can be halted and controlled by the transplantation of purified allogeneic HSC with non-myeloablative conditioning. As detailed in the Examples below, purified allogeneic HSC transplantation with the establishment of mixed chimerism succeeded in reversing established symptoms of lupus in a mouse model.

[0025] In the HSCT field, expansion techniques have been mainly directed towards increasing the population of HSCs for the purposes of transplantation and permanent restoration of hematopoiesis (Devine, S.M. et al., Bone Marrow Transplantation 31 :241-252 (2003); Henschler, R. et al., Blood 84(9): 2898-2903 (1994); Bhatia, M. et al., J. Exp. Med. 186:619-624 (1997)). Combinations of cytokines and growth factors employed generally attempt to cause preferential expansion of HSCs while limiting their differentiation into committed cells of the myeloid and lymphoid lineages. The number of HSCs expanded in the context of HSCT is especially relevant since the engraftment characteristics of infused cells and survival of the transplant recipient is correlated with increasing numbers of infused HSCs, particularly where there is a mismatch at the MHC of the donor and recipient (Ketterer N. et al., Blood 91 :3148-3155 (1998)). Culture conditions that induce differentiation of the stem cells are undesirable because of the lower numbers of HSCs produced. Because HSCs have self-renewing capacity, long-term cultures are used in some instances to select for self-replenishing HSC populations (Piacibillo, W. et al., Blood 93(11): 3736-3749 (1999)).

6.1 Definitions

[0026] In reference to the present disclosure, the technical and scientific terms used in the descriptions herein will have the meanings commonly understood by one of ordinary skill in the art, unless specifically defined otherwise. Accordingly, the following terms are intended to have the following meanings:

[0027] "Allogeneic" refers to deriving from, originating in, or being members of the same species, where the members are genetically related or genetically unrelated but genetically similar. An "allogeneic transplant" refers to transfer of cells or organs from a donor to a recipient, where the recipient is the same species as the donor.

[0028] "Autologous" refers to deriving from or originating in the same subject or patient. An "autologous transplant" refers to the harvesting and reinfusion or transplant of a subject's own cells or organs. Exclusive or supplemental use of autologous cells can eliminate or reduce many adverse effects of administration of the cells back to the host, particular graft versus host reaction.

[0029] "Chemically-defined" as used herein refers to culture media of known chemical composition, both quantitatively and qualitatively, with no deliberately added uncharacterized supplements, even though such a medium may contain trace contaminants in its components. A chemically-defined medium necessarily lacks animal serum, feeder cells such as stromal cells, and cell-based extracellular matrices derived from, e.g., fibroblasts and the like. [0030] "Congenic" refers to deriving from, originating in, or being members of the same species, where the members are genetically identical except for a small genetic region, typically a single genetic locus (i.e., a single gene). A "congenic transplant" refers to transfer of cells or organs from a donor to a recipient, where the recipient is genetically identical to the donor except for a single genetic locus.

[0031] "Cytokine" refers to compounds or compositions that in the natural state are made by cells and affect physiological states of the cells that produce the cytokine (i.e., autocrine factors) or other cells. Cytokine also encompasses any compounds or compositions made by recombinant or synthetic processes, where the products of those processes have identical or similar structure and biological activity as the naturally occurring forms. Lymphokines refer to natural, synthetic, or recombinant forms of cytokines naturally produced by lymphocytes, including, but not limited to, IL-1 , IL-3, IL-4, IL-6, IL-11 , and the like.

[0032] "Graft-versus-host response" or "GVH" or "GVHD" refers to a cellular response that occurs when lymphocytes of a different MHC class are introduced into a host, resulting in the reaction of the donor lymphocytes against the host.

[0033] "Growth factor" refers to a compound or composition that in the natural state affects cell proliferation, cell survival, and/or differentiation. A growth factor, while having the indicated effect on the cell, may also affect other physiological process, such as secretion, adhesion, response to external stimuli, and the like. Although many growth factors are made by cells, growth factors as used herein also encompass any compound or composition made by recombinant or synthetic processes, where the product of those processes have identical or similar structure and biological activity as the naturally occurring growth factor. Examples of growth factors include epidermal growth factor (EGF), fibroblast growth factor (FGF), erythropoietin (EPO), thrombopoietin (TPO), stem cell factor (SCF), and flt-3 ligand (FL), and analogs thereof.

[0034] "Isolated" refers to a product, compound, or composition which is separated from at least one other product, compound, or composition with which it is associated in its naturally occurring state, whether in nature or as made synthetically.

[0035] "Hematopoietic stem cell" or "HSC" refers to a clonogenic, self-renewing pluripotent cell capable of ultimately differentiating into all cell types of the hematopoietic system, including B cells, T cells, NK cells, lymphoid dendritic cells, myeloid dendritic cells, granulocytes, macrophages, megakaryocytes, and erythroid cells. As with other cells of the hematopoietic system, HSCs are typically defined by the presence of a characteristic set of cell markers. "Enriched" when used in the context of HSC refers to a cell population selected based on the presence of a single cell marker, generally CD34+, while "purified" in the context of HSC refers to a cell population resulting from a selection on the basis of two or more markers, preferably CD34+ CD90+. Purified HSC may also be free or substantially free of immune cells, for example, to reduce, eliminate or nearly eliminate the risk of GVHD. "Marker phenotyping" refers to identification of markers or antigens on cells for determining their phenotype (e g , differentiation state and/or cell type) This may be done by immunophenotyping, which uses antibodies that recognize antigens present on a cell The antibodies may be monoclonal or polyclonal, but are generally chosen to have minimal crossreactivity with other cell markers It is to be understood that certain cell differentiation or cell surface markers are unique to the animal species from which the cells are derived, while other cell markers will be common between species These markers defining equivalent cell types between species are given the same marker identification even though there are species differences in structure (e g , amino acid sequence) Cell markers include cell surfaces molecules, also referred to in certain situations as cell differentiation (CD) markers, and gene expression markers The gene expression markers are those sets of expressed genes indicative of the cell type or differentiation state In part, the gene expression profile will reflect the cell surface markers, although they may include non-cell surface molecules

[0036] "Mismatched allogeneic" refers to deriving from, originating in, or being members of the same species having non-identical major histocompatability complex (MHC) antigens (ι e , proteins) as typically determined by standard assays used in the art, such as serological or molecular analysis of a defined number of MHC antigens A "partial mismatch" refers to partial match of the MHC antigens tested between members typically between a donor and recipient For instance, a "half mismatch" refers to 50% of the MHC antigens tested as showing different MHC antigen type between two members The term "half mismatch" includes haplo-identical allogenic cells, as well as haplo-identical allogenic cells having minor histocompatibility loci mis-matches, for example, haplo-identical cells obtained from donors related (e g , parent to child, siblings) or unrelated to the subject recipient A "full" or "complete" mismatch refers to all MHC antigens tested as being different between two members

[0037] "Myeloablative" or "myeloablation" refers to impairment or destruction of the hematopoietic system, including both lymphoid and myeloid cells, typically by exposure to a cytotoxic agent or radiation Myeloablation encompasses complete myeloablation brought on by high doses of cytotoxic agent or total body irradiation that destroys the hematopoietic system It also includes a less than complete myeloablated state caused by non-myeloablative conditioning Thus, non-myeloablative conditioning is treatment that does not completely destroy the subject's hematopoietic system, for example a conditioning regimen that eliminates all or most host T cells and/or NK cells For example, non-myeloablative conditioning can result in at least about 50%, at least about 40%, at least about 25%, at least about 15%, or at least about 10% host T cells remaining, and/or non-myeloablative conditioning can result in at least about 50%, at least about 40%, at least about 25%, at least about 15%, or at least about 10% host B cells remaining

[0038] "Sorting" as it pertains to cells refers to separation of cells based on physical characteristics (such as, e g , elutriation or other size-based techniques) or presence of markers (such as sorting using side scatter (SSC) and forward scatter (FSC), or fluorescence activation cell sorting (FACS) using labeled antibodies), or analysis of cells based on presence of cell markers, e g , FACS without sorting, and including as well immunoabsorption techniques such as, e g , magnetic cell separation systems

[0039] "Substantially pure cell population" refers to a population of cells having a specified cell marker characteristic and differentiation potential that is at least about 50%, preferably at least about 75-80 %, more preferably at least about 85-90%, and most preferably at least about 95% of the cells making up the total cell population Thus, a "substantially pure cell population" refers to a population of cells that contain fewer than about 50%, preferably fewer than about 20-25%, more preferably fewer than about 10-15%, and most preferably fewer than about 5% of cells that do not display a specified marker characteristic and differentiation potential under designated assay conditions

[0040] "Subject" or "patient" are used interchangeably and refer to, except where indicated, mammals such as humans and non-human primates, as well as rabbits, rats, mice, goats, pigs, and other mammalian species

[0041] "Syngeneic" refers to deriving from, originating in, or being members of the same species that are genetically identical, particularly with respect to antigens or immunological reactions These include identical twins having matching MHC types Thus, a "syngeneic transplant" refers to transfer of cells or organs from a donor to a recipient who is genetically identical to the donor

[0042] "Xenogeneic" refers to deriving from, originating in, or being members of different species, e g , human and rodent, human and swine, human and chimpanzee, etc A "xenogeneic transplant" refers to transfer of cells or organs from a donor to a recipient where the recipient is a species different from that of the donor

6.2 Cell Types

[0043] The cell types relevant to the present disclosure are those of the hematopoietic system, particularly hematopoietic stem cells and cells of the myeloid lineage Descriptions of cells herein will use those known to the skilled artisan, with the understanding that these descriptions reflect the current state of knowledge in the art and the invention is not limited thereby to only those phenotypic markers described herein

[0044] The hematopoietic stem cells (HSC) are pluπpotent stem cells capable of self-renewal and are characterized by their ability to give rise under permissive conditions to all cell types of the hematopoietic system HSC self-renewal refers to the ability of an HSC cell to divide and produce at least one daughter cell with the same self renewal and differentiation potential of a HSC, that is, cell division gives rise to additional HSCs Self-renewal provides a continual source of undifferentiated stem cells for replenishment of the hematopoietic system The marker phenotypes useful for identifying HSCs will be those commonly known in the art For human HSCs₁ the cell marker phenotypes preferably include CD34⁺ CD38^" CD90 (Thy1 )⁺ Lm^" For mouse HSCs, an exemplary cell marker phenotype is Sca-1⁺ CD90⁺ (see, e g , Spangrude, G J et al , Science 1 661-673 (1988)) or c- kιt⁺ Thy^lo Lm^' Sca-1⁺ (see, Uchida, N et al . J CIm Invest 101(5) 961-966 (1998)) Alternative HSC markers such as aldehyde dehydrogenase (see Storms et al., Proc. Nat'l Acad. Sci. 96:9118-23 (1999)) and AC133 (see Yin et al., Blood 90:5002-12 (1997)) may also find advantageous use. Other markers that may be used include, e.g., CD117, PE-Cy7; Sca-1 (Ly6A/E), APC; and a lineage cocktail of CD3, CD4, CD5, CD8, Ter119, B220, Mac-1 and GR-1 , PE.

[0045] For the lymphoid lineage, a "committed lymphoid progenitor cell" refers to a cell capable of differentiating into any of the terminally differentiated cells of the lymphoid lineage. Encompassed within the lymphoid progenitor cells are the common lymphoid progenitor cells (CLP), a cell population characterized by limited or non-self-renewal capacity but which is capable of cell division to form T lymphocyte and B lymphocyte progenitor cells, NK cells, and lymphoid dendritic cells. The marker phenotypes useful for identifying CLPs will be those commonly known in the art. For CLP cells of mouse, the cell population is characterized by the presence of markers as described in Kondo, M. et al., Cell 91 :661-672 (1997), while for human CLPs, a marker phenotype of CD34⁺ CD38⁺ CDI O⁺ IL7R+ may be used (GaIy, A et al., Immunity, 3:459-473 (1995); Akashi, K. et al., Int. J. Hematol. 69(4): 217-226 (1999)); publications incorporated herein by reference.

[0046] A summary of preferred murine cell surface markers is provided in Table 1 below, where an approximate indication of staining levels is shown by the cell colors in the tables: white indicates no staining, light gray indicates low level staining and dark grey indicates intermediate or high staining.

Table 1

Lin2: CD3, CD4, CD5, CD8, B220, GM , CD90.1 , CD127, TER119

[0047] A summary of preferred human cell surface markers is provided in Table 2 below, where an approximate indication of staining levels is shown by the cell colors in the tables: white indicates no staining, light gray indicates low level staining and dark grey indicates intermediate or high staining.

Table 2

HSC LiM

CLP CD38/CD90 Lin MP CD90 Lin2 CMP CD90 Lin2 CD123 GMP CD90 Lin2

Lin 1 : CD2, CD3, CD7, CD8, CD10, CD11 b, CD14, CD19, CD56, CD235a

Lin2a: CD2, CD3, CD4, CD7, CD8, CD10, CD11 b, CD14, CD19, CD20, CD56, CD235a

Lin 2b: CD10, CD11 b, CD14, CD19, CD235a

[0048] Numerous other suitable cell surface markers are presently known to the skilled artisan, or will be identified and characterized in due course, and such markers will find advantageous use in the methods and compositions described herein.

[0049] Cells can be obtained from a variety of sources, including bone marrow, peripheral blood, cord blood, and other sources known to harbor hematopoietic cells, including liver, particularly fetal liver. Peripheral and cord blood is a rich source of HSCs. Cells are obtained using methods known and commonly practiced in the art. For example, methods for preparing bone marrow cells are described in Sutherland et al., Bone Marrow Processing and Purging: A Practical Guide (Gee, A.P. ed.), CRC Press Inc. (1991 ). Umbilical cord blood or placental cord blood is typically obtained by puncture of the umbilical vein, in both term or preterm, before or after placental detachment (see, e.g., Turner, CW. et al., Bone Marrow Transplant. 10:89 (1992); Bertolini, F. et al., J. Hematother. 4:29 (1995)). HSCs may also be obtained from peripheral blood by leukapheresis, a procedure in which blood drawn from a suitable subject is processed by continuous flow centrifugation (e.g., Cobe BCT Spectra blood cell separators) to remove white blood cells while the other blood components are returned to the donor. Another type of isolation procedure is centrifugation through a medium of varying density, such as Ficoll-Hypaque (Amersham Pharmacia Biotech, Piscataway, NJ).

[0050] The cells are derived from any animal species with a hematopoietic system, as generally described herein. Preferably, suitable animals will be mammals, including, by way of example and not limitation, rodents, rabbits, canines, felines, pigs, horses, cows, primates (e.g., human), and the like. The cells may be obtained from a single subject or from multiple subjects.

[0051] Where applicable, stem cells may be mobilized from the bone marrow into the peripheral blood by prior administration of cytokines or drugs to the subject (see, e.g., Lapidot, T. et al., Exp. Hematol. 30:973-981 (2002)). Cytokines and chemokines capable of inducing mobilization include, by way of example and not limitation, granulocyte colony stimulating factor (G-CSF), granulocyte macrophage colony stimulating factor (GM-CSF), erythropoietin (Kiessinger, A. et al., Exp. Hematol. 23:609-612 (1995)), stem cell factor (SCF), AMD3100 (AnorMed, Vancouver, Canada), interleukin-8 (IL-8), and variants of these factors (e.g., pegfilgastrim, darbopoietin). Combinations of cytokines and/or chemokines, such as G-CSF and SCF or GM-CSF and G-CSF, can act synergistically to promote mobilization and may be used to increase the number of HSC in the peripheral blood, particularly for subjects who do not show efficient mobilization with a single cytokine or chemokine (Morris, C. et al., J. Haematol. 120:413-423 (2003)).

[0052] Cytoablative agents can be used at inducing doses (i.e., cytoreductive doses) to also mobilize HSCs and are useful either alone or in combination with cytokines. This mode of mobilization is applicable when the subject is to undergo myeloablative treatment, and is carried out prior to the higher dose chemotherapy Cytoreductive drugs for mobilization, include, among others, cyclophosphamide, ifosfamide, etoposide, cytosine arabinoside, and carboplatin (Montillo, M et al , Leukemia 18 57-62 (2004), Dasgupta, A et al , J lnfusional Chemother 6 12 (1996), Wright, D E et al , Blood 97 (8) 2278-2285 (2001))

[0053] Variants as used herein include substitutions, deletions, insertions of any ammo acid in the cytokine or growth factor sequence, where the variant retains the biological activity associated with each cytokine or growth factor Substitutions of one or more ammo acid residues may be made while preserving biological activity, and typically involves substitution of one amino acid with a homologous amino acid, also referred to herein as "conservative substitution " In some instances a non- conservative substitutions may also be made Homologous amino acids may be classified based on the size of the side chain and degree of polarization, including, small non-polar (e g , cysteine, proline, alanine, threonine), small polar (e g , serine, glycine, aspartate, asparagine), intermediate polarity (e g , tyrosine, histidine, tryptophan), large non-polar (e g , phenylalanine, methionine, leucine, isoleucine, valine) Homologous amino acid may also be grouped as follows uncharged polar R groups (e g , glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamme), acidic amino acids (e g , aspartic acid, glutamic acid), and basic amino acids (lysine, arginme, and histidine) Examples of conservative variants include the substitution of one hydrophobic residue such as isoleucine, valine, leucine, or methionine for another, the substitution of one polar residue for another polar residue, such as substitution of one arginme for lysine, glutamic acid for aspartic acid, or glutamme for asparagmes, and the substitution of one hydroxylated amino acid serine or threonine for another

[0054] Deletions range from about 1 to about 20 residues, although in some cases, deletions may be much larger, particularly when the cytokine or growth factor has physically separable structural and/or functional domains For instance, a variant of FL is the cleaved extracellular domain, which, as discussed above, retains biological activity when separated from the sequences containing the transmembrane and cytoplasmic domains In addition, ammo acids may be added to the amino or carboxy terminus, or in the amino acid sequences joining structural domains, such as a peptide region joining alpha helixes or beta sheets present in the cytokine or growth factor Variants for each of the cytokines and growth factors will be apparent to the skilled artisan, exemplary references of which are given above

[0055] HSCs may also be subjected to further selection and purification, which can include both positive and negative selection methods, to obtain an enriched or substantially pure population of cells In one aspect, fluorescence activated cell sorting (FACS), also referred to as flow cytometry, is used to sort and analyze the different cell populations Cells having the cellular markers specific for HSC are tagged with an antibody, or typically a mixture of antibodies, that bind the cellular markers Each antibody directed to a different marker is conjugated to a detectable molecule, particularly a fluorescent dye that can be distinguished from other fluorescent dyes coupled to other antibodies A stream of tagged or "stained" cells is passed through a light source that excites the fluorochrome and the emission spectrum from the cells detected to determine the presence of a particular labeled antibody By concurrent detection of different fluorochromes, also referred to in the art as multicolor fluorescence cell sorting, cells displaying different sets of cell markers may be identified and isolated from other cells in the population Other FACS parameters, including, by way of example and not limitation, side scatter (SSC), forward scatter (FSC), and vital dye staining (e g , with propidium iodide) allow selection of cells based on size and viability FACS sorting and analysis of HSC is described in, among others, U S Patent Nos 5,137,809, 5,750,397, 5,840,580, 6,465,249, Manz, M G et al . Proc Natl Acad Set USA 99 11872-11877 (2002), and Akashi, K et al , Nature 404(6774) 193-197 (2000)) General guidance on fluorescence activated cell sorting is described in, for example, Shapiro, H M , Practical Flow Cytometry, 4th Ed , Wiley-Liss (2003) and Ormerod, M G , Flow Cytometry A Practical Approach, 3rd Ed , Oxford University Press (2000)

[0056] Another method of isolating cell populations uses a solid or insoluble substrate to which is bound antibodies or ligands that interact with specific cell surface markers In immunoadsorption techniques, cells are contacted with the substrate (e g , column of beads, flasks, magnetic particles) containing the antibodies and any unbound cells removed Immunoadsorption techniques can be scaled up to deal directly with the large numbers of cells in a clinical harvest Suitable substrates include, by way of example and not limitation, plastic, cellulose, dextran, polyacrylamide, agarose, and others known in the art (e g , Pharmacia Sepharose 6MB macrobeads) When a solid substrate comprising magnetic or paramagnetic beads is used, cells bound to the beads can be readily isolated by a magnetic separator (see, e g , Kato, K and Radbruch, A , Cytometry 14(4) 384-92 (1993), CD34⁺ direct isolation kit, Miltenyi Biotec, Bergisch, Gladbach, Germany) Affinity chromatographic cell separations typically involve passing a suspension of cells over a support bearing a selective ligand immobilized to its surface The ligand interacts with its specific target molecule on the cell and is captured on the matrix The bound cell is released by the addition of an elution agent to the running buffer of the column and the free cell is washed through the column and harvested as a homogeneous population As apparent to the skilled artisan, adsorption techniques are not limited to those employing specific antibodies, and may use nonspecific adsorption For example, adsorption to silica is a simple procedure for removing phagocytes from cell preparations

[0057] FACS and most batch wise immunoadsorption techniques can be adapted to both positive and negative selection procedures (see, e g , U S Patent No 5,877,299) In positive selection, the desired cells are labeled with antibodies and removed away from the remaining unlabeled/unwanted cells In negative selection, the unwanted cells are labeled and removed Another type of negative selection that can be employed is use of antibody/complement treatment or immunotoxins to remove unwanted cells

[0058] It is to be understood that the enrichment and/or purification of cells also includes combinations of the methods described above A typical combination may comprise an initial procedure that is effective in removing the bulk of unwanted cells and cellular material, for example leukapharesis An additional step providing higher resolution of different cell types, such as FACS sorting with antibodies to a set of specific cellular markers, can be used to obtain substantially pure populations of the desired cells Another combination may involve an initial separation using magnetic beads bound with antι-CD34 antibodies followed by an additional round of purification with FACS

[0059] Cells in the mixture may be completely matched allogeneic, partially mismatched allogeneic, and/or fully mismatched allogeneic cells with respect to the MHC of the transplant recipient, and may be from related donors, usually siblings with the same parental alleles, or unrelated donors Determining the degree of MHC mismatch will employ standard tests known and used in the art

[0060] For instance, there are at least six major categories of MHC genes in humans, identified as being important in transplant biology HLA-A, HLA-B, HLA-C encode the HLA class I proteins while HLA-DR, HLA-DQ, and HLA-DP encode the HLA class Il proteins Genes within each of these groups are highly polymorphic, as reflected in the numerous HLA alleles or variants found in the human population, and differences in these groups between individuals is associated with the strength of the immune response against transplanted cells Standard methods for determining the degree of MHC match examine alleles within HLA-B and HLA-DR, or HLA-A, HLA-B and HLA-DR groups Thus, tests are made of at least 4, and preferably at least 6 MHC antigens within the two or three HLA groups, respectively

[0061] In serological MHC tests, antibodies directed against each HLA antigen type are reacted with cells from one subject (e g , donor) to determine the presence or absence of certain MHC antigens that react with the antibodies This is compared to the reactivity profile of the other subject (e g , recipient) Reaction of the antibody with an MHC antigen is typically determined by incubating the antibody with cells, and then adding complement to induce cell lysis (ι e , lymphocytotoxicity testing) The reaction is examined and graded according to the amount of cells lysed in the reaction (Mickelson, E and Petersdorf, E W , Hematopoietic Cell Transplantation, Thomas, E D et al eds , pg 28-37, Blackwell Scientific, Maiden, MA (1999)) Other cell-based assays include flow cytometry using labeled antibodies or enzyme linked immunoassays (ELISA)

[0062] Molecular methods for determining MHC type generally employ synthetic probes and/or primers to detect specific gene sequences that encode the HLA protein Synthetic oligonucleotides may be used as hybridization probes to detect restriction fragment length polymorphisms associated with particular HLA types (Vaughn, R W , Methods in Molecular Biology MHC Protocols 210 45-60 (2002)) Alternatively, primers may be used for amplifying the HLA sequences (e g , by polymerase chain reaction or ligation chain reaction), the products of which can be further examined by direct DNA sequencing, restriction fragment polymorphism analysis (RFLP), or hydπdization with a series of sequence specific oligonucleotide primers (SSOP) (Petersdorf, E W et al , Blood 92(10) 3515-20 (1998), Moπshima ,Y et al , Blood 99(11) 4200-6 (2002), and Middleton, D and Williams, F , Methods in Molecular Biology MHC Protocols 210 67-112 (2002)) [0063] While description of "matched allogeneic" or "mismatched allogeneic" is given for human MHC, it is to be understood that similar analysis may be conducted for MHCs for various animal species. These include, by way of example and not limitation, mouse, rat (Gill, TJ. et al., Transplant Proc. 27(2): 1495-500 (1995)), cow (Lewin, H.A, et al., Immunol Rev. 167:145-58 (1999)), canine (Wagner, J. L. et al., J. Hered. 90(1):35-8 (1999)), feline (O'Brien, S.J. and Yuhki, N., Immunol Rev. 167: 133-44 (1999)), swine (Chardon, P. et al., Genet SeI Evol. 32(2):109-28 (2000)), horses (Kydd, J. et al., Vet Immunol Immunopathol. 42(1):3-60 (1994), and primates (Heise, E.R. et al., Genetica 73(1- 2):53-68 (1987)).

[0064] Allogeneic mixtures of cells may be made in various ways. In one embodiment, cells are obtained from different donors and mixed prior to their expansion in culture. In some embodiments, cells are obtained from a single donor and not expanded in culture. The use of allogenic HSC provides several advantages in some embodiments of the instant invention. For example, allogenic transplantation can be better suited to patients with autoimmune disease, e.g., since the complications and risks of stem cell mobilization for autologous transplant are increased in some such patients. Mobilization can be associated with flair of autoimmunity and bacteremia caused by severe cytopenias, leading to increased morbidity and mortality (Burt, R. K., Traynor, A., Statkute, L., Barr, W.G., Rosa, R., Schroeder, J., Verda, L., Krosnjar, N., Quigley, K., Yaung, K., et al. 2006. Nonmyeloablative hematopoietic stem cell transplantation for systemic lupus erythematosus. Jama 295:527-535; Traynor, A.E., Barr, W.G., Rosa, R.M., Rodriguez, J., Oyama, Y., Baker, S., Brush, M., and Burt, R. K. 2002. Hematopoietic stem cell transplantation for severe and refractory lupus. Analysis after five years and fifteen patients. Arthritis Rheum 46:2917-2923; Burt, R. K., Fassas, A., Snowden, J., van Laar, J. M., Kozak, T., Wulffraat, N. M., Nash, R.A., Dunbar, C.E., Arnold, R., Prentice, G., et al. 2001. Collection of hematopoietic stem cells from patients with autoimmune diseases. Bone Marrow Transplant 28:1-12; Openshaw, H., Stuve, O., Antel, J. P., Nash, R., Lund, B.T., Weiner, L.P., Kashyap, A., McSweeney, P., and Forman, S. 2000. Multiple sclerosis flares associated with recombinant granulocyte colony-stimulating factor. Neurology 54:2147-2150). Also with autologous HCT there can be a risk of relapse from reinfusion of autoreactive lymphocytes. Although patients with malignancies have been treated by allogeneic HCT, autologous HCT has been favored over allogeneic HCT for treatment of autoimmune disorders due to the increased risks of morbidity and mortality from GVHD. In the treatment of malignancy, GVHD is tolerated due to the related graft- versus-leukemia effect that has been shown to be important in maintaining remission.

[0065] Although some encouraging results have been seen in patients with long-term remissions of autoimmune diseases receiving allogeneic transplant for a coexisting disease, few patients have received allogeneic transplant primarily for autoimmune disease mostly due to fears of fatal graft- versus-host disease (GVHD) (Marmont, A.M. 2004. Stem cell transplantation for autoimmune disorders. Coincidental autoimmune disease in patients transplanted for conventional indications. Best Pract Res Clin Haematol 17:223-232; de Buys, P., Khanna, D., and Furst, D. E. 2005. Hemopoietic stem cell transplantation in rheumatic diseases-an update. Autoimmun Rev 4:442-449). Since mature T cells are the effectors of GVHD, they are exclusion from the graft material in preferred embodiments to prevent its onset. For example, engraftment of purified allogenic HSC can reconstitute a fully functional hematopoietic system without GVHD (Bowers, E., Tamaki, S., Coward, A., Kaneshima, H., and Chao, CC. 2000. Differing functional recovery of donor-derived immune cells after purified haploidentical and fully mismatched hematopoietic stem cell transplantation in mice. Exp Hematol 28:1481-1489; Beilhack, G.F., Scheffold, Y.C., Weissman, I. L, Taylor, C₁ Jerabek, L., Burge, M.J., Masek, MA, and Shizuru, J.A. 2003. Purified allogeneic hematopoietic stem cell transplantation blocks diabetes pathogenesis in NOD mice. Diabetes 52:59-68; Beilhack, G. F., Landa, R. R., Masek, M. A., and Shizuru, J.A. 2005. Prevention of type Idiabetes with major histocompatibility complex-compatible and nonmarrow ablative hematopoietic stem cell transplants. Diabetes 54:1770- 1779; Shizuru, J.A., Jerabek, L., Edwards, C. T., and Weissman, I. L. 1996. Transplantation of purified hematopoietic stem cells: requirements for overcoming the barriers of allogeneic engraftment. Biol Blood Marrow Transplant 2:3-14; Shizuru, J. A., Weissman, LL, Kernoff, R., Masek, M., and Scheffold, Y.C. 2000. Purified hematopoietic stem cell grafts induce tolerance to alloantigens and can mediate positive and negative T cell selection. Proc Natl Acad Sci U S A 97:9555-9560; Shizuru, J. A., Negrin, R. S., and Weissman, LL 2005. Hematopoietic stem and progenitor cells: clinical and preclinical regeneration of the hematolymphoid system. Annu Rev Med 56:509-538; Uchida, N., Tsukamoto, A., He, D., Friera, A.M., Scoliay, R., and Weissman, LL 1998. High doses of purified stem cells cause early hematopoietic recovery in syngeneic and allogeneic hosts. J Clin Invest 101 :961-966). Further, transplantation of SLE resistant HSC can allow for partial or complete replacement of an autoimmune- prone system (Weissman, LL. 2000. Translating stem and progenitor cell biology to the clinic: barriers and opportunities. Science 287:1442-1446).

[0066] Without being bound to a particular theory, the instant invention allows more successful allogenic transplant in patients with autoimmune disease, such as SLE, because of a graft-versus- autoimmunity effect. It is believed that the of effects of allogenic transplantation are mostly attributable to acute and chronic GVHD from the T cells in the graft (Weinberg, K., Blazar, B. R., Wagner, J. E., Agura, E., Hill, B. J., Smogorzewska, M., Koup, R.A., Betts, M. R., Collins, R. H., and Douek, D. C. 2001. Factors affecting thymic function after allogeneic hematopoietic stem cell transplantation. Blood 97:1458-1466; Lapp, W.S., Ghayur, T., Mendes, M., Seddik, M., and Seemayer, T.A. 1985. The functional and histological basis for graft-versus-host-induced immunosuppression. Immunol Rev 88:107-133; Seddik, M., Seemayer, T.A., and Lapp, W.S. 1984. The graft-versus-host reaction and immune function. I. T helper cell immunodeficiency associated with graft-versus-host-induced thymic epithelial cell damage. Transplantation 37:281-286; Ghayur, T., Seemayer, T.A., Xenocostas, A., and Lapp, W.S. 1988. Complete sequential regeneration of graft-vs- host-induced severely dysplastic thymuses. Implications for the pathogenesis of chronic graft-vs-host disease. Am J Pathol 133:39-46; Hollander, G. A., Widmer, B., and Burakoff, S.J. 1994. Loss of normal thymic repertoire selection and persistence of autoreactive T cells in graft vs host disease. J Immunol 152:1609-1617; van den Brink, M. R., Moore, E., Ferrara, J. L, and Burakoff, S.J. 2000. Graft-versus-host-disease-associated thymic damage results in the appearance of T cell clones with anti-host reactivity. Transplantation 69:446-449; Tivol, E., Komorowski, R., and Drobyski, W. R. 2005. Emergent autoimmunity in graft-versus-host disease. Blood 105:4885-4891; Fukushi, N., Arase, H., Wang, B , Ogasawara, K , Gotohda, T , Good, R A , and Onoe, K 1990 Thymus a direct target tissue in graft-versus-host reaction after allogeneic bone marrow transplantation that results in abrogation of induction of self-tolerance Proc Natl Acad Sci U S A 87 6301-6305) These effects can include acute organ toxicity, increase in TRM, delayed reconstitution of the immune system, persistence of autoreactive lymphocytes and an increase in the rate of opportunistic infection However, in preferred embodiments, nonmyeloablative transplantation of purified haploidentical allogeneic HSC alone can restore tolerance to alleviate immune symptoms, even in the presence of host T cells (McCoy, K L , Kendπck, L , and Chused, T M 1986 Tolerance defects in New Zealand Black and New Zealand Black X New Zealand White F1 mice J Immunol 136 1217-1222) Without being bound to a particular theory, the purification of HSC reduces, eliminates, or nearly eliminates T cell contamination, reducing or avoiding GVHD, while immunologically naive donor immune cells can develop within the host environment

[0067] In preferred embodiments, the recipient is subjected to non-myeoablative conditioning, e g , where a portion of host T and/or B cells survive Non-myeloabative conditioning can avoid exceptionally high dose of irradiation required for full myeloablation For example, non-myeloablative conditioning can result in at least about 50%, at least about 40%, at least about 25%, at least about 15%, or at least about 10% host T cells surviving T cells surviving lethal doses of irradiation have been shown able to mediate disease pathogenesis in autoimmune mice (Beilhack, G F , Scheffold, Y C , Weissman, I L , Taylor, C , Jerabek, L , Burge, M J , Masek, M A , and Shizuru, J A 2003 Purified allogeneic hematopoietic stem cell transplantation blocks diabetes pathogenesis in NOD mice Diabetes 52 59-68)

[0068] In some embodiment, non-myeloablative conditioning can result in at least about 50%, at least about 40%, at least about 25%, at least about 15%, or at least about 10% host B cells surviving In the Examples presented below, for instance, the auto-antibodies detected in some allo-transplanted mice may be due to the surviving host B1 cells Large populations of host derived peritoneal B1 cells after non-myeloablative conditioning may be due to radioresistance, poor de novo generation of B1 cells from adult stem cells and the ability of B1 cells to maintain their numbers by self-replenishment (Hayakawa, K , Hardy, R R , and Herzenberg, L A 1985 Progenitors for Ly-1 B cells are distinct from progenitors for other B cells J Exp Med 161 1554-1568, Hayakawa, K , Hardy, R R , and Herzenberg, L A 1986 Peritoneal Ly-1 B cells genetic control, autoantibody production, increased lambda light chain expression Eur J Immunol 16 450-456, Kantor, A B , Stall, A M , Adams, S , and Herzenberg, L A 1992 Differential development of progenitor activity for three B-cell lineages Proc Natl Acad Sci U S A 89 3320-3324) Peritoneal B-1 cells have been linked to the production of auto-antibodies in non-autoimmune and NZBW mice (Ito, T , Ishikawa, S , Sato, T , Akadegawa, K , Yurino, H , Kitabatake, M , Hontsu, S , Ezaki, T , Kimura, H , and Matsushima, K 2004 Defective B1 cell homing to the peritoneal cavity and preferential recruitment of B1 cells in the target organs in a murine model for systemic lupus erythematosus J Immunol 172 3628-3634, Mercolino, T J , Locke, A L , Afshaπ, A , Sasser, D , Travis, W W , Arnold, L W , and Haughton, G 1989 Restricted immunoglobulin variable region gene usage by normal Ly-1 (CD5+) B cells that recognize phosphatidyl choline J Exp Med 169 1869-1877, Hayakawa, K , Hardy, R R , Parks, D R , and Herzenberg, L A 1983 The "Ly-1 B" cell subpopulation in normal immunodefective, and autoimmune mice J Exp Med 157 202-218, Pennell, C A , Mercolino, T J , Grdina, T A , Arnold, L W , Haughton, G , and Clarke, S H 1989 Biased immunoglobulin variable region gene expression by Ly-1 B cells due to clonal selection Eur J Immunol 19 1289-1295, Murakami, M , Yoshioka, H , Shirai, T , Tsubata, T , and Honjo, T 1995 Prevention of autoimmune symptoms in autoimmune-prone mice by elimination of B-1 cells lnt Immunol 7 877-882) Aberrant B cell infiltration of target organs including the thymus, kidneys and lungs is a hallmark of aged NZBW mice and defective homing of B-1 cells due to the over-expression of chemokines by dendritic cells in these tissues is one factor that has been linked to this event in NZBW mice (Ishikawa, S , Sato, T , Abe, M , Nagai, S , Onai, N , Yoneyama, H , Zhang, Y , Suzuki, T , Hashimoto, S , Shirai, T , et al 2001 Aberrant high expression of B lymphocyte chemokine (BLC/CXCL13) by C11 b+CD11c+ dendritic cells in murine lupus and preferential chemotaxis of B1 cells towards BLC J Exp Med 193 1393-1402) In preferred embodiments, however, recipients transplanted with allogeneic HSC show decreased occurrence of B cell infiltration of the thymus

[0069] In preferred embodiments, the combination of purified allogeneic HSC transplant and non- myeloablative conditioning successfully treats SLE with less morbidity and mortality and is more effective than autologous transplant

6.3 Treatment

[0070] Cells prepared by the methods described herein are used for treatment of autoimmune disorders As used herein, "treatment" refers to therapeutic or prophylactic treatment, or a suppressive measure for the disease, disorder or undesirable condition Treatment encompasses administration of the subject cells in an appropriate form prior to the onset of disease symptoms and/or after clinical manifestations, or other manifestations of the disease or condition to reduce disease severity, halt disease progression, or eliminate the disease Prevention of the disease includes prolonging or delaying the onset of symptoms of the disorder or disease, preferably in a subject with increased susceptibility to the disorder

[0071] Conditions suitable for treatment with the cells described herein include SLE Patients with severe SLE refractory to conventional treatment are considered candidates for autologous HSC transplantation where the intent is to re-set the immunological clock Such SLE patients are candidates for allotransplantation with SLE resistant MHC haplotype matched HSC for partial or complete replacement of an autoimmune-prone system, in accordance with various embodiments of the invention described herein For example, depending on their genetic predisposition and the nature of the environmental trigger initiating the disease, some patients will benefit from allogeneic haploidentical HSC transplantation that can offer replacement of their immune system, where cells are obtained from haplotype-matched family members without SLE, or unrelated donors

[0072] Additional conditions suitable for treatment with the cells described herein include other autoimmune diseases These include, but are not limited to, rheumatoid arthritis, juvenile idiopathic (chronic) arthritis, type 1 diabetes mellitus, Grave's disease, multiple sclerosis, pernicious anemia, scleroderma, systemic sclerosis, and/or Crohn's disease.

[0073] Rheumatoid arthritis is an autoimmune disease that can cause chronic inflammation of the joints, as well as inflammation of the tissue around the joints. Symptoms can include fatigue, lack of appetite, low-grade fever, muscle and joint aches, and stiffness. Like SLE, the prior art offers no known cure for rheumatoid arthritis and treatment has focused on reducing joint inflammation and pain, maximizing joint function, and preventing joint destruction and deformity. Juvenile idiopathic (chronic) arthritis is persistent arthritis in one or more joints that begins before age 16 and lasts at least 6 weeks.

[0074] Type 1 diabetes mellitus is usually diagnosed in children and young adults and is a condition where the body does not produce sufficient insulin. Conditions associated with type 1 diabetes include hyperglycemia, hypoglycemia, ketoacidosis and celiac disease. Treatments for type 1 diabetes include insulin, aspirin, controlling blood pressure and cholesterol and making dietary changes.

[0075] Grave's disease is a type of autoimmune disease that causes over-activity of the thyroid gland, causing hyperthyroidism. High levels of thyroid hormones can cause side effects such as weight loss, rapid heart rate and nervousness. Treatments have involved antithyroid drugs to lower the amount of thyroid hormones made by the thyroid; radioactive iodine to damage thyroid cells, shrinking and eventually destroying the thyroid gland in order to reduce hormone levels; and/or surgery, e.g., where the thyroid gland is removed.

[0076] Multiple sclerosis is a chronic, inflammatory, demyelinating disease that affects the central nervous system (CNS). MS affects the neurons in the while matter areas of the brain and spinal cord, destroying oligodendrocytes and results in a thinning or complete loss of myelin and, less frequently, the cutting (transection) of the neuron's extensions or axons. Like SLE, there is hitherto no known cure for multiple sclerosis. Thee primary aims of therapy have been returning function after an attack, preventing new attacks, and preventing disability. Different treatment strategies are used depending on whether the disease is progressive or relapsing. Medications for relapsing MS include betainterferons, glatiramer (Copaxone) and natalizumab (Tysabri); medications for progressive MS include corticosteroids and muscle relaxants.

[0077] Pernicious anemia is a condition in which the body does not make enough red blood cells due to a lack of vitamin B12 and has been treated by administering vitamin B12 supplements. Much higher doses than normally required in order to overcome the impaired absorption that characterizes pernicious anaemia. If oral tablets are not desired, vitamin B12 can also be administered via injection.

[0078] Scleroderma is an autoimmune disease of the connective tissue. Scleroderma is characterized by the formation of scar tissue (fibrosis) in the skin and organs that can lead to thickness and firmness of involved areas. Scleroderma can also be referred to as systemic sclerosis. Treatment of scleroderma is directed towards the individual feature(s) affecting different areas of the body. For example, aggressive treatment of elevated blood pressure, using blood pressure medications such as captopril, can prevent kidney failure. Further, colchicine can be helpful in decreasing the inflammation and tenderness that periodically accompanies the calcinosis nodules in the skin and skin itching can be relieved with lotions (emollients), such as Eucerin and Lubriderm.

[0079] Systemic sclerosis is a clinically heterogeneous, systemic disorder, which affects the connective tissue of the skin, internal organs and the walls of blood vessels. It may be characterized by alterations of the microvasculature, disturbances of the immune system and by massive deposition of collagen and other matrix substances in the connective tissue. Again like SLE, the condition has been treatable, but not curable. Therapy involves immunomodulation as well as the targeting of blood vessel mechanics and fibrosis.

[0080] Crohn's disease is an ongoing disorder that causes inflammation of the gastrointestinal (Gl) tract. Crohn's disease can affect any area of the Gl tract, from the mouth to the anus, but it most commonly affects the lower part of the small intestine, the ileum. Swelling can extend deep into the lining of the affected organ and cause pain and diarrhea. Treatment may include drugs, nutrition supplements, surgery, or a combination of these options, but like SLE, there is hitherto no cure. The goals of treatment have been to control inflammation, correct nutritional deficiencies, and relieve symptoms like abdominal pain, diarrhea, and rectal bleeding. Treatment for Crohn's disease also depends on the location and severity of disease.

[0081] Embodiments of the instant invention find use in treating one or more of the autoimmune conditions described herein, e.g., in conjunction with one or more other treatment approaches known in the art.

[0082] The amount of the cells needed for achieving a therapeutic effect will be determined empirically in accordance with conventional procedures for the particular purpose. Generally, for administering the cells for therapeutic purposes, the cells are given at a pharmacologically effective dose. By "pharmacologically effective amount" or "pharmacologically effective dose" is an amount sufficient to produce the desired physiological effect or amount capable of achieving the desired result, particularly for treating the disorder or disease condition, including reducing or eliminating one or more symptoms or manifestations of the disorder or disease. With respect to SLE, for example, symptoms include renal dysfunction, accelerated atherosclerosis and in some cases almost all organ systems can be affected (Zampieri, S., laccarino, L., Ghirardello, A., Tarricone, E., Arienti, S., Sarzi- Puttini, P., Gambari, P., and Doria, A. 2005. Systemic lupus erythematosus, atherosclerosis, and autoantibodies. Ann N Y Acad Sci 1051 :351-361). Therapeutic benefit also includes halting, reversing, stabilizing or slowing the progression of the underlying disease or disorder, regardless of whether improvement is realized. The benefit may be observed in an appreciable or significant portion of treated patients (or animals in an experimental model), e.g., at least about 60%, at least about 70%, or at least about 80%. Therapeutic benefit also includes a significant increase in overall survival. Pharmacologically effective dose, as defined above, will also apply to therapeutic compounds used in combination with the cells, as further described below. [0083] Cells isolated directly from a donor subject without expansion in culture may be used for therapeutic purposes. Preferably, the isolated cells are an enriched or substantially pure population of cells. The unexpanded cells are preferably allogeneic to the recipient, where the cells have a complete match, or partial or full mismatch with the MHC of the recipient. In preferred embodiments, the HSC cells represent allogeneic haplo-identical HSC. For example, some embodiments use purified haplo-identical but minor histocompatibility loci mis-matched allogeneic HSC.

[0084] Transplantation of cells into an appropriate host is accomplished by methods generally used in the art. The preferred method of administration is intravenous infusion. The number of cells transfused will take into consideration factors such as sex, age, weight, the types of disease or disorder, stage of the disorder, the percentage of the desired cells in the cell population (e.g., purity of cell population), and the cell number needed to produce the desired result. Generally, the numbers of cells infused may be from about 1 x 10⁴ to about 1 x 10⁵ cells/kg, from about 1 x 10⁵ to about 10 x 10⁶ cells/kg, preferably about 1 x 10^δ cells to about 5 x 10⁶ cells/kg of body weight, or more as necessary. In some embodiments, the cells are in a pharmaceutically acceptable carrier at about 1 x10⁹ to about 5 x10⁹ cells. Cells are administered in one infusion, or through successive infusions over a defined time period sufficient to generate a therapeutic effect. Different populations of cells may be infused when treatment involves successive infusions. A pharmaceutically acceptable carrier, as further described below, may be used for infusion of the cells into the patient. These will typically comprise, for example, buffered saline (e.g., phosphate buffered saline) or unsupplemented basal cell culture medium, or medium as known in the art.

[0085] In accordance with the objectives of the present invention the number of cells infused will be sufficient to establish chimerism, that is, mixed chimerism, preferably durable, mixed chimerism. Chimerism is a stable mixture of donor and host immune cells (at any ratio) that can be induced by conditioning followed hematopoietic stem cell transplantation. Conditioning may be fully myeloablative, reduced intensity or non-myeloablative. As discussed above, conditioning is a heterogeneous group of treatments employing varying doses of radiation, chemotherapeutics, and anti-lymphocyte globulins. In preferred embodiments, reduced intensity conditioning and non- myeloablative conditioning are used to minimize transplant related risks.

[0086] In some embodiments, the methods and compositions described herein find use in treating autoimmune diseases before the disease progresses significantly. That is, in such embodiments, the patient receives treatment when her condition is at early stage of disease progression, e.g., before any, one, a few or all of the symptoms typically associated with the autoimmune disease appear or are established. In other embodiments, the methods and compositions described herein find use in treating autoimmune diseases at later stages, e.g., to reverse established symptoms of lupus. For example, the patient may receive treatment after the autoimmune disease is established, such as when many or all of the symptoms typically associated with the autoimmune disease have manifested and/or long-term effects have become apparent. This includes patients being treated for SLE by hematopoietic cell transplantation that have already developed severe symptoms of disease. In preferred embodiments, transplantation is performed before major organ damage occurs in the recipient patient.

6.4 Adjunctive Treatments

[0087] A variety of adjunctive treatments may be used with the cells, expanded or unexpanded, described above. In one aspect, the adjunctive treatments include, among others, anti-fungal agents, anti-bacterial agents, and anti-viral agents.

[0088] In one aspect, the adjunctively administered agent is an anti-fungal agent. Fungal infections are one of the major causes of mortality in patients suffering from neutropenia, being a significant problem in patients who have undergone myeloablative therapy and HSCT. Recipients with delayed engraftment and patients who develop GVHD typically have prolonged neutropenia, and thus are at high risk for fungal infections. Types of fungal infections are varied, and include, among others, candidiasis (e.g., with Candida krusei, Candida glabrata, Candida albicans, Candida tropicalis), aspergillosis (e.g., with aspergillus fumigatus, aspergillus flavus), mucormycosis (e.g., with rhizobium arrhizus, absidia corymbifera, rhizomucor pusillus), cryptococcosis, histoplasma capsulatum, and coccidioides immitis.

[0089] Anti-fungal agents for adjunctive administration will generally be a systemic antifungal agent. One useful antifungal agent of this type is amphotericin B from the family of polyene macrolide antibiotics. Amphotericin B is available in various formulations, including as a complex with deoxycholate; in a colloidal suspension with cholestearyl sulfate; and encapsulated in liposomes made of soy lecithin, cholesterol, and distearoylphosphatidylglycerol. Other formulations are known in the art.

[0090] Another antifungal agent is flucytosine, a fluorinated pyrimidine. Deamination of flucytosine by the fungus generates 5-flurouracil, an anti-metabolite and DNA synthesis inhibitor. Flucytosine is typically used for infections of cryptococcus and candiadosis. Although used alone, flucytosine is generally used in combination with amphotericin B.

[0091] Imidazoles and triazoles represent a broad class of azole based antifungal agents. It is believed that imidazoles and triazoles inhibit sterol 14-.alpha.-demethylase, resulting in impaired biosynthesis of ergosterol and disruption of cell membrane based activities, such as electron transport. Azole based antifungals are effective against certain types of candiadosis, such as Candida albicans, Candida glabrata, and Candida neoformans. Exemplary azole antifungals suitable for systemic administration include, among others, ketoconzaole, itracanazole, fluconazole, econazole, voriconazole, and tercanozole.

[0092] In addition to fungal infections, a patient with neutropenia is susceptible to infection with a variety of bacterial pathogens. Patients undergoing myeloablative regimens and HSCT have high rates of bacterial infection with both Gram positive (e.g., streptococcus and staphylococcus aureus) and Gram negative bacteria (e.g., E. coli. and pseudomonas aeruginosa). Septecemia is a common occurrence. In addition, delayed engraftment and impaired restoration of immune responses against encapsulated bacteria, such as streptococcus pneumoniae or haemophilus influenza, increases the morbidity rate for transplant recipients with GVHD.

[0093] Adjunctive antibacterial therapy can use any known antibiotics suitable for the particular bacterial pathogen. These include both wide spectrum antibiotics and more targeted anti-bacterial compounds. Various classes of anti-bacterial agents suitable with the expanded myeloid cells include, by way of example and not limitation, quinolones and fluoroquinolones, .beta. -lactam antibiotics, aminoglycosides, tetracycline, macrolides, and various cogeners thereof. Exemplary quinolone compounds include ciprofloxacin, ofloxacin, sparfloxacin, lomefloxacin, and moxifloxacin. Exemplary .beta. -lactam antibiotics include penicillins (e.g., penicillin G, penicillin V), ampicillin, carbenicillin, methicillin, carbapenem, and cephalosporins (e.g., cephalothin, cefamandole, cefaclor, cefonicid, cefotetan, cefatoxime, ceftazidime, ceftizoxime, cefepime). Exemplary aminoglycosides include neomycin, streptomycin, kanamycin, gentamicin, tobramycin, amikacin, and netilmicin. Exemplary macrolides include erythromycin, clarithromycin, and azithromycin. Other antibiotics will be apparent to the skilled artisan.

[0094] Viral infections are also problematic in myeloablated patients and HSCTs. Generally the increased risk of viral infection results from impaired cell mediated immunity brought on by the myeloablative therapy. Many of these infections arise from reactivation of latent virus existing in a seropositive patient or in the cells of a seropositive donor. Viruses commonly encountered include, among others, cytomegalovirus, herpes simplex virus, varicella zoster virus, herepesvirus-6, Epstein Barr virus; adenoviruses, and the like. As an adjunct to the cell infusions, anti-viral compounds selected are those appropriate to the viruses encountered by the patient. Useful antiviral compounds include, by way of example and not limitation, acyclovir, cidofovir, ganciclovir, idoxuridine, penciclovir, valganciclovir, valacyclovir, vidarabine, amantadine, rimantadine, zanamivir, fomivirsen, imiquimod, and ribavirin. Therapeutics directed against retroviruses include, among others, nucleoside reverse transcriptase inhibitors (e.g., zidovudine, didanosine, stavudine, zalcitabine, lamividudine), non- nucleoside reverse transcriptase inhibitors (e.g., nevirapine, efavirenz, delvirudine), and protease inhibitors (e.g., saquinivir, indinavir, ritonavir, nelfinavir, amprenavir, and iopinavir).

[0095] The antifungal, antibacterial, and antiviral agents may be used as prophylaxis to reduce the occurrence of the infection, or upon appearance of the disease. Prophylaxis is particularly indicated for fungal infections common in immunosuppressed patients, and for viral infections in seropositive patients or seropositive transplant donors. Accordingly, embodiments for therapeutic purposes include combinations of HSCs and the antifungal, antibacterial, or antiviral compounds.

[0096] A variety of vehicles and excipients and routes of administration may be used for adjunctive therapy, as will be apparent to the skilled artisan. Representative formulation technology is taught in, inter alia, Remington: The Science and Practice of Pharmacy, 19th Ed., Mack Publishing Co., Easton, PA (1995) and Handbook of Pharmaceutical Excipients, 3rd Ed, Kibbe, A.H. ed., Washington DC, American Pharmaceutical Association (2000); hereby incorporated by reference in their entirety. 6.5 Pharmaceutical Compositions

[0097] The pharmaceutical compositions will generally comprise a pharmaceutically acceptable carrier and a pharmacologically effective amount of the compounds, or mixture of thereof, or suitable salts thereof. The pharmaceutical compositions may be formulated as powders, granules, solutions, suspensions, aerosols, solids, pills, tablets, capsules, gels, topical cremes, suppositories, transdermal patches, and other formulations known in the art.

[0098] As used herein, "pharmaceutically acceptable carrier" comprises any of standard pharmaceutically accepted carriers known to those of ordinary skill in the art in formulating pharmaceutical compositions. Thus, the cells or compounds, by themselves, such as being present as pharmaceutically acceptable salts, or as conjugates, may be prepared as formulations in pharmaceutically acceptable diluents; for example, saline, phosphate buffer saline (PBS), aqueous ethanol, or solutions of glucose, mannitol, dextran, propylene glycol, oils (e.g., vegetable oils, animal oils, synthetic oils, etc.), microcrystalline cellulose, carboxymethyl cellulose, hydroxylpropyl methyl cellulose, magnesium stearate, calcium phosphate, gelatin, polysorbate 80 or the like, or as solid formulations in appropriate excipients.

[0099] The pharmaceutical compositions will often further comprise one or more buffers (e.g., neutral buffered saline or phosphate buffered saline), carbohydrates (e.g., glucose, mannose, sucrose or dextrans), mannitol, proteins, polypeptides or amino acids such as glycine, antioxidants (e.g., ascorbic acid, sodium metabisulfite, butylated hydroxytoluene, butylated hydroxyanisole, etc.), bacteriostats, chelating agents such as EDTA or glutathione, solutes that render the formulation isotonic, hypotonic or weakly hypertonic with the blood of a recipient, suspending agents, thickening agents, preservatives, flavoring agents, sweetening agents, and coloring compounds as appropriate.

[00100] While any suitable carrier known to those of ordinary skill in the art may be employed in the compositions, the type of carrier will typically vary depending on the mode of administration. The therapeutic compositions may be formulated for any appropriate manner of administration, including for example, oral, nasal, mucosal, rectal, vaginal, topical, intravenous, intraperitoneal, intradermal, subcutaneous, and intramuscular administration.

[00101] For parenteral administration, the compositions can be administered as injectable dosages of a solution or suspension of the substance in a physiologically acceptable diluent with a pharmaceutical carrier that can be a sterile liquid such as sterile pyrogen free water, oils, saline, glycerol, polyethylene glycol or ethanol. Additionally, auxiliary substances, such as wetting or emulsifying agents, surfactants, pH buffering substances and the like can be present in compositions. Other components of pharmaceutical compositions are those of petroleum, animal, vegetable, or synthetic origin, for example, non-aqueous solutions of peanut oil, soybean oil, corn oil, cottonseed oil, ethyl oleate, and isopropyl myristate. [00102] The pharmaceutical compositions described herein may be presented in unit-dose or multi- dose containers, such as sealed ampoules or vials Such containers are typically sealed in such a way to preserve the sterility and stability of the formulation until use In general, formulations may be stored as suspensions, solutions or emulsions in oily or aqueous vehicles, as indicated above Alternatively, a pharmaceutical composition may be stored in a freeze-dπed condition requiring only the addition of a sterile liquid carrier immediately prior to use

6.6 Kits

[00103] Other embodiments of the compositions described herein are kits comprising the HSCs, cytokines and growth factors (e g , G-CSF, GM-CSF, TPO) and/or adjunctive therapeutic compounds A label typically accompanies the kit, and includes any writing or recorded material, which may be electronic or computer readable form (e g , disk, optical disc, memory chip, or tape) providing instructions or other information for use of the kit contents

[00104] The following examples serve to more fully describe the manner of using the above-described invention It is understood that these examples in no way serve to limit the true scope of this invention, but rather are presented for illustrative purposes

[00105] All reference publications cited herein are incorporated by reference in their entirety

7. EXAMPLES

[00106] In these Examples, the inventors investigated the use of highly enriched, haplo-identical allogeneic hematopoietic stem cells to both prevent the development and reverse already established symptoms of autoimmune disease and treat established autoimmune pathology

[00107] The experiments of Example 1 were designed to compare the outcome of allogeneic HSC transplantation to syngeneic HSC and syngeneic whole bone marrow (WBM) transplants for the treatment of lupus-like symptoms Briefly, the inventors transplanted young, fully ablated, female NZBW mice with purified haplo-identical allogeneic HSC, syngeneic HSC or syngeneic whole bone marrow from age matched donors The experiments of Example 2 were designed to determine if lupus-like disease in older NZBW mice with established autoimmune disease could be reversed by nonmyeloablative transplantation of purified haplo-identical allogeneic HSC

[00108] The mice transplanted with allogeneic HSC in both studies had increased overall survival, and a stabilization or reversal of their lupus symptoms as measured by a reduction of proteinuria and a lower frequency of mice with CIC and auto-antibodies, as compared to untreated control mice, lymphoablated mice or mice transplanted with syngeneic cells, demonstrating that transplant can prevent and reverse the disease These results demonstrate the use of purified allogeneic HSC alone to treat established autoimmune disease in animals, that is, using highly purified haplo-identical allogeneic HSC with nonmyeloablative conditioning with a goal of durable mixed chimeπsm can treat established lupus Example 1 -Transplantation of young NZBW mice and establishment of donor chimerism to compare allogeneic HSC transplants to syngeneic HSC and WBM transplants when myeloablative conditioning is used

[00109] The inventors compared the onset and severity of lupus-like disease in female NZBW mice transplanted with either purified haplo-identical allogeneic HSC or syngeneic (pseudo-autologous) HSC. The inventors also compared transplantation of purified syngeneic HSC with syngeneic WBM in delaying or preventing disease onset.

[00110] Mouse Strains. Eight-week old New Zealand Black x New Zealand White (NZBW, H-2d/z) female mice were purchased from Jackson Laboratories (Barharbor, ME). NZBW mice develop a complex, spontaneous autoimmune disease involving the misregulation of many aspects of the immune system that is very similar to SLE (Theofilopoulos, A. N., and Dixon, F.J. 1985. Murine models of systemic lupus erythematosus. Adv Immunol 37:269-390). The lupus-like disease in these mice is 100% lethal, with female mice living an average of 280 days, and few surviving more than 390 days (Andrews, B. S., Eisenberg, R. A., Theofilopoulos, A.N., Izui, S., Wilson, C. B., McConahey, P.J., Murphy, E. D., Roths, J. B., and Dixon, FJ. 1978. Spontaneous murine lupus-like syndromes. Clinical and immunopathological manifestations in several strains. J Exp Med 148:1198-1215; Yoshida, S., Dorshkind, K., and Gershwin, M. E. 1987. Hemopoietic cell dysfunction in murine lupus. Clin Exp Rheumatol 5:79-87). Mice were maintained and aged in the Stem Cells Inc. animal facility (Palo Alto, CA) following Institutional Animal Care and Use Committee approved protocols and procedures.

[00111]/\/7f/όod/es and Cell Preparation. Hematopoietic stem cells were isolated as c-Kit⁺Sca-1⁺lin^' "^ow. Femurs and tibia from donor mice were crushed, and the suspension was filtered through nylon screen. Red blood cells were lysed during 3-minute incubation in 0.15 M ammonium chloride, 0.01 M potassium bicarbonate solution on ice. c-Kit-positive cells were enriched by positive selection using CD117-congugated magnetic beads and an autoMACS cell separator (Miltenyi Biotec, Auburn, CA) according to manufacturer instructions. All antibody incubations were performed on ice for 25 minutes with an appropriate concentration of antibody. The monoclonal antibodies used in the immunofluorescence staining for HSC isolation included CD117, PE-Cy7; Sca-1 (Ly6A/E), APC; and a lineage cocktail of CD3, CD4, CD5, CD8, Ter119, B220, Mac-1 and GR-1 , PE (eBioscience, San Diego, CA). Prior to FACS sorting or analysis, cells were resuspended in 1 μg/ml of propidium iodine to detect and exclude dead ceils. Cell sorting and analysis was performed using a 3-laser FACSAria (BD Biosciences, San Jose, CA). Cells used in this study were double sorted to ensure purity.

[00112] Conditioning. For the myeloablative studies, recipient mice were lethally irradiated with 14.5 Gy radiation using a cesium source irradiator (J. L. Shepard, Los Angeles, CA), delivering 171 rads/min (maintained by Stem Cells Inc., Palo Alto, CA). Radiation was delivered in a split dose, given 3-4 hours apart. Three cohorts of mice were lethally irradiated and transplanted with allogeneic HSC, syngeneic HSC or syngeneic WBM. A fourth group was studied as unmanipulated age matched controls. [00113] Mice were anesthetized with inhaled isoflurane (Baxter Pharmaceutical, Deerfield, IL) prior to delivery of cells Cells were administered with a 27 gauge needle in 100 ml PBS, 2% FCS and injected into the posterolateral venous sinus of the orbital cavity All mice were maintained on acidified water Antibiotics (106 U/L polymixin B sulfate and 1 1 g/L neomycin sulfate) were added to the water of transplanted mice for 6 weeks following irradiation

[00114] Transplants Recipient mice used in the myeloablative studies were transplanted at 73 and 79 days of age NZBW donor mice for the syngeneic HSC and WBM were age matched to the recipients Donor mice for the allogeneic HSC, DBA2 x C57/BI6 (DBF1 , H-2d/b), were purchased from Charles River Laboratories (Hollister, CA) These are H2 haploidentical and minor histocompatibility loci mismatched to the NZBW hosts DBF1 donor mice were 10 weeks of age at time of bone marrow collection Three separate cohorts of mice were transplanted, in total 28 mice received 25-40x10³ allogeneic HSC, 33 mice received 1 5-7 5x10³ syngeneic HSC and 26 mice received 0 8-1 5x10⁶ WBM cells Fifteen mice were not transplanted

[00115] Recipient mice were bled monthly via the tail vein and peripheral blood was analyzed for donor chimeπsm using antibodies to the H-2K^b and H-2K^d loci (BD Bioscience, San Jose, CA), and the lineage markers B220, CD3, Mac-1 , and GR-1 (eBioscience, San Deigo, CA, BD Biosciences, San Jose, CA) Cell suspensions were analyzed using a FACSAria (BD Biosciences, San Jose, CA) Data were analyzed using FIoJo software (Tree Star lnc , San Carlos, CA)

[00116] Rest//te Mice receiving haplo-identical allogeneic HSC had stable donor chimeπsm exceeding 95% (Figure 1A), as measured in the peripheral blood by means of MHC differences in the host and donor strains Fig 1 A illustrates results with NZBW mice conditioned with lethal irradiation and transplanted with purified allogeneic HSC As detailed above, the blood was analyzed periodically for donor chimeπsm by flow cytometry Lower levels of T cell chimeπsm were detected than B cell and myeloid chimeπsm (P< 0 00001) The circles depict individual mice Horizontal lines represent mean chimerism of each group

[00117] As Fig 1A illustrates, a significant number of host T cells remained in circulation after transplantation with purified allogeneic HSC, as previously described (Beilhack, G F , Scheffold, Y C , Weissman, I L , Taylor, C , Jerabek, L , Burge, M J , Masek, M A , and Shizuru, J A 2003 Purified allogeneic hematopoietic stem cell transplantation blocks diabetes pathogenesis in NOD mice Diabetes 52 59-68, Shizuru, J A , Jerabek, L , Edwards, C T , and Weissman, I L 1996 Transplantation of purified hematopoietic stem cells requirements for overcoming the barriers of allogeneic engraftment Biol Blood Marrow Transplant 2 3-1) These host T cells most likely survived radiation, perhaps due to the relative resistance gained by antigen activation (Adkms, B , Gandour, D , Strober, S , and Weissman, I 1988 Total lymphoid irradiation leads to transient depletion of the mouse thymic medulla and persistent abnormalities among medullary stromal cells J Immunol 140 3373-3379 ) [00118] High donor chimeπsm was also observed in the hematopoietic organs of the surviving allogeneic transplanted mice (Figure 1 B) Thymuses were obtained from mice when moribund or at the conclusion of the myeloablative study and analyzed for B cell infiltration Cells were dispersed by grinding the tissue between frosted slides in HANKS' Balanced Salt Solution

[00119] To collect peritoneal B cells, the animal was euthanized and immediately injected IP (intraperitoneal) with 10 ml HANKS' Balanced Salt Solution supplemented with 2% FCS and 2mM EDTA The abdomen of the mouse was massaged to evenly disperse the media throughout the cavity The media was then aspirated with the syringe The peritoneal cavity was then exposed, and any remaining fluid from the cavity was removed The remaining volume was added to the original sample

[00120] Cells collected from the thymus and peritoneal cavity of transplanted mice were analyzed for donor chimerism using antibodies to the H-2K^b and H-2K^d loci (Caltag- Invitrogen), and various combination of the B cell markers B220, PECy7, CD5, biotin, IgM, FITC, IgD, PE, Mac-1 , APC (eBioscience, San Deigo, CA, BD Biosciences, San Jose, CA) Streptavidin was conjugated to pacific blue fluorochrome (Molecular Probes, Eugene, OR)

[00121] As Fig 1 Billustrates, tissues of transplanted mice were analyzed by flow cytometry when moribund or at the conclusion of the study T cell (Green) chimerism was lower than B cell (Blue) and myeloid (Red) chimerism in the blood, bone marrow, lymph nodes and spleen Donor B cell chimerism was significantly lower in the peritoneal cavity than in the other tissues (P≤ 0 0007) (Data combined from 3 experiments)

[00122] As noted, in the peripheral blood, donor T cell chimerism in the hematopoietic tissues was lower than B cell and myeloid chimersim However, the highest frequency of host cells was observed in the B cell population in the peritoneal cavity, which was composed almost exclusively of CD5⁺ B1 cells

[00123] Effect of transplantation on the clinical course of lupus-like disease Detection of Lupus Symptoms Mice were monitored for approximately 350 days post transplantation for the onset of lupus-like symptoms weight loss, proteinuria (urine protein > 100 mg/dL), and death Proteinuria was monitored using a Chemstrip (Roche Pharmaceutical, Indianapolis, IN) Urine was collected from each animal into a sterile tube A volume of 20-30 μl was applied to a Chemstrip Urine protein (mg/dL) was determined according to manufacturer's instructions Proteinuria was defined as urine protein ≥ 100 mg/dL in concurrence with published studies (Wofsy, D , and Seaman, W E 1985 Successful treatment of autoimmunity in NZB/NZW F1 mice with monoclonal antibody to L3T4 J Exp Med 161 378-391) During the development of lupus-like disease in NZBW mice the levels of urine protein fluctuates to some extent during disease progression, and stabilizes with severe disease Peak levels of proteinuria were observed [00124] Serum was collected monthly beginning at 8 weeks post transplant, the final time point before death was screened for CIC and auto-antibodies to dsDNA, nuclear antigens (ANA) and histones. Antigen test kit and Anti-Histone test kits were performed according to manufacturer's instructions. Circulating Immune Complexes (CIC) and auto-antibodies were tested by ELISA using test kits (Alpha Diagnostics, San Antonio, TX). Serum was collected monthly from all mice during the studies. The last time point before death was used for serology. The donor strain, DBF, was used to establish baseline levels of auto-antibodies. Circulating Immune Complexes test kit, Anti-dsDNA test kit, Anti- Nuclear. Serology was not performed for mice not surviving 8 weeks post transplant.

[00125] As Fig. 2 and Table 3 show, transplantation with either syngeneic HSC or WBM accelerated mortality in these mice, resulting in a rate of death exceeding age matched controls (P ≤ 0.0001).

Table 3: Summary of survival, proteinuria and serology of mice receiving fully ablative conditioning.

[00126] Statistical Analysis. Statistical significance was evaluated using 2-sample T test. For survival curves, statistical significance was assessed using Kaplan Meyer survival analysis, with differences between groups analyzed by the log-rank test (Graphpad Prism 4.0). Results were considered statistically significant with a P less than 0.05.

[00127] Fig. 2 illustrates results obtained with NZBW mice lethally irradiated and transplanted with either allogeneic HSC (N=28), syngeneic HSC (N=28) or syngeneic WBM (N=21 ). Mice were either 73 or 79 days of age at the time of transplant (arrow approximate, showing that mice were on average 75 days of age at the time of transplant). A subset of mice was reserved as unmanipulated age matched controls (N=15). Survival was monitored for approximately 350 days post transplantation (420 days of age). Data is presented as the percent mice with peak urine protein levels > 100 mg/dL. Mice were analyzed monthly for proteinuria beginning at 8-12 weeks post transplantation. Mice dying before that time are not included. The last time point before death or at the conclusion of the study was used for serology. Data shown indicates the percentage of NZBW mice with serum autoantibody titers greater than levels detected in the DBF mice. (Data combined from 3 experiments).

[00128] Mice transplanted with purified allogeneic HSC (red) had significantly improved overall survival compared to the age matched control mice (gray) (P= 0.0243) or the mice receiving syngeneic cells (P≤ 0.0001). Mice transplanted with either syngeneic HSC (blue) or syngeneic WBM (green) had significantly poorer survival than the age matched control mice (P≤ 0.0001). Survival curves illustrate age of mice at death. (Data combined from 3 experiments).

[00129] In Fig. 3, serum was screened for titers of CIC or auto-antibodies (as described above) by ELISA from the final draw before death or at the conclusion of the study (3A: circulating immune complexes (CIC); 3B: anti-dsDNA; 3C: anti-nuclear antigen (ANA); and 3D: anti-histone). The levels of CIC and all auto-antibodies analyzed was significantly lower in the mice transplanted with allogeneic HSC than mice transplanted with syngeneic HSC or WBM, and the age matched controls (P≤ 0.0001 ). Horizontal lines represent mean of each group. Horizontal lines represent mean chimerism of each group. Serum from donor strain mice, DBF, was used to establish baseline levels of auto-antibodies. (Data combined from 3 experiments).

[00130] Most of the mice in these three groups developed severe proteinuria, urine protein ≥ 500 mg/dL. Mice transplanted with syngeneic HSC or WBM lived an average of 182 and 193 days post transplant, with no mice living until the conclusion of the study. There was no significant difference in survival between mice transplanted syngeneic HSC or WBM (P = 0.6367). The age matched control mice lived an average of 269 days, with 2 of 15 mice alive at the conclusion of the study. The occurrence and severity of proteinuria, CIC and autoantibodies measured in the syngeneic transplanted groups was comparable to the age matched controls (Table 3) (Figure 3).

[00131] In contrast, the mice transplanted with allogeneic HSC had improved survival (P = 0.0243), 14 of 28 survived to the end of the study, with lower incidence of proteinuria, CIC and auto-antibodies (P ≤ 0.0001) than the age matched controls (Table 3). Two of the 28 mice died shortly after transplantation and were not analyzed for urine protein or auto-antibodies. Of the 26 mice analyzed, only 4 of the mice transplanted with allogeneic HSC developed proteinuria, only one of these progressed to severe proteinuria (urine protein > 500 mg/dL). Of those mice transplanted with allogeneic HSC that developed positive titers of CIC or auto-antibodies, the serum levels were generally lower than the levels measured in the syngeneic transplanted groups or age matched controls (Figure 3). These mice were on average, 100 days older than the syngeneic transplanted mice when the serum for the serology was collected. [00132] Effect of transplantation on the aberrant accumulation of B cells in the thymus, B cell abnormalities are one of the most recognized elements in the immunologic deregulation in NZBW mice. While B cells are a normal component of the thymic microenvironment, aged NZBW have an aberrant increase of B cells in the thymus as well as other target organs (Ito, T., Ishikawa, S., Sato, T., Akadegawa, K., Yurino, H., Kitabatake, M., Hontsu, S., Ezaki, T., Kimura, H., and Matsushima, K. 2004. Defective B1 cell homing to the peritoneal cavity and preferential recruitment of B1 cells in the target organs in a murine model for systemic lupus erythematosus. J Immunol 172:3628-3634; Farinas, M. C, Adkins, B., Stall, A.M., Weissman, I., and Strober, S. 1990. B cell infiltration of the thymic medulla in New Zealand black, New Zealand white, and (New Zealand black x New Zealand white) F1 mice. Effect of total lymphoid irradiation. Arthritis Rheum 33:702-710). At death or at the conclusion of this study, the inventors analyzed the thymuses of transplanted and control mice for thymic B cell accumulation (as described above). To establish a baseline, the thymuses of young, female NWBW mice, ≤ 6 months of age, were analyzed by flow cytometry for B cells.

[00133] The frequency of B cells in the thymuses of these mice ranged from 0.5-3.8%, with a mean of 1.6%. In comparison, the older age matched control mice had levels of thymic B cells ranging from 4.7 to 70.4%, with a mean of 27.5% at the time of death. Of this group, 2 of 8 or 25% had a B cell frequency of approximately 5%, near baseline levels. The mice transplanted with either syngeneic HSC or WBM had a marked increase of thymic B cells, ranging from 21-68.8% with a mean of 39.4%. In contrast, the mice transplanted with allogeneic HSC had thymic B cell numbers ranging from 1.3- 36.8%, with a mean of 14.1 %. Of the allo-transplant group, 8/17 or 47%, had approximately 5% or fewer thymic B cells, a significantly lower frequency than the age matched controls (P=O.0416), and mice transplanted with syngeneic cells (P ≤ 0.001).

[00134] Discussion - These studies show that transplantation of purified, haplo-identical but minor histocompatibility loci mis-matched allogeneic HSC can prevent the occurrence of the symptoms of auto-immune disease, successfully blocking the strong genetic predisposition for development of the lupus-like disorder in this strain of mice and improving overall survival. Of the young mice transplanted with allogeneic HSC, only 1 developed severe proteinuria. The mice transplanted with allogeneic HSC also had a significantly reduced tendency towards the generation of auto-antibodies. These mice were able to maintain tolerance even with the presence of an average of 25% host T cells (McCoy, K.L., Kendrick, L., and Chused, T.M. 1986. Tolerance defects in New Zealand Black and New Zealand Black X New Zealand White F1 mice. J Immunol 136:1217-1222).

[00135] These studies demonstrate further that young NZBW mice transplanted with haplo-identical allogeneic HSC had a significant decrease in autoantibody production and proteinuria and reduced accumulation of B cells in the thymus, indicating that replacement of the faulty immune system is effective in controlling several aspects of this complex disease. In contrast, NZBW mice treated by transplantation of purified syngeneic HSC or WBM experienced acceleration in the rate of death. No difference was observed in the disease course whether the T and B cells were eliminated from the syngeneic graft prior to transplantation. The ablative conditioning may have further deregulated the immune response in these mice, allowing for increased expansion of radioresistant auto-reactive cells and a hastening of the disease course Allogeneic HSC transplantation can offer a curative treatment for SLE through replacement of the abnormal immune system, immune modulation, or induction and maintenance of tolerance

Example 2 - Nonmyeloablative transplantation in eight month old NZBW mice leads to reversal of lupus symptoms

[00136] The inventors also sought to determine if nonmyeloablative transplantation of haplo-identical allogeneic HSC could halt progression or reverse auto-immune disease in older NZBW mice with established lupus-like disease Because nonmyeloablative conditioning is also lymphoablative, the inventors compared the progression of lupus-like disease in transplanted mice to mice receiving lymphoablative conditioning alone To achieve lymphoablation and eliminate cells that posed a barrier to engraftment, the mice received a nonmyeloablative conditioning regimen with radiation, anti- thymocyte serum and anti-asialo GM 1

[00137] Mice - Recipient mice used in the nonmyeloablative studies were 227 and 255 days of age DBF1 donor mice were 10 weeks of age at time of bone marrow collection Two separate cohorts of mice were treated, mice that were transplanted received 30x10³ allogeneic HSC

[00138] Conditioning Eight-month old NZBW mice were conditioned by sub-lethal doses of irradiation, and administration of anti-thymocyte serum and anti-asialo GM-1 For the nonmyeloablative studies, mice received two doses of 5 Gy irradiation given 24 hours apart on days -2, and -1 Mice were additionally given IP injections of 200 ml of rabbit anti-mouse anti-thymocyte serum (Fitzgerald Industries International, Concord, MA) on days -5, and -3, and 100 mg of anti-Asialo GM1 polyclonal antibody (Wako Chemical USA, Richmond, VA) on days -7 and -2 prior to transplantation

[00139] Transplants - The conditioned mice were divided into two groups, one receiving haplo- identical allogeneic HSC and the other receiving conditioning only Since the conditioning regimen was truly non-myeloablative, syngeneic stem cell rescue was unnecessary in the group receiving conditioning alone A third group of mice was reserved as unmanipulated age matched controls At the time of conditioning the mice were on average 240 days of age with established symptoms of lupus, some of the age-matched control mice died from lupus-like disease prior to the day of treatment Mice were treated at an average of 241 days of age The nonmyeloablative conditioning was well tolerated, no animals died in the 4 weeks after conditioning The mice transplanted with the allogeneic HSC developed an average mixed chimeπsm of approximately 50% (Figure 4)

[00140] Results - Fig 4 illustrates results obtained with NZBW mice receiving nonmyeloablative conditioning and purified allogeneic HSC The blood was analyzed periodically for donor chimeπsm by flow cytometry Circles depict individual mice (Data combined from 2 experiments) [00141] Attenuation of autoimmune disease by transplantation of allogeneic HSC. Following the conditioning regimen, the mice were closely monitored for progression of lupus-like disease. Mice were analyzed monthly for proteinuria beginning before transplantation. Data presented as the percent mice with peak urine protein levels ≥ 100 mg/dL both pre-treatment and post-therapy. Serum was collected monthly beginning at 4 weeks post transplant. Mice dying before that time were not analyzed. The last time point before death or just prior to 335 days post treatment was used for serology. Data shown indicates the percentage of NZBW mice with serum auto-antibody titers greater than levels detected in the DBF mice. (Data combined from 2 experiments).

[00142] The mice that received the conditioning treatment without stem cell transplant had a slight survival advantage over the age matched controls (P< 0.0001), living to an average of 407 days of age versus an average of 350 days of age (Table 4 below) (Figure 5).

Table 4: Summary of survival, proteinuria and serology of mice receiving nonmyeloablative conditioning.

[00143] Fig. 5 illustrates disease progression in eight month old NZBW mice conditioned by sub-lethal doses of irradiation, and administration of anti-thymocyte serum and anti-asialo GM-1 , where approximately half of the mice received allogeneic HSC (N=33), while the others received the conditioning regimen only (N=30). As described above, mice were treated at 227 or 255 days of age (arrow approximate). A subset of mice was reserved as unmanipulated age matched controls (N=13). The mice receiving conditioning only (blue) had a survival advantage over the age matched control mice (gray) (P< 0.0001). The mice transplanted with allogeneic HSC (red) had far better overall survival than the other two groups (P< 0.0001). Survival curves illustrate age of mice at death. (Data combined from 2 experiments).

[00144] The longest surviving mouse from the group that received conditioning only, lived to 518 days of age. In contrast, the mice transplanted with haplo-identical allogeneic HSC had far greater overall survival as compared to both groups (P< 0.0001), with 60% still alive at 575 days of age, 335 days post transplant. All the age matched controls developed proteinuria before death. The frequency of mice with proteinuria increased following treatment in the group receiving conditioning only, from 47% having proteinuria (≥ 100 mg/dL) before treatment to 70% with proteinuria at time of death. The mice transplanted with allogeneic HSC showed a reversal or stabilization of their lupus symptoms. The incidence of transplanted mice with proteinuria declined from 45% pre-transplantation to 30% posttransplantation, a significant difference from both the mice receiving conditioning only (P= 0.014895) and the age matched controls (P< 0.0001).

[00145] The frequency of mice with elevated levels of CIC or auto-antibodies was also lower in the mice transplanted with allogeneic HSC than in the mice receiving the conditioning regiment alone (Table 4). One cohort of mice was analyzed for auto-antibodies just prior to conditioning or transplantation and again before death or at 508 days of age (Figure 6A).

[00146] As Fig. 6A illustrates, eight month old NZBW mice transplanted with haplo-identical allogeneic HSC had a reversal of their disease as measured by a decrease in the frequency of mice with anti- histone auto-antibody titers post transplant. In contrast, the frequency of mice receiving only the conditioning regimen showed no change. Bar grafts indicate the frequency of mice with positive anti- histone titers before and post treatment. Serum was collected the week before treatment and again prior to death or at 335 days post treatment. Frequency of mice with positive titers was not significant between these groups before treatment (P = 0.249), but was significant post-treatment (P = 0.0076).

[00147] Of the mice receiving allogeneic HSC, 13 of 18 mice (72%) had a positive titer of anti-histone auto-antibodies before transplant. Post transplant, 7 of 18 (39%) mice had positive titers, a decrease in frequency of 33%. In contrast, the mice given the conditioning regiment only, 10 of 12 mice (83%) had positive anti-histone titers pre-treatment, post conditioning no change was detected, 10 of 12 mice (83%) had positive titers. The difference between the transplanted and conditioned groups post treatment was significant (P = 0.0076). Differences in CIC or dsDNA titers were not significantly different before and following treatment.

[00148] Four of the transplanted animals and one conditioned animal were euthanized while on study because they developed abscesses. Most of the abscesses occurred late in the study, >427 days of age, after most of the conditioned animals and age-matched controls had died from lupus related indications. At necropsy we noted that 5 of the 11 moribund mice from the group receiving only the conditioning regiment had developed thymic lymphomas (Kaplan, H. S., Brown, M. B., Hirsch, B. B., and Carries, W H 1956 Indirect induction of lymphomas in irradiated mice Il Factor of irradiation of the host Cancer Res 16 426-428, Lieberman, M , Hansteen, G A , McCune, J M , Scott, M L , White, J H , and Weissman, I L 1987 Indirect induction of radiation lymphomas in mice Evidence for a novel, transmissible leukemogen J Exp Med 166 1883-1893) No thymic lymphomas in the allo- transplanted mice were observed Progression of lupus as related to disease course - In Fig 6B, bar grafts indicate the frequency of NZBW mice with proteinuria, urine protein ≥ 100 mg/dL, before and post treatment Eight-month old NZBW mice with mild to moderate disease before treatment, urine protein ≤ 100 mg/dL, were monitored for 335 days post treatment or until death NZBW mice transplanted with allogeneic HSC had a reversal or stabilization of their disease quantified as a decrease in the frequency of mice with proteinuria post-transplant Mice receiving the conditioning regiment only had an increase in frequency of mice with proteinuria post-treatment Frequency of mice with proteinuria was not significant between these groups before treatment (P = 0 415), but was significant post-treatment (P = 0 0008) Average age at the final measurement was 468 days of age for the transplanted mice or 332 days of age for the mice receiving conditioning only (Data combined from 2 experiments)

[00149] In this group, the frequency of mice with proteinuria decreased from 37% pre-transplant to 21% post-transplantation In contrast, the frequency of mice with proteinuria in the conditioning only group increased, from 32% with proteinuria pre-treatment to 61 % with proteinuria before death The difference between the transplanted and conditioned mice after treatment was significant (P = 0 0008) All age matched control mice progressed to severe proteinuria

[00150] Stabilization of disease symptoms and long-term survival was associated with the severity of disease at the time of transplantation - Eight-month old NZBW mice with severe proteinuria at the time of allogeneic HSC transplantation, ≥ 500 mg/dL urine protein, had poor overall survival of 20% In comparison, mice from the same cohort with mild to moderate lupus symptoms at the time of stem cell transplantation, ≤ 100 mg/dL urine protein, had superior overall survival with 64% alive and healthy at 575 days of age Eight month old NZBW mice with mild to moderate lupus symptoms at the time of transplant, had a stabilization or reversal of their disease and 79% did not have measurable proteinuria post-transplantation (Figure 6B)

[00151] Discussion - These studies show that NZBW mice with established symptoms of lupus-like disease can be treated by non-myeloablative allogeneic HSC transplantation Transplantation of eight-month old NZBW mice with allogeneic haplotype-matched HSC successfully halted or reversed the progression of lupus-like disease in approximately 80% of mice with mild to moderate disease and resulted in a significant increase in overall survival The inventors found the benefits of lymphoablative conditioning alone was transient, with greater than 65% of the mice that received conditioning without HSC transplant demonstrating a worsening, continuance or development of proteinuria Mice with severe proteinuria at the time of treatment did not respond well to either therapy The data in this nonmyeloablative model, demonstrates that the induction of mixed chimeπsm is sufficient to control the autoreactive mechanisms in these mice [00152] Moreover, survival of the 8-month old mice by allogeneic transplant with nonmyeloablative conditioning resulted in a greater survival rate than allotransplantation of fully ablated young mice While the number of mice with positive anti-histone titers decreased post transplant, a higher percentage of aged mice transplanted with allogeneic HSC had positive titers than those mice transplanted at a younger age Similarly, aged BXSB mice transplanted with allogeneic WBM also maintained positive levels of auto-antibodies post transplant (Jones, O Y , Steele, A , Jones, J M , Maπkar, Y , Chang, Y , Feliz, A , Cahill, R A , and Good, R A 2004 Nonmyeloablative bone marrow transplantation of BXSB lupus mice using fully matched allogeneic donor cells from green fluorescent protein transgenic mice J Immunol 172 5415-5419) Without being limited to a particular hypothesis, these titers may be due to surviving host auto-reactive B, plasma or B-1 cells (Hayakawa, K , Hardy, R R , Parks, D R , and Herzenberg, L A 1983 The "Ly-1 B" cell subpopulation in normal immunodefective, and autoimmune mice J Exp Med 157 202-218, Hoyer, B F , Moser, K , Hauser, A E , Peddinghaus, A , Voigt, C , Eilat, D , Radbruch, A , Hiepe, F , and Manz, R A 2004 Short-lived plasmablasts and long-lived plasma cells contribute to chronic humoral autoimmunity in NZB/W mice J Exp Med 199 1577-15), as discussed above

[00153] In sum, young, NZBW mice transplanted with puπfied allogeneic HSC had improved overall survival, decreased appearance of proteinuria, of circulating immune complexes (CIC), and of autoantibodies to nuclear antigens than untreated mice or mice given NZBW HSC NZBW mice with established lupus-like disease that received nonmyeloablative conditioning and transplantation with haplo-identical allogeneic HSC also had greatly increased overall survival Transplanted mice exhibited stabilization or reversal of their lupus symptoms, stabilization of or decreased proteinuria, and the frequency of mice with elevated CIC or auto-antibodies was lower in the transplanted mice than various control mice These results establish that induction of durable mixed chimeπsm by transplantation of purified allogeneic HSC after nonmyeloablative conditioning can reverse symptoms of established NZBW lupus

Claims

What is claimed is

1 A method of treating a patient suffering from systemic lupus erythematosus comprising subjecting said patient to myeloablation and administering to said patient allogeneic hematopoietic stem cells in an amount sufficient to establish mixed chimeπsm in said patient

2 The method of claim 1 wherein said allogeneic hematopoietic stem cells are enriched hematopoietic stem cells

3 The method of claim 1 wherein said allogeneic hematopoietic stem cells are purified hematopoietic stem cells

4 The method of claim 3 wherein said purified allogeneic hematopoietic stem cells are CD34+ CD90+ cells

5 The method of claim 1 , wherein said allogeneic hematopoietic stem cells are from a haplo- identical allogenic donor

6 The method of claim 1 , further comprising administering to said patient at least one of an antiviral compound, an anti-fungal compound, an anti-bacterial compound, a cytokine or a growth factor

7 The method of claim 1 wherein said systemic lupus erythematosus is at an early stage of disease progression

8 The method of claim 1 wherein said systemic lupus erythematosus is at a late stage of disease progression