LYMPHOCYTES FROM DONOR STEM CELL OR LYMPHOCYTE GRAFTS TO PREVENT GRAFT- VERSUS-HOST DISEASE

CROSS REFERENCE TO RELATED APPLICATION

This claims the benefit of U.S. Provisional Application No. 62/153,174, filed April 27, 2015, which is incorporated herein by reference. FIELD

This disclosure relates to alloreactive T cells and in particular, to methods of depleting alloreactive T cells from donor grafts to prevent graft-versus-host disease (GVHD).

BACKGROUND

Allogeneic hematopoietic stem cell transplant (Allo-HSCT) is a treatment that can cure malignant and nonmalignant hematopoietic diseases. However, the efficacy and safety of this treatment is limited by GVHD, a multi-organ destructive process caused by allogeneic donor T cells, leading to morbidity and mortality. Immune suppressive agents are used to prevent and treat GVHD but are not always effective and can cause immunodeficiency after HSCT increasing the risk of infectious complications and relapse of malignant disease after transplantation.

SUMMARY

Disclosed herein is a method to specifically remove the donor T cells that cause GVHD from a graft before it is given to a recipient. This approach prevents, reduces or inhibits GVHD while conserving the stem cells and providing beneficial donor immunity against infection and the malignant disease being treated. In some embodiments, the disclosed method uses the ex vivo coculture of donor graft cells with the antigen presenting cells (APC) of the recipients. The recipient's APC stimulate and activate the alloreactive (GVHD causing) donor T lymphocytes. These activated cells can be eliminated specifically by the addition of adenosine or an adenosine- like molecule, which only targets and kills the activated T cells. The resulting cells are then washed to remove adenosine or adenosine-like molecule and are given to the recipient.

In one embodiment, a method is provided for selective depleting alloreactive T cells that includes culturing donor cells comprising donor T cells with recipient antigen presenting cells (APCs) to form a mixture of cells under conditions sufficient for recipient APCs to activate donor T cells. The mixture of cells is contacted with an effective concentration of adenosine or adenosine- like molecule to decrease or inhibit viability of the activated donor T-cells, and then the adenosine or adenosine-like molecule is removed from the mixture of cells.

In another embodiment, method is provided for inhibiting, preventing or reducing the risk of acquiring graft-versus-host disease (GVHD) in a subject receiving donor stem cells, donor immune cells, donor tissue or donor organ transplant. The method includes obtaining donor cells comprising donor T cells from a donor, and obtaining recipient antigen presenting cells (APCs) from the subject receiving the donor stem cells, the donor immune cells, the donor tissue or the donor organ transplant. The donor cells including donor T cells are contacted with the recipient APCs to form a mixture of cells under conditions sufficient for recipient APCs to activate donor T cells. The mixture of cells is contacted with an effective concentration of adenosine or adenosine- like molecule to decrease or inhibit viability of the activated donor T-cells. The adenosine or adenosine-like molecule is then removed from the mixture of cells; and the resulting cell mixture is administered to the recipient thereby preventing or inhibiting GVHD, or reducing the risk of developing GVHD.

The foregoing and other features and advantages of the disclosure will become more apparent from the following detailed description, which proceeds with reference to the

accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic illustrating an exemplary method for the depletion of alloreactive T cells using adenosine.

FIG. 2 is a series of plots and histograms illustrating 2 mM adenosine kills CD3/CD28 bead stimulated T cells. Healthy donor lymphocytes were thawed and stained with 2μΜ

Carboxyfluorescein succinimidyl ester (CFSE, Life technologies) at room temperature in PBS and washed. The lymphocytes were then co-cultured with Dynabeads Human T- Activator CD3/CD28 beads (Life technologies) at a 3:1 cell to bead ratio in human culture media. The beads were washed with PBS before use. Half of the cultures were treated with a 2mM Adenosine

(Adenoscan®, Astellas Pharma US, Inc.) in 2X solution on days 1, 2, and 5 after the cultures were established. Two days after the last treatment the cells were harvested and the CFSE signal on the cells was acquired on a BD LSR Fortessa flow cytometer (BD Biosciences) and analyzed using Flowjo software. CFSE is used to track cell proliferation; the intensity of CFSE signal is diluted and decreases as cells divide. FIG. 3 is a series of plots and histograms demonstrating adenosine treatment selectively eliminated activated alloreactive T cells (CD39 ⁺ CD25 ⁺ CD45RO ⁺ ) in mixed lymphocyte reaction (MLR) culture. MLR assay was established with healthy donor lymphocytes and allogeneic donor dendritic cells (DC) in a 5: 1 ratio. Dendritic cells were generated from healthy donor monocytes cultured in human culture media (AIM V and RPMI (Life technologies) supplemented with 5% normal human AB serum, 1,000 IU/ml GMCSF and 1,000 IU/ml IL-4 (both from (Peprotech) for 4 days, and then matured with 15ng/ml lipopolysaccharide (LPS, Sigma chemicals) overnight. Half of the cultures were treated with a 2mM Adenosine in 2X solution on days 1, 2, and 5 after the cultures were established. Two days after the last treatment the cells were harvested and stained with fluorochrome conjugated monoclonal antibodies as indicated in the figure. Data were acquired on a BD LSR Fortessa and analyzed using Flowjo software.

FIG. 4 provides a schematic of an exemplary product validation proliferation assay. The product validation assay tests the efficiency of allodepletion. MLR was established between healthy donor lymphocytes and allogeneic dendritic cells at a 5:1 ratio. Half of the cultures were treated with a 2mM adenosine in 2X solution on days 1, 2, and 5 after the cultures were established. Two days after the last treatment T cells were harvested from the adenosine treated and untreated cultures and stained with 2μΜ CFSE at room temperature in PBS and then cocultured separately against 4 targets at 5:1 ratio: autologous DCs, allogeneic DCs from the donor from the initial MLR DCs, a mix of DCs from three 3 ^rd party donors, and Dynabeads Human T- Activator CD3/CD28 beads at a 3:1 cell to bead ratio in human culture media. After 5 days, proliferation by CFSE dilution was measured on a BD LSR Fortessa and data were analyzed using Flowjo software.

FIGS. 5A and 5B are a series of plots and histograms demonstrating the efficacy of adenosine-mediated selective depletion of alloreactive. In particular, FIGS. 5A-5B illustrate representative donor results from the study described in FIG. 4.

FIG. 6 is a bar graph illustrating the results of quality control testing of post alloreactive T cell depleted cell products. In particular, FIG. 6 illustrates compiled data of 6 donor and recipient pairs from the study described in FIG. 4.

FIG. 7 is a series of bar graphs illustrating CMV virus-specific memory T cells were retained in the cell product post depletion of alloreactive T cells with adenosine (Top panel) and CMV virus-specific memory T cells were retained in the cell product post depletion of alloreactive T cells with adenosine (Bottom panel). Top panel: MLRs for three donors were established as described in FIG. 4. For each donor, frozen lymphocytes from three CMV positive donors were cocultured separately with mature DCs from two allogeneic donors in a 5 : 1 ratio. Half of the cultures were treated with a 2mM adenosine in 2X solution on days 1, 2, and 5 after the cultures were established. Two days after the final treatment, the cells were harvested and rested overnight. The next day the cells were pulsed with a mixture of CMV IE-1 and pp65 peptide libraries, a WT1 peptide library, or no peptide (control). The protein transport inhibitors Brefeldin A

(GOLGIPLUG™) or Monensin (GOLGISTOP™) that prevent the secretion of cytokines thus trapping them in intracellular compartments were added to the cultures 1 hour after initiation of the coculture. The cultures were then incubated for another 5 hours and then stained with fluorescent labeled antibodies against T cell surface markers and intracellular cytokines as per the

manufacturer's instructions. The flow cytometry data were acquired on a BD LSR Fortessa and analyzed using Flowjo software. The graphs represent IFNy ^4" , TNFa ⁺ , and IFNy ^4" and TNFa ⁺ cells gated on live CD3 ⁺ T cells for three representative samples. Bottom panel: MLRs for three donors were established as described in top panel. For each donor, frozen lymphocytes were cocultured separately with mature DCs from two allogeneic donors in a 5:1 ratio. Half of the cultures were treated with a 2mM adenosine in 2X solution on days 1, 2, and 5 after the cultures were established. Two days after the final treatment, the cells were harvested and washed with PBS and primed twice at weekly interval with autologous mature dendritic cells pulsed with CMV IE-1 and pp65 peptides at a responder to stimulator ratio of 5: 1. Cultures received IL-7 (10 ng/ml) and IL-15 (10 ng/ml) during first priming and IL-2 (lOU/ml) in addition to IL-7 and IL-15 during second priming step. No cytokines were present during the initial 3 days of both priming steps. After two rounds of priming the cells were harvested, washed and rested overnight in media with no cytokines and then tested for CMV- specific cytokine release as described previously.

FIG. 8 is a series of bar graphs illustrating leukemia antigen-specific T cells were efficiently generated from donor cell product post adenosine mediated-depletion of alloreactive T cells. In particular, MLRs for three donors were established as described in FIG. 4. For each donor, frozen lymphocytes were cocultured separately with mature DCs from two allogeneic donors in a 5:1 ratio. Half of the cultures were treated with a 2mM adenosine in 2X solution on days 1, 2, and 5 after the cultures were established. Two days after the final treatment, the cells were harvested and washed with PBS and primed twice at weekly interval with lentivirally engineered autologous DC expressing PRAME at a responder to stimulator ratio of 5:1. Cultures received IL-7 (10 ng/ml) and IL-15 (10 ng/ml) during first priming and IL-2 (lOU/ml) in addition to IL-7 and IL-15 during second priming step. No cytokines were present during the initial 3 days of both priming steps. Resulting effector T cell cultures were tested for antigen-specific cytokine release in response to autologous DC target expressing the relevant (PRAME) or irrelevant (WT1) or untransduced DC in a 6 hour coculture assay as described in FIG. 7. The graphs represent IFNy ^1" , TNFoc ⁺ , and IFNy ^4" and TNFa ⁺ cells gated on live CD3 ⁺ T cells for three representative samples.

FIG. 9 is a series of bar graphs showing the effect of selective depletion with adenosine in HLA-matched and haploidentical donor-recipient pairs. Greater than 90% depletion was achieved in these pairs.

DETAILED DESCRIPTION

/. Introduction

Acute graft versus host disease (GVHD) occurs in 20-40% of recipients of HLA-identical sibling donor grafts and up to 70-80% of recipients of unrelated donor grafts. Therefore, approaches to prevent this complication of bone marrow transplantation are needed. Ex vivo removal of T-cells from the donor graft before transfer can prevent GVHD but it can increase graft failure, relapse, and infectious complications, and delays immune recovery. Several methods for depletion of donor anti-host GVHD-mediating T cells have been developed. These methods target ex vivo alloantigen-activated T cells (from a mixed lymphocyte culture with irradiated alio host antigen presenting cells) based on the expression of various T cell activation markers (such as CD25, CD69, CD71, HLA-DR, CD137, and CD134) the dilution of CFSE or the uptake of the dye 4,5-dibromorhodamine 123 (TH9402) by activated cells, making them sensitive to light. These approaches are not effective at preventing GVHD and some are associated with poor immune recovery.

Methods and reagents are disclosed herein for the selective removal of alloreactive T cells from a donor graft to decrease the rate and severity of GVHD while preserving specific immunity against infections and malignant cells in patients with malignant and nonmalignant diseases undergoing hematopoietic stem cell transplantation (HSCT), lymphocyte infusions, or organ transplant. These data illustrate that the presently disclosed methods are superior to alternative approaches to selective depletion. Furthermore the disclosed process eliminates several steps, so that the methods are inexpensive and simple to apply clinically.

Specifically, the present disclosure is directed to a method for exposing donor grafts ex vivo to the recipient cells to activate the host reacting cells which are then targeted for elimination as adenosine selectively kills the activated T cells. In some examples, this method is used to prevent, reduce or inhibit GVHD while retaining useful immunity in the graft as well as the stem cells. Adenosine is an FDA approved drug used to treat cardiac failure. This novel strategy can serve as a platform to generate third party off-the shelf antigen-specific T cells for the safe (GVHD-free) treatment of microbial infections, malignancies, and other human diseases that could be treated by T cells. This strategy could revolutionize the practice of stem cell transplantation and immunotherapy.

//. Terms

The following explanations of terms and methods are provided to better describe the present disclosure and to guide those of ordinary skill in the art in the practice of the present disclosure. The singular forms "a," "an," and "the" refer to one or more than one, unless the context clearly dictates otherwise. For example, the term "comprising a nucleic acid molecule" includes single or plural nucleic acid molecules and is considered equivalent to the phrase "comprising at least one nucleic acid molecule." The term "or" refers to a single element of stated alternative elements or a combination of two or more elements, unless the context clearly indicates otherwise. As used herein, "comprises" means "includes." Thus, "comprising A or B," means "including A, B, or A and B," without excluding additional elements.

Unless explained otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. The materials, methods, and examples are illustrative only and not intended to be limiting.

Adenosine: A ribonucleotide consisting of the nitrogenous base, adenine, linked to the sugar, ribose. The IUPAC name for adenosine is (2R,3R,45,5R)-2-(6-amino-9H-purin-9-yl)-5- (hydroxymethyl)oxolane-3,4-diol, which has a molecular mass of 267.241 and a chemical formula of C10H13N5O4, as shown in the structure below:

Adenosine modulates diverse physiological functions including induction of sedation, vasodilatation, suppression of cardiac rate and contractility, inhibition of platelet aggregability, stimulation of gluconeogenesis and inhibition of lipolysis. In addition, adenosine and some analogs of adenosine that nonselectively activate adenosine receptor subtypes have been shown to decrease neutrophil production of inflammatory oxidative products. At least four subtypes of adenosine receptor exist (Adoral, Adora2a, Adora2b, and Adora3, also called Al, A2a, A2b, and A3, respectively) which have been cloned from animal or human sources. Adenosine receptors are members of the G-protein coupled receptor (GPCR) superfamily, and are typically thought to mediate stimulation or inhibition of adenylyl cyclase activity, and hence cyclic AMP levels.

Adenosine receptors are of note since they represent the smallest of the GPCR superfamily yet cloned. Stimulant properties of the methylxanthine antagonists of adenosine receptors (caffeine, theophylline - present in tea, coffee and cocoa) are known.

The Al receptor is chiefly linked to inhibition of adenylyl cyclase activity. However, there is also good evidence for coupling (via G-proteins) to ion channels, and phospholipase C. In the nervous system, the Al adenosine receptor is thought to mediate the inhibition of transmitter release and the reduction in neuronal activity. Blockade of this receptor in the heart leads to the accelerated, pronounced "pounding" observed after drinking large amounts of strong coffee (due to the caffeine and theophylline). In one embodiment, Al is shown as GENBANK® Accession No. L22214.

The A2a is almost exclusively coupled to stimulation of adenylyl cyclase activity. Its distribution in the CNS is very discrete, being heavily localized in the caudate and putamen bodies, and the nucleus accumbens and olfactory tubercle. There is growing interest in this receptor as a means of influencing dopamine-mediated responses in these brain regions. In the periphery, the A2a receptor is present on platelets and is anti-aggregatory. In one embodiment, A2A is shown as GENBANK® Accession No. AH003248, and A2b is shown as GENBANK® Accession No.

NM000676. A2a and A2b receptors cause similar cellular effects, but have different tissue distribution and requirements for the levels of extracellular adenosine needed for their activation. It appears that A2b receptor is activated by higher levels of adenosine then A2a receptor (see Linden, Ann. Rev Pharmacol. Toxicol. 2001;41:775-87).

A3 couples to inhibition of adenylyl cyclase activity and has a proposed pro-inflammatory role in mast cells. In one embodiment, A3 is shown as GENBANK® Accession No. AH003597.

Adenosine-like molecules: Adenosine analogs and other molecules which have the same effect on alloreactive active donor T cells as adenosine. Adenosine-like molecules include adenosine analogs such as adenosine agonists and adenosine receptor agonists, and compounds that increase intracellular or extracellular adenosine levels. Adenosine-like molecules are suitable for use in the disclosed methods.

Allograft: A transplant of an organ, tissue, bodily fluid or cell from one individual to a genetically nonidentical individual of the same species. As used herein, "allogeneic" encompasses a genetically different phenotype present in non-identical individuals of the same species. Allogeneic examples include blood group phenotypes and immunoantigeneic allotypes. An "alloantigen" encompasses any antigen recognized by different individuals of the same species. Organisms, cells, tissues, organs, and the like from, or derived from, a single individual, or from a genetically identical individual are "autologous." 'Transplant rejection" refers to a partial or complete immune response to a transplanted cell, tissue, organ, or the like on or in a recipient of the transplant due to an immune response to an allogeneic graft. "Alloreactive T cells" are stimulated by donor APCs which express both the allogeneic MHC and costimulatory activity.

Animal: Living multi-cellular vertebrate organisms, a category that includes, for example, mammals and birds. The term mammal includes both human and non-human mammals. Similarly, the term "subject" includes both human and veterinary subjects.

Antigen: A compound, composition, or substance that can stimulate the production of antibodies or a T cell response in an animal, including compositions that are injected or absorbed into an animal. An antigen reacts with the products of specific humoral or cellular immunity, including those induced by heterologous immunogens. The term is used interchangeably with the term "immunogen." The term "antigen" includes all related antigenic epitopes. An "antigenic polypeptide" is a polypeptide to which an immune response, such as a T cell response or an antibody response, can be stimulated. "Epitope" or "antigenic determinant" refers to a site on an antigen to which B and/or T cells respond. T cells can respond to the epitope when the epitope is presented in conjunction with an MHC molecule. Epitopes can be formed both from contiguous amino acids (linear) or noncontiguous amino acids juxtaposed by tertiary folding of an antigenic polypeptide (conformational). Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. Normally, a B-cell epitope will include at least about 5 amino acids but can be as small as 3-4 amino acids. A T-cell epitope, such as a CTL epitope, will include at least about 7-9 amino acids, and a helper T-cell epitope at least about 12-20 amino acids.

Normally, an epitope will include between about 5 and 15 amino acids, such as, 9, 10, 12 or 15 amino acids. The amino acids are in a unique spatial conformation. In one example, the donor antigen includes antigens from lymphocytes, leukocytes, such as peripheral blood leukocytes (including monocytes or monocyte-derived cells, such as dendritic cells) or a combination thereof. In some examples, donor antigen includes lysed cell membranes from donor peripheral blood leukocytes, spleen cells, or bone marrow cells. In an example, donor antigen can be provided from a subject that had similar HLA-A, HLA-B, or HLA-DR loci profile as the donor. A third-party antigen is an antigen that was not obtained from the organ donor or organ recipient and has no similarity to the recipient or donor (as indicated by measuring HLA-A, HLA-B and HLA-DR loci). Exemplary third-party antigen samples include lymphocytes, leukocytes, such as peripheral blood leukocytes (including monocytes or monocyte-derived cells, such as dendritic cells) or a combination thereof. For example, third-party antigen samples include lysed cell membranes from donor peripheral blood leukocytes (including monocytes or monocyte-derived cells, such as dendritic cells), spleen cells, or bone marrow cells. An autoantigen is an antigen that under normal conditions would not be a target of the immune system. However, the normal immunological tolerance for such an antigen is lost in a subject suffering from a specific autoimmune disease and stimulates the production of autoantibodies.

Antigen Presenting Cells (APCs): Highly specialized cells that can process antigens and display their peptide fragments on the cell surface together with molecules required for lymphocyte activation. For example, an APC is a cell that can present antigen bound to MHC class I or class II molecules to T cells. APCs include, but are not limited to, monocytes, macrophages, dendritic cells, B cells, T cells and Langerhans cells. A T cell that can present antigen to other T cells (including CD4+ and/or CD8+ T cells) is an antigen presenting T cell (T-APC).

Autoimmune disorder: A disorder in which the immune system produces an immune response (e.g. a B cell or a T cell response) against an endogenous antigen, with consequent injury to tissues.

Carriers or Vehicles: Acceptable carriers or vehicles useful in this disclosure are conventional. Remington 's Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, PA, 19th Edition (1995), describes compositions and formulations suitable for delivery of adenosine or adenosine-like molecules utilized herein. For example, adenosine or adenosine-like molecules can be administered in the presence of one or more acceptable carriers, including a non- natural or natural acceptable carrier molecule.

In general, the nature of the carrier depends on the particular mode of administration being employed. For instance, pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle may be utilized. In addition to biologically-neutral carriers, compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate. Embodiments of other compositions can be prepared with conventional

pharmaceutically acceptable carriers, adjuvants, and counter-ions, as would be known to those of skill in the art. Contacting: Placement in direct physical association; includes both in solid and liquid form. Contacting can occur in vitro with isolated cells or in vivo by administering the agent to a subject.

Control: A reference standard. A control can be a known value indicative of a non-anemic or an anemic subject. A difference between a test sample and a control can be a decrease or conversely an increase. The difference can be a qualitative difference or a quantitative difference, for example a statistically significant difference. In some examples, a difference is an increase or decrease, relative to a control, of at least about 5%, such as at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 350%, at least about 400%, at least about 500%, or greater than 500%.

Effective Amount or Effective Concentration: An amount sufficient to achieve the desired effect. With regard to adenosine or an adenosine-like molecule of use in the disclosed methods, this is the amount sufficient to decrease viability.

Graft- Versus-Host Disease (GVHD): A common and serious complication of bone marrow or other tissue transplantation wherein there is a reaction of donated immunologically competent lymphocytes against a transplant recipient's own tissue. GVHD is a possible complication of any transplant that uses or contains stem cells from either a related or an unrelated donor.

There are two kinds of GVHD, acute and chronic. Acute GVHD appears within the first three months following transplantation. Signs of acute GVHD include a reddish skin rash on the hands and feet that may spread and become more severe, with peeling or blistering skin. Acute GVHD can also affect the stomach and intestines, in which case cramping, nausea, and diarrhea are present. Yellowing of the skin and eyes (jaundice) indicates that acute GVHD has affected the liver. Chronic GVHD is ranked based on its severity: stage/grade 1 is mild; stage/grade 4 is severe. Chronic GVHD develops three months or later following transplantation. The symptoms of chronic GVHD are similar to those of acute GVHD, but in addition, chronic GVHD may also affect the mucous glands in the eyes, salivary glands in the mouth, and glands that lubricate the stomach lining and intestines.

Immune cell: Any cell involved in a host defense mechanism. These can include, for example, T cells, B cells, natural killer cells, neutrophils, mast cells, macrophages, antigen- presenting cells, basophils, eosinophils, and neutrophils. Immune response: A response of a cell of the immune system, such as a B cell, or a T cell, to a stimulus. In one embodiment, the response is specific for a particular antigen (an "antigen- specific response").

A "parameter of an immune response" is any particular measurable aspect of an immune response, including, but not limited to, cytokine secretion (IL-6, IL-10, IFN-γ, etc.),

immunoglobulin production, dendritic cell maturation, and proliferation of a cell of the immune system. One of skill in the art can readily determine an increase in any one of these parameters, using known laboratory assays. In one specific non-limiting example, to assess cell proliferation, incorporation of ³ H-thymidine can be assessed. A "substantial" increase in a parameter of the immune response is a significant increase in this parameter as compared to a control. Specific, non-limiting examples of a substantial increase are at least about a 50% increase, at least about a 75% increase, at least about a 90% increase, at least about a 100% increase, at least about a 200% increase, at least about a 300% increase, and at least about a 500% increase. Similarly, an inhibition or decrease in a parameter of the immune response is a significant decrease in this parameter as compared to a control. Specific, non-limiting examples of a substantial decrease are at least about a 50% decrease, at least about a 75% decrease, at least about a 90% decrease, at least about a 100% decrease, at least about a 200% decrease, at least about a 300% decrease, and at least about a 500% decrease. A statistical test, such as a non-paramentric ANOVA, can be used to compare differences in the magnitude of the response induced by one agent as compared to the percent of samples that respond using a second agent. In some examples, p <_0.05 is significant, and indicates a substantial increase or decrease in the parameter of the immune response. One of skill in the art can readily identify other statistical assays of use.

Immunocompromised subject: A subject who is incapable of developing or unlikely to develop a robust immune response, usually as a result of disease, malnutrition, or

immunosuppressive therapy. An immunocompromised immune system is an immune system that is functioning below normal. Immunocompromised subjects are more susceptible to opportunistic infections, for example viral, fungal, protozoan, or bacterial infections, prion diseases, and certain neoplasms. Those who can be considered to be immunocompromised include, but are not limited to, subjects with AIDS (or HIV positive), subjects with severe combined immune deficiency (SCID), diabetics, subjects who have had transplants and who are taking immunosuppressives, and those who are receiving chemotherapy for cancer. Immunocompromised individuals also includes subjects with most forms of cancer (other than skin cancer), sickle cell anemia, cystic fibrosis, those who do not have a spleen, subjects with end stage kidney disease (dialysis), and those who have been taking corticosteroids on a frequent basis by pill or injection within the last year.

Subjects with severe liver, lung, or heart disease also may be immunocompromised.

Immunoglobulins: A class of proteins found in plasma and other body fluids that exhibits antibody activity and binds with other molecules with a high degree of specificity; divided into five classes (IgM, IgG, IgA, IgD, and IgE) on the basis of structure and biological activity.

Immunoglobulins and certain variants thereof are known and many have been prepared in recombinant cell culture (e.g. see U.S. Patent No. 4,745,055; U.S. Patent No. 4,444,487; WO 88/03565; EP 256,654; EP 120,694; EP 125, 023; Faoulkner et al, Nature 298:286, 1982;

Morrison, /. Immunol. 123:793, 1979; Morrison et al., Ann Rev. Immunol 2:239, 1984).

A native (naturally occurring) immunoglobulin is made up of four polypeptide chains.

There are two long chains, called the "heavy" or "H" chains which weigh between 50 and 75 kilodaltons and two short chains called "light" or "L" chains weighing in at 25 kilodaltons. They are linked together by disulfide bonds to form a "Y" shaped molecule. Each heavy chain and light chain can be divided into a variable region and a constant region. An Fc region includes the constant regions of the heavy and the light chains, but not the variable regions.

Infectious disease: Any disease caused by an infectious agent. Examples of infectious pathogens include, but are not limited to: viruses, bacteria, mycoplasma and fungi. In a particular example, it is a disease caused by at least one type of infectious pathogen. In another example, it is a disease caused by at least two different types of infectious pathogens. Infectious diseases can affect any body system, be acute (short-acting) or chronic/persistent (long-acting), occur with or without fever, strike any age group, and overlap each other. Infectious diseases can be

opportunistic infections, in that they occur more frequently in immunocompromised subjects

Viral diseases commonly occur after immunosuppression due to re-activation of viruses already present in the recipient. Particular examples of viral infections include, but are not limited to, cytomegalovirus (CMV) pneumonia, enteritis and retinitis; Epstein-Barr virus (EBV) lymphoproliferative disease; chicken pox/shingles (caused by varicella zoster virus, VZV); HSV-1 and -2 mucositis; HSV-6 encephalitis, BK-virus hemorrhagic cystitis; viral influenza; pneumonia from respiratory syncytial virus (RSV); AIDS (caused by HIV); and hepatitis A, B or C.

Opportunistic infections occur in a subject with a compromised immune system, such as a subject who has been immuno-depleted and recently received a bone marrow transplant or a hematopoietic stem cell transplant. These infections include, but are not limited to cytomegalovirus, Candida albicans, human immunodeficiency virus, Staphlococcus aureus, Steptococcus pyogenes,

Pseudomas aeruginosa, Acinteobacter baumanni, Toxoplasma gondii, Pneumocystitis carinii, or Aspergillus infections. Additional examples of infectious virus include: Retroviridae; Picornaviridae (for example, polio viruses, hepatitis A virus; enteroviruses, human coxsackie viruses, rhinoviruses, echo viruses); Calciviridae (such as strains that cause gastroenteritis); Togaviridae (for example, equine encephalitis viruses, rubella viruses); Flaviridae (for example, dengue viruses, encephalitis viruses, yellow fever viruses); Coronaviridae (for example, corona viruses); Rhabdoviridae (for example, vesicular stomatitis viruses, rabies viruses); Filoviridae (for example, ebola viruses); Paramyxoviridae (for example, parainfluenza viruses, mumps virus, measles virus, respiratory syncytial virus); Orthomyxoviridae (for example, influenza viruses); Bungaviridae (for example, Hantaan viruses, bunga viruses, phleboviruses and Nairo viruses); Arena viridae (hemorrhagic fever viruses); Reoviridae (e.g., reoviruses, orbiviurses and rotaviruses); Birnaviridae;

Hepadnaviridae (Hepatitis B virus); Parvoviridae (parvoviruses); Papovaviridae (papilloma viruses, polyoma viruses); Adenoviridae (most adenoviruses); Herpesviridae (herpes simplex virus (HSV) 1 and HSV-2, varicella zoster virus, cytomegalovirus (CMV), herpes viruses); Poxviridae (variola viruses, vaccinia viruses, pox viruses); and Iridoviridae (such as African swine fever virus); and unclassified viruses (for example, the etiological agents of Spongiform

encephalopathies, the agent of delta hepatitis (thought to be a defective satellite of hepatitis B virus), the agents of non-A, non-B hepatitis (class l=internally transmitted; class 2=parenterally transmitted (i.e., Hepatitis C); Norwalk and related viruses, and astroviruses).

Examples of fungal infections include but are not limited to: aspergillosis; thrush (caused by Candida albicans); cryptococcosis (caused by Cryptococcus); and histoplasmosis. Thus, examples of infectious fungi include, but are not limited to, Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Chlamydia trachomatis, Candida albicans.

Examples of infectious bacteria include: Helicobacter pyloris, Borelia burgdorferi, Legionella pneumophilia, Mycobacteria sps (such as. M. tuberculosis, M. avium, M. intracellulare, M. kansaii, M. gordonae), Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitidis, Listeria monocytogenes, Streptococcus pyogenes (Group A Streptococcus), Streptococcus agalactiae (Group B Streptococcus), Streptococcus (viridans group), Streptococcus faecalis, Streptococcus bovis, Streptococcus (anaerobic sps.), Streptococcus pneumoniae, pathogenic Campylobacter sp., Enterococcus sp., Haemophilus influenzae, Bacillus anthracis,

corynebacterium diphtheriae, corynebacterium sp., Erysipelothrix rhusiopathiae, Clostridium perfringers, Clostridium tetani, Enterobacter aerogenes, Klebsiella pneumoniae, Pasturella multocida, Bacteroides sp., Fusobacterium nucleatum, Streptobacillus moniliformis, Treponema pallidium, Treponema pertenue, Leptospira, and Actinomyces israelii. Other infectious organisms (such as protists) include: Plasmodium falciparum and Toxoplasma gondii.

Leukocyte: Cells in the blood, also termed "white cells," that are involved in defending the body against infective organisms and foreign substances. Leukocytes are produced in the bone marrow. There are 5 main types of white blood cell, subdivided between 2 main groups:

polymorphonuclear leukocytes (neutrophils, eosinophils, basophils) and mononuclear leukocytes (monocytes and lymphocytes).

Lymphocytes: A type of white blood cell that is involved in the immune defenses of the body. The main types of lymphocytes are: B cells, T cells and natural killer cells (NK cells). "T lymphocytes" or "T cells" are non-antibody producing lymphocytes that constitute a part of the cell-mediated arm of the immune system. T cells arise from immature lymphocytes that migrate from the bone marrow to the thymus, where they undergo a maturation process under the direction of thymic hormones. Here, the mature lymphocytes rapidly divide increasing to very large numbers. The maturing T cells become immunocompetent based on their ability to recognize and bind a specific antigen. Activation of immunocompetent T cells is triggered when an antigen binds to the lymphocyte's surface receptors. T cells include, but are not limited to, CD4 ⁺ T cells and CD8 ⁺ T cells. A CD4 ⁺ T lymphocyte is an immune cell that carries a marker on its surface known as "cluster of differentiation 4" (CD4). These cells, also known as helper T cells, help orchestrate the immune response, including antibody responses as well as killer T cell responses. CD8 ⁺ T cells carry the "cluster of differentiation 8" (CD8) marker. In one embodiment, a CD8 T cell is a cytotoxic T lymphocyte. In another embodiment, a CD8 cell is a suppressor T cell.

Major Histocompatability Complex (MHC): A generic designation meant to encompass the histocompatability antigen systems described in different species, including the human leukocyte antigens ("HLA").

Monocyte: A type of mononuclear leukocyte. Monocytes are produced by the bone marrow from hematopoietic stem cell precursors called monoblasts. Monocytes circulate in the bloodstream for about one to three days and then typically move into tissues throughout the body. Monocytes which migrate from the bloodstream to other tissues will then differentiate into tissue resident macrophages or dendritic cells. They constitute between three to eight percent of the leukocytes in the blood. Monocytes and monocyte-derived cells such as dendritic cells have three main functions in the immune system: phagocytosis, antigen presentation and cytokine production. Thus, monocytes and monocyte-derived dendritic cells are APCs. Monocyte cells express CD 14. Some types of monocytes express CD 16 in addition to CD 14. Organ rejection or transplant rejection: Functional and structural deterioration of an organ due to an active immune response expressed by the recipient, and independent of non- immunologic causes of organ dysfunction.

Preventing: Inhibiting the full development of a condition or disease, for example in a person who is known to have a predisposition to a condition or disease.

Receptor: A molecular structure within a cell or on the surface of a cell, characterized by selective binding of a specific substance and a specific physiological effect that accompanies the binding, for example, cell surface receptors for peptide hormones, neurotransmitters,

immunoglobulins, small molecules, and cytoplasmic receptors for steroid hormones. An adenosine receptor is a cell surface receptor for adenosine, and includes, but is not limited to, the A2 or A3 receptors.

Sample (or biological sample): A biological specimen containing genomic DNA, RNA (including mRNA and microRNA), protein, cells, tissues or combinations thereof, obtained from a subject. Examples include, but are not limited to, peripheral blood (including monocytes or monocyte-derived cells, such as dendritic cells), urine, saliva, tissue biopsy, fine needle aspiration samples, surgical specimen, and autopsy material. In one example, a sample is blood sample which includes lymphocytes, leukocytes, such as peripheral blood leukocytes, or a combination thereof with or without red blood cells.

Stem cell: A cell that can generate a fully differentiated functional cell of a more than one given cell type and can self-renew. The role of stem cells in vivo is to replace cells that are destroyed during the normal life of an animal. Generally, stem cells can divide without limit and are totipotent or pluripotent. After division, the stem cell may remain as a stem cell, become a precursor cell, or proceed to terminal differentiation.

A nervous system (NS) stem cell is, for example, a cell of the central nervous system that can self -renew and can generate astrocytes, neurons and oligodendrocytes.

A hematopoetic stem cell (HSC) is, for example, a cell that gives rise to all other blood cells. They give rise to the myeloid (monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes/platelets, dendritic cells), and lymphoid lineages (T- cells, B-cells, NK-cells). The definition of hematopoietic stem cells has changed in the last two decades. The hematopoietic tissue contains cells with long-term and short-term regeneration capacities and committed multipotent, oligopotent, and unipotent progenitors. HSCs constitute 1 : 10.000 of cells in myeloid tissue. HSCs are a heterogeneous population. Three classes of stem cells exist, distinguished by their ratio of lymphoid to myeloid progeny (L/M) in blood. Myeloid- biased (My-bi) HSC have low L/M ratio (>0, <3), whereas lymphoid-biased (Ly-bi) HSC show a large ratio (>10). The third category consists of the balanced (Bala) HSC for which 3 < L/M < 10. Only the myeloid-biased and -balanced HSCs have durable self-renewal properties. In addition, serial transplantation studies have shown that each subtype preferentially re-creates its blood cell type distribution, suggesting an inherited epigenetic program for each subtype. HSCs can be identified and characterized based upon morphology, presence and/or absence of particular markers and functional assays, such as the cobblestone area- forming cell (CAFC) assay.

A bone marrow stem cell is, for example, an adult, mesoderm-derived cell that is capable of generating cells of mesenchymal lineages, typically of two or more mesenchymal lineages, e.g., osteocytic (bone), chondrocytic (cartilage), myocytic (muscle), tendonocytic (tendon), fibroblastic (connective tissue), adipocytic (fat) and stromogenic (marrow stroma) lineage. Bone marrow stem cells are present in or (partly) isolated from a sample of bone marrow. A sample of bone marrow may be obtained, e.g., from iliac crest, femora, tibiae, spine, rib or other medullar spaces of a subject. Bone marrow stem cells encompass any and all subtypes thereof, such as without limitation, "rapidly self -renewing cells" RS-1 or RS-2 as described in Colter et al. 2000 (PNAS 97(7): 3213-8); "side population" (SP) cells as described by Goodell et al. 1997 (Nat Med 3(12): 1337-45); osteogenic precursor (OP) cells which are initially identified by their low density (e.g., upon density gradient centrifugation), non-adherent nature and low-level of expression of osteogenic markers (as described by Long et al. 1995. J Clin Invest. 95(2): 881-7; U.S. Pat. No. 5,972,703); primitive precursor cells which can generate cells of both the haematopoietic and non- haematopoietic lineages as described by Krause et al. 2001 (Cell 105: 369-377) and Dominici et al. 2004. (PNAS 101(32): 11761-6); and others.

Reduced: A statistically significant decrease, such as in a cell population. "Elimination" indicates that an element, such as a cell, has been reduced to such an extent that it cannot be detected.

Subject: Living multi-cellular vertebrate organisms, a category that includes human and non-human mammals. In one particular example, the subject is a child. As used herein, a "child" refers to a person under the age of 18.

T-Cell: A white blood cell critical to the immune response. T cells include, but are not limited to, CD4 ⁺ T cells and CD8 ⁺ T cells. A CD4 ⁺ T lymphocyte is an immune cell that carries a marker on its surface known as "cluster of differentiation 4" (CD4). These cells, also known as helper T cells, help orchestrate the immune response, including antibody responses as well as killer T cell responses. CD8 ⁺ T cells carry the "cluster of differentiation 8" (CD8) marker. In one embodiment, a CD8 T cells is a cytotoxic T lymphocytes. In another embodiment, a CD8 cell is a suppressor T cell. An "alloreactive T cell" is stimulated by a donor APC which expresses both allogeneic MHC and a costimulatory molecule. In one embodiment, an activated alloreactive T cell is CD39 ⁺ CD25 ⁺ CD45RO ⁺ . A "memory" T cell is a T cell that previously encountered and responded to its cognate antigen, and thus can mount a secondary response to that cognate antigen. These cells can be for example, TCM cells express L-selectin and the CCR7, they secrete IL-2, TSCM cells which are CD45RO ^" , CCR7 ⁺ , CD45RA ⁺ , CD62L ⁺ , CD27+, CD28 ⁺ and IL-7Ra ⁺ , and also express CD95, IL-2R , CXCR3, and LFA-1, or an effector memory TEM cell, which do not express L-selectin or CCR7 but produce effector cytokines, such as IFNy and IL-4.

Therapeutic agent: A chemical compound, small molecule, or other composition, such as an antisense compound, antibody, protease inhibitor, hormone, chemokine, cytokine, radioactive agent, or anti-inflammatory agent, capable of inducing a desired therapeutic or prophylactic effect when properly administered to a subject.

Therapeutically effective amount: A quantity of a specified pharmaceutical or therapeutic agent sufficient to achieve a desired effect in a subject, or in a cell, being treated with the agent. The effective amount of the agent will be dependent on several factors, including, but not limited to the subject or cells being treated, and the manner of administration of the therapeutic composition.

Under conditions sufficient to: A phrase that is used to describe any environment that permits the desired activity.

Viable: Alive. The number of viable cells can be determined using standard techniques, such as, but not limited to, trypan blue exclusion or fluorescence activated cell sorting.

III. Method for Depletion of Alloreactive T cells

Disclosed herein is a method for selective depletion of alloreactive T lymphocytes, such as from donor stem cells or lymphocyte grafts. The disclosed method specifically removes the donor T cells that cause GVHD from the graft before it is infused. This approach prevents, reduces or inhibits GVHD while conserving the stem cells and providing beneficial donor immunity against infection and the malignant disease being treated. In some embodiments, the disclosed method uses the ex vivo coculture of donor graft cells with the antigen presenting cells (APC) of the recipients. The recipient's APC stimulate and activate the alloreactive (GVHD causing) donor T lymphocytes. The activated cells are then eliminated specifically by the addition of adenosine, which only targets and kills the activated T cells. The adenosine is then removed. For example, the remaining cells can be washed free of adenosine using a buffer, such as phosphate buffered saline (PBS). This can occur after the final treatment, such as about two days after the treatment, and the cells can be given to the recipient immediately or frozen until use per transfusion medicine guidelines/standard procedures. In some embodiments, the resulting cells possess the following characteristics: contain no or minimal alloreactivity to recipients; react against third party alloantigens; virus-specific memory T cells retained; and generate new immune response against viral and tumor antigens.

In additional embodiments, the method includes culturing donor cells, such as donor graft cells including donor T cells with recipient APCs to form a mixture and under conditions sufficient for recipient APCs to stimulate and activate donor T cells and contacting the mixture of cells with an effective concentration of adenosine or adenosine-like molecule (such as an adenosine analog) to decrease or inhibit viability of the activated donor T-cells. In some examples, the method further includes removing the adenosine or adenosine-like molecule, such as by washing the cell mixture. The method may further include administering the resulting cell mixture to the recipient.

a. APC of a recipient

The APC is a cell that can present antigen bound to MHC class I or class II molecules to T cells. APCs include, but are not limited to, monocytes, macrophages, dendritic cells, B cells, T cells and Langerhans cells. A T cell that can present antigen to other T cells (including CD4+ and/or CD8+ T cells) is an antigen presenting T cell (T-APC). In some examples, recipient APCs include B cells, T cells, monocytes or monocyte-derived cells (such as dendritic cells) obtained from the subject who is to receive an organ transplant.

b. Mixture of Donor T cells and Recipient APCs

Donor T cells are co-cultured with recipient APC under conditions sufficient for the recipient APCs to activate the donor T cells. In some examples, the ratio of donor T cells to recipient APC is about 1 :1. In other examples, the ratio is about 2: 1, 3: 1, 4:1, 5: 1, 6: 1, 7: 1, 8: 1, 9: 1 or 10: 1 donor T cells, such as from donor lymphocytes to recipient APCs, such as recipient dendritic cells. Thus a ratio of about 1: 1 to about 10: 1 donor lymphocytes, such as donor T cells to APCs, can be used, such as a ratio of about 1 : 1 to about 5: 1. In some specific example, a mixture includes donor lymphocytes and recipient dendritic cells at a about 5: 1 ratio, respectively. In some examples, the mixture is about 50% donor lymphocytes, such as donor T cells, and about 50% recipient APCs, such as recipient dendritic cells. In some examples, the mixture includes about 60% donor T cells and about 40% recipient APCs, about 70% donor T cells and about 30% recipient APCs, about 80% donor T cells and about 20% recipient APCs, or about 90% donor T cells and 10% recipient APCs.

c. Co-culture conditions and Effective Amount of Adenosine

In some examples, the method includes ex-vivo co-culturing donor lymphocytes, such as donor T cells and recipient APCs, such as recipient dendritic cells, for a sufficient period of time for the recipient APCs to activate the donor T cells. Activated Alloreactive T cells can be CD39 ⁺ CD25 ⁺ CD45RO ⁺ T cells. In some examples, a sufficient period of time for the recipient APCs to activate the donor T cells includes incubating the cells together for a minimum of 24 hours and a maximum of 5 days, such as about 2 days to about 4 days. Thus, the cells can be incubated together for about 1 day, about 2 days, about 3 days, about 4 days or about 5 days. Once the donor T cells are activated, then the co-culture is contacted with adenosine or an adenosine-like molecule to decrease or eliminate the alloreactive donor T cells about every 12 hours, 24 hours, 36 hours, 48 hours, 72 hours, 96 hours or a combination thereof. In some examples, the mixture is treated with adenosine over a period of about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days, (e.g., from about 1 to about 10 days, such as about 2 to about 5 days), in which adenosine is added to the co-culture every day, every other day, every 3 days, every four days, or every 5 days. In some non-limiting examples, the co-culture is exposed to three treatments of an effective amount of adenosine or adenosine-like molecule over a 5 day period.

Adenosine and suitable adenosine-like molecules, such as adenosine analogs, are suitable for reducing or eliminating alloreactive donor T cells in the disclosed method. Suitable adenosine- like molecules include those that have the same resulting effect on alloreactive active donor T-cells as adenosine - reduction or elimination of alloreactive active donor T cells while still reacting against third party alloantigens, having memory T cells (such as virus-specific memory T cells) retained and being able to generate new immune response against viral and/or tumor antigens. Adenosine analogs such as adenosine agonists, adenosine receptor agonists, and compounds that increase intracellular or extracellular adenosine levels are suitable for use in the method.

Agonists of adenosine include 2'-deoxyadenosine; 2',3'-isopropoylidene adenosine;

toyocamycin; 1-methyladenosine; N-6-methyladenosine; adenosine N-oxide; 6- methylmercaptopurine riboside; 6-chloropurine riboside, 5'-adenosine monophosphate, 5'- adenosine diphosphate, or 5 '-adenosine triphosphate. Adenosine receptor agonists specifically bind an adenosine receptor and induce a biological activity of adenosine. Adenosine receptor agonists include phenylisopropyl-adenosine ("PIA"), 1-Methylisoguanosine, ENBA (S(-), N ⁶ - Cyclohexyladenosine (CHA), N ⁶ -Cyclopentyladenosine (CPA), 2-Chloro-N6-cyclopentyladenosine, 2-chloroadenosine, and adenosine amine congener (AD AC), all of which are agonists for the adenosine Ai receptor. Other receptor agonists include 2-p-(2-carboxy-ethyl) phenethyl-amino-5'- N-ethylcarboxamido-adenosine (CGS-21680), N-ethylcarboxamido-adenosine (NECA) and napthyl-substituted aralkoxyadenosine (SHA-082), 5'(N-Cyclopropyl)-carboxamidoadenosine, DPMA (PD 129,944), Metrifudil, which are agonists for the adenosine A2 receptor. Adenosine receptor agonists that preferentially the Ai receptor relative to the A2 receptor include 2- Chloroadenosine, N ⁶ -Phenyladenosine, and N ⁶ -Phenylethyladenosine. Adenosine receptor agonist that preferentially bind the A2 receptor relative to the Ai receptor include 2-Phenylaminoadenosine and MECA. All of these adenosine receptor agonists are of use in any of the disclosed methods.

Adenosine agonists also include compounds that increase intracellular adenosine concentration by enhancing its cellular uptake or inhibiting its breakdown by both the extracellular or intracellular mechanisms. One pathway of adenosine metabolism is the conversion of adenosine to inosine by adenosine deaminase. An example of an adenosine deaminase inhibitor is erythro-9- (2-hydroxy-3-nonyl) adenine ("EHNA"). Adenosine kinase inhibitors can also be used. Adenosine kinase converts adenosine to adenosine monophosphate by adenosine kinase. An example of an adenosine kinase inhibitor is iodotubercidin. Other suitable compounds include those that inhibit the dipyridamole-sensitive nucleoside transporter, which exports adenosine from the cytoplasm, and agents that promote the activity of a 5 '-nucleotidase, e.g., the ATP-activated 5 '-nucleotidase, which forms adenosine. Compounds that increase tissue adenosine and ATP levels include acadesine (AICA-riboside), which is described in Gruber et al., Circulation 80: 1400-1411 (1989). All of these adenosine agonists are of use in any of the disclosed methods.

An effective amount of adenosine or adenosine-like molecule (such as an adenosine analog) is one when administered induces the desired response (e.g. , to decrease or inhibit viability of the activated donor T-cells). In some examples, an effective amount of adenosine is between 0.1 mM adenosine and 2 mM adenosine. In some examples, an effective amount of adenosine is between 0.1 mM and 10 mM, such 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM, 3.5 mM, 4 mM, 4.5 mM, 5 mM, 5.5 mM, 6 mM, 6.5 mM, 7 mM, 7.5 mM, 8 mM, 8.5 mM, 9 mM, or 9.5 mM, provided to the co-culture every 12 hours, 24 hours, 36 hours, 48 hours, 72 hours, 96 hours or 120 hours.

In addition examples, the concentration is greater than about lmM, greater than about 2 mM, greater that about 3mM, or greater than about 5 mM. In further examples the concentration is about 2 mM to about 5mM, such as about 3 mM to about 5 mM, or 4 mM to about 5 mM. In other exmaples, the concentration is about 1 mM to about 4 mM, such as about 1 mM to about 3 mM, or 1 mM to about 2 mM. The adenosine can be provided, for example, over about a 3 day, about a 4 day, or about a 5 day period. In some examples, between 1 mM and 5 mM is provided to the co- culture over a 5 day period.

For example, the co-culture is provided at least one treatment of 1 mM to 5 mM over a 5 day period, such as 2 treatments, 3 treatments, 4 treatments or 5 treatments. In one specific example, adenosine treatment includes exposing the co-culture to 3 treatments of 1 mM adenosine over a 5 day period. In some examples, clinical grade adenosine is resuspended in saline. In some examples, adenosine is suspended in 1 M NH4OH (50 mg/ml), with heat as needed or in water (7 mg/100 ml) (see The Merck Index, 12th ed., New York: Merck ISBN 0911910-12-3, Entry# 152 which is hereby incorporated by reference).

The concentration of adenosine that is effective for use can readily be determined by one of skill in the art by evaluating the viability of T cells in a culture. Too low of a concentration of adenosine will not reduce the viability of alloreactive T cells, and thus cannot be used in the claimed methods.

Adenosine can be also be administered with a second compound. The second compound can enhance the action of adenosine or the adenosine analog, e.g., by enhancing binding of adenosine or an adenosine analog to an adenosine receptor. An example of such a compound is PD 81,728 (see Kollias-Baker et al., J. Pharmacol. Exp. Ther. 281:761-68 which is hereby incorporated by reference in its entirety).

Following exposure to the adenosine or the adenosine-like molecule, the adenosine or adenosine like molecule is removed from the culture. This can be accomplished by washing techniques, such as by centrifuging the cells, and then re-suspending cells in a buffer, such as phosphate buffered saline or any other suitable buffer. The washing can be repeated one to ten times, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times. The T cells, which have been depleted for alloreactive T cells, and are thus "allodepleted" can then be administered to the recipient. The alloreactive T cells are significantly reduced, such as about a 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% reduction in the number of alloreactive T cells. In some embodiments, the alloreactive T are reduced by about 95%, 96%, 97%, 98% or 99%. The alloreactive T cells can be eliminated, such that they cannot be detected using standard biological methods. In specific non- limiting examples, the alloreactive T cells are CD39 ⁺ CD25 ⁺ CD45RO ⁺ T cells.

The disclosed method can reduce graft versus host disease. For example, a decrease in the GVHD symptoms so that the patient may be assigned a lower level stage, or, for example, a reduction of a symptom of graft versus host disease by at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%. A reduction in the effect of graft versus host disease may also be measured by detection of a reduction in activated T cells involved in a GVHD reaction. A decreased requirement for immune suppression may also be an indication of GVHD.

Once prepared, the allodepleted T cells are typically administered to the recipient in a pharmaceutically acceptable carrier, such as by intravenous infusion. Carriers for the allodepleted cells can include but are not limited to solutions of phosphate buffered saline (PBS) containing a mixture of salt in physiologic concentrations. The number of cells that are administered to a specific recipient will vary dependent upon the nature of the condition being treated, the condition and age of the recipient and the number of cells available. One of ordinary skill can readily determine the proper dosage of cells that should be administered to a specific recipient. Some dosages for stem cell transplantation are, for example, about 1 X 10 ⁸ T cells/kg. This range is nevertheless only exemplary and dosage ranges include about 1 X 10 ² cells/kg to about 1 X 10 ⁹ T cells/kg.

d. Particular Uses of Disclosed Method

The disclosed methods may be used to prevent, reduce or inhibit GVHD or tissue/organ transplant rejection. For example, the method is used to prevent, reduce, or inhibit GVHD after solid organ, bone-marrow, stem cell transplantation or a combination thereof. In some examples, the method is used to prevent, reduce or inhibit acute GVHD after solid organ, bone-marrow, stem cell transplantation or a combination thereof. In some examples, the method is used to prevent, reduce or inhibit chronic GVHD after solid organ, bone-marrow, stem cell transplantation or a combination thereof. It is contemplated that the transplant can be any organ. Examples of solid organs include, but are not limited to, liver, intestine, kidney, heart, lung, pancreas and skin. In the context of the present disclosure, a transplanted organ need not be the entire organ, but can be a portion or section of the organ. In particular examples, the subject is in need of receiving, multiple organs, or portions of multiple organs. In some cases, the subject is a candidate for a transplant of a combination of two or more of a solid organ, bone marrow and stem cells. In some examples, the method is used for the selective removal of alloreactive T cells from a donor graft in a subject receiving stem cell, immune cell, tissue or organ transplant. In some cases, the subject is undergoing treatment, including immunosuppressive therapy.

The disclosed methods can be clinically applied for any diseases in which alloreactive immune responses are possible. Exemplary diseases that are specifically contemplated to be targeted by the disclosed approaches include, but are not limited to: acute myeloid leukemia, chronic myeloid leukemia, non-Hodgkin's lymphoma, Hodgkin's disease, acute lymphoid leukemia, chronic lymphocytic leukemia, multiple myeloma, Waldenstrom's macroglobulinemia,

myelodysplasia, myelofibrosis, aplastic anemia, renal cell carcinoma, hemoglobinopathies

(including thallasemias and sickle cell diseases). Other diseases where allogeneic stem cell transplantation may have clinical relevancy to include: breast carcinoma, ovarian carcinoma, autoimmune diseases (including scleroderma, Sjogren's syndrome and systemic lupus

erythematosus), as well as common neoplasms like lung cancer, colon cancer, and a broader range of hematological, immunodeficiency (such as SCID) and autoimmune diseases.

In some examples, the disclosed method is utilized to produce third party antigen specific T cells which can be used for the treatment of microbial infections, malignancies and other human disease. For example, the same technique that is used to generate allodepleted T cells as described herein is followed, but a third party partially matched donor would be substituted. After the allodepletion, the donor cells are exposed to antigen presenting cells presenting one or more antigens of choice and further cultured in vitro to expand antigen specific T cells recognizing chosen pathogens or malignant cells bearing a defined tumor antigen. A bank of "off the shelf allodepleted T cell products can be created from volunteer donors of a known HLA type and cryopreserved. The generated cells can be used to treat particular infections of malignant diseases. The choice of cell product would be based on the "best fit" for example donor and recipient matched at least at one MHC class I molecule and one MHC class II molecule. Microbial infections can include any allergen, bacteria, fungus or virus including those described herein. "Off-the-shelf third party antigen- specific effector T cells can be generated as illustrated in FIG. 4 and the Example section below.

i. Viral pathogens

Specific examples of viral pathogens include without limitation any one or more of (or any combination of) Arenaviruses (such as Guanarito virus, Lassa virus, Junin virus, Machupo virus and Sabia), Arteriviruses, Roniviruses, Astroviruses, Bunyaviruses (such as Crimean-Congo hemorrhagic fever virus and Hantavirus), Barnaviruses, Birnaviruses, Bornaviruses (such as Borna disease virus), Bromoviruses, Caliciviruses, Chrysoviruses, Coronaviruses (such as Coronavirus and SARS), Cystoviruses, Closteroviruses, Comoviruses, Dicistroviruses, Flaviruses (such as Yellow fever virus, West Nile virus, Hepatitis C virus, and Dengue fever virus), Filoviruses (such as Ebola virus and Marburg virus), Flexi viruses, Hepeviruses (such as Hepatitis E virus), human adenoviruses (such as human adenovirus A-F), human astroviruses, human BK polyomaviruses, human bocaviruses, human coronavirus (such as a human coronavirus HKU1, NL63, and OC43), human enteroviruses (such as human enterovirus A-D), human erythro virus V9, human foamy viruses, human herpesviruses (such as human herpesvirus 1 (herpes simplex virus type 1), human herpesvirus 2 (herpes simplex virus type 2), human herpesvirus 3 (Varicella zoster virus), human herpesvirus 4 type 1 (Epstein-Barr virus type 1), human herpesvirus 4 type 2 (Epstein-Barr virus type 2), human herpesvirus 5 strain AD169, human herpesvirus 5 strain Merlin Strain, human herpesvirus 6A, human herpesvirus 6B, human herpesvirus 7, human herpesvirus 8 type M, human herpesvirus 8 type P and Human Cyotmegalo virus), human immunodeficiency viruses (HIV) (such as HIV 1 and HIV 2), human metapneumoviruses, human papillomaviruses, human parainfluenza viruses (such as human parainfluenza virus 1-3), human parechoviruses, human parvoviruses (such as human parvovirus 4 and human parvovirus B 19), human respiratory syncytial viruses, human rhinoviruses (such as human rhinovirus A and human rhinovirus B), human spumaretroviruses, human T-lymphotropic viruses (such as human T-lymphotropic virus 1 and human T-lymphotropic virus 2), Human polyoma viruses, Hypoviruses, Leviviruses, Luteoviruses, Lymphocytic choriomeningitis viruses (LCM), Marnaviruses, Narnaviruses, Nidovirales, Nodaviruses,

Orthomyxoviruses (such as Influenza viruses), Partitiviruses, Paramyxoviruses (such as Measles virus and Mumps virus), Picornaviruses (such as Poliovirus, the common cold virus, and Hepatitis A virus), Potyviruses, Poxviruses (such as Variola and Cowpox), Sequiviruses, Reoviruses (such as Rotavirus), Rhabdoviruses (such as Rabies virus), Rhabdoviruses (such as Vesicular stomatitis virus, Tetraviruses, Togaviruses (such as Rubella virus and Ross River virus), Tombusviruses, Totiviruses, Tymoviruses, and Noroviruses among others.

it Allergens

Exemplary allergens (which are nonparasitic antigens capable of stimulating a type-I hypersensitivity reaction) include those derived from plants, such as trees, for example Betula verrucosa allergens Bet v 1, Bet v 2, and Bet v 4; Juniperous oxycedrus allergen Jun o 2; Castanea sativa allergen Cas s 2; and Hevea brasiliensis allergens Hev b 1, Hev b 3, Hev b 8, Hev b 9, Hev b 10 and Hev b 11; grasses, such as Phleum pretense allergens Phi p 1, Phi p 2, Phi p 4, Phi p 5a, Phi p 5, Phi p 6, Phi p 7, Phi p 11, and Phi p 12; weeds, such as Parietaria judaica allergen Par j

2.01011; and Artemisia vulgaris allergens Art v 1 and Art v 3; Mites, such as Dermatophagoides pteronyssinus allergens Der p 1, Der p 2, Der p 5, Der p 7, Der p 8, and Der p 10; Tyrophagu putrescentiae allergen Tyr p 2; Lepidoglyphus destructor allergens Lep d 2.01 and Lep d 13; and Euroglyphus maynei allergen Eur m 2.0101 ; animals, such as cats, for example Felis domesticus allergen Fel d 1 ; Penaeus aztecus allergen Pen a 1; Cyprinus carpo allergen Cyp c 1 ; and albumin from cat, dog, cattle, mouse, rat, pig, sheep, chicken, rabbit, hamster, horse, pigeon, and guinea pig; Fungi, such as Penicillium citrinum allergens Pen c 3 and Pen c 19; Penicillium notatum allergen Pen n 13; Aspergillus fumigatus allergens Asp f 1, Asp f3, Asp f 4, Asp f 6, Asp f 7 and Asp f 8; Alternaria alternata allergens Alt a 1 and Alt a 5; Malassezia furfur allergen Mai f 1, Mai f 5, Mai f 6, Mai f 7, Mai f 8, and Mai f 9; insects, such as Blatella germanica allergens Bla g 2, Bla g 4, and Bla g 5; Apis mellifera allergens Api m 2 and Api m 1 ; Vespula vulgaris allergen Ves v 5; Vespula germanica allergen Ves g 5; and Polstes annularis allergen Pol a 5; food, such as Malus domestica allergens Mai d 1 and Mai d 2; Apium graveolens allergend Api g 1 and Api g 1.0201 ; Daucus carota allergen Dau c 1 ; and Arachis hypogaea allergens Ara h 2 and Ara h 5 and the like.

Hi. Bacterial pathogen

Specific examples of bacterial pathogens include without limitation any one or more of (or any combination of) Acinetobacter baumanii, Actinobacillus sp., Actinomycetes, Actinomyces sp. (such as Actinomyces israelii and Actinomyces naeslundii), Aeromonas sp. (such as Aeromonas hydrophila, Aeromonas veronii biovar sobria (Aeromonas sobria), and Aeromonas caviae), Anaplasma phagocytophilum, Alcaligenes xylosoxidans, Acinetobacter baumanii, Actinobacillus actinomycetemcomitans, Bacillus sp. (such as Bacillus anthracis, Bacillus cereus, Bacillus subtilis, Bacillus thuringiensis, and Bacillus stearothermophilus), Bacteroides sp. (such as Bacteroides fragilis), Bartonella sp. (such as Bartonella bacillijormis and Bartonella henselae, Bifidobacterium sp., Bordetella sp. ( such as Bordetella pertussis, Bordetella parapertussis, and Bordetella bronchiseptica), Borrelia sp. (such as Borrelia recurrentis, and Borrelia burgdorferi), Brucella sp. (such as Brucella abortus, Brucella canis, Brucella melintensis and Brucella suis), Burkholderia sp. (such as Burkholderia pseudomallei and Burkholderia cepacia), Campylobacter sp. (such as Campylobacter jejuni, Campylobacter coli, Campylobacter lari and Campylobacter fetus), Capnocytophaga sp., Cardiobacterium hominis, Chlamydia trachomatis, Chlamydophila pneumoniae, Chlamydophila psittaci, Citrobacter sp. Coxiella burnetii, Corynebacterium sp. (such as, Corynebacterium diphtheriae, Corynebacterium jeikeum and Corynebacterium), Clostridium sp. (such as Clostridium perfringens, Clostridium difficile, Clostridium botulinum and Clostridium tetani), Eikenella corrodens, Enterobacter sp. (such as Enterobacter aerogenes, Enterobacter agglomerans, Enterobacter cloacae and Escherichia coli, including opportunistic Escherichia coli, such as enterotoxigenic E. coli, enteroinvasive E. coli, enter opatho genie E. coli, enterohemorrhagic E. coli, enteroaggregative E. coli and uropathogenic E. coli) Enterococcus sp. (such as

Enterococcus faecalis and Enterococcus faecium) Ehrlichia sp. (such as Ehrlichia chafeensia and Ehrlichia canis), Erysipelothrix rhusiopathiae, Eubacterium sp., Francisella tularensis,

Fusobacterium nucleatum, Gardnerella vaginalis, Gemella morbillorum, Haemophilus sp. (such as Haemophilus influenzae, Haemophilus ducreyi, Haemophilus aegyptius, Haemophilus

parainfluenzae, Haemophilus haemolyticus and Haemophilus parahaemolyticus, Helicobacter sp. (such as Helicobacter pylori, Helicobacter cinaedi and Helicobacter fennelliae), Kingella kingii, Klebsiella sp. ( such as Klebsiella pneumoniae, Klebsiella granulomatis and Klebsiella oxytoca), Lactobacillus sp., Listeria monocytogenes, Leptospira interrogans, Legionella pneumophila, Leptospira interrogans, Peptostreptococcus sp., Moraxella catarrhalis, Morganella sp.,

Mobiluncus sp., Micrococcus sp., Mycobacterium sp. (such as Mycobacterium leprae,

Mycobacterium tuberculosis, Mycobacterium intracellulare, Mycobacterium avium,

Mycobacterium bovis, and Mycobacterium marinum), Mycoplasm sp. (such as Mycoplasma pneumoniae, Mycoplasma hominis, and Mycoplasma genitalium), Nocardia sp. (such as Nocardia asteroides, Nocardia cyriacigeorgica and Nocardia brasiliensis), Neisseria sp. (such as Neisseria gonorrhoeae and Neisseria meningitidis), Pasteurella multocida, Plesiomonas shigelloides.

Prevotella sp., Porphyromonas sp., Prevotella melaninogenica, Proteus sp. (such as Proteus vulgaris and Proteus mirabilis), Providencia sp. (such as Providencia alcalifaciens, Providencia rettgeri and Providencia stucirtii), Pseudomonas aeruginosa, Propionibacterium acnes,

Rhodococcus equi, Rickettsia sp. (such as Rickettsia rickettsii, Rickettsia akari and Rickettsia prowazekii, Orientia tsutsugamushi (formerly: Rickettsia tsutsugamushi) and Rickettsia typhi), Rhodococcus sp., Serratia marcescens, Stenotrophomonas maltophilia, Salmonella sp. (such as Salmonella enterica, Salmonella typhi, Salmonella paratyphi, Salmonella enteritidis, Salmonella cholerasuis and Salmonella typhimurium), Serratia sp. (such as Serratia marcesans and Serratia liquifaciens), Shigella sp. (such as Shigella dysenteriae, Shigella flexneri, Shigella boydii and Shigella sonnei), Staphylococcus sp. (such as Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus hemolyticus, Staphylococcus saprophyticus), Streptococcus sp. (such as

Streptococcus pneumoniae (for example chloramphenicol-resistant serotype 4 Streptococcus pneumoniae, spectinomycin-resistant serotype 6B Streptococcus pneumoniae, streptomycin- resistant serotype 9V Streptococcus pneumoniae, erythromycin-resistant serotype 14 Streptococcus pneumoniae, optochin-resistant serotype 14 Streptococcus pneumoniae, rifampicin-resistant serotype 18C Streptococcus pneumoniae, tetracycline-resistant serotype 19F Streptococcus pneumoniae, penicillin-resistant serotype 19F Streptococcus pneumoniae, and trimethoprim- resistant serotype 23F Streptococcus pneumoniae, chloramphenicol-resistant serotype 4

Streptococcus pneumoniae, spectinomycin-resistant serotype 6B Streptococcus pneumoniae, streptomycin-resistant serotype 9V Streptococcus pneumoniae, optochin-resistant serotype 14 Streptococcus pneumoniae, rifampicin-resistant serotype 18C Streptococcus pneumoniae, penicillin-resistant serotype 19F Streptococcus pneumoniae, or trimethoprim-resistant serotype 23F Streptococcus pneumoniae), Streptococcus agalactiae, Streptococcus mutans, Streptococcus pyogenes, Group A streptococci, Streptococcus pyogenes, Group B streptococci, Streptococcus agalactiae, Group C streptococci, Streptococcus anginosus, Streptococcus equismilis, Group D streptococci, Streptococcus bovis, Group F streptococci, and Streptococcus anginosus Group G streptococci), Spirillum minus, Streptobacillus moniliformi, Treponema sp. (such as Treponema carateum, Treponema petenue, Treponema pallidum and Treponema endemicum, Tropheryma whippelii, Ureaplasma urealyticum, Veillonella sp., Vibrio sp. (such as Vibrio cholerae, Vibrio parahemolyticus, Vibrio vulnificus, Vibrio parahaemolyticus, Vibrio vulnificus, Vibrio

alginolyticus, Vibrio mimicus, Vibrio hollisae, Vibrio fluvialis, Vibrio metchnikovii, Vibrio damsela and Vibrio furnisii), Yersinia sp. (such asYersinia enterocolitica, Yersinia pestis, and Yersinia pseudotuberculosis) and Xanthomonas maltophilia among others.

iv. Fungal pathogens

Exemplary fungal pathogens include one or more of Trichophyton rubrum, T.

mentagrophytes, Epidermophyton floccosum, Microsporum canis, Pityrosporum orbicular e (Malassezia furfur), Candida sp. (such as Candida albicans), Aspergillus sp. (such as Aspergillus jumigatus, Aspergillus flavus and Aspergillus clavatus), Cryptococcus sp. (such as Cryptococcus neoformans, Cryptococcus gattii, Cryptococcus laurentii and Cryptococcus albidus), Histoplasma sp. (such as Histoplasma capsulatum), Pneumocystis sp. (such as Pneumocystis jirovecii), and Stachybotrys (such as Stachybotrys chartarum).

v. Parasites

Exemplary parasitic organisms include Malaria (Plasmodium falciparum, P. vivax, P. malariae), Schistosomes, Trypanosomes, Leishmania, Filarial nematodes, Trichomoniasis, Sarcosporidiasis, Taenia (T. saginata, T. solium), Leishmania, Toxoplasma gondii, Trichinelosis (Trichinella spiralis) or Coccidiosis (Eimeria species).

vi. Malignancies including Tumors

Exemplary tumors and their tumor antigens (antigens produced by tumor cells that can stimulate tumor- specific T-cell immune responses) are shown below.

Exemplary tumors and their tumor antigens

Tumor Tumor Associated Target Antigens

Colon cancer CEA

Cervical Cancer SCC, CA125, CEA, Cytokeratins (TPA, TPS,

Cyfra21-1)

Renal cell carcinoma (RCC) Fibroblast growth factor 5

Germ cell tumors AFP

The disclosure is further illustrated by the following non-limiting Example.

EXAMPLES

Example 1

Selective Depletion of Alloreactive T cells with Adenosine

This Example describes a method of depleting alloreactive T cells by treatment with adenosine.

Hematopoietic stem cell transplantation (SCT) is a potentially curative treatment for patients with hematologic malignancies. Relapse remains the major failure after allogeneic SCT. Donor lymphocyte infusion (DLI) has been shown to induce remissions post-transplant in patients with relapsed hematologic malignancies. However, the efficacy and safety of the SCT and DLI are limited by Graft Versus Host Disease (GVHD), a multi-organ destructive process caused by allogeneic donor T cells in the transplant product, leading to graft failure, morbidity, and mortality.

Selective removal of alloreactive T cells from donor graft can decrease the rate and severity of GVHD as well as preserve specific immunity against infections and tumors. Selectively depleted T cell transplants can serve as an ideal platform for the generation of "off-the-shelf third party antigen- specific T cells for the treatment of microbial infections, malignancies, and other human diseases. Previous selective depletion of alloreactive T cells in transplant medicine including ex vivo depletion (such as depletion of cells expressing activation surface markers (e.g., CD25, CD69, CD71, CD134, CD137, or HLA-DR), immunotoxin: antibody against activation marker CD25 conjugated with dgA (ricin), photodepletion with rhodamine-like dye, Fas-mediated apoptosis (use of FasL) or engineering stem cell grafts that are depleted of naive T cells) or in vivo depletion (such as the 'Kill Switch' (HSV-tk, iC9), blocking APC co-stimulatory molecules CD80, CD86 or T-Reg, MSc Infusion) are not sufficient to prevent GVHD and some are associated with poor immune recovery.

Disclosed herein is the use of adenosine for the selective removal of alloreactive T cells from donor graft in patients receiving stem cell, immune cell, tissue or organ transplant. Adenosine is an FDA approved drug used in myocardial perfusion imaging (stress test) as well as in the treatment of PSVT (paroxysmal supra ventricular tachycardia). Adenosine signaling induces immune tolerance. Immune regulatory cells produce adenosine. Tumors use adenosine as a mechanism of immune evasion.

In particular, the method used an ex vivo co-culture of donor graft cells with the antigen presenting cells (APC) of the recipients. The recipient's APC stimulated the alloreactive (GVHD causing) donor T lymphocytes. These activated cells were eliminated specifically by the addition of adenosine, which selectively kills the activated T cells. The graft cells were then washed free of adenosine and given to the recipient.

Some of the advantages of the disclosed method include the following: donor T cell product after allo-depletion with adenosine contains no or minimal alloreactivity to recipients; donor T cell product can react against third party alio antigens; virus-specific memory T cells were retained; and the product was able to generate new immune response against viral and leukemia antigens.

Additionally, the method eliminates several steps in the process and would be therefore cheaper and simpler to apply clinically.

i. Material and Methods

a. Cells

Peripheral blood mononuclear cells (PBMC) from healthy donors were collected by apheresis (Amicus Separator; Fenwal, Lake Zurich, IL, USA). Human PBMCs were obtained from healthy donor leukopacs and leukocytes enriched by centrifugation over Ficoll. Monocytes were obtained from healthy donors by leukopheresis and elutriation (Transfusion Medicine Department, Clinical Center, National Institutes of Health, Bethesda, MD). Monocytes and lymphocytes were isolated from the PBMC concentrates on the day of collection by elutriation (Elutra, Gambro BCT, Lakewood, CO, USA), according to the manufacturer's recommendations. Cells were enriched by centrifugation over Ficoll and cryopreserved using a controlled rate freezer (Kryosave; Integra, Planer pic, Sunbury-on- Thames, UK) and media containing 5% dimethyl sulfoxide (Edwards Lifesciences, Irvine, CA, USA), 6% pent starch (Pharmaceutical Development Section, Pharmacy Department, Clinical Center, National Institutes of Health) and 4% human serum albumin (Baxter Health Care Corporation, LosAngeles, CA, USA). Before use, cells were thawed, washed, and suspended in complete medium, (CM) supplemented with 5% human AB serum (HS) and rested overnight. The cells were thawed and washed in 50:50 mix of AIM V and RPMI 1640 media supplemented with 5% heat-inactivated normal AB serum (Gemini Bio-Product, Woodland, CA) and 2 mM L-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin (Invitrogen, Carlsbad, CA) CM and 30U/ml DNase I (Sigma). b. Preparation of monocyte-derived dendritic cells (DC)

Elutriated monocytes were cultured at lxlO ⁶ cell/ml in 12- well plates in complete media Recombinant human rIL-4 (800 U/ml) and GM-CSF (1000 U/ml, PeproTech) were added at Days 0 and 2 and cultured at 37 °C in 5% CO2 and 95% humidity. Four days after culture, cells were phenotypically characterized and used in subsequent experiments as described below. On Day 4, MDDCs were transduced with lentiviral vector expressing the indicated matured with LPS (30 ng/ml) for 24-48 h before use as APCs.

c. Lentiviral vectors

The human immunodeficiency virus (HIV)- 1 -based gene transfer vectors used in this study are pRRLsinl8.PPT.PGK.PRAME.Wpre (Lenti-PRAME) and pRRLsinl8.PPT.MSCV.WTl.Wpre (Lenti-WTl). The vesicular stomatitis virus G protein (VSV-G) envelope encoding construct pMD.G, and the packaging construct pCMV AR8.91 were kindly provided by Prof. D. Trono, Department of Genetics and Microbiology, CMU, Geneva, Switzerland. The lentiviral vector particles were produced in 293T cells (American Type Culture Collection, Manassas, VA) by three- plasmid transfection using a calcium phosphate transfection kit (Invitrogen) as previously described (Chinnasamy et al. Blood. 2000;96: 1309-1316), except that Opti-MEM-1 containing 5% HS was used as culture medium during transfection. The viral supernatants were concentrated to 50x by ultracentrifugation at 50,000g for 1.5 hours at 4°C). Viral pellets were resuspended in CM and stored frozen at -80°C until use.

d. Generation of DC-base antigen presenting cells for ex vivo generation of antigen- specific T cells ex vivo

Immature DC cultures (Day 4) were transduced with lentiviral vectors at a multiplicity of infection (MOI) of approximately 30, in the presence of protamine sulfate (10μg/ml, Sigma), in 24- well plate containing 1 ml/well of DC culture medium supplemented with rhGM-CSF (lOOOU/ml) and rhIL-4(800U/ml). After 48 hours transduction, cells were washed and cultured in fresh DC culture medium containing GM-CSF (lOOOU/ml) and IL-4 (lOOOU/ml) in the presence of 15 ng/ml endotoxin-free lipopolyschharide (Lipopolysaccharide, Sigma) for 24 hours. In some conditions, the untransduced were DCs matured with LPS and pulsed with peptide libraries derived from the target peptides and used to stimulate antigen- specific T cells

e. Ex vivo generation of "off-the-shelf third party antigen-specific effector T cells

To generate and expand effector T cells with specificity against the selected antigens ex vivo, MLRs for three donors were established as described in FIG. 4. For each donor, frozen lymphocytes were cocultured separately with mature DCs from two allogeneic donors in a 5 : 1 ratio. Half of the cultures were treated with a 2mM adenosine in 2X solution on days 1, 2, and 5 after the cultures were established. Two days after the final treatment, the cells were harvested and washed with PBS and primed twice at weekly interval with autologous DC lentivirally engineered to express target antigen or pulsed with peptide libraries of the target antigen of interest at a responder to stimulator ratio of 5: 1. Cultures received IL-7 (10 ng/ml) and IL-15 (10 ng/ml) during first priming and IL-2 (lOU/ml) in addition to IL-7 and IL-15 during second priming step. No cytokines were present during the initial 3 days of both priming steps. Resulting effector T cell cultures were tested for reactivity against autologous DC target expressing the relevant or irrelevant or untransduced DC. Briefly, effector cells were cocultured for 6 hours with target cells at 5: 1 ratio. Autologous Dc transduced to express the relevant or irrelevant (control) antigen or pulsed with peptide libraries of the target or irrelevant antigen (control). The protein transport inhibitors

Brefeldin A (BD GolgiPlug) or Monensin (BD GolgiStop) that prevent the secretion of cytokines thus trapping them in intracellular compartments were added to the cultures 1 hour after initiation of the coculture. The cultures were then incubated for another 5 hours and then stained with fluorescent labeled antibodies against T cell surface markers and intracellular cytokines as per the manufacturer's instructions.

Detection of cytokines by intracellular staining

To determine the intracellular cytokines in various subsets of antigen-experienced CD3+ T cells aliquots of cells were stained with the following fluorochrome labeled mouse monoclonal antibodies: anti-CD3 brilliant violet 605 (Biolegend), anti-CD4 V500 (BD biosciences), and anti- CD8 Cy7-allophycocyanin, anti-CD45RO allophycocyanin (BD biosciences), and anti-CD27 Cy5- Phycoerythrin. Subsequently cells were fixed and permeabilized with Cytofix/Cytoperm (BD PharMingen) solution according to the manufacturer' s instructions and stained with the following mouse anti-human cytokine antibodies: anti-TNFoc Cy7-Phycoerythrin, and anti-IFNy Alexa Fluor® 700 (all from eBioscience). Flow cytometry data were acquired using the BD Fortessa™ flow cytometer (BD biosciences) and Flowjo™ software.

ii. Results and Conclusions

FIG. 1 illustrates the protocol for the depletion of alloreactive T cells using adenosine. As illustrated in FIG. 1, donor lymphocytes and recipient dendritic cells were mixed at a 5: 1 lymphocyte reaction. The mixture was then exposed to 3 treatments with 1 mM adenosine over 5 days. Untreated cells were treated with 9% saline media. FIG. 2 illustrates 2 mM adenosine kills CD3/CD28 bead stimulated T cells. FIG. 3 illustrates adenosine treatment selectively eliminated activated alloreactive T cells (CD39 ⁺ CD25 ⁺ CD45RO ⁺ ) in MLR culture. FIG. 4 provides a schematic of a product validation proliferation assay. FIG. 5 demonstrates the efficacy of adenosine-mediated selective depletion of alloreactive. FIG. 6 illustrates the results of quality control testing of post alloreactive T cell depleted cell products. FIG. 7 illustrates CMV virus- specific memory T cells were retained in the cell product post depletion of alloreactive T cells with adenosine (Top panel) and CMV virus-specific memory T cells were retained in the cell product post depletion of alloreactive T cells with adenosine (Bottom panel). FIG. 8 illustrates leukemia antigen-specific T cells were efficiently generated from donor cell product post adenosine mediated-depletion of alloreactive T cells.

Example 2

Selective Depletion with Adenosine in Matched and Haplo Pairs This example shows that adenosine can be used for selective depletion (SD) in

haploidentical and HLA-matched sibling donor-recipient pairs. SD with adenosine reduced alloreactivity significantly below that of untreated controls in both the haploidentical and HLA- matched sibling cocultures. Materials and Methods

Cell Processing and Collection: Recipient leukapheresis (LP) products were obtained from patients with acute myeloblastic leukemia (AML) in remission 20-30 days prior to transplantation. Responder (donor) LP products were obtained from HLA matched sibling donors, and

haploidentical mother or sibling of the corresponding recipient patients. Dendritic cells (DC) were generated from monocytes immunomagnetically purified by CD 14 positive selection using EASYSEP™ Human CD14 Positive Selection Kit (Catalog* 18058, (Stemcell Technologies, Vancouver, BC, Canada). HLA typing of the matched and haplodentical donor-recipients is indicated in corresponding data figure.

Allodepletion with Adenosine: Donor peripheral blood mononuclear cells (PBMCs) from processed LP products were cocultured with matched sibling or haploidentical monocyte-generated recipient DCs in 24 well plates. Selective depletion of alloreactive T cells was performed on half of the cultures using methods disclosed above, specifically using 2mM adenosine treatments on days 1, 2, and 5 of coculture.

Proliferation Assay to Test Efficacy of Depletion: On day 7, allodepleted cultures and non- depleted control cultures were harvested, washed, and stained with CFSE. After staining, the cells were cocultured with a panel of stimulators: DCs autologous to responder T cells to measure background proliferation, stimulator DCs to measure alloreacting cells, and a mix of DCs from 3 unrelated donors to measure response to de novo antigens. Stimulation of T cells with anti- CD3/CD28 Beads (DYNABEADS® Human T-Activator CD3/CD28; Invitrogen) was used to measure maximum proliferative capacity. Responder-DC cocultures were established at a 10:1 ratio and responder-bead cocultures were established at a 3:1 ratio.

Results

SD was performed in haploidentical and HLA-matched sibling cocultures to verify the feasibility of this method in these more clinically relevant pairings. Adenosine selectively depleted cultures were compared to unmanipulated, not depleted control cultures. In three experiments, SD with adenosine reduced alloreactivity significantly below that of untreated controls in both haploidentical and matched sibling cocultures. 90-98% depletion was achieved in the

Haploidentical setting, and 98-100% depletion was achieved in the matched sibling setting, see

FIG. 9. Proliferative response to anti-CD3-CD28 bead stimulation and 3 ^rd party antigen stimulation was retained in depleted cultures, but decreased relative to controls in 2/3 haploidentical pairs and 1/3 matched sibling pairs. Reported % "Proliferating T Cell" values are normalized by subtracting background proliferation of corresponding CFSE-stained, unstimulated cultures over the same time period.

In view of the many possible embodiments to which the principles of the disclosed invention may be applied, it should be recognized that the illustrated embodiments are only preferred examples of the invention and should not be taken as limiting the scope of the invention. Rather, the scope of the invention is defined by the following claims. We therefore claim as our invention all that comes within the scope and spirit of these claims.