AN ANTI-CD45RC ANTIBODY FOR USE AS DRUG

Abstract

The invention relates to an isolated anti-CD45RC antibody for use in preventing or treating transplant rejection, autoimmune diseases, unwanted immune responses against proteins expressed in the course of gene therapy and/or therapeutic proteins, allergy as well as lymphoma or cancer which are associated with CD45RC.sup.+ cells. The invention relates to an isolated anti-CD45RC antibody for use in expanding and/or potentiating regulatory T cells.

Claims

1. A method of i) preventing or reducing allogeneic transplant rejection or 2) preventing or treating an autoimmune disease, an unwanted immune response against therapeutic protein, an allergy, or a lymphoma or cancer which is associated with CD45RC+ cells in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of an anti-CD45RC antibody sufficient to prevent or reduce the allogeneic transplant rejection or prevent or treat the autoimmune disease, unwanted immune response against therapeutic protein, allergy and lymphoma or cancer which is associated with CD45RC+ cells.

2. The method according to claim 1, wherein said allogeneic transplant rejection is selected from the group consisting of allogeneic hematopoietic stem cell transplant rejection (GVHD), cardiac transplant rejection, pancreatic islet transplant rejection, vascular tissue transplant rejection, kidney transplant rejection, lung transplant rejection and liver transplant rejection.

3. The method according to claim 1, wherein said allogeneic transplant rejection is GVHD.

4. The method according to claim 2, wherein said allogeneic transplant rejection is cardiac allotransplant rejection.

5. The method according to claim 1, wherein the autoimmune disease is selected from the group consisting of rheumatoid arthritis, juvenile oligoarthritis, collagen-induced arthritis, adjuvant-induced arthritis, Sjogren's syndrome, multiple sclerosis, experimental autoimmune encephalomyelitis, inflammatory bowel disease, autoimmune gastric atrophy, pemphigus vulgaris, psoriasis, vitiligo, type 1 diabetes, non-obese diabetes, myasthenia gravis, Grave's disease, Hashimoto's thyroiditis, sclerosing cholangitis, sclerosing sialadenitis, systemic lupus erythematosis, autoimmune thrombocytopenia purpura, Goodpasture's syndrome, Addison's disease, systemic sclerosis, polymyositis, dermatomyositis, acquired hemophilia and thrombotic thrombocytopenic purpura.

6. The method according to claim 1, wherein said anti-CD45RC antibody is an anti-CD45RC monoclonal antibody or a fragment thereof.

7. The method according to claim 6, wherein said anti-CD45RC monoclonal antibody is an anti-human CD45RC monoclonal antibody.

8. The method according to any one claim 1, wherein said anti-CD45RC antibody is a humanized antibody or a fully human antibody.

9. A pharmaceutical composition or a kit-of-part composition comprising an isolated anti-human CD45RC monoclonal antibody and a pharmaceutically acceptable excipient.

10. The pharmaceutical composition or a the kit-of-part composition according to claim 9, further comprising an immunosuppressive drug.

11. The pharmaceutical composition or a the kit-of-part composition according to claim 10, wherein the immunosuppressive drug is selected from the group consisting of cytostatics; alkylating agents, antimetabolites, therapeutic antibodies, calcineurin inhibitors, glucocorticoids and mycophenolate mofetil.

12. A method of preventing or treating allogeneic transplant rejection, autoimmune diseases, unwanted immune response against therapeutic proteins allergies and lymphoma or cancer which is associated with CD45RC+ cells in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of the pharmaceutical composition or the kit-of-part composition according to claim 9 sufficient to prevent or treat the allogeneic transplant rejection, autoimmune diseases, unwanted immune response against therapeutic proteins, allergies and lymphoma or cancer.

13. A method for expanding and/or potentiating regulatory T cells in a patient in need thereof, comprising a step of administering to said patient a therapeutically effective amount of an anti-CD45RC antibody sufficient to expand and/or potentiate the regulatory T cells.

14. (canceled)

15. A method for improving transplant survival in a transplant patient, comprising a step of administering to said patient a therapeutically effective amount of an anti-CD45RC antibody sufficient to improve the transplant survival of the patient.

16. The pharmaceutical composition or the kit-of-part composition according to claim 11, wherein the cytostatic is a mammalian target of rapamycin (mTOR) inhibitor or rapamycin (sirolimus).

17. The pharmaceutical composition or the kit-of-part composition according to claim 11, wherein the alkylating agent is cyclophosphamide.

18. The pharmaceutical composition or the kit-of-part composition according to claim 11, wherein the antimetabolites is azathioprine, mercaptopurine or methotrexate.

19. The pharmaceutical composition or the kit-of-part composition according to claim 11, wherein the therapeutic antibody is an anti-CD40L monoclonal antibody, an anti-IL-2R monoclonal antibody, an anti-CD3 monoclonal antibody, an anti-lymphocyte globulin (ALG) or an anti-thymocyte globulin (ATG).

20. The pharmaceutical composition or the kit-of-part composition according to claim 11, wherein the calcineurin inhibitor is cyclosporine.

Description

FIGURES

[0131] FIG. 1: Tolerance induction after anti-CD45RC short-term treatment. Recipients were either untreated (n=9), treated 5 days (n=3), 10 days (n=3) or 20 days (n=3) in the LEW.1W/LEW.1A strain combination or untreated (n=4) or treated 20 days in the LEW.1A/LEW.1W strain combination. Anti-CD45RC mAbs (OX22) was given i.v at 0.8 mg/kg/injection. **p<0.01 versus untreated.

[0132] FIG. 2: Lymphocyte phenotyping in the blood during and following 10 days of anti-CD45RC treatment. PBMCs were recovered at day 0, 4, and 10 from anti-CD45RC-treated recipients and analyzed by flow cytometry for absolute number of sub-populations.

[0133] FIG. 3: Regulatory cells are increased in the spleen of long-surviving anti-CD45RC-treated recipients. Splenocytes were recovered at day 120 from anti-CD45RC-treated recipients compared to naive splenocytes and analyzed by flow cytometry for absolute numbers of sub-populations.

[0134] FIG. 4: Adoptive transfer of tolerance. LEW.1A recipients were sublethally irradiated (4.5 Gy) at day −1 and received heart allografts and i.v. injections at day 0 of splenocytes or sorted-subpopulations from long-surviving anti-CD45RC treated recipients. Graft survival was monitored by abdominal palpation.

[0135] FIG. 5: Increased suppressive capacity of CD8.sup.+CD45RC.sup.−/low Tregs following anti-CD45RC-treatment. CFSE-labeled LEW.1A dividing CD4.sup.+CD25.sup.− T cells stimulated with donor LEW.1W pDCs were analyzed after 6 days of culture in the absence or presence of d120 LEW.1A naive or anti-CD45RC-treated CD8.sup.+CD45RC.sup.−/low Tregs at different effector/suppressor ratio.

[0136] FIG. 6: Anti-CD45RC mAb treatment reduces lethality and weight loss in a model of GVHD. Rats receiving 200.10.sup.6 splenocytes were treated with anti-CD45RC mAb or isotype control to assess potency for preventing GVHD. Average weight loss (percentage of initial) for rats surviving on a given day was shown +/−SEM. ***, p<0.001. n=3.

EXAMPLE 1

Short-Term anti-CD45RC Antibody Treatment Results in Transplantation Tolerance Associated with Induction of Potent Regulatory Cells

[0137] Material & Methods

[0138] Animals and cardiac transplantation models: Heart allotransplantation were performed between whole MHC incompatible male LEW-1W and LEW-1A rats as previously described (16, 22, 23). Heart survival was evaluated by palpation through the abdominal wall and heart beating was graded from +++ to.

[0139] The experiments were approved by the regional ethical committee for animal experimentation.

[0140] Anti-CD45RC administration: The OX22 (IgG1 anti-rat CD45RC) mouse hybridoma was used to produce the anti-CD45RC monoclonal antibody. Anti-CD45RC mAbs were given at a dose of 0.8 mg/kg/2.5days i.v. from day 0 to 5, 10 or 20 post-transplantation. Control IgG1 (3G8, anti-human CD16, with no cross-reaction with the rat) was used at the same dose.

[0141] Histopathology studies: Tissue specimens were analyzed for chronic rejection at day 120 post-transplantation as previously described. Briefly, tissues were fixed in paraformaldhéhyde, paraffin embedded, cut in 5μm-thick sections and stained with hematoxylin-eosin-saffron. (24)

[0142] Lymphocyte preparation, isolation of subpopulations and adoptive transfert: Spleen were harvested at day 120 and splenocytes or sub-populations were purified as previously described (16). Cell transfers were performed by i.v. injection of total splenocytes or purified sub-populations on the day of transplantation of LEW.1W onto LEW.1A sublethally irradiated (4.5 Gy day −1) recipient.

[0143] Monoclonal antibodies and staining: T cells and pDC were purified and sorted as previously described (20). Flow cytometry and cell sorting was performed using antibodies directed to T cells (TCRαβ, clone R7/3), CD4.sup.+CD25.sup.−T cells (clones OX35 and OX39), CD8.sup.+ T cells (clone OX8), CD8.sup.+ or CD4.sup.− CD45RC.sup.low T cells (clones OX8 and OX22), and CD4.sup.+CD45R.sup.+85C7.sup.+ pDCs (clones His24, OX35 and 85C7), CD11b and CD45RA.sup.+ B cells (OX33) obtained from the European Collection of Cell Culture (Salisbury, UK). All biotin-labelled mAbs were visualized using Strepavidin-PE-Cy7 (BD Biosciences) or Streptavidin-Alexa405. A Canto II cytometer (BD Biosciences, Mountain View, Calif.) was used to measure fluorescence, and data were analyzed using the FLOWJO software (Tree Star, Inc. USA). Cells were first gated by their morphology excluding dead cells by selecting DAPI viable cells.

[0144] Mixed lymphocyte reaction: Naive Lewis 1A CD4.sup.+ T cells, naive Lewis 1W allogeneic pDC, and treated-syngeneic Lewis 1A CD8.sup.+CD45RC.sup.low Tregs subsets were sorted as previously described (19). Proliferation of CFSE-labelled CD4.sup.+CD25.sup.− T cells was analyzed by flow cytometry 6 days later, by gating on TCR.sup.+CD4.sup.+ cells (R7/3-APC, Ox35-PECY7) among living cells (DAPI negative).

[0145] Antibody detection: Alloantibodies. Donor spleens were digested by collagenase D, stopped with 400 μl EDTA 0.1 mM, and red cells were lysed. Serum of recipients were added to donor splenocytes at a dilution 1/8, and incubated with either anti-rat IgG-FITC (Jackson ImmunoResearch Labs INC, Baltimore, USA), anti-rat IgG1 (MCA 194, Serotec), anti-rat IgG2a (MCA 278, Serotec), or anti-rat IgG2b (STAR114F, Serotec) and anti-Ms Ig-FITC (115-095-164, Jackson ImmunoResearch). A FACS Canto (BD Biosciences, Mountain View, Calif.) was used to measure fluorescence, and data were analyzed using the FLOWJO software (Tree Star, Inc. USA). Geometric mean of fluorescence (MFI) of tested sera was divided by mean of 5 naive Lewis 1A sera MFI as control.

[0146] Immunization and detection of anti-KLH antibodies: Keyhole limpet hemocyanin (KLH, Sigma Aldrich, St. Louis, Mo.) was injected at >100 days after transplantation in the footpad (50 μg emulsified in 200 μl of complete freund adjuvant). Anti-KLH IgM and IgG Abs were detected repectively at day 4 and day 13 post-immunization by ELISA as previously described (18).

[0147] Statistical analysis: One Way ANOVA Kruskal Wallis test and Dunn's posttest was used for coculture experiments, Two-Way ANOVA test and Bonferroni posttests was applied for donor-directed antibodies, and splenocytes phenotype characterization, and Mantel Cox test was used to analyse survival curves.

[0148] Results

[0149] Tolerance induction following short-term anti-CD45RC antibody treatment: As we previously demonstrated the presence and potential of CD8.sup.+ T cells with absent or low expression of the CD45RC surface marker, we speculated that a targeting strategy using an anti-CD45RC MAb would promote tolerance in cardiac transplanted recipients. Thus, we have administered an anti-CD45RC antibody (OX22, 0.8 mg/kg/i.v.) every 2.5 days during 20, 10 or 5 days to transplanted rat recipients (cardiac allograft MHC mismatched, LEW.1W grafted onto LEW.1A), starting the day of the transplantation. We obtained indefinite allograft survival in recipients treated 20 or 10 days with the anti-CD45RC antibody compared to control untreated recipients and recipients treated 5 days (FIG. 1), demonstrating for the first time that short-term therapy with the anti-CD545RC antibody efficiently promote indefinite allograft survival.

[0150] To further test the tolerogenic potential of the anti-CD45RC antibody in vivo, recipients were grafted in a stronger combination of allograft rejection (LEW.1A grafted onto LEW.1W) and administered the anti-CD45RC antibody for 20 days. In this graft combination, we also observed indefinite allograft survival in all recipients (FIG. 1), demonstrating the high suppressive effect of the anti-CD45RC treatment.

[0151] Cardiac histopathology analysis revealed a complete absence of fibrosis, infiltrate and vessel sclerosis at day 120 in 20-days anti-CD45RC-treated recipients, demonstrating that this short-term treatment efficiently inhibits both acute and chronic rejection.

[0152] Finally, we analyzed in these recipients the presence of anti-donor antibodies and the capacity to mount antibody responses against exogenous antigens (KLH) at day 120 post-transplantation. We observed a complete absence of anti-donor IgG, IgG1, IgG2a and IgG2B antibodies in recipients treated 20 days with the anti-CD45RC antibody compared to controls. In contrast, we observed that immunization at day 120 of 20-days treated long-term surviving recipients with exogenous antigen (KLH mixed with CFA) resulted in high-level production of both IgM and IgG antibodies compared to naive recipients.

[0153] Altogether, we demonstrated that anti-CD45RC antibody treatment resulted in inhibition of both acute and chronic rejection, without compromising immunity and capacity of the recipients to mount antibody responses against cognate antigens.

[0154] Short-term anti-CD45RC antibody treatment resulted in temporary lymphocyte reduction but increased regulatory populations: To analyze the effect of the anti-CD45RC antibody on the phenotype of the cell subsets in the blood of 10-days treated-recipients at day 0, 4 and 10 after transplantation, we analyzed the different cell subset (FIG. 2). We observed at day 4 a complete absence of CD45RC.sup.+ cells and a slight decreased of T and B cells (as CD45RC is expressed by both populations). At day 10, we observed all subset had return to normal level and we observed a strong increased in T CD4.sup.+CD45RC.sup.−, T CD8.sup.+CD45RC.sup.− and B.sup.+CD45RC.sup.− populations, as well as MDSCs (FIG. 2).

[0155] Given the long-term tolerance induction that resulted from the 10 and 20-days anti-CD45RC-treatment, we analyzed the proportion of regulatory T.sup.+CD4.sup.+CD45RC.sup.−/low, T.sup.+CD8+CD45RC.sup.−/low or B.sup.+CD45RC.sup.−/low cells in the spleen. At day 120 after treatment, analysis of the phenotype of the recipients in the spleen revealed a strong enrichment in absolute number of T cells and particularly of T CD4.sup.+CD45RC.sup.int/−, T CD8.sup.+CD45RC.sup.−, while other cell subsets were not modified (FIG. 3).

[0156] Altogether, we demonstrated that short-term anti-CD45RC treatment resulted in temporary decreased of CD45RC.sup.+, T and B cells, while T.sup.+CD4.sup.+CD45RC.sup.−/low, T.sup.+CD8.sup.+CD45RC.sup.−/low and B.sup.+CD45RC.sup.−/low were increased early and at day 120 suggesting a role for regulatory cells in tolerance induction.

[0157] Short-term anti-CD45RC antibody treatment resulted in potentiation of CD8.sup.+CD45RC.sup.−/low Tregs and transferable tolerance: To confirm in vivo that short-term treatment with anti-CD45RC antibodies resulted in induction of potent suppressive regulatory cells, we performed adoptive cell transfer of splenocytes or sub-populations into secondary irradiated cardiac grafted recipients (FIG. 4). We observed that adoptive cell transfer of 150.10e.sup.6 splenocytes resulted in indefinite allograft survival in all recipients compared to recipients transferred with naive splenocytes or non-transferred. To further determine the sub-population responsible of the tolerance induction, we sorted CD8.sup.+CD45RC.sup.−/low, CD4.sup.+CD45RC.sup.−/low and B+CD45RC.sup.−/low cells and performed adoptive cell transfer. While we observed a slight prolongation of allograft survival in recipients adoptively transferred with B cells, we observed 100% allograft survival in recipients transferred with CD8.sup.+CD45RC.sup.low Tregs and, CD4.sup.+CD45RC.sup.low T cells, demonstrating that the depletion of CD45RC.sup.+ cells from day 0 to 20 has induced and activated regulatory cells with a strong suppressive capacity.

[0158] As we had previously described an ex vivo assay to characterize the suppressive activity of CD8.sup.+CD45RC.sup.low Tregs (19) and to further evaluate and compare the suppressive capacity of CD8.sup.+CD45RC.sup.low Tregs induced with the anti-CD45RC Ab to naive CD8.sup.+CD45RC.sup.low Tregs, CD8.sup.+CD45RC.sup.low Tregs were sorted at day 120 and added in a dose dependent manner in an assay were CFSE-labeled CD4.sup.−CD25.sup.− effector T cells were cocultured with donor-derived plasmacytoid dendritic cells (pDC) (FIG. 5). In absence of Tregs, we observed a strong proliferation of the CD4.sup.+CD25.sup.− Teff cells. This proliferation was reduced in presence of naive CD8.sup.+CD45RC.sup.low Tregs as we have previously described (19). Interestingly, in presence of anti-CD45RC-induced CD8.sup.+CD45RC.sup.low Tregs from d120 recipients, we observed an almost total inhibition of proliferation of the CD4.sup.+CD25.sup.− Teff cells in a dose dependent manner until at least an effector:suppressor ratio of 16:1, demonstrating the strong potential of the Ab therapy in inducing strong suppressive CD8.sup.+CD45RC.sup.low Tregs.

EXAMPLE 2

Anti-CD45RC mAb Treatment Reduces Lethality and Weight Loss in a Model of GVHD

[0159] Material & Methods

[0160] Graft-versus-host disease induction in vivo: 200.10.sup.6 splenocytes were injected intravenously in syngeneic or allogeneic rats treated 24 hours earlier with whole-body sublethal irradiation of 7.8 Gy. Recipients received anti-CD45RC mAbs (OX22) or an irrelevant control mAbs starting day 0 and every 3 days at 2 mg/kg/injection. Recipients were weighted every day and sacrifice when percentage of weight loss was >20% of their initial weight.

[0161] Results

[0162] Anti-CD45RC mAb treatment reduces lethality and weight loss in a model of GVHD: To determine whether administration of the anti-CD45RC could prevent the development of acute graft versus host disease (GVHD), recipients received either syngeneic or allogeneic splenocytes following whole-body sublethal irradiation. Anti-CD45RC or irrelevant control mAbs were then administered i.p. every 3 days. We observed that recipients that received syngeneic splenocytes started to lose weight but then recovered from the whole-body irradiation and gained weight until the end of the experiment. Interestingly, we observed that recipients that were administered allogeneic splenocytes and the anti-CD45RC antibody lost significatively less weight than the recipients administered the control irrelevant antibody and even started to gain weight by day 10, while the control group had lost >20% of their weight and was sacrificed by day 12, altogether demonstrating the potential of the anti-CD45RC therapy in controlling the GVHD (FIG. 6).

REFERENCES

[0163] Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure. [0164] 1 Gurkan, S., Luan, Y., Dhillon, N., Allam, S. R., Montague, T., Bromberg, J. S., Ames, S., Lerner, S., Ebcioglu, Z., Nair, V., et al. 2010. Immune reconstitution following rabbit antithymocyte globulin. Am J Transplant 10:2132-2141. [0165] 2. Mai, H. L., Boeffard, F., Longis, J., Danger, R., Martinet, B., Haspot, F., Vanhove, B., Brouard, S., and Soulillou, J. P. 2014. IL-7 receptor blockade following T cell depletion promotes long-term allograft survival. J Clin Invest 124:1723-1733. [0166] 3. Streuli, M., Hall, L. R., Saga, Y., Schlossman, S. F., and Saito, H. 1987. Differential usage of three exons generates at least five different mRNAs encoding human leukocyte common antigens. J Exp Med 166:1548-1566. [0167] 4. Thomas, M. L. 1989. The leukocyte common antigen family. Annu Rev Immunol 7:339-369. [0168] 5. Ordonez, L., Bernard, I., L'Faqihi-Olive, F. E., Tervaert, J. W., Damoiseaux, J., and Saoudi, A. 2009. CD45RC isoform expression identifies functionally distinct T cell subsets differentially distributed between healthy individuals and AAV patients. PLoS One 4:e5287. [0169] 6. Birkeland, M. L., Johnson, P., Trowbridge, I. S., and Pure, E. 1989. Changes in CD45 isoform expression accompany antigen-induced murine T-cell activation. Proc Natl Acad Sci USA 86:6734-6738. [0170] 7. Hermiston, M. L., Xu, Z., and Weiss, A. 2003. CD45: a critical regulator of signaling thresholds in immune cells. Annu Rev Immunol 21:107-137. [0171] 8. Hargreaves, M., and Bell, E. B. 1997. Identical expression of CD45R isoforms by CD45RC+ ‘revertant’ memory and CD45RC+ naive CD4 T cells. Immunology 91:323-330. [0172] 9. Lazarovits, A. I., Poppema, S., Zhang, Z., Khandaker, M., Le Feuvre, C. E., Singhal, S. K., Garcia, B. M., Ogasa, N., Jevnikar, A. M., White, M. H., et al. 1996. Prevention and reversal of renal allograft rejection by antibody against CD45RB. Nature 380:717-720. [0173] 10. Basadonna, G. P., Auersvald, L., Khuong, C. Q., Zheng, X. X., Kashio, N., Zekzer, D., Minozzo, M., Qian, H., Visser, L., Diepstra, A., et al. 1998. Antibody-mediated targeting of CD45 isoforms: a novel immunotherapeutic strategy. Proc Natl Acad Sci USA 95:3821-3826. [0174] 11. Abu-Hadid, M. M., Lazarovits, A. I., and Madrenas, J. 2000. Prevention of diabetes mellitus in the non-obese diabetic mouse strain with monoclonal antibodies against the CD45RB molecule. Autoimmunity 32:73-76. [0175] 12. Rogers, P. R., Pilapil, S., Hayakawa, K., Romain, P. L., and Parker, D. C. 1992. CD45 alternative exon expression in murine and human CD4+ T cell subsets. J Immunol 148:4054-4065. [0176] 13. Spickett, G. P., Brandon, M. R., Mason, D. W., Williams, A. F., and Woollett, G. R. 1983. MRC OX-22, a monoclonal antibody that labels a new subset of T lymphocytes and reacts with the high molecular weight form of the leukocyte-common antigen. J Exp Med 158:795-810. [0177] 14. Xystrakis, E., Bernard, I., Dejean, A. S., Alsaati, T., Druet, P., and Saoudi, A. 2004. Alloreactive CD4 T lymphocytes responsible for acute and chronic graft-versus-host disease are contained within the CD45RChigh but not the CD45RClow subset. Eur J Immunol 34:408-417. [0178] 15. Xystrakis, E., Dejean, A. S., Bernard, I., Druet, P., Liblau, R., Gonzalez-Dunia, D., and Saoudi, A. 2004. Identification of a novel natural regulatory CD8 T-cell subset and analysis of its mechanism of regulation. Blood 104:3294-3301. [0179] 16. Guillonneau, C., Hill, M., Hubert, F. X., Chiffoleau, E., Herve, C., Li, X. L., Heslan, M., Usal, C., Tesson, L., Menoret, S., et al. 2007. CD40Ig treatment results in allograft acceptance mediated by CD8CD45RC T cells, IFN-gamma, and indoleamine 2,3-dioxygenase. J Clin Invest 117:1096-1106. [0180] 17. Powrie, F., and Mason, D. 1990. OX-22high CD4+ T cells induce wasting disease with multiple organ pathology: prevention by the OX-221ow subset. J Exp Med 172:1701-1708. [0181] 18. Guillot, C., Guillonneau, C., Mathieu, P., Gerdes, C. A., Menoret, S., Braudeau, C., Tesson, L., Renaudin, K., Castro, M. G., Lowenstein, P. R., et al. 2002. Prolonged blockade of CD40-CD40 ligand interactions by gene transfer of CD40Ig results in long-term heart allograft survival and donor-specific hyporesponsiveness, but does not prevent chronic rejection. J Immunol 168:1600-1609. [0182] 19. Li, X. L., Menoret, S., Bezie, S., Caron, L., Chabannes, D., Hill, M., Halary, F., Angin, M., Heslan, M., Usal, C., et al. 2010. Mechanism and localization of CD8 regulatory T cells in a heart transplant model of tolerance. J Immunol 185:823-833. [0183] 20. Picarda, E., Bezie, S., Venturi, V., Echasserieau, K., Merieau, E., Delhumeau, A., Renaudin, K., Brouard, S., Bernardeau, K., Anegon, I., et al. 2014. MHC-derived allopeptide activates TCR-biased CD8+ Tregs and suppresses organ rejection. J Clin Invest 124:2497-2512. [0184] 21. Ordonez, L., Bernard, I., Chabod, M., Augusto, J. F., Lauwers-Cances, V., Cristini, C., Cuturi, M. C., Subra, J. F., and Saoudi, A. 2013. A higher risk of acute rejection of human kidney allografts can be predicted from the level of CD45RC expressed by the recipients' CD8 T cells. PLoS One 8:e69791. [0185] 22. Haspot, F., Seveno, C., Dugast, A. S., Coulon, F., Renaudin, K., Usal, C., Hill, M., Anegon, I., Heslan, M., Josien, R., et al. 2005. Anti-CD28 antibody-induced kidney allograft tolerance related to tryptophan degradation and TCR class II B7 regulatory cells. Am J Transplant 5:2339-2348. [0186] 23. Josien, R., Heslan, M., Brouard, S., Soulillou, J. P., and Cuturi, M. C. 1998. Critical requirement for graft passenger leukocytes in allograft tolerance induced by donor blood transfusion. Blood 92:4539-4544. [0187] 24. Guillonneau, C., Louvet, C., Renaudin, K., Heslan, J. M., Heslan, M., Tesson, L., Vignes, C., Guillot, C., Choi, Y., Turka, L. A., et al. 2004. The role of TNF-related activation-induced cytokine-receptor activating NF-kappa B interaction in acute allograft rejection and CD40L-independent chronic allograft rejection. J Immunol 172:1619-1629. [0188] 25. Bedke T, Baars W, Schwinzer R. Modulation of human anti-pig T cell responses by monoclonal antibodies directed to porcine CD45 molecules. Ann Transplant. 2003; 8(3):35-8.