Biomarkers of transplantation tolerance induced by donor apoptotic leukocytes

Abstract

In certain embodiments, the present invention provides methods for identifying and treating transplant recipient patients with transplant tolerance induced by donor apoptotic leukocytes infused in conjunction with transient immunotherapy, comprising: (a) assaying a first blood sample from the patient to detect a baseline incidence of target cells, wherein the first blood sample is obtained before tolerization, before transplantation, and before the initiation of transient immunotherapy; (b) assaying a second blood sample from the patient to detect a post-procedure incidence of target cells, wherein the second blood sample is obtained after tolerization, after transplantation, and after the initiation of transient immunotherapy; and (c) identifying the patient as having transplant tolerance / acceptance induced by the donor antigen if the post-procedure incidence is at least two-fold higher than the baseline incidence.

Description

[Technical field]

[0001] Related Applications This application claims priority to U.S. Provisional Application No. 62 / 834,798, filed April 16, 2019. The entire contents of the above-referenced applications are hereby incorporated by reference in their entirety.

[0002] STATEMENT REGARDING FEDERALLY FUNDED RESEARCH This invention was made with Government support under AI102463 awarded by the National Institutes of Health. The Government has certain rights in this invention. [Background technology]

[0003] For many patients with end-stage organ failure, transplantation has become the most effective treatment option. Current immunosuppressive regimens effectively prevent acute rejection, but chronic rejection remains a serious problem due to the high morbidity and lack of efficacy of these regimens in preventing chronic rejection. Although the number of chronically immunosuppressed transplant recipients continues to increase, these recipients continue to suffer from these problems, which adversely affect recipient survival. Inducing immune tolerance to allografts would eliminate the need for maintenance immunotherapy and improve long-term allograft survival. However, despite the clinical significance of immune tolerance, which was first demonstrated in small animal models more than 65 years ago, this has only been achieved in a very small number of patients through mixed hemopoietic chimerism, which requires extensive conditioning therapy. Similarly, in translational models in monkeys, only mixed chimerism induced immune habituation to same-donor kidney allografts with near consistency.

[0004] In non-human primate studies, the donor apoptotic leukocyte regimen was consistently effective with little need for intensive short-term immunotherapy. Because of its efficacy and highly favorable safety profile, this regimen is the first clinically applicable non-chimeric transplantation tolerance regimen. Biomarkers are needed to monitor the induction, maintenance, and loss of transplantation tolerance in human recipients. Summary of the Invention [Means for solving the problem]

[0005] The present invention identifies biomarkers for monitoring the induction, maintenance, and loss of immune tolerance of solid organ, tissue, and cellular allografts in human recipients.

[0006] In certain embodiments, the present invention provides a method of identifying a graft recipient patient having transplant tolerance induced by donor antigens administered in conjunction with transient immunotherapy, the method comprising: (a) assaying a first blood sample from the patient to detect a baseline frequency of target cells, where the first blood sample is obtained before tolerization, before transplant, and before initiation of transient immunotherapy; (b) assaying a second blood sample from the patient to detect a post-procedure frequency of target cells, where the second sample is obtained after tolerization, after transplant, and after initiation of transient immunotherapy; and (c) identifying the patient as having donor antigen induced transplant tolerance / acceptance if the post-procedure frequency is at least two-fold higher than the baseline frequency, where the target cells are regulatory T cells type 1 (Tr1) with markers CD49b+, LAG-3+, CD4+. In certain embodiments, transplant recipient patients with transplant tolerance induced by donor antigens administered in conjunction with transient immunotherapy maintain transplant tolerance. In certain embodiments, the donor antigens are donor apoptotic leukocytes (ADL), donor-specific transfusion (DST) nanoparticles conjugated or encapsulated with donor peptides, and / or recipient apoptotic leukocytes conjugated with donor peptides. In certain embodiments, the target cells are regulatory T cells type 1 (Tr1) with markers CD49b+, LAG-3+, CD4+, have indirect specificity for at least one mismatched donor MHC class I peptide, have a transcriptomic signature indicative of antigen-specific signaling, and have a transcriptomic signature indicative of an activation state.

[0007] In certain embodiments, the transplant recipient patient has maintained transplant tolerance. In certain embodiments, the transplant recipient patient has had tolerance induced but failed. In certain embodiments, tolerance has not been induced in the transplant recipient patient.

[0008] In certain embodiments, the present invention provides a method comprising: (a) obtaining a first blood sample from a graft recipient patient to detect a baseline occurrence of target cells, where the first blood sample is obtained pre-tolerization, pre-transplant, and prior to initiation of transient immunotherapy; (b) obtaining a second blood sample from the patient to detect a post-procedure occurrence of target cells, where the second sample is obtained post-tolerization, post-transplant, and after initiation of transient immunotherapy; (c) assaying the first blood sample and the second blood sample to detect target cell levels before and after immune tolerization; and (d) identifying the graft recipient patient as having transplant tolerance / acceptance induced by donor antigens infused in conjunction with transient immunotherapy if the post-procedure occurrence is at least 2-fold higher than the baseline occurrence, wherein the target cells are regulatory T cells type 1 (Tr1) with markers CD49b+, LAG-3+, CD4+. In certain embodiments, the donor antigen is a donor apoptotic leukocyte (ADL), a donor-specific transfusion (DST) nanoparticle conjugated or encapsulated with a donor peptide, and / or a recipient apoptotic leukocyte conjugated with a donor peptide. In certain embodiments, the target cell is a regulatory T cell type 1 (Tr1) with markers CD49b+, LAG-3+, CD4+, has indirect specificity for at least one mismatched donor MHC class I peptide, has a transcriptomic signature indicative of antigen-specific signaling, and has a transcriptomic signature indicative of an activation state.

[0009] In certain embodiments, the transplant recipient patient has maintained transplant tolerance. In certain embodiments, the transplant recipient patient has had tolerance induced but failed. In certain embodiments, tolerance has not been induced in the transplant recipient patient.

[0010] In certain embodiments, the present invention provides a method for identifying a graft recipient patient having transplant tolerance induced by peri-transplant infusions (i.e., infusions around the time of transplant, with at least one infusion occurring several days before transplant) of donor apoptotic leukocytes coupled with transient immunotherapy, the method comprising: (a) assaying a first blood sample from the patient to detect a baseline incidence of target cells, where the first blood sample is obtained before tolerization, before transplant, and before initiation of transient immunotherapy; (b) assaying a second blood sample from the patient to detect a post-procedure incidence of target cells, where the second blood sample is obtained after tolerization, after transplant, and after initiation of transient immunotherapy; and (c) identifying the patient as having transplant tolerance / acceptance induced by donor apoptotic leukocytes if the post-procedure incidence is at least two-fold higher than the baseline incidence, where the target cells are CD49b + , LAG-3 + , CD4 + Regulatory T cells type 1 (Tr1) are defined as cells that have indirect specificity for at least one mismatched donor MHC class I peptide (as verified using recipient-specific MHC class II tetramers added with the MHC class I peptide), have a transcriptomic signature indicative of antigen-specific signaling, and / or have a transcriptomic signature indicative of an activation state. In certain embodiments, target cells express the marker CD49b + , LAG-3 + , CD4 + Regulatory T cells type 1 (Tr1) having indirect specificity for at least one mismatched donor MHC class I peptide, having a transcriptomic signature indicative of antigen-specific signaling, and having a transcriptomic signature indicative of an activation state. In certain embodiments, transplant recipient patients with transplant tolerance induced by donor antigen administered in conjunction with transient immunotherapy maintain transplant tolerance.

[0011] As used herein, the term "embarking on a transient immunotherapy" means that the recipient is transiently administered an immunotherapeutic agent, such as an immunosuppressant that targets, among other cells, antigen-presenting cells and their activation of donor-reactive T cells, any CD40-expressing cells, and directly T and B cells. As used herein, "transient" means that the therapeutic effect lasts for only a short period of time, for example, a few days (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days), or a few weeks (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks), or a few months (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months). As used herein, "immunosuppression" means partial or complete suppression of the immune response, in which the body's immune system has been purposely shut down or is less effective than if the body were not administered an immunosuppressant drug. In certain embodiments, the immunotherapy also includes the transient administration of an anti-inflammatory therapeutic agent.

[0012] In certain embodiments, the invention provides a method comprising: (a) obtaining a first blood sample from a graft recipient patient to detect a baseline occurrence of target cells, where the first blood sample is obtained pre-tolerization, pre-transplant, and prior to initiation of transient immunotherapy; (b) obtaining a second blood sample from the patient to detect a post-procedure occurrence of target cells, where the second sample is obtained post-tolerization, post-transplant, and after initiation of transient immunotherapy; (c) assaying the first blood sample and the second blood sample to detect target cell levels pre- and post-tolerization; and (d) identifying the graft recipient patient as having transplant tolerance / acceptance induced by donor apoptotic leukocytes if the post-procedure occurrence is at least two-fold higher than the baseline occurrence, wherein the target cells are CD49b + , LAG-3 + , CD4 +The target cells are regulatory T cells type 1 (Tr1), defined as cells that have indirect specificity for at least one mismatched donor MHC class I peptide, have a transcriptomic signature indicative of antigen-specific signaling, and / or have a transcriptomic signature indicative of an activation state. In certain embodiments, the target cells express the marker CD49b + , LAG-3 + , CD4 + Regulatory T cells type 1 (Tr1) with indirect specificity for at least one mismatched donor MHC class I peptide and with a transcriptomic signature indicative of antigen-specific signaling, as well as a transcriptomic signature indicative of an activation state.

[0013] In certain embodiments, the present invention provides a method for treating a transplant recipient, comprising: (a) identifying a transplant recipient patient having transplant tolerance / acceptance induced by infused donor apoptotic leukocytes (ADLs) in conjunction with transient immunotherapy using the above-described method, and (b) treating the transplant recipient patient by withdrawing administration of immunosuppressants. In an embodiment of the present invention, for example, the following items are provided: (Item 1) 1. A method for identifying a transplant recipient patient having transplant tolerance induced by a donor antigen administered in conjunction with transient immunotherapy, comprising: (a) assaying a first blood sample from said patient to detect a baseline frequency of target cells, said first blood sample being obtained prior to immune tolerization, prior to transplantation, and prior to initiation of transient immunotherapy; (b) assaying a second blood sample from the patient to detect a post-procedure incidence of target cells, wherein the second blood sample is obtained after immune tolerization, after transplantation, and after initiation of transient immunotherapy; and (c) identifying the patient as having transplant tolerance / acceptance induced by the donor antigen if the post-procedure incidence is at least two-fold higher than the baseline incidence; Including, The target cells express the marker CD49b + , LAG-3 + , CD4 + Regulatory T cells type 1 (Tr1) The method. (Item 2) below: (a) obtaining a first blood sample from a transplant recipient patient to detect a baseline frequency of target cells, wherein said first blood sample is obtained prior to immune tolerization, prior to transplantation, and prior to initiation of transient immunotherapy; (b) obtaining a second blood sample from said patient to detect a post-procedure incidence of target cells, said second blood sample being obtained after immune tolerization, after transplantation, and after initiation of transient immunotherapy; (c) assaying the first blood sample and the second blood sample to detect levels of target cells before and after immune tolerization; (d) identifying said graft recipient patient as having transplant tolerance / acceptance induced by donor antigens injected with transient immunotherapy if said post-procedure incidence is at least two-fold higher than said baseline incidence; A method comprising: The target cells are + , LAG-3 + , CD4 + Regulatory T cells type 1 (Tr1) The method. (Item 3) The method according to claim 1 or 2, wherein the donor antigen is donor apoptotic leukocytes (ADL), donor-specific transfusion (DST) nanoparticles conjugated with or encapsulating a donor peptide, and / or recipient apoptotic leukocytes conjugated with a donor peptide. (Item 4) 4. The method of any one of items 1 to 3, wherein the transplant recipient patient maintains transplant tolerance. (Item 5) 4. The method according to any one of items 1 to 3, wherein said graft recipient patient has had immune tolerance induced but subsequently failed. (Item 6) 4. The method according to any one of items 1 to 3, wherein immune tolerance has not been induced in the graft recipient patient. (Item 7) 1. A method of treating a transplant recipient patient comprising: (a) identifying said graft recipient patient by using the method according to any one of items 1 to 6; (b) treating said graft recipient patient by withdrawing immunosuppressive medication; The method comprising: (Item 8) 8. The method of any one of items 1 to 7, wherein the Tr1 cells exhibit indirect specificity for at least one mismatched donor MHC class I peptide. (Item 9) 9. The method of any one of items 1 to 8, wherein the Tr1 cells have a transcriptomic signature indicative of antigen-specific signaling. (Item 10) 10. The method of claim 9, wherein the transcriptomic signature indicative of antigen-specific signaling is SH2D2a. (Item 11) 11. The method of any one of items 1 to 10, wherein the Tr1 cells have a transcriptome signature indicative of an activation state. (Item 12) 12. The method of claim 11, wherein the transcriptomic signature indicative of activation status is the mitochondrial respiration-related transcript NDUFS4. (Item 13) The target cells express the marker CD49b + , LAG-3 + , CD4 + 13. The method of any one of items 1 to 12, wherein the regulatory T cells are type 1 (Tr1) having indirect specificity for at least one mismatched donor MHC class I peptide, have a transcriptomic signature indicative of antigen-specific signaling, and have a transcriptomic signature indicative of an activation state. (Item 14) Total CD4 in step (a) + CD49b in T cells+ LAG-3 + CD4 + 14. The method according to any one of items 1 to 13, wherein said frequency of (Tr1) cells is determined by multiparametric flow cytometry. (Item 15) Total CD4 in step (a) + CD49b in T cells + LAG-3 + CD4 + 15. The method according to any one of items 1 to 14, wherein said occurrence of (Tr1) cells is identified by CyTOF mass cytometry. (Item 16) 16. The method of any one of items 1 to 15, wherein at least a two-fold increase in said frequency of target cells is indicative of immune tolerance / acceptance induced by said peri-transplant infusion of donor apoptotic leukocytes. (Item 17) 16. The method of any one of items 1 to 15, wherein at least a three-fold increase in said incidence is indicative of immune tolerance / acceptance induced by said peri-transplant infusion of donor apoptotic leukocytes. (Item 18) 16. The method of any one of items 1 to 15, wherein at least a 5-fold increase in said incidence is indicative of immune tolerance / acceptance induced by said peri-transplant infusion of donor apoptotic leukocytes. (Item 19) 19. The method of any one of items 1 to 18, wherein the patient has received two intravenous infusions of donor apoptotic leukocytes around the time of transplantation. (Item 20) 20. The method of any one of items 1 to 19, wherein the transient immunotherapy comprises at least one immunosuppressant. (Item 21) 21. The method of item 20, wherein the immunosuppressant is an inhibitor of CD40:CD40L costimulation, an mTOR inhibitor, and a combination anti-inflammatory therapeutic agent targeting pro-inflammatory cytokines. (Item 22) 22. The method of claim 21, wherein the inhibitor of CD40:CD40L costimulation is an antagonist anti-CD40 antibody, an Fc-modified (disabled, silenced) anti-CD40L antibody, a Fab' anti-CD40L antibody, or a peptide that interferes with CD40:CD40L costimulation. (Item 23) 22. The method of claim 21, wherein the inhibitor of CD40:CD40L costimulation is the antagonist anti-CD40 mAb 2C10R4. (Item 24) 22. The method of claim 21, wherein the mTOR inhibitor is rapamycin. (Item 25) 25. The method of any one of items 1 to 24, wherein the transient immunotherapy comprises an anti-inflammatory agent. (Item 26) 26. The method of claim 25, wherein the anti-inflammatory agent is αIL-6R and / or sTNFR. (Item 27) 27. The method of claim 26, wherein the anti-inflammatory agent is αIL-6R, and the αIL-6R is tocilizumab. (Item 28) 27. The method of claim 26, wherein the anti-inflammatory agent is a sTNFR, and the sTNFR is etanercept. (Item 29) 29. The method of any one of items 1 to 28, wherein the graft is an allograft. (Item 30) 30. The method of claim 29, wherein the allograft is a solid organ allograft. (Item 31) 31. The method of claim 30, wherein the solid organ allograft is a pancreas, liver, intestine, heart, kidney, lung, or uterus transplant. (Item 32) 31. The method of item 30, wherein the solid organ allograft is a kidney graft. (Item 33) 31. The method of claim 30, wherein the kidney graft is a living donor kidney graft. (Item 34) 30. The method of claim 29, wherein the allograft is a tissue allograft. (Item 35) 35. The method of claim 34, wherein the tissue is adipose tissue, amniotic tissue, chorionic tissue, connective tissue, dura mater, facial tissue, intestinal tissue, glandular tissue, liver tissue, muscle tissue, nervous tissue, ocular tissue, pancreatic tissue, pericardium, skeletal tissue, skin tissue, urogenital tissue, or vascular tissue. (Item 36) 30. The method of claim 29, wherein the allograft is a cell graft. (Item 37) 37. The method of claim 36, wherein the allograft is a pancreatic islet, a hepatocyte, a myoblast, an embryonic stem cell derived differentiated cell graft, or an induced pluripotent stem cell derived differentiated cell graft, or a bone marrow graft. (Item 38) 38. The method of claim 37, wherein the embryonic stem cell derived differentiated cell graft is a pancreatic islet or pancreatic islet beta cell graft. (Item 39) 38. The method of claim 37, wherein the induced pluripotent stem cell derived differentiated cell graft is a pancreatic islet or pancreatic islet beta cell graft. [Brief description of the drawings]

[0014] [Figure 1A] Flow gating of Tr1 cells: (a) Tr1 cells (gating on CD49b+LAG-3+ versus CD4+CD45RA- CD3+ T cells), excluding doublets and dead cells. [Figure 1B] Flow gating of tetramer staining. Flow gating shows enumeration of tetramer+ total CD4 T cells, Tr1 cells, and Treg cells. [Figure 1C] Transcriptional profile of Tr1 cells. Left panel: Transcription levels of XBP1, SUMO2, and SH2D2 are presented as scatter plots in PBL obtained at termination. Right panel: Relative expression profiles of NDUFS4 and NDUFS5 in PBL obtained at termination in recipients of cohorts B and C are presented as scatter plots. [Diagram 2]Increased frequency of Tr1 cells in immune-tolerant animals. ADL infusion increases the frequency and enhances the function of immune cells with a regulatory phenotype. Relative numbers of circulating cells with a regulatory phenotype in monkeys from cohort B (n=7, circles) and cohort C (n=5, squares). Tr1 cells in PBL, LMNC, and LN at termination. [Diagram 3] Depletion of Tr1 cells in tolerant animals restores donor-specific proliferation. Depletion of Tr1, Treg, and Breg cells in PBL from cohort C (n=3) collected 12 months after transplantation restored donor-specific proliferation of CD4+, CD8+, and CD20+ cells in the CFSE-MLR. [Figure 4] Silencing SH2D2a by siRNA in Tr1 cells abolishes the suppression of donor-specific proliferation. RNA silencing of SH2D2 in Tr1 cells abolishes the suppressive capacity of Tr1 cells. Fold change in donor-specific proliferation in CD4+, CD8+, and CD20+ cells, respectively, without Tr1 cells, with Tr1 cells + vehicle, and with Tr1 cells treated with siRNA targeting the SH2D2 transcription molecule, compared to donor-treated recipient PBLs alone. [Diagram 5] Flow gating of tetramer staining. Increased frequency of tetramer+ donor-specific Tr1 cells in tolerant animals. Percentage of Treg cells (CD25+CD127-) within gated tetramer+CD4+lym in monkeys from cohorts B, C, and D. [Figure 6]ADL infusion added to transient immunosuppression promotes stable tolerance of islet allografts in monkeys. (A) Immunotherapy protocol including therapeutic product, dosage, route, and timeline in monkeys from cohorts B and C. sTNFR, soluble tumor necrosis factor receptor (etanercept); anti-IL-6R, anti-IL-6 receptor (tocilizumab); IE, islet equivalent. (B) Kaplan-Meier estimates of rejection-free islet allograft survival rates supported by histology show superior sustained allograft survival in cohort C (with ADL; n = 5; solid line) compared with cohort B (without ADL; n = 7; dashed line; P = 0.021, Mantel-Cox). [Figure 7] The absence of immune tolerance biomarkers correlates with early loss of transplanted graft function in recipients sensitized to donor antigens at baseline pre-transplant. The frequency of Tr1 cells in the peripheral circulation (before and after ADL+TIS) was analyzed by flow cytometry. Pre-transplant ADL infusion and TIS in donor antigen-sensitized recipients resulted in a significant decrease, rather than an increase, in the frequency of Tr1 cells 14 days after transplantation. At 28 days after transplantation, the frequency of circulating Tr1 cells reached the level observed in the naive state in the same recipients. In these recipients, pre-transplant sera showed evidence of baseline sensitization to donor antigens. Monitoring of Tr1 cells in these recipients strongly suggested that ADL infusion and TIS failed to induce immune tolerance to donor alloantigens in these recipients, even when we did not specifically monitor Tr1 cells with indirect specificity for mismatched donor MHC class I peptides and a highly defined transcriptome profile. [Figure 8]Loss of immune tolerance biomarkers precedes loss of transplanted graft function. The frequency of Tr1 cells in the peripheral circulation (before and after ADL+TIS) was analyzed by flow cytometry. ADL infusion and TIS led to a significant increase in the frequency of Tr1 cells early after transplantation. The frequency of circulating Tr1 cells began to decrease at 180 days after transplantation and reached the level observed in the naive state at 300 days. This indicates that loss of immune tolerance biomarkers precedes loss of graft function. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] Negative vaccination with donor apoptotic leukocytes (ADL) has become a promising non-chimeric strategy to induce donor antigen-specific immune tolerance in transplantation. Leukocytes treated ex vivo with the chemical crosslinker ethylcarbodiimide (ECDI) rapidly underwent apoptosis after intravenous injection. In a mouse allograft model, intravenous injection of ECDI-treated donor apoptotic splenocytes on days -7 and +1 (relative to day 0 transplantation) induced robust alloantigen-specific immune tolerance to minor antigen-mismatched skin grafts, to fully major histocompatibility complex (MHC)-mismatched pancreatic islet allografts, and, in the case of short-term rapamycin combination, to cardiac allografts. Most of the donor ECDI-treated splenocytes were rapidly internalized by splenic marginal zone antigen-presenting cells (APCs), and maturation of APCs following uptake of apoptotic bodies was arrested, resulting in selective upregulation of negative, but not positive, costimulatory molecules.

[0016] After encountering recipient APCs, T cells with indirect allospecificity rapidly expanded in number and subsequently underwent severe clonal contraction. The remaining T cells were sequestered in the spleen and did not traffic to the allograft or allograft-draining lymph nodes. The remaining donor ECDI-treated splenocytes that were not internalized by host phagocytes weakly activated T cells with direct allospecificity, rendering them resistant to subsequent stimulation (anergy). ECDI-treated splenocytes also activated and expanded the numbers of regulatory T (Treg) cells and myeloid-derived suppressor cells (MDSCs). Thus, in a mouse allotransplantation model, the mechanism of graft protection induced by alloantigen delivery via ECDI-treated splenocytes involved clonal anergy of anti-donor CD4+ T cells with direct specificity, clonal depletion of anti-donor CD4+ T cells with indirect specificity, and regulation by CD4+ Treg cells and MDSCs.

[0017] Intravenous delivery of antigens cross-linked with ECDI to the surface of syngeneic leukocytes restored antigen-specific immune tolerance in mass models of autoimmunity and allergy. Importantly, this strategy prevented both priming of naive T cells and effective regulation of pre-existing memory / effector CD4+ and CD8+ T cell responses. Clinical trials involving multiple sclerosis patients confirmed the safety of coupling of autologous leukocytes with ECDI followed by intravenous delivery of encephalitogenic peptides and provided preliminary evidence of efficacy.

[0018] In this study, we significantly expand on findings with ECDI-treated donor splenocytes in mouse allografts, demonstrating stable immune tolerance to islet allografts in rhesus macaques (referred to as monkeys) administered two ADL infusions under transient immunosuppression. We found that sustained immune tolerance in our model was associated with depletion of donor-specific T and B cell clones. This was most pronounced in recipients of ADL and allografts matched for one MHC class II (MHC-II) allele, a potent and persistent regulation. Multiple immune cell subsets, including antigen-specific Tr1 cells, were involved in immune regulation, suppressing the post-transplant expansion of donor-reactive T cells and their recruitment to the allograft.

[0019] ADL-induced transplant tolerance involves regulatory immune cell subsets, including Tr1 cells, with distinct specificities and transcriptomic signatures, identifying biomarkers for monitoring the induction, maintenance, and loss of regulatory tolerance induced by intravenously infused ADLs on transient immunosuppression. Circulating CD4+ cells exhibiting indirect specificity for at least one mismatched donor MHC class I peptide as well as a transcriptomic signature indicative of antigen-specific signaling (e.g., SH2D2a) and a transcriptomic signature indicative of mitochondrial respiration associated with an activation state (e.g., NDUFS4) between baseline and post-procedure blood samples. + CD49b among T cells (Tr1 cells) + LAG-3 + A greater than two-fold increase in the incidence of is indicative of transplant tolerance.

[0020] In certain embodiments, the present invention provides a method for identifying a graft recipient patient having transplantation tolerance induced by peri-transplant infusion of donor apoptotic leukocytes in conjunction with transient immunotherapy, the method comprising: (a) assaying a first blood sample from the patient to detect a baseline frequency of target cells, the first blood sample being obtained pre-tolerization, pre-transplant, and prior to initiation of transient immunotherapy; (b) assaying a second blood sample from the patient to detect a post-procedure frequency of target cells, the second blood sample being obtained post-tolerization, post-transplant, and after initiation of transient immunotherapy; and (c) identifying the patient as having transplantation tolerance / acceptance induced by donor apoptotic leukocytes if the post-procedure frequency is at least two-fold higher than the baseline frequency, the target cells being CD49b + , LAG-3 + , CD4 + Regulatory T cells type 1 (Tr1) are defined as cells that, in certain embodiments, have indirect specificity for at least one mismatched donor MHC class I peptide (as verified using recipient-specific MHC class II tetramers added with the MHC class I peptide), have a transcriptomic signature indicative of antigen-specific signaling, and / or have a transcriptomic signature indicative of an activation state.

[0021] In certain embodiments, the invention provides a method comprising: (a) obtaining a first blood sample from a patient to detect a baseline incidence of target cells, where the first blood sample is obtained pre-tolerization, pre-transplant, and prior to initiation of transient immunotherapy; (b) obtaining a second blood sample from the patient to detect a post-procedure incidence of target cells, where the second sample is obtained post-tolerization, post-transplant, and after initiation of transient immunotherapy; (c) assaying the first blood sample and the second blood sample to detect target cell levels pre- and post-tolerization; and (d) identifying the patient as having transplant tolerance / acceptance induced by donor apoptotic leukocytes if the post-procedure incidence is at least two-fold higher than the baseline incidence, wherein the target cells are CD49b + , LAG-3 + , CD4 + Regulatory T cells type 1 (Tr1) are defined as cells that have indirect specificity for at least one mismatched donor MHC class I peptide, have a transcriptomic signature indicative of antigen-specific signaling, and / or have a transcriptomic signature indicative of an activation state.

[0022] In certain embodiments, the present invention provides a method of treating a transplant recipient, the method comprising: (a) identifying a transplant recipient patient as described herein; and (b) treating the transplant recipient patient by discontinuing administration of immunosuppressive agents.

[0023] It is important to determine whether the graft recipient patient has acquired and maintained immune tolerance to the graft. In certain embodiments, the graft that the patient receives will be an allograft. As used herein, the term "allograft" is defined as a cell, tissue, or organ that is transplanted from a donor of the same species but not genetically identical (i.e., separate) to a recipient. The graft may be referred to as an allograft, an allogeneic graft, or a homograft. In certain embodiments, the allograft is a solid organ allograft, such as a kidney, pancreas, liver, intestine, heart, lung, or uterus transplant. In certain embodiments, the allograft is a tissue allograft, including, but not limited to, adipose tissue, amniotic tissue, chorionic tissue, connective tissue, dura mater, facial tissue, intestinal tissue, glandular tissue, liver tissue, muscle tissue, nervous tissue, ocular tissue, pancreatic tissue, pericardium, skeletal tissue, skin tissue, urogenital tissue, and vascular tissue. In certain embodiments, the allograft is a cellular allograft, such as a pancreatic islet, hepatocyte, myoblast, embryonic stem cell derived differentiated cell graft (e.g., pancreatic islet or islet beta cell or hepatocyte transplant), or an induced pluripotent stem cell derived differentiated cell graft (e.g., pancreatic islet or islet beta cell transplant), a hematopoietic stem cell graft, or a bone marrow graft.

[0024] As used herein, "immune acceptance," "immune tolerance," "immunological tolerance," or "immunotolerance" refers to a state of unresponsiveness of the immune system in a given organism to substances or tissues capable of eliciting an immune response. The term "transplant tolerance" is a form of immune tolerance. "Transplant tolerance" is long-term allograft survival in the absence of maintenance immunosuppressive therapy. Implicit in this definition is that a tolerant recipient of an organ transplant is unresponsive to donor antigens but maintains responsiveness to other (third-party) antigens. Organ transplant recipients who have been successfully weaned off immunosuppression and have maintained stable graft function for more than one year are said to be functionally or operationally tolerant.

[0025] immunotherapy In certain circumstances, the graft recipient patient will receive immunotherapy prior to, concurrently with, or following the graft in order to induce transplant tolerance, in this case the immunotherapy being the administration of donor apoptotic leukocytes (ADL).

[0026] In certain embodiments, the patient undergoes immunotherapy prior to, concurrently with, or following the transplant. In certain embodiments, the donor apoptotic leukocytes can be administered with or in addition to one or more immunomodulatory molecules and a transient anti-inflammatory therapeutic agent, the immunomodulatory molecule being, for example, an antagonist anti-CD40 mAb antibodies, Fc-modified anti-CD40L antibodies, peptides interfering with CD40:CD40L costimulation, mTOR inhibitors (e.g., sirolimus, everolimus), as well as transient anti-inflammatory therapeutics such as compstatin (e.g., compstatin derivative APL-2), cytokine antagonists (e.g., anti-IL-6 receptor mAb (tocilizumab), anti-IL-6 antibodies (sarilumab, olokizumab), soluble TNF receptors (etanercept), anti-TNF-alpha antibodies (e.g., infliximab (Remicade), adalimumab (Humira)), α1-antitrypsin, nuclear factor-κB inhibitors (e.g., dehydroxymethylepoxyquinomycin (DHMEQ)), ATG (antithymocyte globulin) and other polyclonal T cell depleting antibodies, alemtuzumab (Campus), anti-IL-2R Abs (basiliximab), B cell targeting strategies (e.g., B cell depleting biologics, e.g., biologics targeting CD20, CD19, or CD22, and / or B cell modulating biologics, e.g., biologics targeting BLyS, BAFF, BAFF / APRIL, CD40, IgG4, ICOS, IL-21, B7RP1), mycophenolate mofetil, mycophenolic acid, downregulators that downregulate sphingosine-1 phosphate receptors (e.g., FTY720), JAK inhibitors (e.g., Tofa citinib), immunoglobulins (e.g., IVIg), CTLA4-Ig (abatacept / Orencia), belatacept (LEA29Y, Nulojix), tacrolimus (Prograf), cyclosporine A, leflunomide, anti-CXCR3 antibodies, anti-ICOS antibodies, anti-OX40 antibodies, and anti-CD122 antibodies, deoxyspergualin, soluble complement receptor 1, cobra venom factor, complement inhibitors (e.g., C1 inhibitor, compstatin), anti-C5 antibodies (eculizumab / Soliris), methylprednisolone, and azathioprine.Examples, without limitation, of biologics that target B cells include rituximab and anti-CD20 antibodies.

[0027] In certain embodiments, the transient immunotherapy comprises at least one immunosuppressant. In certain embodiments, the immunosuppressant is an inhibitor of CD40:CD40L costimulation, an mTOR inhibitor, and a combination anti-inflammatory therapeutic targeting a pro-inflammatory cytokine. In certain embodiments, the inhibitor of CD40:CD40L costimulation is an antagonist anti-CD40 antibody, an Fc-modified (disabled, silent) or Fab' anti-CD40L antibody, or a peptide that interferes with CD40:CD40L costimulation. In certain embodiments, the inhibitor of CD40:CD40L costimulation is the antagonist anti-CD40 mAb 2C10R4. In certain embodiments, the at least one immunosuppressant is rapamycin. In certain embodiments, the transient immunotherapy comprises an anti-inflammatory agent. In certain embodiments, the anti-inflammatory agent is anti-IL-6R (tocilizumab) and / or sTNFR (etanercept).

[0028] In certain embodiments, to prevent activation of the immune system and induction of anti-donor immunity by injecting donor apoptotic leukocytes on days -7 and +1 relative to transplantation on day 0, the recipient was transiently immunosuppressed with drugs that directly target, among other cells, antigen presenting cells and their resulting donor-reactive T cell activation, other CD40 expressing cells, or T and B cells. Methods for preparation and administration of donor apoptotic leukocytes are known in the art. Luo X, Pothoven KL, McCarthy D, DeGutes M, Martin A, Getts DR, Xia G, He J, Zhang X, Kaufman DB, Miller SD, ECDI-fixed allogeneic splenocytes induce donor-specific tolerance for long-term survival of islet transplants via two distinct mechanisms. Proc Natl Acad Sci US A. 2008 Sep 23;105(38): 14527-32; Miller et al. U.S. Patent No. 8,734,786. The first dose of each immunosuppressant was administered on days -8 or -7, based on day 0 of transplantation. Antagonist anti-CD40 mAb 2C10R4 was administered at 50 mg kg on days -8, -1, 7, and 14. -1 Rapamycin (Rapamune®) was administered PO from day −7 until day 21 after transplantation. Target trough levels were 5–12 ng mL -1 Concomitant anti-inflammatory treatment consisted of i) αIL-6R (tocilizumab, Actemra®) at 10 mg kg on days −7, 0, 7, 14, and 21; -1 IV at 1 mg kg on days −7 and 0, and ii) sTNFR (etanercept, Enbrel®) at 1 mg kg on days −7 and 0. -1 IV and 0.5 mg kg on days 3, 7, 10, 14, and 21. -1 The study consisted of SC administration at 100 mg / kg / day.

[0029] In certain embodiments, the first dose of immunosuppressant is administered to the patient 7 to 14 days (e.g., days -1, -2, -3, -4, -5, -6, -7, -8, -9, -10, -11, -12, -13, -14) prior to transplant. In certain embodiments, a second dose of immunosuppressant is administered to the graft recipient patient several days after transplant (e.g., days +1, +2, +3, +4, +5, +6, +7, +8, +9, +10, +11, +12, +13, +14, +15, +16, +17, +18, +19+, or +20). In certain embodiments, multiple doses of immunosuppressant are administered to the graft recipient over the span of a treatment period ranging from several days to several months (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 doses).

[0030] Therapeutics can be administered via a selected route of administration. Therapeutics can be administered intravenously, intraperitoneally, or intramuscularly by infusion or injection.

[0031] target cell In certain embodiments of the invention, a first (baseline) biological sample, e.g., a blood sample, is taken from the patient prior to immunotherapy and transplantation ("pre-tolerization"). In certain embodiments, on day -7, the patient receives an infusion of donor apoptotic cells, on day 0, the graft recipient patient receives the transplant, and on day +1, the graft recipient patient receives a second infusion of donor apoptotic cells. A second biological sample is taken after transplantation ("post-transplant"), and a third sample is taken after the second infusion of donor apoptotic cells. In certain embodiments, a fourth biological sample is taken after the first infusion of cells and transplantation. From these samples, highly specific target cells are isolated, i.e., CD49b + , LAG-3 + , CD4 +Regulatory T cells type 1 (Tr1) are defined as cells that have indirect specificity for at least one mismatched donor MHC class I peptide, have a transcriptomic signature indicative of antigen-specific signaling, and / or have a transcriptomic signature indicative of an activation state. Tolerogenic Tr1 cells express CD4+, which are believed to be important mediators of immune tolerance / acceptance induced by peri-transplant infusion of donor apoptotic leukocytes. + It is a T cell subset.

[0032] Antigen-specific CD4 defined by major histocompatibility complex (MHC) class II tetramer staining + This allows for the characterization, quantification, and selection of T cells. The MHC tetramer binds antigen-specific CD4 + It is an essential tool for characterizing T cells. Relatively rare antigen-specific CD4 +Protocols for ex vivo tetramer staining of T cells have provided an important tool for the analysis of helper T cells in basic and clinical immunology. (Uchtenhagen, H. et al. Efficient ex vivo analysis of CD4+ T-cell responses using combinatorial HLA class II tetramer staining. Nat. Commun. 7, 12614 (2016); Day, CL et al. Ex vivo analysis of human memory CD4 T cells specific for hepatitis C virus using MHC class II tetramers. J. Clin. Invest. 112, 831-842 (2003); Kwok, WW et al. Direct ex vivo analysis of allergen-specific CD4t T cells. J. Allergy Clin. Immunol. 125, 1407-1409.e1401 (2010); Moon, JJ et al. Naive CD4+ T cell frequency varies for different epitopes and predicts repertoire diversity and response magnitude. Immunity 27, 203-213 (2007)). MHC class II tetramer staining has therefore become a valuable approach in immunology, allowing direct interrogation of naturally occurring T cell repertoires, assessment of changes in T cell responses caused by perturbations such as vaccination and disease, as well as providing a means to confirm the translational relevance of findings in model systems.

[0033] Using flow cytometry and gating, CD49b + and LAG-3 + CD4 + Identifying T cells is known in the art. + LAG-3 +CD4 + The T cells are referred to as Tr1 cells. In certain embodiments, the prevalence of Tr1 cells in a sample is identified by multiparametric flow cytometry. A subset of Tr1 cells with indirect specificity is identified using MHC class II tetramers added with mismatched donor MHC class I peptides. In certain embodiments, this powerful tetramer technology tracks these rare donor peptide-specific cell subsets. In certain embodiments, the prevalence of Tr1 cells is identified by CyTOF mass cytometry.

[0034] Then, specific target cells, i.e., CD49b + and LAG-3 + CD4 + The T cells were analyzed to determine whether they have indirect specificity for at least one mismatched donor MHC class I peptide. The determination of the presence of at least one mismatched donor MHC class I peptide is effected using multiparametric flow cytometry. In a particular embodiment, the mismatched donor MHC class I peptide is APVALRNLRGYYNQS, a 14-mer peptide in the variable region of the MHC class I molecule (28-114 amino acids). A t-BLAST analysis was performed on the NCBI website with the Mamu DRB sequence and the human genome to identify the human homolog. HLA DRB1 * 13 (accession no. CDP32905.1) was 92% identical, 96% positive and 0% gapped to Mamu DRB03a with an e-value of 6e-178. * 14 (accession number ABN54683.1) was 93% identical, 94% positive and 0% gaps to Mamu DRB03a with an e-value of 2e-175.

[0035] Using the Immune Epitope Database Analysis information resource, HLA DRB1 was identified from Mamu MHC class I and class II sequences. *13 or HLA DRB1 * Peptides with high binding affinity to 14 were identified (Table 1). [Table 1]

[0036] Specific target cells were also analyzed to identify the transcriptome signature indicative of antigen-specific signaling. As used herein, the "transcriptome signature" of a cell is the expression level of an RNA in a cell population. Briefly, RNA from the sorted target cells is analyzed by quantitative real-time PCR using a set of primers and probes selected and defined by an unbiased RNAseq analysis previously performed on cells from a reported stable tolerant graft recipient. In certain embodiments of the present invention, quantitative real-time PCR was performed on RNA obtained from flow-sorted Tr1 cells. In certain embodiments, the transcriptome signature indicative of antigen-specific signaling is SH2 domain-containing 2A (SH2D2a). [Table 2-1] [Table 2-2] [Table 2-3]

[0037] In certain embodiments, the transcriptomic signature is indicative of an activation state. In certain embodiments, the transcriptomic signature is indicative of an activation state is the mitochondrial respiration-related transcript NADH:ubiquinone oxidoreductase subunit S4 (NDUFS4).

[0038] graft In certain embodiments, the graft is an allograft. In certain embodiments, the allograft is a solid organ allograft. In certain embodiments, the allograft is a solid organ allograft, such as a kidney, pancreas, liver, intestine, heart, lung, or uterus transplant. In certain embodiments, the solid organ allograft is a kidney transplant. In certain embodiments, the allograft is a tissue allograft, including, but not limited to, adipose tissue, amniotic tissue, chorionic tissue, connective tissue, dura mater, facial tissue, intestinal tissue, glandular tissue, liver tissue, muscle tissue, nervous tissue, ocular tissue, pancreatic tissue, pericardium, skeletal tissue, skin tissue, urogenital tissue, and vascular tissue. In certain embodiments, the allograft is a cellular allograft, such as a pancreatic islet, hepatocyte, myoblast, embryonic stem cell derived differentiated cell graft (e.g., pancreatic islet or islet beta cell or hepatocyte transplant), or an induced pluripotent stem cell derived differentiated cell graft (e.g., pancreatic islet or islet beta cell transplant), a hematopoietic stem cell graft, or a bone marrow graft.

[0039] In certain embodiments, the graft is a living donor graft, hi certain embodiments, the allograft is a cell graft.

[0040] Assay Method In certain embodiments, the present invention comprises the steps of: (a) assaying a first blood sample from the patient to detect a baseline incidence of target cells, where the first blood sample is obtained before immune tolerization, before transplantation, and before initiation of transient immunotherapy; (b) assaying a second blood sample from the patient to detect a post-procedure incidence of target cells, where the second blood sample is obtained after immune tolerization, after transplantation, and after initiation of transient immunotherapy; and (c) identifying the patient as having transplant tolerance / acceptance induced by donor apoptotic leukocytes if the post-procedure incidence is at least two-fold higher than the baseline incidence, where in the present invention the target cells are CD49b + , LAG-3 + , CD4 +Regulatory T cells type 1 (Tr1) are defined as cells that have indirect specificity for at least one mismatched donor MHC class I peptide, have a transcriptomic signature indicative of antigen-specific signaling, and / or have a transcriptomic signature indicative of an activation state.

[0041] In certain embodiments, the incidence of target cells in a first (baseline) sample is compared to the incidence of target cells in a second (post-procedure) sample and subsequent samples. In certain embodiments, a determination that the incidence is increased by at least 2-fold is indicative of immune tolerance / acceptance induced by peri-transplant infusion of donor apoptotic leukocytes. In certain embodiments, a determination that the incidence is increased by at least 3-fold is indicative of immune tolerance / acceptance induced by peri-transplant infusion of donor apoptotic leukocytes. In certain embodiments, the incidence is increased by at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, at least 100-fold, or more, between the first and second samples.

[0042] In certain embodiments, the frequency of target cells is determined by multi-parametric flow cytometry. In certain embodiments, the frequency of target cells is determined by CyTOF mass cytometry.

[0043] In certain embodiments, the graft recipient patient receives two infusions of intravenous donor apoptotic leukocytes around the time of transplant.

[0044] In certain embodiments, peripheral blood mononuclear cells from graft recipient patients are stained with a limited cocktail of fluorescently conjugated antibodies and markers (anti-CD4, anti-CD49b, anti-LAG3, MHC class II tetramers added with mismatched donor MHC class I peptide) to facilitate sorting of labeled cells using microfluidic technology, and the labeled cells are characterized by subsequent quantification of immune tolerance-associated transcripts using quantitative real-time PCR.

[0045] CD4+ / -expressing transcripts indicative of antigen-specific TCR signaling (SH2D2a) and metabolically active state (NDUFS4) with indirect specificity for mismatched donor peptides, as defined above + CD49b + LAG3 + The percentage of T cells (Tr1 cells) is extremely low at baseline. A more than 2-3 fold increase in the percentage of this circulating T cell subset can only be explained by the presence of an antigen-specific immune tolerance state.

[0046] The invention will now be illustrated by the following non-limiting examples. EXAMPLES

[0047] Example 1 Transplant tolerance has been pursued as a clinically relevant goal for decades. Here, we demonstrate that a regimen of two peri-transplant ADL infusions under short-term immunotherapy safely induced long-term (≥1 year) tolerance to islet allografts in 5 of 5 unsensitized monkeys matched for one MHC-II DRB allele. These findings, obtained in a rigorous preclinical allograft NHP model, are unique and point for the first time to a clinically applicable route toward nonchimeric transplant tolerance in humans.

[0048] Previous NHP studies reported immune tolerance in kidney allografts, but not in heart or islet allografts, when donor bone marrow was administered under nonmyeloablative conditioning including CD154 blockade. Of eight monkeys so treated in one of these studies, six maintained kidney allograft function for one year in the absence of maintenance immunosuppression. Moreover, three of these maintained long-term function without developing chronic rejection. Another study used a nonchimeric strategy to achieve kidney allograft tolerance, but only inconsistently, in three of five monkeys that received donor-specific transfusions in conjunction with anti-CD40L for eight weeks and rapamycin for 90 days. Similar NHP strategies, as well as other previously studied strategies, extended islet allograft survival after cessation of maintenance immunosuppression or rapamycin monotherapy, but unlike our study, none of those protocols induced sustained immune tolerance. In contrast to other cell-mediated tolerance strategies currently being investigated, our regimen did not require adoptive transfer of regulatory cells. Instead, we found that peri-transplant ADL infusion under short-term immunosuppression established strong and sustained immune regulation in vivo involving multiple regulatory cell types. With regard to safety, unlike mixed chimerism strategies, our regimen effectively induced stable immune tolerance without the need for irradiation, indiscriminate universal T cell depletion, hematopoietic stem cell co-transplantation, or a process using either calcineurin inhibitors or anti-CD8 depleting antibodies to control early post-transplant direct pathway activation. Finally, unlike other antigen-specific strategies involving soluble peptide and modified peptide ligand therapy, our ECDI fixed leukocyte infusion was not associated with the risk of anaphylaxis or any other safety concerns in our preclinical studies or in multiple sclerosis clinical trials.

[0049] Multiple distinct immune mechanisms were associated with immune tolerance to one DRB-matched ADL infusion and islet allografts under our transient immunosuppression. The regimen depleted alloreactive effector T and B cells early after two peri-transplant ADL infusions, as supported in cohort A by our observations tracking Ki67+ proliferating cells, alloreactive proliferation in MLRs, proliferating TCRβ clones, and CD4+ T cells with indirect specificity for mismatched MHC class I allopeptides in our tetramer assays. A previous mouse study showed that uptake of apoptotic bodies by APCs after ADL infusion substantially increased PD-L1 / 2 expression while downregulating positive co-stimulation.12 APCs exhibiting such a pattern rapidly (but transiently) activated T cells producing IFN-γ and IL-10, but not IL-2, IL-6, and TNF-α. This is a cytokine microenvironment known to promote apoptotic exhaustion of antigen-specific T cells. Rapamycin, part of our combination immunotherapy, enhances activation-induced cell death triggered by donor antigens under CD40:CD40L blockade.

[0050] In the present study, in vitro, post-transplant expansion of circulating CD4+ and CD8+ TEM cells, their recruitment to the graft, and expansion of donor-reactive CD4+ and CD8+ T cells, as well as suppression of these, were recorded in cohort C (but not in cohorts B and D). These findings suggest that our ADL infusion and one DRB match played an important role in the induction and maintenance of immune tolerance. The restoration of T cell proliferation to the donor after depletion of regulatory subsets that we observed in vitro suggests that donor-specific T cell clones were neither deleted nor anergized, but rather regulated to control their post-transplant expansion and effector functions.

[0051] To further support this interpretation, we showed that adding ADL infusion to short-term immunosuppression in cohort C established a regulatory network characterized by a significant and sustained increase in circulating MDSCs as well as Tr1, Treg, NS, Breg, and B10 cells. At the end, Tr1 cells were significantly more prevalent in the liver and lymph nodes of recipients with allografts in cohort C (compared to cohort B). The detailed mechanism underlying the formation of this regulatory network remains to be elucidated. Even so, it is likely that infusion of our one DRB-matched ADL provided a large amount of shared MHC-II peptides for presentation by MHC-II molecules in host splenic marginal zone APCs and in host liver sinusoidal endothelial cells. It is known that after trogocytosis into activated T cells, such peptide-MHC-II complexes may deliver a strong activation signal to thymus-derived Treg (tTreg) cells with TCR repertoires skewed to self-recognize. Treg cells are known to promote the generation of IL-10-producing Tr1 cells,46 but it remains to be seen whether the expansion of Tr1 cells in our study was due to the influence of activated tTregs, leading to de-novo formation and / or conversion of donor-reactive effector T cells. In autoimmune models, Tr1-like cells generated by nanoparticles coated with autoimmune disease-associated peptides bound to self-MHC-II are known to contribute to the formation of regulatory networks by driving the differentiation of cognate B cells into disease-suppressing regulatory B cells. Consistent with the notion that matched MHC-II peptides promoted regulatory networks in our study, the frequency of circulating Treg, Tr1, and Breg cells in one DRB-matched ADL Cohort C recipients was significantly higher than that in fully mismatched Cohort D recipients.

[0052] Among the regulatory subsets, Tr1 cells were found to exhibit the most potent suppression of donor-specific proliferation of T and B cells, which was partially mediated by IL-10. In contrast, third-party responses were not affected by sorted Tr1 cells, indicating their antigen specificity. Our tetramer studies revealed that circulating Treg and Tr1 cells with indirect specificity for mismatched donor MHC-I peptides were persistently increased after transplantation in cohort C (but not in cohorts B and D). This finding demonstrated their antigen specificity and was consistent with previous studies in mouse and human allograft recipients, which showed regulation induced by mismatched MHC-I peptide presentation by shared self-MHC-II molecules after transfusions matched for one MHC-II allele. ADL increased regulatory subsets in fully mismatched mouse allograft recipients6. The effect of MHC-II matching in these models remains to be studied. In cohort C recipients, Tr1 cells exhibited unparalleled immune cell signaling, including a marked increase in SH2D2 levels. The gene product of Sh2D2a, T cell-specific adaptor protein (TSAd), regulates TCR signaling through its interaction with Lck51; however, its absence promotes systemic autoimmunity. In cohort C, Tr1 cell transcriptome profiles also demonstrated increased mitochondrial respiratory activity and energy utilization in Tr1 cells, revealing their activated state.

[0053] In cohort B (no ADL infusion), two of seven recipients maintained allograft survival without immunosuppression for 1 year after transplantation, and all seven avoided acute rejection. This confirmed that favorable allograft survival can be achieved when early direct pathway activation is suppressed with strong induction in MHC-II-matched recipients. However, this regimen failed to control indirect pathway activation, as evidenced by the occurrence of de-novo DSA in most cohort B recipients. The tolerogenic efficacy of infusion of one DRB-matched ADL under transient immunosuppression was limited to primed cohort E recipients, especially those with preformed DSA, in whom memory T cells, including those that do not secrete IFN-γ, are essential for DSA responses, and in these recipients, APCs activated by uptake of DSA-opsonized ADL likely stimulated donor-reactive T cells instead of tolerizing them. Figures 7 and 8 present data from islet graft recipients who were part of a cohort that was sensitized to donor antigens at baseline (Figure 7) and from recipient monkeys that were fully mismatched with the donor in MHC class I and class II (including DRB alleles) (Figure 8).

[0054] method Test animals The cohorts included Indian-origin, purpose-bred macaque (Macaca mulatta) donors and recipients, which were obtained from the National Institute of Health Infectious Disease Colony Laboratory, AlphaGenesis, Inc, Yemassee, SC. ​​The exploratory cohort included three males, age 7.3 ± 0.1 and weight 12.5 ± 1.5 kg. The control cohort included eight males, age 4.3 ± 2.1 and weight 6.2 ± 1.6 kg. The experimental cohort included seven males and one female, age 4.1 ± 1.7 and weight 5.2 ± 1.2 kg. The donor cohort included 19 males, age 6.7 ± 3.3 and weight 11.7 ± 3.6 kg. The animals were free of herpesvirus-1 (B virus), simian immunodeficiency virus (SIV), type D simian retrovirus (SRV), and simian T-lymphotropic virus (STLV-1). Eligibility additionally included ABO compatibility and study-defined MHC matching (donor-recipient pairs matched for MHC-I-alien and one MHC-II DRB allele). All animals underwent high-resolution MHC-I and -II genotyping by 454 pyrosequencing (Wisconsin National Primate Research Center Genetics Services Division). Animals had free access to water and were fed biscuits (Harlan Primate Diet 2055C, Harlan Teklad, Madison, WI) based on body weight (BW). Animal diets were enriched daily with fresh fruits, vegetables, grains, legumes, nuts, and a multivitamin preparation. Semiannual veterinary physical examinations were performed on all animals. The animals were socially housed and participated in an environmental enrichment program designed to encourage sensory engagement, enhance feeding behavior, promote novelty exploration, mental stimulation, increase exploration, play, and activity levels, and reinforce social behaviors, while providing the animals with the opportunity to increase the allocation of time spent on species-specific behaviors. The monkeys were trained to cooperate with medical procedures, which included hand-feeding and drinking, transfer into transport cages, and positioning for examinations, medication, metabolic testing, and blood sampling. The animals were also instrumented with indwelling central and intraportal vascular access devices.Diabetes was induced with STZ (100 mg / kg IV) and confirmed by basal C-peptide <0.3 ng / mL and a negative C-peptide response to an intravenous glucose load.61 Monitoring of recipient monkeys included daily clinical evaluations by research staff, regular examinations by veterinary staff, and weekly hematology and chemistry laboratory testing. All animal care and procedures in this study were approved by the University of Minnesota Animal Care and Use Committee and conformed to the recommendations in the Guide for the Care and Use of Laboratory Animals (Office of Laboratory Animal Resources, National Council of Science, U.S. Department of Health and Human Services).

[0055] Flow cytometric analysis of immune cell phenotypes Multicolor flow cytometry analysis was performed on cryopreserved peripheral blood mononuclear cell (PBL) samples, liver-derived tissue-infiltrating mononuclear cell (LMNC) samples, and lymph node (LN) samples from monkeys in cohorts B–E. 6 Cells were stained with viability stains (Aqua; Life Technologies) to distinguish live cells from cell debris. Cells were stained with antibodies, fluorescence minus one (FMO), and / or isotype controls for 25 min at room temperature, followed by fixation (eBiosciences) and washing. To assess regulatory T cells, as well as proliferating T and B cells, and intracellular cytokines, PBLs were stained with antibodies recognizing extracellular epitopes (CD3, CD4, CD8, CD25, and CD127), followed by fixation / permeabilization with FoxP3 fixation / permeabilization kit (eBioscience) and staining with anti-FoxP3, Ki67, IFN-γ, IL-10, and TGF-β antibodies. A minimum of 200,000 events were acquired using FACSDIVA 6.1.3 on a 3-laser BD Canto II (BD Bioscience). The relative percentages of each of these subpopulations were determined using FlowJo10.1 software (TreeStar).

[0056] Gating Method Cells were first gated on FSC-H vs. FSC-A, then on SSC-H vs. SSC-A to identify doublets (Figures 1A-1C, Figure 2). Lymphocytes were then gated based on well-characterized SSC-A and FSC-A signatures. Dead cells were excluded based on viability staining. The following phenotypic characteristics were used to define immune cell populations: T cells: CD3 + Lymphocytes; CD4 + T cells: CD4 + / CD3 + / CD8 - ;CD8 + T cells: CD8 + / CD3 + / CD4 - ;CD4 or CD8 T cells express CD2 hi / CD28 - The expression of PD-1, Tbet, CD40, and Ki67 was significantly higher in CD4 + , CD8 + Both T cells and CD20 + Identified in B cells. Chemokine receptor (CXCR-5) expression was examined in CD4+ T cells, and Tfh cells: CXCR5 + CD4 + T cells were enumerated. Regulatory T cells were Tr1 cells: gated CD4vCD45RA - CD49b in lymphocytes + LAG-3 + What are Treg cells: gated CD4 + CD25 + CD127-FoxP3 in lymphocytes + Natural suppressor (NS) cells: CD8 lymphocytes among gated CD8 lymphocytes + CD122 + and for Breg cells: regulatory B cells (CD24 hi CD38 hi ), B10 cells: CD3 - CD19 + / CD20 + Based on the expression of CD24, CD27, and CD38 antigens in lymphocytes (CD24 hi CD27+ ) is defined as the gated Lin - (CD3 - CD20 - )HLA-DR - CD14 + Analyze the cells to identify myeloid-derived suppressor cells (MDSC): CD14 + Lin - HLA-DR - CD11b cells hi CD33 hi I have enumerated the things that are.

[0057] Donor-specific T and B cell responses Mixed lymphocyte reactions (MLR) were performed with frozen stored PBL samples from islet donors and graft recipients. Responder PBL (300,000 cells) samples from recipient monkeys were labeled with 2.5 μM CFSE (Invitrogen, Cat. No. C34554) and cocultured with stimulator PBL (300,000 cells) from islet donors and unrelated MHC-mismatched donors (third parties) that were VPD-450 labeled (BD, Cat. No. 562158) and irradiated (3000 cGy). In another set of experiments, CFSE-labeled PBL from naive responder monkeys were cocultured (300,000 cells) with ECDI-fixed PBL (ADL) from islet donors. Donor apoptotic leukocytes (ADL) were prepared. On day 6 of the MLR, CD4 + , CD8 + , and CD20 + Cells were counted and CFSE low cells were defined as proliferating cells and expressed as a percentage of this.

[0058] Evaluation of the polyfunctional cytokine profile of donor-stimulated Tr1 cells. T cells from peripheral blood of monkeys in cohort B (n=3), cohort C (n=3), and cohort E (n=2) were harvested at termination. Briefly, PBL (1×10 6Cells) were cultured in the presence of donor PBLs (VPD-450 labeled) at a 1:1 ratio for 48 hours. These donor stimulated cells were briefly activated with a small amount of PMA / ionomycin in the presence of Brefeldin-A (10ug / ml) for 4 hours. Cells were surface stained for CD4, CD49b, and LAG-3, followed by permeabilization and intracellular staining for IL-10 and TGF-β. Gating methods to identify Tr1 cells were performed as described above.

[0059] ELISPOT. For IFN-γ ELISPOT assays, longitudinally collected PBLs from cohort B and cohort C monkeys were thawed, washed, and preincubated with donor PBLs in 12-well culture plates at 37° C. and 5% CO2 in a final volume of 1 ml CRPMI medium. After 48 h, cells were harvested, washed twice with PBS, and resuspended in 200 μl culture medium. Cells were transferred to two ELISPOT wells coated with anti-IFN-γ antibodies and incubated at 37° C. for 5 h in a final volume of 100 μl per well. ELISPOT assays (U-Cytech Biosciences) were subsequently performed according to the manufacturer's protocol. Spot analysis was performed with an Immune Spot ELISPOT reader (CTL).

[0060] Sensitization screening (donor-specific antibodies, DSA). Serum was collected from recipient RMs at various time points and the presence of DSA was detected by flow cytometry. Briefly, stored donor PBLs were thawed, washed with complete RPMI, and then transferred to FACS buffer (PBS containing 2% FBS) at 4 × 10 6The cells were resuspended at 100 μl / ml. 50 μl of the prepared cell suspension was seeded into each well of a U-shaped 96-well plate together with 50 μl of completely inactivated (56° C. for 45 min) recipient serum, followed by incubation at room temperature for 30 min and washing three times with PBS. Finally, the cells were resuspended in 100 μl of FACS buffer together with FITC-anti-IgG, PE-anti-CD20, PE Cy7-anti-CD3, and LIVE / DEAD™ Fixable Aqua dye, followed by incubation at room temperature for 20 min. After incubation, the cells were washed twice, fixed with paraformaldehyde, and analyzed on a BD FACS Canto II flow cytometer. CD3 + The anti-IgG levels detected in cells gated on A represent the amount of DSA in each recipient serum.

[0061] Suppression assays to examine immune modulation All planned regulatory subpopulations were selected from cohort C monkeys. PBLs obtained from freshly drawn blood were transfected with Tr1 cells (CD4 + Of these, LAG-3 + CD49b + ) for selection of CD4, CD49b, and LAG-3, and Breg cells (CD19 + Of these, CD24 + CD38 + ) for selection of Treg cells (CD4 + Of these, CD25hi + CD127 - ) were labeled with CD4, CD25, and CD127 for sorting, followed by washing. The BD FACSAria II system was configured for sorting using an 85 μm nozzle (45 psi at a frequency of 47 kHz). All sorts were performed at 8,000-10,000 events per second. Sorted cells were collected into 12 x 75 mm round-bottom tubes containing CRPMI. Post-sort analysis was performed to assess purity.

[0062] In the depletion assay, Treg, Breg, and Tr1 cells were depleted from PBLs of cohort C monkeys collected 12 months after transplantation. Figure 3. Identical numbers of CFSE-labeled total PBLs (undepleted) or Treg-depleted (non-CD4 + Lym plus CD127 - CD25 hi CD4 + lym) PBL, Breg depleted (non-CD19 + lym plus CD24 - CD38 - CD19 + lym) PBL, and Tr1 depleted (non-CD4 + Lym plus CD49b - LAG3 - CD4 + lym) PBLs were cultured with an equal number of irradiated VPD450-labeled donor PBLs in a 1-way CFSE Flow-MLR for 6 days. For all CFSE-MLR proliferation assays, the ratio of responder to stimulator cells was maintained at 1:1. Proliferating cells (CFSE - During the flow analysis of the total donor population, VPD450 + Excluded based on positivity.

[0063] To confirm the suppressive capacity of the selected cells, naïve recipient PBLs collected at baseline before vaccination and transplantation were challenged with irradiated VPD450-labeled donor PBLs (1:1 ratio) in a 1-way CFSE Flow-MLR for 3–4 days, and subsequently challenged again with irradiated donor PBLs in the presence or absence of immune cells with different regulatory phenotypes (Tr1 cells, Treg cells, and Breg cells) at a ratio (1:50). These cells were selected from immune-tolerant recipients between 9 and 12 months after transplantation. For all suppressive assays examining Tr1 cells, a ratio of 1:50 Tr1 to total PBLs was used in the presence of donor and third-party donors. Transwell experiments were set up to examine whether Tr1-mediated suppression is contact-dependent. In this experimental setup, CFSE-labeled Tr1-depleted PBLs were seeded (300,000 cells) on the bottom of plates together with irradiated VPD450-labeled donor cells (1:1 ratio) in the presence or absence of Tr1 cells, which were separated by transmembrane technique (4 μm pore size, Corning, Ref#3391) in the presence or absence of isotype-matched anti-human IL-10 neutralizing Abs known to cross-react with monkey IL-10 (10 μg / ml).

[0064] siRNA-mediated SH2D2 inhibition in Tr1 cells Flow-sorted Tr1 + cells (CD49b + LAG-3 + CD4 + ) and Tr1-lym cells (CD4 + Of these, CD4 - Lym + CD49b - LAG-3 -Tr1-lym cells (pool of 100,000) were sorted from PBL of cohort C monkeys collected 12 months after transplantation. CFSE-labeled Tr1-lym cells (300,000) were cultured in MLR with or without irradiated VPD-450-labeled donor cells (300,000) for 6 days. First, sorted Tr1 cells were placed in CRPMI for the first 3 days, after which Accell siRNA stock solution and Accell delivery medium (GE Healthcare, Cat. No. B-005000-500) were added directly to the sorted Tr1 cells. + They were treated with 100 μM Accell human SH2D2A siRNA (Dharmacon Accell, Cat. No. E-017851-00-0005) by mixing with the cells. Tr1 cells treated with SH2D2A siRNA or Accell delivery medium alone + Cells were added back into the MLR for the final 3 days. To measure the effect of siRNA-mediated SH2D2 inhibition on Tr1-mediated suppression of donor-specific T and B cells, all cultures were harvested on day 6 and stained to assess T and B cell proliferation (Figure 4).

[0065] Tetramer preparation and staining In these monkeys, MHC class II tetramers were used to identify CD4 + To allow tracking of T cells, Tr1 cells, and Treg cells with indirect allopeptide specificity, we exploited the high similarity observed in the peptide-binding motifs of MHC class II molecules from rhesus, cynomolgus, and humans. St-BLAST analysis of the Mamu DRB sequence was performed with the human genome at the NCBI website to identify the human homologue. HLA DRB1 * 13 (accession no. CDP32905.1) is 92% identical to Mamu DRB03a, with 96% positives and 0% gaps, and an e-value of 6e-178. HLA DRB1 * 14 (accession no. ABN54683.1) is 93% identical to Mamu DRB04, with 94% positives and 0% gaps, and an e-value of 2e-175. HLA DRB1 * 13 or HLA DRB1 *Peptides derived from Mamu MHC class I and class II sequences with high binding affinity to 14 were identified using the Immune Epitope Database Analysis resource. Synthetic peptides (Genscript USA Inc) were cloned into the HLA DRB1 * 13 or HLA DRB1 * PBLs were incubated with 0.5 or 1 μg / ml HLA class II tetramer PE in combination with antibodies for specific cell surface markers for 20 min. Stained cells were washed with cold PBS / 1% FCS, fixed with 1% PBS / formaldehyde, attached to a BD Canto II, and data analysis was performed using FlowJo version 10.2 (Tree Star, Ashland, OR). Figure 5.

[0066] TCR sequencing to track donor reactive T cells RNA-based high-throughput sequencing of the TCR β-chain CDR3 region was used to compare the entire repertoire of T cell clones in the pre- and post-ADL infusion intervals in cohort A monkeys. This approach has advantages over genomic methods that require the design and optimization of multiple primer sets spanning all V-gene segments. V-gene segments are still poorly defined in monkeys. Total RNA was extracted from frozen PBLs using RNeasy Plus Universal (Qiagen), and first-strand cDNA was generated from the polyA-tailed fraction of total RNA. Briefly, cDNA was synthesized using specially designed oligo-dT and template switch primers in a reverse transcriptase reaction, and this cDNA was used as a template for selective PCR enrichment of the TCR VDJ region using primers specific for the M. mulatta TCR constant region and template switch sequences. The enriched VDJ amplicons from each sample were uniquely double-indexed by PCR to multiplex the matches during sequencing. All PCR amplifications were performed using KAPA HiFi HotStart DNA Polymerase Ready Mix Kit (Roche). Indexed amplicon libraries were pooled in equimolar amounts and cleaned up with SPRI beads (Ampure XP, Beckman Coulter). The final pool was sequenced on a MiSeq (Illumina) 300bp paired-end run (v3 kit). TCR NGS library preparation and sequencing were performed at the University of Minnesota Genome Sciences Center. Raw data of QC-ed short reads were cleaned with trimmomatic68 using set parameters (ILLUMINACLIP:all_illumina_adapters.fa:2:30:10 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:70). Preprocessed reads were input directly into MiXCR69 with default settings for TCR profiling (https: / / mixcr.readthedocs.io / en / latest / rnaseq.html). Known TRB clonotypes in rhesus monkeys were used as references.The resulting TRB clonotypes were further filtered using a customized threshold with a clonal proportion of ≥ 0.5%. The frequency of clonal expansion was calculated by dividing the clonal prevalence at each time point by the average prevalence of all identified and mapped TCR clones. Since most of the donor-specific TCR clones at baseline were very low or undetectable, we analyzed the fate of these expanded T cell clones using peak expansion as the baseline.

[0067] RNASeq method to examine gene expression in sorted Tr1 cells RNA samples were sequenced using 50bp paired-end reads on an Illumina Hiseq 2500 platform. Raw sequence data passed the CASAV 1.8 P / F filter and were evaluated with fastqc (http: / / www.bioinformatics.babraham.ac.uk / projects / fastqc). Read mapping was performed with Hisat2 (v2.0.2) using the UCSC human genome (hg38) as a reference. Expression levels of each known gene were quantified in FPKM (mapped fragments per kilobase of exon per million mapped reads) using Cuffdiff2.2.1. Differentially expressed genes were identified using the read count raw data using the edgeR (negative binomial) function in CLCGWB (Qiagen, Valencia, CA). DEGs are represented by a color scale. Expressed transcripts were annotated using the non-human primate or human genome databases. The results were ranked by absolute fold change to identify DEGs between cohort B and cohort C. The resulting list was filtered based on a minimum absolute fold change of 1.5-fold and a raw P-value of <0.01. These DEGs were imported into Ingenuity Pathway Analysis software (Qiagen, Valencia, CA) for pathway identification. Figure 1C.

[0068] ADL treatment, release test, and injection On day −7 relative to islet transplantation, splenocytes were isolated from donor monkey spleens, RBC lysed, and B cells enriched from remaining cells using nylon wool columns (Polysciences, Inc.). Cells (80%) were incubated with ECDI (3.2 × 10 8 The cells were stirred with 0.25 × 10 ECDI-fixed splenocytes per kilogram of recipient body weight (30 mg / ml, AppliChem), washed to remove necrotic cells and microaggregates, and assessed for viability / necrosis by AO / PI fluorescence microscopy. 9 The desired dose of cells at a maximum concentration of 20 x 10 6 Cells / mL were added to a cold syringe (n=9) or IV bag (n=2) for IV infusion and kept on ice until recipient administration. ECDI-fixed cells were incubated at 37°C for 4–6 h and induction of apoptosis was monitored in vitro by labeling with Annexin V / PI (Invitrogen) and analyzed by fluorescence microscopy.

[0069] To match the desired dose of ECDI-immobilized ADL for infusion on day +1, the remaining 20% ​​of blood and pancreatic cells collected from donor monkeys on days -15 and -7 based on islet transplantation were enriched for B cells by magnetic sorting using non-human primate CD20 beads (Miltenyi Biotech) and expanded ex-vivo in GREX100M flasks (Wilson Wolf) in the presence of rhIL-10 (10 ng / ml), rIL-4 (10 ng / ml), rhBAFF (30 ng / ml), rhTLR9a (10 ng / ml), and either or both of rhCD40L-MEGA or rhCD40L multimers (500 ng / ml), and rhAPRIL (50 ng / ml) until day +1. Expanded cells were stimulated with rhIL-21 (5 ng / ml) 24 hours prior to harvest. Recipients were pretreated with a combination of diphenhydramine 12.5 mg, acetaminophen 160 mg, and ondansetron 4 mg PO prior to infusion.

[0070] Transient immunosuppression Immunosuppressants were administered to all recipient monkeys in cohorts A-E. To encompass all ADL infusions in monkeys in cohorts A, C, D, and E, the first dose of each drug was administered to all recipients in cohorts A-E on days -8 or -7 relative to day 0 islet transplantation. Antagonist anti-CD40 mAb 2C10R4, provided by the NIH Nonhuman Primate Reagent Resource, was administered IV at 50 mg / kg on days -8, -1, 7, and 14. Rapamycin (Rapamune®) was administered PO from day -7 until day 21 post-transplant with target trough levels of 5-12 ng / ml. Concomitant anti-inflammatory treatment consisted of: i) αIL-6R (tocilizumab, Actemra®) at 10 mg / kg IV on days -7, 0, 7, 14, and 21, and ii) sTNFR (etanercept, Enbrel®) at 1 mg / kg IV on days -7 and 0, and 0.5 mg / kg / SC on days 3, 7, 10, 14, and 21. The exploratory cohort RM was terminated on day +7. Thus, the final dose of immunosuppressant was administered on day +7 in these RMs.

[0071] Pancreas procurement, islet processing and release studies, islet transplantation, and evaluation of islet graft function Total pancreatectomy was performed in donor monkeys from cohorts C-E, and islets were isolated, purified, and cultured for 7 days to minimize direct pathway stimulation capacity and for quality control. On day 0, target numbers ≥ 5,000 IE / kg by DNA64 were transplanted nonoperatively using intraportal vascular access ports placed in STZ diabetic RMs with endotoxin content ≤ 1.0 EU / kg of recipient BW. Protective exogenous insulin was stopped 21 days after transplant in animals with intact graft function. Metabolic monitoring included daily am / pm blood glucose, weekly C-peptide, monthly HbA1c, mixed meal testing, and bimonthly IVGTT with identification of acute C-peptide in response to glucose and glucose disappearance rate (Kg).

[0072] Histopathological examination of islet grafts Liver specimens were obtained from 10 different anatomical regions of each recipient, fixed in 10% formalin, and processed for routine histology. Sections from each of the 10 blocks were stained with hematoxylin & eosin (H&E) or immunostained for insulin to score transplanted islets. Rejection-free islet allograft survival was confirmed at autopsy by graft histopathology showing significant numbers of intact type A and mildly infiltrated type B islets, and few or no type C-F islets (mildly to severely infiltrated islets and islets partially or completely replaced by infiltrate or fibrosis).

[0073] ADL induces defective proliferation of donor-specific T and B cell clones Several findings were obtained by monitoring cellular immunity early after ADL infusion in three non-transplanted, non-diabetic, single DRB-matched monkeys from cohort A under short-term immunosuppression. The incidence of circulating MDSCs was significantly elevated, beginning 1 day after the first ADL infusion (day -6) and remaining elevated throughout follow-up on day +7. Ki67 + CD4 + T cell frequency increased 2.6-fold on day -5, followed by a 90% decline 3 days later and an almost complete absence beginning 3 days after the second ADL infusion. + CD8 + The frequency of T cells increased 19-fold after the first ADL infusion, followed by a sharp decline beginning 4 days after the first ADL infusion and an almost complete absence shortly after the second ADL infusion. + B cells showed similar kinetics and degrees of proliferation and contraction. Interferon-gamma (IFN-γ)-secreting CD4 + The frequency of T cells was significantly reduced, and interleukin (IL)-10-secreting CD4 + The prevalence of T cells remained unchanged. + , CD8 + , and CD20 +Donor-specific proliferation of cells was significantly reduced, while proliferation in response to a third-party donor remained unchanged in a carboxyfluorescein diacetate succinimidyl ester mixed lymphocyte reaction (CFSE-MLR) assay.

[0074] CD4 with indirect specificity for mismatched donor HC-I Mamu A00427-41 peptide + To follow the fate of T cells, we performed this in three monkeys from cohort A by using HLA DRB1 * 13 (human homologue of Mamu-DR03) tetramer. The cells increased 5.6-fold on day -5, then decreased 3.6-fold on day 0. Then, 2 days after the second ADL infusion, tetramer-positive CD4 + The frequency of T cells increased 1.24-fold but was significantly reduced compared to naive monkeys by day 7.

[0075] Clonotype analysis of the VDJ region in these monkeys demonstrated that the frequency of approximately 30 T cell clones was altered after ADL infusion. Alterations in multiple T cell clones with different Vβ chains (4-Vβ5 and 3 each of Vβ4, Vβ7, Vβ9, Vβ11, Vβ12, and Vβ28) indicated that ADL infusion targeted multiple alloreactive clones, consistent with the idea that alloreactivity is polyclonal. Analysis of individual T cell clones demonstrated incomplete expansion and subsequent 5- to 8-fold contraction of multiple clones. Thus, multiple lines of evidence indicated that ADL infusion caused the expansion and subsequent contraction of donor-specific T and B cells.

[0076] ADL promotes stable islet allograft immune tolerance in one DRB-matched RM Intraportal transplantation of islet allografts cultured for 8 days resulted in survival for more than 365 days in 2 of 7 streptozotocin (STZ) diabetic, DRB-matched cohort B monkeys on short-term immunosuppression (Fig. 6a and 6b). The addition of ADL infusion to short-term immunosuppression was associated with significant improvement in survival in 5 of 5 cohort C monkeys (P=0.021). All 5 monkeys exhibited post-transplant immune tolerance of the islet allograft for more than 365 days after transplantation (Fig. 6a and 6b). Cohort C monkey #13EP5 became normoglycemic soon after transplantation and maintained it even after immunosuppression and exogenous insulin were discontinued 21 days after transplantation. This recipient's HbA1C level became and remained normal after transplantation. Sustained weight gain after transplantation was also observed in other cohort C monkeys, consistent with the overall safety of the treatment regimen. Pre-transplant serum C-peptide levels and responses to glucose stimulation were negative in all five recipients. Strongly positive fasting and unregulated serum C-peptide levels after transplantation and their elevation after stimulation throughout the 1-year follow-up in monkey #13EP5 confirmed stable islet allograft function. This recipient showed a stable blood glucose disappearance rate (Kg) after intravenous glucose loading after transplantation that was comparable to the pre-STZ rate. C-peptide levels from matching tests showed a substantial increase of >1 ng / ml throughout the post-transplant course. Histopathological analysis of this recipient's liver at necropsy revealed numerous intact islets with little or no peri-islet infiltration. Transplanted intrahepatic islets showed strong positive staining for insulin. The absence of insulin-positive islet beta cells in the native pancreas at necropsy indicated that post-transplant normoglycemia reflected graft function and was not due to remission from STZ-induced diabetes. Cohort C monkey #15CP1 was not sacrificed 1 year after transplantation. Islet allograft function continued in this recipient for more than 2 years after cessation of immunosuppression. At necropsy of monkey #15CP1, histopathological examination confirmed the presence of a rejection-free islet allograft in the native pancreas and the absence of insulin-positive beta cells.In comparison, although cohort B monkey #15CP3 became normoglycemic after transplantation, a decline in graft function became evident starting 4 months after transplantation. Necropsy one month later confirmed rejection, as evidenced by the presence of a small number of insulin-positive islet beta cells heavily infiltrated by mononuclear cells. Collectively, these results demonstrate the long-term functional and histological survival of a single DRB-matched islet allograft in ADL-treated RM, even after cessation of immunosuppression, indicating robust immune tolerance in a rigorous translational model.

[0077] ADL suppresses effector cell proliferation and donor-specific antibody (DSA) induction Effector cell and antibody responses were compared in recipients of cohorts B and C. CD3 + , CD4 + , and CD8 + T cells and CD20 + The incidence of circulating B cells was not affected by ADL infusion in cohort C. However, in contrast to cohort B monkeys that did not receive ADL, peri-transplant ADL infusion in cohort C significantly increased the number of circulating, liver mononuclear cells (LMNC), mesenteric lymph nodes (LN), and anti-donor CD4 + and CD8 + This was accompanied by a long-term suppression of T effector memory (TEM) cell expansion. Throughout the 12-month post-transplant follow-up, ADL infusion was associated with a lower incidence of circulating T follicular helper (Tfh) cells in cohort C compared with cohort B monkeys. PD-1 + CD4 + Like T cells, PD-1 + CD8 + The proportion of T cells was higher in cohort C compared to cohort B after transplantation, suggesting that ADL depletes T cells for phenotypic induction and elimination. Our analysis also revealed that circulating Tbet in cohort C monkeys was significantly higher than that in cohort C monkeys. + CD4 + and CD40 + CD4 + T cells were persistently suppressed and CD4 +It was also shown that Tbet had no effect on T cell proliferation. + CD8 + , CD40 + CD8 + , and CD107 + CD8 + At 3 months after transplant, the incidence of circulating T cells was lower in cohort C monkeys than in cohort B monkeys, and the third-party CD8 + There was no defective T cell expansion. Enzyme-linked immunosorbent spot (ELISPOT) analysis revealed no clear differences in the frequency of IFN-γ-secreting T cells with direct and indirect specificity in response to irradiated donor peripheral blood lymphocytes (PBL) at 1 month and at sacrifice between cohorts B and C, and when compared to baseline. Circulating CD20 + The incidence of B cells was similar in cohorts B and C, but circulating Tbet at 3 and 12 months post-transplant was significantly higher. + Percentage of B cells and CD19 in LMNCs at sacrifice + The proportion of B cells was significantly lower in Cohort C compared to Cohort B monkeys. Only in Cohort B, but not in Cohort C, did recipients develop high DSA levels (expressed as mean fluorescence intensity (mfi)). We did not measure DSA frequently enough to determine whether it was present prior to clinical rejection. In each of the DSA-positive recipients, rejection was confirmed by histopathological analysis. In summary, peri-transplant ADL infusion disrupted post-transplant activation and proliferation of effector T and B cells and their recruitment to the allograft in one DRB-matched monkey on short-term immunosuppression.

[0078] ADL expands antigen-specific regulatory networks Next, we compared the frequency of lymphoid myeloid cells with a regulatory phenotype in cohorts B and C monkeys. We found that the frequency of circulating Tr1 cells at 3, 6, and 12 months post-transplant, as well as in LMNC and LN at sacrifice, was significantly higher in ADL-treated cohort C monkeys than in untreated cohort B monkeys. We also found that the percentage of circulating natural suppressor (NS) and Treg cells was significantly higher in ADL-treated cohort C monkeys than in untreated cohort B monkeys throughout the post-transplant follow-up period. Regulatory B (Breg) cells, B10 cells, and MDSCs were also significantly more abundant in the circulation, and, except for MDSCs, in the liver and LN at sacrifice, in cohort C monkeys than in cohort B monkeys. In PBL from cohort C at 9 and 12 months post-transplant (compared to unmodified recipient PBL), depletion of Treg, Breg, and Tr1 cells was associated with CD4 + T (4.9x, 2.1x, and 8.1x), CD8 + During revalidation, Tr1 cells sorted from immune-tolerant Cohort C recipients 12 months after transplantation were added back to PBL collected from the recipients at baseline, which was associated with increased CD4 + , CD8 + , and CD20 + There was a significant inhibition of donor-specific proliferation of Tr1 cells, but no discernible effect on T and B cell proliferation in response to the third-party donor. Isolation of Tr1 cells in Transwell experiments did not block the suppression of donor-specific responses, indicating that Tr1 cells suppressed immune responses through soluble factors. Addition of neutralizing IL-10, but not a control isotype antibody, in the one-way CFSEMLR assay significantly abrogated the suppression of donor-specific responses.

[0079] By analyzing differentially expressed genes (DEGs) in flow-sorted Tr1 cells from monkeys in cohorts B and C, 258 genes were identified. Grouping of DEGs revealed that immune cell signaling and mitochondrial respiration were the two major biological pathways activated in sorted Tr1 cells from cohort C, but not in RMs from cohort B. Our heatmap analysis of the Z-scores of DEGs demonstrated that there was a significant upregulation of immune signaling intermediates in Tr1 cells only in cohort C. The related transcripts of the top three regulators of immune cell signaling, namely SH2D2, XBP1, and SUMO2, were significantly upregulated in Tr1 cells from cohort C when compared to cohort B, indicating that Tr1 cells from cohort C were in an activated state. Our heatmap Z-score analysis of DEGs mapping mitochondrial respiration showed that Tr1 cells from Cohort C clustered at one end, demonstrating that the cells were metabolically highly active. NDUFS4 and NDUFS5, members of the NDUSF family that regulate mitochondrial respiration, were significantly upregulated in Tr1 cells from Cohort C. Twelve months after transplantation, treatment of Tr1 cells sorted from immune-tolerant Cohort C monkeys with small interfering RNA (siRNA) targeting the SH2D2 transcription molecule reduced CD4 expression in response to the donor. + (59%), CD8 + (53%), and CD20 + (80.5%) decreased the ability of Tr1 cells to suppress cell proliferation. Thus, ADL expanded a regulatory network involving antigen-specific Tr1 cells that exhibited unique immune cell signaling and metabolic profiles.

[0080] The tolerogenic efficacy of ADL appears to be blunted in fully mismatched RM. ADL infusion in fully mismatched cohort D monkeys was associated with prolonged allograft function in two of three recipients. However, in the third recipient, the graft was rejected between 120 and 150 days after transplantation. In this recipient, the proliferation of T cells was not suppressed. After ADL infusion, the proliferation of Treg (1.4-fold) and Tr1 (0.98-fold) cells at 6 months after transplantation in cohort D was not as great as in cohort C monkeys (2.3-fold and 2.1-fold, P = 0.04). Moreover, in cohort D recipients whose grafts were rejected, the proliferation of donor-specific T cells was not suppressed. The frequency of three categories of Tr1 cells (IL-10, transforming growth factor beta (TGF-β), and IL-10 plus TGF-β dual producers) was significantly elevated in Cohort C, but not in Cohort D, compared with Cohort B. Tr1 cells isolated at sacrifice from two Cohort D recipients with long-term allograft function reduced donor-reactive proliferation of T and B cells by >45% compared with >75% in Tr1 cells from Cohort C when added at the same ratio to CFSE-MLR. Depletion of Tr1 cells from PBL obtained at sacrifice increased donor-specific proliferation of both T and B cells by ≥45%. Thus, infusion of fully mismatched ADL can also establish donor-specific regulation.

[0081] A single DRB-matched ADL expands alloantigen-specific Treg and Tr1 cells Using MHC-II tetramers, we identify circulating CD4+ cells with indirect specificity for self (shared) MHC-II and mismatched donor MHC-II and MHC-I peptides. + T cell subsets were monitored. At baseline, CD4 + Among the T cell subsets, CD4 has indirect specificity for these peptides +The frequency of T cells varied from 1.72 ± 1.2% to 5.23 ± 3.0%. Compared to baseline, the frequency of non-regulatory CD4+ T cells with indirect specificity for self (shared) MHC-II peptides did not increase after transplant in cohorts B, C, and D. In contrast, CD4 + A sustained increase from baseline in Treg cells (up to 2.43 ± 0.35-fold) and Tr1 cells (up to 5.4 ± 1.2-fold) occurred in cohort C, but not in cohorts B and D. In cohort D, CD4 + No changes in the frequency of T cells, either non-regulatory or regulatory, were observed.

[0082] Conversely, in cohort C, non-regulatory CD4 + The frequency of T cells did not change after transplantation, whereas the frequency of Treg cells with this specificity (maximum 1.93 ± 0.4-fold) and Tr1 cells (maximum 3.9 ± 1.2-fold) increased. + The frequency of T cells was elevated (1.6±0.9-fold) only in cohort B after transplantation, without any changes in the corresponding subsets of Treg and Tr1 cells. Taken together, ADL expanded Treg and Tr1 cells with indirect specificity for shared (self) MHC-II and mismatched MHC-I peptides in one MHC-II-matched RM, which likely contributes to the induction and maintenance of immune tolerance.

[0083] Example 2 Absence of biomarkers correlates with loss of transplanted islet function The sensitization or presence of pre-transplant donor-specific immune responses has been shown in humans and animal models to seriously impede the induction of long-term function or immune tolerance to transplanted solid organ or cell grafts. In the present preclinical model, the presence of donor-specific antibodies led to accelerated antibody-mediated rejection of transplanted islets. Serial peripheral blood samples were analyzed for the presence of Tr1 cells after administration of immune tolerance regimes in non-human primates with pre-existing donor-specific antibodies. Analysis of peripheral blood samples demonstrated that administration of immune tolerance regimes resulted in a reduction in the incidence of Tr1 cells after transplantation (day 14) to levels lower than those observed in the naive state (mean 0.59±0.24), and thus an absence of an increase in Tr1 cells. An increase in Tr1 cells indicates a failure to induce donor-specific immune tolerance and correlates with the loss of graft function of islet allografts. FIG. 7.

[0084] Loss of immune tolerance biomarkers precedes loss of graft function In the present non-human primate preclinical transplantation model, administration of ADL+TIS to fully mismatched islet allograft recipients resulted in loss of graft function approximately 300 days after transplantation, whereas transplantation in one DRB-matched recipients resulted in indefinite (≧365 days) survival and function of transplanted islets. To test whether loss of Tr1 cells precedes loss of graft function in this subset of recipients, the frequency of Tr1 cells was serially analyzed by flow cytometry in peripheral blood samples collected pre- and post-transplant from fully mismatched islet graft recipients. Similar to the one DRB-matched group, administration of ADL+TIS resulted in a significant increase in the fold change in the frequency of Tr1 cells at day 90 (3.98±0.98), followed by a decline at day 180 (1.98±0.75), reaching levels observed in the naive state at day 300 (1.19±0.1). The decline in the incidence of circulating Tr1 cells correlated with the complete loss of graft function. These observations strongly suggest that the loss of Tr1 cells, indicative of a loss of immune tolerance biomarkers, precedes the loss of graft function by approximately 120 days. Figure 8.

[0085] While the above specification and examples fully disclose and enable the invention, they are not intended to limit the scope of the invention, which is defined by the appended claims.

[0086] All publications, patents, and patent applications are incorporated herein by reference. In the foregoing specification, the invention has been described with respect to certain specific embodiments thereof, and while many details have been set forth for purposes of illustration, it will be apparent to those skilled in the art that the invention is susceptible to additional embodiments, and certain of the details described herein may be varied considerably without departing from the fundamental principles of the invention.

[0087] The use of the terms "a" and "an" and "the" and similar referents, in the context of describing the present invention, should be construed to encompass both the singular and the plural, unless otherwise stated herein or clearly contradicted by the context. The terms "comprising," "having," "including," and "containing" should be construed as open-ended terms (i.e., meaning "including, but not limited to"), unless otherwise stated. The description of ranges of values ​​herein is intended only to serve as a shorthand method of individually referring to each individual value falling within the range, unless otherwise stated herein, and each individual value is incorporated herein as if the value were individually stated herein. All methods described herein can be performed in any suitable order, unless otherwise stated herein or clearly contradicted by the context. Any and all examples provided herein, or the use of illustrative language (e.g., "such as"), are intended only to better highlight the invention, and do not limit the scope of the invention unless specifically claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0088] Embodiments of the present invention are described herein, including the best mode known to the inventors for carrying out the invention. Modifications of these embodiments will become apparent to those skilled in the art upon reading the above description. The inventors expect that such modifications will be adopted by those skilled in the art, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter described in the claims appended hereto as permitted by applicable law. Moreover, unless otherwise indicated herein or clearly contradicted by context, any combination of the above-described elements in all possible variations thereof is encompassed by the present invention.

Claims

1. A method for using the occurrence rate of regulatory T cell type 1 (Tr1) cells as an indicator of whether a graft recipient patient has transplant tolerance induced by one or more donor antigens administered in conjunction with transient immunotherapy, comprising: (a) assaying a first blood sample from said transplant recipient patient to detect a baseline prevalence of Tr1 cells, said first blood sample being obtained prior to immune tolerization, prior to transplantation, and prior to the initiation of transient immunotherapy; and (b) assaying a second blood sample from the transplant recipient patient to detect the prevalence of Tr1 cells, wherein the second blood sample is obtained after immune tolerization, after transplantation, and after initiation of transient immunotherapy. Including, if the prevalence of Tr1 cells in the second blood sample is at least two-fold higher than the baseline prevalence of Tr1 cells, the transplant recipient patient is indicated as having transplant tolerance induced by the one or more donor antigens; The Tr1 cells express the marker CD49b ＋ , LAG-3 ＋ , CD4 ＋ having the occurrence rate of Tr1 cells is the proportion of Tr1 cells in a blood sample; The method, wherein the one or more donor antigens include donor apoptotic leukocytes (ADLs), donor-specific transfusion (DST) nanoparticles bound to a donor peptide, DST nanoparticles encapsulating a donor peptide, and / or recipient apoptotic leukocytes bound to a donor peptide.

2. A method for using the occurrence rate of regulatory T cell type 1 (Tr1) cells as an indicator of whether a graft recipient patient has transplant tolerance induced by one or more donor antigens, comprising: (a) assaying a first blood sample obtained from a transplant recipient patient to detect a baseline prevalence of Tr1 cells, wherein said first blood sample is obtained prior to immune tolerization, prior to transplantation, and prior to initiation of transient immunotherapy; (b) assaying a second blood sample obtained from the patient to detect the prevalence of Tr1 cells, wherein the second blood sample is obtained after immune tolerization, after transplantation, and after initiation of transient immunotherapy; Including, if the prevalence of Tr1 cells in the second blood sample is at least two-fold higher than the baseline prevalence, the transplant recipient patient is declared as having transplant tolerance induced by one or more donor antigens infused in conjunction with transient immunotherapy; The Tr1 cells express the marker CD49b ＋ , LAG-3 ＋ , CD4 ＋ having the occurrence rate of Tr1 cells is the proportion of Tr1 cells in a blood sample; The aforementioned methods, wherein the one or more donor antigens comprise donor apoptotic leukocytes (ADLs).

3. The method of claim 1 or 2, wherein if the graft recipient patient is indicated as having transplant tolerance induced by the one or more donor antigens, the graft recipient patient is indicated to be treated by discontinuing administration of immunosuppressants.

4. The method of claim 1 , further comprising determining indirect specificity of the Tr1 cells for at least one mismatched donor MHC class I peptide.

5. The method of claim 1 , further comprising identifying a transcriptomic signature of the Tr1 cells, the transcriptomic signature comprising markers indicative of antigen-specific signaling.

6. 6. The method of claim 5, wherein the transcriptomic signature indicative of antigen-specific signaling is SH2D2a.

7. The method of claim 1 , further comprising identifying a transcriptomic signature of the Tr1 cells that is indicative of an activation state.

8. 8. The method of claim 7, wherein the transcriptomic signature indicative of activation state is the mitochondrial respiration-related transcript NDUFS4.

9. Total CD4 in step (a) ＋ CD49b in T cells ＋ LAG-3 ＋ CD4 ＋ The method of any one of claims 1 to 8, wherein the incidence of (Tr1) cells is determined by multiparametric flow cytometry.

10. Total CD4 in step (a) ＋ CD49b in T cells ＋ LAG-3 ＋ CD4 ＋ 10. The method of claim 1, wherein the incidence of (Tr1) cells is determined by CyTOF mass cytometry.

11. 11. The method of any one of claims 1 to 10, wherein the graft recipient patient receives two intravenous infusions of donor apoptotic leukocytes seven days prior to transplantation.

12. 12. The method of any one of claims 1 to 11, wherein the transient immunotherapy comprises at least one immunosuppressant.

13. 13. The method of claim 12, wherein the immunosuppressant is an inhibitor of CD40:CD40L costimulation, an mTOR inhibitor, and a combination anti-inflammatory therapeutic targeting pro-inflammatory cytokines.

14. The method of claim 13, wherein the inhibitor of CD40:CD40L costimulation is an antagonist anti-CD40 antibody, an Fc-modified anti-CD40L antibody, a Fab' anti-CD40L antibody, or a peptide that interferes with CD40:CD40L costimulation.

15. The method of claim 13, wherein the inhibitor of CD40:CD40L costimulation is the antagonist anti-CD40 mAb 2C10R4.

16. 14. The method of claim 13, wherein the mTOR inhibitor is rapamycin.

17. 17. The method of any one of claims 1 to 16, wherein the transient immunotherapy comprises an anti-inflammatory agent.

18. The method of claim 17, wherein the anti-inflammatory agent is αIL-6R and / or sTNFR.

19. The method of claim 18, wherein the anti-inflammatory agent is αIL-6R, and the αIL-6R is tocilizumab.

20. 20. The method of claim 18, wherein the anti-inflammatory agent is sTNFR, and the sTNFR is etanercept.

21. 21. The method of any one of claims 1 to 20, wherein the transplant recipient patient is receiving an allogeneic transplant.

22. 22. The method of claim 21, wherein the allograft is a solid organ allograft.

23. 23. The method of claim 22, wherein the solid organ allograft comprises a pancreas transplant, a liver transplant, an intestinal transplant, a heart transplant, a kidney transplant, a lung transplant, or a uterus transplant.

24. 23. The method of claim 22, wherein the solid organ allograft is a kidney graft.

25. 25. The method of claim 24, wherein the kidney graft is a living donor kidney graft.

26. 22. The method of claim 21, wherein the allograft is a tissue allograft.

27. 27. The method of claim 26, wherein the tissue allograft comprises adipose tissue, amniotic tissue, chorionic tissue, connective tissue, dura mater, facial tissue, intestinal tissue, glandular tissue, liver tissue, muscle tissue, nervous tissue, ocular tissue, pancreatic tissue, pericardium, skeletal tissue, skin tissue, urogenital tissue, or vascular tissue.

28. 22. The method of claim 21, wherein the allograft is a cell graft.

29. 30. The method of claim 28, wherein the cell graft comprises pancreatic islet cells, hepatocytes, myoblasts, embryonic stem cell derived differentiated cell grafts, induced pluripotent stem cell derived differentiated cell grafts, or bone marrow grafts.

30. 30. The method of claim 29, wherein the embryonic stem cell derived differentiated cell graft is a pancreatic islet graft or a pancreatic islet beta cell graft.

31. 30. The method of claim 29, wherein the induced pluripotent stem cell derived differentiated cell graft is a pancreatic islet graft or a pancreatic islet beta cell graft.

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