Amnion‐Derived Multipotent Progenitor Cells Support Allograft Tolerance Induction

Donor‐specific immunological tolerance using high doses of bone marrow cells (BMCs) has been demonstrated in mixed chimerism‐based tolerance induction protocols; however, the development of graft versus host disease remains a risk. Here, we demonstrate that the co‐infusion of limited numbers of donor unfractionated BMCs with human amnion‐derived multipotent progenitor cells (AMPs) 7 days post–allograft transplantation facilitates macrochimerism induction and graft tolerance in a mouse skin transplantation model. AMPs + BMCs co‐infusion with minimal conditioning led to stable, mixed, multilineage lymphoid and myeloid macrochimerism, deletion of donor‐reactive T cells, expansion of CD4⁺CD25⁺Foxp3⁺ regulatory T cells (T_regs) and long‐term allograft survival (>300 days). Based on these findings, we speculate that AMPs maybe a pro‐tolerogenic cellular therapeutic that could have clinical efficacy for both solid organ and hematopoietic stem cell transplant applications.     

Contrasting effects of B cell depletion on CD4+ and CD8+ memory T cell responses generated after transplantation

Alloreactive memory T cells play a key role in transplantation by accelerating allograft rejection and preventing tolerance induction. Some studies using µMT mice, which are constitutionally devoid of B cells, showed that B cells were required for the generation of memory T cells after allotransplantation. However, whether B cell depletion in normal adult mice has the same effect on memory responses by CD4⁺ and CD8⁺ T cells activated after transplantation has not been thoroughly investigated. In this study, we tested the effect of anti‐CD20 antibody‐mediated B cell depletion on CD4⁺ and CD8⁺ memory T cell alloresponses after skin transplantation in wild‐type mice. We found that B cell depletion prevented the development of memory alloresponses by CD4⁺ T cells but enhanced that of CD8⁺ memory T cells. Next, we tested the influence of B cell depletion on hematopoietic chimerism. In OT‐II CD4⁺ anti‐OVA TCR transgenic mice sensitized to ovalbumin antigen, B cell depletion also impaired allospecific memory T cell responses and thereby enhanced donor hematopoietic chimerism and T cell deletion after bone marrow transplantation. This study underscores the complexity of the relationships between B and T cells in the generation and reactivation of different memory T cell subsets after transplantation.     

Flt3L‐mobilized dendritic cells bearing H2‐Kbm1 apoptotic cells do not induce cross‐tolerance to CD8+ T cells across a class I MHC mismatched barrier

Tolerization of allogeneic CD8⁺ T cells is still a pending issue in the field of transplantation research to achieve long‐term survival. To test whether dendritic cells (DC) bearing allogeneic major histocompatibility complex (MHC) class I mismatched apoptotic cells could induce cross‐tolerance to alloreactive CD8⁺ T cells, the following experimental strategy was devised. Rag2/γ_c KO B6 mice were treated with Fms‐like tyrosine kinase 3 ligand (Flt3L)‐transduced B16 melanoma cells to drive a rapid expansion and mobilization of DC in vivo. Of all DC populations expanded, splenic CD11c⁺CD103⁺CD8α⁺ DC were selectively involved in the process of antigen clearance of X‐ray irradiated apoptotic thymocytes in vivo. Considering that CD11c⁺CD103⁺CD8α⁺ DC selectively take up apoptotic cells and that they are highly specialized in cross‐presenting antigen to CD8⁺ T cells, we investigated whether B6 mice adoptively transferred with Flt3L‐derived DC loaded with donor‐derived apoptotic thymocytes could induce tolerance to bm1 skin allografts. Our findings on host anti‐donor alloresponse, as revealed by skin allograft survival and cytotoxic T lymphocyte assays, indicated that the administration of syngeneic DC presenting K^bm1 donor‐derived allopeptides through the indirect pathway of antigen presentation was not sufficient to induce cross‐tolerance to alloreactive CD8⁺ T cells responding to bm1 alloantigens in a murine model of skin allograft transplantation across an MHC class I mismatched barrier.     

Alefacept Promotes Immunosuppression‐Free Renal Allograft Survival in Nonhuman Primates via Depletion of Recipient Memory T Cells

Renal allograft tolerance has been achieved in MHC‐mismatched primates via nonmyeloablative conditioning beginning 6 days prior to planned kidney and donor bone marrow transplantation (DBMT). To extend the applicability of this approach to deceased donor transplantation, we recently developed a novel‐conditioning regimen, the “delayed protocol” in which donor bone marrow (DBM) is transplanted several months after kidney transplantation. However, activation/expansion of donor‐reactive CD8⁺ memory T cells (TMEM) occurring during the interval between kidney and DBM transplantation impaired tolerance induction using this strategy. In the current study, we tested whether, Alefacept, a fusion protein which targets LFA‐3/CD2 interactions and selectively depletes CD2^highCD8⁺ effector memory T cells (TEM) could similarly induce long‐term immunosuppression‐free renal allograft survival but avoid the deleterious effects of anti‐CD8 mAb treatment. We found that Alefacept significantly delayed the expansion of CD2^high cells including CD8⁺ TEM while sparing naïve CD8⁺ T and NK cells and achieved mixed chimerism and long‐term immunosuppression‐free renal allograft survival. In conclusion, elimination of CD2^high T cells represents a promising approach to prevent electively the expansion/activation of donor‐reactive TEM and promotes tolerance induction via the delayed protocol mixed chimerism approach.

     

Clonal Deletion Established via Invariant NKT Cell Activation and Costimulatory Blockade Requires In Vivo Expansion of Regulatory T Cells

Recently, the immune‐regulating potential of invariant natural killer T (iNKT) cells has attracted considerable attention. We previously reported that a combination treatment with a liposomal ligand for iNKT cells and an anti‐CD154 antibody in a sublethally irradiated murine bone marrow transplant (BMT) model resulted in the establishment of mixed hematopoietic chimerism through in vivo expansion of regulatory T cells (Tregs). Herein, we show the lack of alloreactivity of CD8⁺T cells in chimeras and an early expansion of donor‐derived dendritic cells (DCs) in the recipient thymi accompanied by a sequential reduction in the donor‐reactive Vβ‐T cell receptor repertoire, suggesting a contribution of clonal deletion in this model. Since thymic expansion of donor DCs and the reduction in the donor‐reactive T cell repertoire were precluded with Treg depletion, we presumed that Tregs should preform before the establishment of clonal deletion. In contrast, the mice thymectomized before BMT failed to increase the number of Tregs and to establish CD8⁺T cell tolerance, suggesting the presence of mutual dependence between the thymic donor–DCs and Tregs. These results provide new insights into the regulatory mechanisms that actively promote clonal deletion.     

Chimerism,Graft Survival,and Withdrawal of Immunosuppressive Drugs in HLA Matched and Mismatched Patients After Living Donor Kidney and Hematopoietic Cell Transplantation

Thirty‐eight HLA matched and mismatched patients given combined living donor kidney and enriched CD34⁺ hematopoietic cell transplants were enrolled in tolerance protocols using posttransplant conditioning with total lymphoid irradiation and anti‐thymocyte globulin. Persistent chimerism for at least 6 months was associated with successful complete withdrawal of immunosuppressive drugs in 16 of 22 matched patients without rejection episodes or kidney disease recurrence with up to 5 years follow up thereafter. One patient is in the midst of withdrawal and five are on maintenance drugs. Persistent mixed chimerism was achieved in some haplotype matched patients for at least 12 months by increasing the dose of T cells and CD34⁺ cells infused as compared to matched recipients in a dose escalation study. Success of drug withdrawal in chimeric mismatched patients remains to be determined. None of the 38 patients had kidney graft loss or graft versus host disease with up to 14 years of observation. In conclusion, complete immunosuppressive drug withdrawal could be achieved thus far with the tolerance induction regimen in HLA matched patients with uniform long‐term graft survival in all patients.     

Rapamycin and CTLA4Ig Synergize to Induce Stable Mixed Chimerism Without the Need for CD40 Blockade

The mixed chimerism approach achieves donor‐specific tolerance in organ transplantation, but clinical use is inhibited by the toxicities of current bone marrow (BM) transplantation (BMT) protocols. Blocking the CD40:CD154 pathway with anti‐CD154 monoclonal antibodies (mAbs) is exceptionally potent in inducing mixed chimerism, but these mAbs are clinically not available. Defining the roles of donor and recipient CD40 in a murine allogeneic BMT model, we show that CD4 or CD8 activation through an intact direct or CD4 T cell activation through the indirect pathway is sufficient to trigger BM rejection despite CTLA4Ig treatment. In the absence of CD4 T cells, CD8 T cell activation via the direct pathway, in contrast, leads to a state of split tolerance. Interruption of the CD40 signals in both the direct and indirect pathway of allorecognition or lack of recipient CD154 is required for the induction of chimerism and tolerance. We developed a novel BMT protocol that induces mixed chimerism and donor‐specific tolerance to fully mismatched cardiac allografts relying on CD28 costimulation blockade and mTOR inhibition without targeting the CD40 pathway. Notably, MHC‐mismatched/minor antigen‐matched skin grafts survive indefinitely whereas fully mismatched grafts are rejected, suggesting that non‐MHC antigens cause graft rejection and split tolerance.     

The Programmed Death (PD)‐1/PD‐Ligand 1 Pathway Regulates Graft‐Versus‐Host‐Reactive CD8 T Cells After Liver Transplantation

Acute graft‐versus‐host disease (aGVHD) is a life‐threatening complication after solid‐organ transplantation, which is mediated by host‐reactive donor T cells emigrating from the allograft. We report on two liver transplant recipients who developed an almost complete donor chimerism in peripheral blood and bone marrow‐infiltrating T cells during aGVHD. By analyzing these T cells directly ex vivo, we found that they died by apoptosis over time without evidence of rejection by host T cells. The host‐versus‐donor reactivity was selectively impaired, as anti‐third‐party and antiviral T cells were still detectable in the host repertoire. These findings support the acquired donor‐specific allotolerance concept previously established in animal transplantation studies. We also observed that the resolution of aGVHD was not accompanied by an expansion of circulating immunosuppressive CD4/CD25/FoxP3‐positive T cells. In fact, graft‐versus‐host‐reactive T cells were controlled by an alternative negative regulatory pathway, executed by the programmed death (PD)‐1 receptor and its ligand PD‐L1. We found high PD‐1 expression on donor CD4 and CD8 T cells. In addition, blocking PD‐L1 on host‐derived cells significantly enhanced alloreactivity by CD8 T cells in vitro. We suggest the interference with the PD‐1/PD‐L1 pathway as a therapeutic strategy to control graft‐versus‐host‐reactive T cells in allograft recipients.     

CD8+ T‐Cell Depletion and Rapamycin Synergize with Combined Coreceptor/Stimulation Blockade to Induce Robust Limb Allograft Tolerance in Mice

The growing development of composite tissue allografts (CTA) highlights the need for tolerance induction protocols. Herein, we developed a mouse model of heterotopic limb allograft in a stringent strain combination in which potentially tolerogenic strategies were tested taking advantage of donor stem cells in the grafted limb. BALB/c allografts were transplanted into C57BL/6 mice treated with anti‐CD154 mAb, nondepleting anti‐CD4 combined to either depleting or nondepleting anti‐CD8 mAbs. Some groups received additional rapamycin. Both depleting and nondepleting mAb combinations without rapamycin only delayed limb allograft rejection, whereas the addition of rapamycin induced long‐term allograft survival in both combinations. Nevertheless, robust donor‐specific tolerance, defined by the acceptance of a fresh donor‐type skin allograft and simultaneous rejection of third‐party grafts, required initial CD8⁺ T‐cell depletion. Mixed donor‐recipient chimerism was observed in lymphoid organs and recipient bone marrow of tolerant but not rejecting animals. Tolerance specificity was confirmed by the inability to produce IL‐2, IFN‐γ and TNF‐α in MLC with donor antigen while significant alloreactivity persisted against third‐ party alloantigens. Collectively, these results show that robust CTA tolerance and mixed donor‐recipient chimerism can be achieved in response to the synergizing combination of rapamycin, transient CD8⁺ T‐cell depletion and costimulation/coreceptor blockade.     

Different Mechanisms Control Peripheral and Central Tolerance in Hematopoietic Chimeric Mice

Regulatory T cells (Treg) are important in peripheral tolerance, but their role in establishing and maintaining hematopoietic mixed chimerism and generating central tolerance is unclear. We now show that costimulation blockade using a donor‐specific transfusion and anti‐CD154 antibody applied to mice given bone marrow and simultaneously transplanted with skin allografts leads to hematopoietic chimerism and permanent skin allograft survival. Chimeric mice bearing intact skin allografts fail to generate effector/memory T cells against allogeneic targets as shown by the absence of IFNγ‐producing CD44^highCD8⁺ T cells and in vivo cytotoxicity. Depletion of Tregs by injection of anti‐CD4 or anti‐CD25 antibody prior to costimulation blockade prevents chimerism, shortens skin allograft survival and leads to generation of effector/memory cytotoxic T cells. Depletion of Tregs by injection of anti‐CD4 or anti‐CD25 antibody two months after transplantation leads to loss of skin allografts even though mice remain chimeric and exhibit little in vivo cytotoxicity. In contrast, chimerism is lost, but skin allografts survive following naïve T‐cell injection. We conclude that hematopoietic chimerism and peripheral tolerance may be maintained by different mechanisms in mixed hematopoietic chimeras.     

Graft‐Versus‐Host Disease Following Liver Transplantation: Development of a High‐Incidence Rat Model and a Selective Prevention Method

Graft‐versus‐host disease (GvHD) following liver transplantation (LT) is a rare but serious complication with no presently available animal model and no preventive measures. To develop a rat model of GvHD after LT (LT‐GvHD), we preconditioned hosts with sublethal irradiation plus reduction of natural killer (NK) cells with anti‐CD8α mAb treatment, which invariably resulted in acute LT‐GvHD. Compared with those in the peripheral counterpart, graft CD4⁺CD25^? passenger T cells showed lower alloreactivities in mixed leukocyte culture. Immunohistology revealed that donor CD4⁺ T cells migrated and formed clusters with host dendritic cells in secondary lymphoid organs, with early expansion and subsequent accumulation in target organs. For selectively preventing GvHD, donor livers were perfused ex vivo with organ preservation media containing anti‐TCRαβ mAb. T cell–depleted livers almost completely suppressed clinical GvHD such that host rats survived for >100 days. Our results showed that passenger T cells could develop typical LT‐GvHD if resistant cells such as host radiosensitive cells and host radioresistant NK cells were suppressed. Selective ex vivo T cell depletion prevented LT‐GvHD without affecting host immunity or graft function. This method might be applicable to clinical LT in prediagnosed high‐risk donor–recipient combinations and for analyzing immunoregulatory mechanisms of the liver.     

Anti‐LFA‐1 or rapamycin overcome costimulation blockade‐resistant rejection in sensitized bone marrow recipients

While costimulation blockade‐based mixed chimerism protocols work well for inducing tolerance in rodents, translation to preclinical large animal/nonhuman primate models has been less successful. One recognized cause for these difficulties is the high frequency of alloreactive memory T cells (Tmem) found in the (pre)clinical setting as opposed to laboratory mice. In the present study, we therefore developed a murine bone marrow transplantation (BMT) model employing recipients harboring polyclonal donor‐reactive Tmem without concomitant humoral sensitization. This model was then used to identify strategies to overcome this additional immune barrier. We found that B6 recipients that were enriched with 3 × 10⁷ T cells isolated from B6 mice that had been previously grafted with Balb/c skin, rejected Balb/c BM despite costimulation blockade with anti‐CD40L and CTLA4Ig (while recipients not enriched developed chimerism). Adjunctive short‐term treatment of sensitized BMT recipients with rapamycin or anti‐LFA‐1 mAb was demonstrated to be effective in controlling Tmem in this model, leading to long‐term mixed chimerism and donor‐specific tolerance. Thus, rapamycin and anti‐LFA‐1 mAb are effective in overcoming the potent barrier that donor‐reactive Tmem pose to the induction of mixed chimerism and tolerance despite costimulation blockade.     

CD8 T cell of donor splenocyte mixed with bone marrow cells is more effective than CD4 T cell for induction of donor-specific tolerance in sublethally irradiated mice

BACKGROUND: We previously demonstrated that even a low dose of bone marrow cells (BMCs) established donor-specific tolerance if mixed with splenocytes (SPLCs). In this study, T-cell subsets CD4 (CD4SP) and CD8 (CD8SP) of donor SPLCs were investigated for their contribution to the enhancement of BMC engraftment leading to donor-specific tolerance in sublethally irradiated mice. METHODS: Sublethally irradiated C57BL/6 recipient mice were intravenously injected BMCs mixing with CD4SP or CD8SP harvested from BALB/c donor mice. The degree of chimerism in the peripheral blood lymphocytes (PBLs) and in the SPLCs was analyzed using FACS, mixed lymphocyte reaction, and skin graft transplantation 3 months after injection. RESULTS: Recipients injected with 3 x 10(6) donor BMCs admixed with 10 x 10(6) donor CD8SP established chimerism. However, recipients injected with the same dose of BMCs admixed with 5 x 10(6) CD4SP, 10 x 10(6) CD4SP, and 5 x 10(6) CD8SP did not established chimerism. CD8SP contained 44% of Ly6A/E (Stem Cell Antigen-1 (Sca-1))-positive cells based on FACS analysis, whereas only 6% of CD4SP were positive for Ly6A/E. MLR supernates of donor SPLCs chimeric mice using admixture with CD8SP dominated by Th2 cytokines. In contrast, mixting with MLR supernates from failed chimera showed dominant Th1 cytokines. CONCLUSIONS: CD8SP seems to make a major contribution to enhance BMC engraftment and induce donor-specific tolerance. Ly6A/E (Sca-1)-positive cells need to be further investigated for their contribution to the establishment of chimerism.     

Efficacy of donor splenocytes mixed with bone marrow cells for induction of tolerance in sublethally irradiated mice

BACKGROUND: High dose of bone marrow cells (BMCs) has been reported to be essential to establish donor-specific tolerance. In clinical settings, a large quantity of BMCs is very difficult to be obtained. Our previous report demonstrated that even a low dose of BMCs could establish donor-specific tolerance if mixed with splenocytes (SPLCs). In the present study, various components of SPLCs were purified or removed and were investigated their contribution for enhancement of bone marrow engraftment leading to donor-specific tolerance in sublethally irradiated mice. METHODS: Sublethally irradiated C57BL/6 recipient mice were intravenously injected 3 x 10(6) BMCs mixed with various components and various numbers of SPLCs harvested from BALB/c donor mice. One week after injection, skin grafting was performed. The degree of chimerism in peripheral blood lymphocytes (PBLs) and in SPLCs was analyzed by FACS 3 months after transplantation. RESULTS: Recipients receiving 3 X 106 BMCs mixed with 10 x 10(6) T cell-enriched SPLCs established chimerism. Recipients receiving BMCs mixed with macrophage-depleted SPLCs also showed chimeirism and donor-specific tolerance. B cell-enriched SPLCs did not help small dose of BMCs to establish chimerism. Irradiated SPLCs were not effective to induce tolerance even with additional infusion to recipients. CONCLUSIONS: Active effects of splenic T cells were more important to help engraftment of small dose of BMCs than B cells, but the interaction between T and B cells might play some roles to enhance BMC engraftment. Splenic macrophages or dendritic cells might have some adverse effects against tolerance induction. Fatal graft-versus-host disease (GVHD) might be avoided by depleting adherent cells from SPLCs, so macrophages or dendritic cells were also considered as key components to induce donor-specific tolerance and prevent GVHD in this model.     

A Novel Xenogeneic Graft‐Versus‐Host Disease Model for Investigating the Pathological Role of Human CD4+ or CD8+ T Cells Using Immunodeficient NOG Mice

Graft‐versus‐host disease (GVHD) is a major complication of allogenic bone marrow transplantation and involves the infiltration of donor CD4⁺ and/or CD8⁺ T cells into various organs of the recipient. The pathological role of human CD4⁺ and CD8⁺ T cells in GVHD remains controversial. In this study, we established two novel xenogeneic (xeno)‐GVHD models. Human CD4⁺ or CD8⁺ T cells were purified from peripheral blood and were transplanted into immunodeficient NOD/Shi‐scid IL2rg^null (NOG) mice. Human CD8⁺ T cells did not induce major GVHD symptoms in conventional NOG mice. However, CD8⁺ T cells immediately proliferated and induced severe GVHD when transferred into NOG mice together with at least 0.5 × 10⁶ CD4⁺ T cells or into NOG human interleukin (IL)‐2 transgenic mice. Human CD4⁺ T cell–transplanted NOG mice developed skin inflammations including alopecia, epidermal hyperplasia, and neutrophilia. Pathogenic T helper (Th)17 cells accumulated in the skin of CD4⁺ T cell–transplanted NOG mice. Further, an anti‐human IL‐17 antibody (secukinumab) significantly suppressed these skin pathologies. These results indicate that pathogenic human Th17 cells induce cutaneous GVHD via IL‐17–dependent pathways. This study provides fundamental insights into the pathogenesis of xeno‐GVHD, and these humanized mouse models may be useful as preclinical tools for the prevention of GVHD.     

Alloreactive natural killer cells promote haploidentical hematopoietic stem cell transplantation by expansion of recipient‐derived CD4+CD25+ regulatory T cells

Alloreactive NK cells (Allo‐NKs) have been shown to exert advantageous effects on the outcomes of haploidentical hematopoietic stem cell transplantation (Haplo‐HSCT) for cancer treatment. However, the mechanisms of action of Allo‐NKs remain unclear. We established a novel Haplo‐HSCT conditioning regimen composed of Allo‐NKs and a low dose of immunosuppressive drugs (Allo‐NKs + Chemo) to investigate alternative mechanisms besides direct cytotoxicity. The inhibitory effects of different cell subsets on the donor–recipient mixed lymphocyte reactions (MLRs) were evaluated after Haplo‐HSCT. The quantities and functions of CD4⁺CD25⁺ regulatory T cells (Tregs) and dendritic cells (DCs) in the spleen and the thymus were examined. Our results showed that the Allo‐NKs + Chemo regimen induced systemic tolerance, and that CD4⁺CD25⁺ Tregs played a significant role in inducing and maintaining systemic tolerance after Haplo‐HSCT. Alloreactive NK cells promoted the expansion of recipient‐derived CD4⁺CD25⁺CD127^? Tregs in the thymus and the spleen which could be amplified in vitro by the immature donor‐derived DC subset isolated from the thymus of Allo‐NKs + Chemo‐treated mice. Our findings suggested that Allo‐NKs are capable of inducing systemic tolerance after Haplo‐HSCT by assembling donor‐derived immature DCs to expand recipient‐derived Treg cells in the thymus.     

UVB‐induced depletion of donor‐derived dendritic cells prevents allograft rejection of immune‐privileged hair follicles in humanized mice

Dendritic cells (DCs) are key targets for immunity and tolerance induction; they present donor antigens to recipient T cells by donor‐ and recipient‐derived pathways. Donor‐derived DCs, which are critical during the acute posttransplant period, can be depleted in graft tissue by forced migration via ultraviolet B light (UVB) irradiation. Here, we investigated the tolerogenic potential of donor‐derived DC depletion through in vivo and ex vivo UVB preirradiation (UV) combined with the injection of anti‐CD154 antibody (Ab) into recipients in an MHC‐mismatched hair follicle (HF) allograft model in humanized mice. Surprisingly, human HF allografts achieved long‐term survival with newly growing pigmented hair shafts in both Ab‐treated groups (Ab‐only and UV plus Ab) and in the UV‐only group, whereas the control mice rejected all HF allografts with no hair regrowth. Perifollicular human CD3⁺ T cell and MHC class II⁺ cell infiltration was significantly diminished in the presence of UV and/or Ab treatment. HF allografts in the UV‐only group showed stable maintenance of the immune privilege in the HF epithelium without evidence of antigen‐specific T cell tolerance, which is likely promoted by normal HFs in vivo. This immunomodulatory strategy targeting the donor tissue exhibited novel biological relevance for clinical allogeneic transplantation without generalized immunosuppression.     

Recipient Dendritic Cells,But Not B Cells,Are Required Antigen‐Presenting Cells for Peripheral Alloreactive CD8+ T‐Cell Tolerance

Induction of mixed allogeneic chimerism is a promising approach for achieving donor‐specific tolerance, thereby obviating the need for life‐long immunosuppression for solid organ allograft acceptance. In mice receiving a low dose (3Gy) of total body irradiation, allogeneic bone marrow transplantation combined with anti‐CD154 tolerizes peripheral CD4 and CD8 T cells, allowing achievement of mixed chimerism with specific tolerance to donor. With this approach, peripheral CD8 T‐cell tolerance requires recipient MHC class II, CD4 T cells, B cells and DCs. Recipient‐type B cells from chimeras that were tolerant to donor still promoted CD8 T‐cell tolerance, but their role could not be replaced by donor‐type B cells. Using recipients whose B cells or DCs specifically lack MHC class I and/or class II or lack CD80 and CD86, we demonstrate that dendritic cells (DCs) must express CD80/86 and either MHC class I or class II to promote CD8 tolerance. In contrast, B cells, though required, did not need to express MHC class I or class II or CD80/86 to promote CD8 tolerance. Moreover, recipient IDO and IL‐10 were not required. Thus, antigen presentation by recipient DCs and not by B cells is critical for peripheral alloreactive CD8 T cell tolerance.     

Ex Vivo Costimulatory Blockade to Generate Regulatory T Cells From Patients Awaiting Kidney Transplantation

Short‐term outcomes of kidney transplantation have improved dramatically, but chronic rejection and regimen‐related toxicity continue to compromise overall patient outcomes. Development of regulatory T cells (Tregs) as a means to decrease alloresponsiveness and limit the need for pharmacologic immunosuppression is an active area of preclinical and clinical investigation. Nevertheless, the immunomodulatory effects of end‐stage renal disease on the efficacy of various strategies to generate and expand recipient Tregs for kidney transplantation are incompletely characterized. In this study, we show that Tregs can be successfully generated from either freshly isolated or previously cryopreserved uremic recipient (responder) and healthy donor (stimulator) peripheral blood mononuclear cells using the strategy of ex vivo costimulatory blockade with belatacept during mixed lymphocyte culture. Moreover, these Tregs maintain a CD3⁺CD4⁺CD25⁺CD127^lo surface phenotype, high levels of intracellular FOXP3 and significant demethylation of the FOXP3 Treg‐specific demethylation region on allorestimulation with donor stimulator cells. These data support evaluation of this simple, brief Treg production strategy in clinical trials of mismatched kidney transplantation.     

Receptor tyrosine kinase MerTK suppresses an allogenic type I IFN response to promote transplant tolerance

Recipient infusion of donor apoptotic cells is an emerging strategy for inducing robust transplantation tolerance. Daily clearance of billions of self‐apoptotic cells relies on homeostatic engagement of phagocytic receptors, in particular, receptors of the tyrosine kinase family TAM (Tyro3, Axl, and MerTK), to maintain self‐tolerance. However, an outstanding question is if allogeneic apoptotic cells trigger the same receptor system for inducing allogeneic tolerance. Here, we employed allogeneic apoptotic splenocytes and discovered that the efferocytic receptor MerTK on recipient phagocytes is a critical mediator for transplantation tolerance induced by this strategy. Our findings indicate that the tolerogenic properties of allogeneic apoptotic splenocytes require MerTK transmission of intracellular signaling to suppress the production of inflammatory cytokine interferon α (IFN‐α). We further demonstrate that MerTK is crucial for subsequent expansion of myeloid‐derived suppressor cells and for promoting their immunomodulatory function, including maintaining graft‐infiltrating CD4⁺CD25⁺Foxp3⁺ regulatory T cells. Consequently, recipient MerTK deficiency resulted in failure of tolerance by donor apoptotic cells, and this failure could be effectively rescued by IFN‐α receptor blockade. These findings underscore the importance of the efferocytic receptor MerTK in mediating transplantation tolerance by donor apoptotic cells and implicate MerTK agonism as a promising target for promoting transplantation tolerance.