Donor‐specific chimeric antigen receptor Tregs limit rejection in naive but not sensitized allograft recipients

Cell therapy with autologous donor‐specific regulatory T cells (Tregs) is a promising strategy to minimize immunosuppression in transplant recipients. Chimeric antigen receptor (CAR) technology has recently been used successfully to generate donor‐specific Tregs and overcome the limitations of enrichment protocols based on repetitive stimulations with alloantigens. However, the ability of CAR‐Treg therapy to control alloreactivity in immunocompetent recipients is unknown. We first analyzed the effect of donor‐specific CAR Tregs on alloreactivity in naive, immunocompetent mice receiving skin allografts. Tregs expressing an irrelevant or anti‐HLA‐A2‐specific CAR were administered to Bl/6 mice at the time of transplanting an HLA‐A2⁺ Bl/6 skin graft. Donor‐specific CAR‐Tregs, but not irrelevant‐CAR Tregs, significantly delayed skin rejection and diminished donor‐specific antibodies (DSAs) and frequencies of DSA‐secreting B cells. Donor‐specific CAR‐Treg–treated mice also had a weaker recall DSA response, but normal responses to an irrelevant antigen, demonstrating antigen‐specific suppression. When donor‐specific CAR Tregs were tested in HLA‐A2‐sensitized mice, they were unable to delay allograft rejection or diminish DSAs. The finding that donor‐specific CAR‐Tregs restrain de novo but not memory alloreactivity has important implications for their use as an adoptive cell therapy in transplantation.     

Expression of a Chimeric Antigen Receptor Specific for Donor HLA Class I Enhances the Potency of Human Regulatory T Cells in Preventing Human Skin Transplant Rejection

Regulatory T cell (Treg) therapy using recipient‐derived Tregs expanded ex vivo is currently being investigated clinically by us and others as a means of reducing allograft rejection following organ transplantation. Data from animal models has demonstrated that adoptive transfer of allospecific Tregs offers greater protection from graft rejection compared to polyclonal Tregs. Chimeric antigen receptors (CAR) are clinically translatable synthetic fusion proteins that can redirect the specificity of T cells toward designated antigens. We used CAR technology to redirect human polyclonal Tregs toward donor‐MHC class I molecules, which are ubiquitously expressed in allografts. Two novel HLA‐A2‐specific CARs were engineered: one comprising a CD28‐CD3ζ signaling domain (CAR) and one lacking an intracellular signaling domain (ΔCAR). CAR Tregs were specifically activated and significantly more suppressive than polyclonal or ΔCAR Tregs in the presence of HLA‐A2, without eliciting cytotoxic activity. Furthermore, CAR and ΔCAR Tregs preferentially transmigrated across HLA‐A2‐expressing endothelial cell monolayers. In a human skin xenograft transplant model, adoptive transfer of CAR Tregs alleviated the alloimmune‐mediated skin injury caused by transferring allogeneic peripheral blood mononuclear cells more effectively than polyclonal Tregs. Our results demonstrated that the use of CAR technology is a clinically applicable refinement of Treg therapy for organ transplantation.     

In Vivo Development of Transplant Arteriosclerosis in Humanized Mice Reflects Alloantigen Recognition and Peripheral Treg Phenotype of Lung Transplant Recipients

Experimentally, regulatory T cells inhibit rejection. In clinical transplantations, however, it is not known whether T cell regulation is the cause for, or an epiphenomenon of, long‐term allograft survival. Here, we study naïve and alloantigen‐primed T cell responses of clinical lung transplant recipients in humanized mice. The pericardiophrenic artery procured from human lung grafts was implanted into the aorta of NODrag^?/?/IL‐2rγc^?/? mice reconstituted with peripheral blood mononuclear cells (PBMCs) from the respective lung recipient. Naïve or primed allogeneic PBMCs procured 21 days post–lung transplantation with or without enriching for CD4⁺CD25^high T cells were used. Transplant arteriosclerosis was assessed 28 days later by histology. Mice reconstituted with alloantigen‐primed PBMCs showed significantly more severe transplant arteriosclerosis than did mice with naïve PBMCs (p = 0.005). Transplant arteriosclerosis was equally suppressed by enriching for autologous naïve (p = 0.012) or alloantigen‐primed regulatory T cells (Tregs) (p = 0.009). Alloantigen priming in clinical lung recipients can be adoptively transferred into a humanized mouse model. Transplant arteriosclerosis elicited by naïve or alloantigen‐primed PBMCs can be similarly controlled by potent autologous Tregs. Cellular therapy with expanded autologous Tregs in lung transplantation might be a promising future strategy.     

Siplizumab selectively depletes effector memory T cells and promotes a relative expansion of alloreactive regulatory T cells in vitro

Siplizumab, a humanized anti‐CD2 monoclonal antibody, has been used in conditioning regimens for hematopoietic cell transplantation and tolerance induction with combined kidney‐bone marrow transplantation. Siplizumab‐based tolerance induction regimens deplete T cells globally while enriching regulatory T cells (Tregs) early posttransplantation. Siplizumab inhibits allogeneic mixed‐lymphocyte reactions (MLRs) in vitro. We compared the impact of siplizumab on Tregs versus other T cell subsets in HLA‐mismatched allogeneic MLRs using PBMCs. Siplizumab predominantly reduced the percentage of CD4⁺ and CD8⁺ effector memory T cells, which express higher CD2 levels than naïve T cells or resting Tregs. Conversely, siplizumab enriched proliferating CD45RA^? FoxP3^HI cells in MLRs. FoxP3 expression was stable over time in siplizumab‐containing cultures, consistent with enrichment for bona fide Tregs. Consistently, high‐throughput TCRβ CDR3 sequencing of sorted unstimulated and proliferating T cells in MLRs revealed selective expansion of donor‐reactive Tregs along with depletion of donor‐reactive CD4⁺ effector/memory T cells in siplizumab‐containing MLRs. These results indicate that siplizumab may have immunomodulatory functions that may contribute to its success in tolerance‐inducing regimens. Our studies also confirm that naïve in addition to effector/memory T cells contribute to the allogeneic MLR and mandate further investigation of the impact of siplizumab on alloreactive naïve T cells.     

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.     

Rejection of xenogeneic porcine islets in humanized mice is characterized by graft‐infiltrating Th17 cells and activated B cells

Xenogeneic porcine islet transplantation is a promising potential therapy for type 1 diabetes (T1D). Understanding human immune responses against porcine islets is crucial for the design of optimal immunomodulatory regimens for effective control of xenogeneic rejection of porcine islets in humans. Humanized mice are a valuable tool for studying human immune responses and therefore present an attractive alternative to human subject research. Here, by using a pig‐to‐humanized mouse model of xenogeneic islet transplantation, we described the human immune response to transplanted porcine islets, a process characterized by dense islet xenograft infiltration of human CD45⁺ cells comprising activated human B cells, CD4⁺CD44⁺IL‐17⁺ Th17 cells, and CD68⁺ macrophages. In addition, we tested an experimental immunomodulatory regimen in promoting long‐term islet xenograft survival, a triple therapy consisting of donor splenocytes treated with ethylcarbodiimide (ECDI‐SP), and peri‐transplant rituximab and rapamycin. We observed that the triple therapy effectively inhibited graft infiltration of T and B cells as well as macrophages, promoted transitional B cells both in the periphery and in the islet xenografts, and provided a superior islet xenograft protection. Our study therefore indicates an advantage of donor ECDI‐SP treatment in controlling human immune cells in promoting long‐term islet xenograft survival.     

Pancreatic islets engineered with a FasL protein induce systemic tolerance at the induction phase that evolves into long‐term graft‐localized immune privilege

We have previously shown that pancreatic islets engineered to transiently display a modified form of FasL protein (SA‐FasL) on their surface survive indefinitely in allogeneic recipients without a need for chronic immunosuppression. Mechanisms that confer long‐term protection to allograft are yet to be elucidated. We herein demonstrated that immune protection evolves in two distinct phases; induction and maintenance. SA‐FasL‐engineered allogeneic islets survived indefinitely and conferred protection to a second set of donor‐matched, but not third‐party, unmanipulated islet grafts simultaneously transplanted under the contralateral kidney capsule. Protection at the induction phase involved a reduction in the frequency of proliferating alloreactive T cells in the graft‐draining lymph nodes, and required phagocytes and TGF‐β. At the maintenance phase, immune protection evolved into graft site‐restricted immune privilege as the destruction of long‐surviving SA‐FasL‐islet grafts by streptozotocin followed by the transplantation of a second set of unmanipulated islet grafts into the same site from the donor, but not third party, resulted in indefinite survival. The induced immune privilege required both CD4⁺CD25⁺Foxp3⁺ Treg cells and persistent presence of donor antigens. Engineering cell and tissue surfaces with SA‐FasL protein provides a practical, efficient, and safe means of localized immunomodulation with important implications for autoimmunity and transplantation.     

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.     

CMV‐infected kidney grafts drive the expansion of blood‐borne CMV‐specific T cells restricted by shared class I HLA molecules via presentation on donor cells

We aimed to determine the role of cytomegalovirus (CMV)‐infected donor cells in the development of a CMV‐specific immune response in kidney transplant recipients. We assessed the CMV pp65‐specific immune response by using interferon‐? ELISPOT and dextramers in peripheral blood mononuclear cells from 115 recipients (D+R? 31, D+R + 44, D?R + 40) late after transplantation (mean 59 ± 42 months). Receiving a kidney from a D+ donor resulted in a higher number of IFN‐?‐producing anti‐CMV T cells (P = .004). This effect disappeared with the absence of shared HLA class I specificities between donors and recipients (P = .430). To confirm the role of donor cells in stimulating the expansion of newly developed CMV‐specific CD8⁺ T cells after transplantation, we compared the number of HLA‐A2–restricted CMV‐specific CD8⁺ T cells in primo‐infected recipients who received an HLA‐A2 or non–HLA‐A2 graft. The median of anti‐CMV pp65 T cells restricted by HLA‐A2 was very low for patients who received a non–HLA‐A2 graft vs an HLA‐A2 graft (300 [0‐14638] vs. 17972 [222‐85594] anti‐CMV pp65 CD8⁺ T cells/million CD8⁺ T cells, P = .001). This adds new evidence that CMV‐infected kidney donor cells present CMV peptides and drive an inflation of memory CMV‐specific CD8⁺ T cells, likely because of frequent CMV replications within the graft.     

Origin of Enriched Regulatory T Cells in Patients Receiving Combined Kidney–Bone Marrow Transplantation to Induce Transplantation Tolerance

We examined tolerance mechanisms in patients receiving HLA‐mismatched combined kidney–bone marrow transplantation (CKBMT) that led to transient chimerism under a previously published nonmyeloablative conditioning regimen (Immune Tolerance Network study 036). Polychromatic flow cytometry and high‐throughput sequencing of T cell receptor‐β hypervariable regions of DNA from peripheral blood regulatory T cells (Tregs) and CD4 non‐Tregs revealed marked early enrichment of Tregs (CD3⁺CD4⁺CD25^highCD127^lowFoxp3⁺) in blood that resulted from peripheral proliferation (Ki67⁺), possibly new thymic emigration (CD31⁺), and, in one tolerant subject, conversion from non‐Tregs. Among recovering conventional T cells, central memory CD4⁺ and CD8⁺ cells predominated. A large proportion of the T cell clones detected in posttransplantation biopsy specimens by T cell receptor sequencing were detected in the peripheral blood and were not donor‐reactive. Our results suggest that enrichment of Tregs by new thymic emigration and lymphopenia‐driven peripheral proliferation in the early posttransplantation period may contribute to tolerance after CKBMT. Further, most conventional T cell clones detected in immunologically quiescent posttransplantation biopsy specimens appear to be circulating cells in the microvasculature rather than infiltrating T cells.     

The Leuven Immunomodulatory Protocol Promotes T‐Regulatory Cells and Substantially Prolongs Survival After First Intestinal Transplantation

Intestinal transplantation (ITx) remains challenged by frequent/severe rejections and immunosuppression‐related complications (infections/malignancies/drug toxicity). We developed the Leuven Immunomodulatory Protocol (LIP) in the lab and translated it to the clinics. LIP consists of experimentally proven maneuvers, destined to promote T‐regulatory (Tregs)‐dependent graft‐protective mechanisms: donor‐specific blood transfusion (DSBT); avoiding high‐dose steroids/calcineurin‐inhibitors; and minimizing reperfusion injury and endotoxin translocation. LIP was tested in 13 consecutive ITx from deceased donors (2000–2014) (observational cohort study). Recipient age was 37 years (2.8–57 years). Five‐year graft/patient survival was 92%. One patient died at 9 months due to aspergillosis, another at 12 years due to nonsteroidal anti‐inflammatory drug–induced enteropathy. Early acute rejection (AR) developed in two (15%); late AR in three (23%); all were reversible. No chronic rejection (CR) occurred. No malignancies developed and estimated glomerular filtration rate remained stable post‐Tx. At last follow‐up (3.5 years [0.5–12.5 years]), no donor‐specific antibodies were detected and 11 survivors were total parenteral nutrition free with a Karnofsky score >90% in 8 recipients (follow‐up >1 years). A high frequency of circulating CD4⁺CD45RA^‐Foxp3^hi memory Tregs was found (1.8% [1.39–2.21]), comparable to tolerant kidney transplant (KTx) recipients and superior to stable immunosuppression (IS)‐KTx, KTx with CR, and healthy volunteers. In this ITx cohort we show that DSBT in a low‐inflammatory/pro‐regulatory environment activates Tregs at levels similar to tolerant‐KTx, without causing sensitization. LIP limits rejection under reduced IS and thereby prolongs long‐term survival to an extent not previously attained after ITx.     

Rapamycin‐Conditioned Donor Dendritic Cells Differentiate CD4+CD25+Foxp3+ T Cells In Vitro with TGF‐β1 for Islet Transplantation

Dendritic cells (DCs) conditioned with the mammalian target of rapamycin (mTOR) inhibitor rapamycin have been previously shown to expand naturally existing regulatory T cells (nTregs). This work addresses whether rapamycin‐conditioned donor DCs could effectively induce CD4⁺CD25⁺Foxp3⁺ Tregs (iTregs) in cell cultures with alloantigen specificities, and whether such in vitro‐differentiated CD4⁺CD25⁺Foxp3⁺ iTregs could effectively control acute rejection in allogeneic islet transplantation. We found that donor BALB/c bone marrow‐derived DCs (BMDCs) pharmacologically modified by the mTOR inhibitor rapamycin had significantly enhanced ability to induce CD4⁺CD25⁺Foxp3⁺ iTregs of recipient origin (C57BL/6 (B6)) in vitro under Treg driving conditions compared to unmodified BMDCs. These in vitro‐induced CD4⁺CD25⁺Foxp3⁺ iTregs exerted donor‐specific suppression in vitro, and prolonged allogeneic islet graft survival in vivo in RAG^?/‐ hosts upon coadoptive transfer with T‐effector cells. The CD4⁺CD25⁺Foxp3⁺ iTregs expanded and preferentially maintained Foxp3 expression in the graft draining lymph nodes. Finally, the CD4⁺CD25⁺Foxp3⁺ iTregs were further able to induce endogenous naïve T cells to convert to CD4⁺CD25⁺Foxp3⁺ T cells. We conclude that rapamycin‐conditioned donor BMDCs can be exploited for efficient in vitro differentiation of donor antigen‐specific CD4⁺CD25⁺Foxp3⁺ iTregs. Such in vitro‐generated donor‐specific CD4⁺CD25⁺Foxp3⁺ iTregs are able to effectively control allogeneic islet graft rejection.     

Adoptive Transfer of Tracer‐Alloreactive CD4+ T Cell Receptor Transgenic T Cells Alters the Endogenous Immune Response to an Allograft

T cell receptor transgenic (TCR‐Tg) T cells are often used as tracer populations of antigen‐specific responses to extrapolate findings to endogenous T cells. The extent to which TCR‐Tg T cells behave purely as tracer cells or modify the endogenous immune response is not clear. To test the impact of TCR‐Tg T cell transfer on endogenous alloimmunity, recipient mice were seeded with CD4⁺ or CD8⁺ TCR‐Tg or polyclonal T cells at the time of cardiac allograft transplantation. Only CD4⁺ TCR‐Tg T cells accelerated rejection and, unexpectedly, led to a dose‐dependent decrease in both transferred and endogenous T cells infiltrating the graft. In contrast, recipients of CD4⁺ TCR‐Tg T cells exhibited enhanced endogenous donor‐specific CD8⁺ T cell activation in the spleen and accelerated alloantibody production. Introduction of CD4⁺ TCR‐Tg T cells also perturbed the intragraft accumulation of innate cell populations. Transferred CD4⁺ TCR‐Tg T cells alter many aspects of endogenous alloimmunity, suggesting that caution should be used when interpreting experiments using these adoptively transferred cells because the overall nature of allograft rejection may be altered. These results also may have implications for adoptive CD4⁺ T cell immunotherapy in tumor and infectious clinical settings because cell infusion may have additional effects on natural immune responses.     

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.     

Allergic Conjunctivitis Renders CD4+ T Cells Resistant to T Regulatory Cells and Exacerbates Corneal Allograft Rejection

Allergic diseases rob corneal allografts of immune privilege and increase immune rejection. Corneal allograft rejection in BALB/c allergic hosts was analyzed using a short ragweed (SWR) pollen model of allergic conjunctivitis. Allergic conjunctivitis did not induce exaggerated T‐cell responses to donor C57BL/6 (B6) alloantigens or stimulate cytotoxic T lymphocyte (CTL) responses. Allergic conjunctivitis did affect T regulatory cells (Tregs) that support graft survival. Exogenous IL‐4, but not IL‐5 or IL‐13, prevented Treg suppression of CD4⁺ effector T cells isolated from naïve mice. However, mice with allergic conjunctivitis developed Tregs that suppressed CD4⁺ effector T‐cell proliferation. In addition, IL‐4 did not inhibit Treg suppression of IL‐4Rα^?/? CD4⁺ T‐cell responses, suggesting that IL‐4 rendered effector T cells resistant to Tregs. SRW‐sensitized IL‐4Rα^?/? mice displayed the same 50% graft survival as nonallergic WT mice, that was significantly less than the 100% rejection that occurred in allergic WT hosts, supporting the role of IL‐4 in the abrogation of immune privilege. Moreover, exacerbation of corneal allograft rejection in allergic mice was reversed by administering anti‐IL‐4 antibody. Thus, allergy‐induced exacerbation of corneal graft rejection is due to the production of IL‐4, which renders effector T cells resistant to Treg suppression of alloimmune responses.     

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.     

PD‐1 expression on CD8+ T cells regulates their differentiation within lung allografts and is critical for tolerance induction

Immunological requirements for rejection and tolerance induction differ between various organs. While memory CD8⁺ T cells are considered a barrier to immunosuppression‐mediated acceptance of most tissues and organs, tolerance induction after lung transplantation is critically dependent on central memory CD8⁺ T lymphocytes. Here we demonstrate that costimulation blockade‐mediated tolerance after lung transplantation is dependent on programmed cell death 1 (PD‐1) expression on CD8⁺ T cells. In the absence of PD‐1 expression, CD8⁺ T cells form prolonged interactions with graft‐infiltrating CD11c⁺ cells; their differentiation is skewed towards an effector memory phenotype and grafts are rejected acutely. These findings extend the notion that requirements for tolerance induction after lung transplantation differ from other organs. Thus, immunosuppressive strategies for lung transplant recipients need to be tailored based on the unique immunological properties of this organ.     

Combined Anti‐CD154/CTLA4Ig Costimulation Blockade‐Based Therapy Induces Donor‐Specific Tolerance to Vascularized Osteomyocutaneous Allografts

Tolerance induction by means of costimulation blockade has been successfully applied in solid organ transplantation; however, its efficacy in vascularized composite allotransplantation, containing a vascularized bone marrow component and thus a constant source of donor‐derived stem cells, remains poorly explored. In this study, osteomyocutaneous allografts (alloOMCs) from Balb/c (H2^d) mice were transplanted into C57BL/6 (H2^b) recipients. Immunosuppression consisted of 1 mg anti‐CD154 on day 0, 0.5 mg CTLA4Ig on day 2 and rapamycin (RPM; 3 mg/kg per day from days 0–7, then every other day for 3 weeks). Long‐term allograft survival, donor‐specific tolerance and donor–recipient cell trafficking were evaluated. Treatment with costimulation blockade plus RPM resulted in long‐term graft survival (>120 days) of alloOMC in 12 of 15 recipients compared with untreated controls (median survival time [MST] ≈10.2 ± 0.8 days), RPM alone (MST ≈33 ± 5.5 days) and costimulation blockade alone (MST ≈45.8 ± 7.1 days). Donor‐specific hyporesponsiveness in recipients with viable grafts was demonstrated in vitro. Evidence of donor‐specific tolerance was further assessed in vivo by secondary donor‐specific skin graft survival and third‐party graft rejection. A significant increase of Foxp3⁺ regulatory T cells was evident in tolerant animals. Donor cells populated peripheral blood, thymus, and both donor and recipient bone marrow. Consequently, combined anti‐CD154/CTLA4Ig costimulation blockade‐based therapy induces donor‐specific tolerance in a stringent murine alloOMC transplant model.