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.     

Pathogen Stimulation History Impacts Donor‐Specific CD8+ T Cell Susceptibility to Costimulation/Integrin Blockade–Based Therapy

Recent studies have shown that the quantity of donor‐reactive memory T cells is an important factor in determining the relative heterologous immunity barrier posed during transplantation. Here, we hypothesized that the quality of T cell memory also potently influences the response to costimulation blockade‐based immunosuppression. Using a murine skin graft model of CD8⁺ memory T cell–mediated costimulation blockade resistance, we elicited donor‐reactive memory T cells using three distinct types of pathogen infections. Strikingly, we observed differential efficacy of a costimulation and integrin blockade regimen based on the type of pathogen used to elicit the donor‐reactive memory T cell response. Intriguingly, the most immunosuppression‐sensitive memory T cell populations were composed primarily of central memory cells that possessed greater recall potential, exhibited a less differentiated phenotype, and contained more multi‐cytokine producers. These data, therefore, demonstrate that the memory T cell barrier is dependent on the specific type of pathogen infection via which the donor‐reactive memory T cells are elicited, and suggest that the immune stimulation history of a given transplant patient may profoundly influence the relative barrier posed by heterologous immunity during transplantation.     

Selective CD28 Blockade Results in Superior Inhibition of Donor‐Specific T Follicular Helper Cell and Antibody Responses Relative to CTLA4‐Ig

Donor‐specific antibodies (DSAs) are a barrier to improved long‐term outcomes after kidney transplantation. Costimulation blockade with CTLA4‐Ig has shown promise as a potential therapeutic strategy to control DSAs. T follicular helper (Tfh) cells, a subset of CD4⁺ T cells required for optimal antibody production, are reliant on the CD28 costimulatory pathway. We have previously shown that selective CD28 blockade leads to superior allograft survival through improved control of CD8⁺ T cells relative to CTLA4‐Ig, but the impact of CD28‐specific blockade on CD4⁺ Tfh cells is unknown. Thus, we identified and characterized donor‐reactive Tfh cells in a murine skin transplant model and then used this model to evaluate the impact of selective CD28 blockade with an anti‐CD28 domain antibody (dAb) on the donor‐specific Tfh cell–mediated immune response. We observed that the anti‐CD28 dAb led to superior inhibition of donor‐reactive CXCR5⁺PD‐1^high Tfh cells, CD95⁺GL7⁺ germinal center B cells and DSA formation compared with CTLA4‐Ig. Interestingly, donor‐reactive Tfh cells differentially upregulated CTLA4 expression, suggesting an important role for CTLA4 in mediating the superior inhibition observed with the anti‐CD28 dAb. Therefore, selective CD28 blockade as a novel approach to control Tfh cell responses and prevent DSA after kidney transplantation warrants further study.     

The Effect of MHC Antigen Matching Between Donors and Recipients on Skin Tolerance of Vascularized Composite Allografts

The emergence of skin‐containing vascularized composite allografts (VCAs) has provided impetus to understand factors affecting rejection and tolerance of skin. VCA tolerance can be established in miniature swine across haploidentical MHC barriers using mixed chimerism. Because the deceased donor pool for VCAs does not permit MHC antigen matching, clinical VCAs are transplanted across varying MHC disparities. We investigated whether sharing of MHC class I or II antigens between donors and recipients influences VCA skin tolerance. Miniature swine were conditioned nonmyeloablatively and received hematopoietic stem cell transplants and VCAs across MHC class I (n = 3) or class II (n = 3) barriers. In vitro immune responsiveness was assessed, and VCA skin‐resident leukocytes were characterized by flow cytometry. Stable mixed chimerism was established in all animals. MHC class II–mismatched chimeras were tolerant of VCAs. MHC class I–mismatched animals, however, rejected VCA skin, characterized by infiltration of recipient‐type CD8⁺ lymphocytes. Systemic donor‐specific nonresponsiveness was maintained, including after VCA rejection. This study shows that MHC antigen matching influences VCA skin rejection and suggests that local regulation of immune tolerance is critical in long‐term acceptance of all VCA components. These results help elucidate novel mechanisms underlying skin tolerance and identify clinically relevant VCA tolerance strategies.     

Vascularized Osteomyocutaneous Allografts Are Permissive to Tolerance by Induction‐Based Immunomodulatory Therapy

Vascularized composite allografts (VCAs) are unique among transplanted organs in that they are composed of multiple tissues with disparate antigenic and immunologic properties. As the predominant indications for VCAs are non‐life‐threatening conditions, there is an immediate need to develop tolerance induction strategies and to elucidate the mechanisms of VCA rejection and tolerance using VCA‐specific animal models. In this study, we explore the effects of in vitro induced donor antigen‐specific CD4^?CD8^? double negative (DN) Treg‐based therapy, in a fully MHC mismatched mouse VCA such as a vascularized osteomyocutaneous as compared to a non‐VCA such as a full thickness skin (FTS) transplantation model to elucidate the unique features of VCA rejection and tolerance. We demonstrate that combined therapy with antigen‐induced CD4 derived DN Tregs and a short course of anti‐lymphocyte serum, rapamycin and IL‐2/Fc fusion protein results in donor‐specific tolerance to VCA, but not FTS allografts. Macrochimerism was detected in VCA but not FTS allograft recipients up to >60 days after transplantation. Moreover, a significant increase of CD4⁺Foxp3⁺ Tregs was found in the peripheral blood of tolerant VCA recipients. These data suggest that VCA are permissive to tolerance induced by DN Treg‐based induction therapy.

     

Characterization of Human CD8+TCR− Facilitating Cells In Vitro and In Vivo in a NOD/SCID/IL2rγnull Mouse Model

CD8⁺/TCR^? facilitating cells (FCs) in mouse bone marrow (BM) significantly enhance engraftment of hematopoietic stem/progenitor cells (HSPCs). Human FC phenotype and mechanism of action remain to be defined. We report, for the first time, the phenotypic characterization of human FCs and correlation of phenotype with function. Approximately half of human FCs are CD8⁺/TCR^?/CD56 negative (CD56^neg); the remainder are CD8⁺/TCR^?/CD56 bright (CD56^bright). The CD56^neg FC subpopulation significantly promotes homing of HSPCs to BM in nonobese diabetic/severe combined immunodeficiency/IL‐2 receptor γ‐chain knockout mouse recipients and enhances hematopoietic colony formation in vitro. The CD56^neg FC subpopulation promotes rapid reconstitution of donor HSPCs without graft‐versus‐host disease (GVHD); recipients of CD56^bright FCs plus HSPCs exhibit low donor chimerism early after transplantation, but the level of chimerism significantly increases with time. Recipients of HSPCs plus CD56^neg or CD56^bright FCs showed durable donor chimerism at significantly higher levels in BM. The majority of both FC subpopulations express CXCR4. Coculture of CD56^bright FCs with HSPCs upregulates cathelicidin and β‐defensin 2, factors that prime responsiveness of HSPCs to stromal cell–derived factor 1. Both FC subpopulations significantly upregulated mRNA expression of the HSPC growth factors and Flt3 ligand. These results indicate that human FCs exert a direct effect on HSPCs to enhance engraftment. Human FCs offer a potential regulatory cell‐based therapy for enhancement of engraftment and prevention of GVHD.

     

Fc‐Silent Anti‐CD154 Domain Antibody Effectively Prevents Nonhuman Primate Renal Allograft Rejection

The advent of costimulation blockade provides the prospect for targeted therapy with improved graft survival in transplant patients. Perhaps the most effective costimulation blockade in experimental models is the use of reagents to block the CD40/CD154 pathway. Unfortunately, successful clinical translation of anti‐CD154 therapy has not been achieved. In an attempt to develop an agent that is as effective as previous CD154 blocking antibodies but lacks the risk of thromboembolism, we evaluated the efficacy and safety of a novel anti‐human CD154 domain antibody (dAb, BMS‐986004). The anti‐CD154 dAb effectively blocked CD40‐CD154 interactions but lacked crystallizable fragment (Fc) binding activity and resultant platelet activation. In a nonhuman primate kidney transplant model, anti‐CD154 dAb was safe and efficacious, significantly prolonging allograft survival without evidence of thromboembolism (Median survival time 103 days). The combination of anti‐CD154 dAb and conventional immunosuppression synergized to effectively control allograft rejection (Median survival time 397 days). Furthermore, anti‐CD154 dAb treatment increased the frequency of CD4⁺CD25⁺Foxp3⁺ regulatory T cells. This study demonstrates that the use of a novel anti‐CD154 dAb that lacks Fc binding activity is safe without evidence of thromboembolism and is equally as potent as previous anti‐CD154 agents at prolonging renal allograft survival in a nonhuman primate preclinical model.     

Anti‐CD40 antibody 2C10 binds to a conformational epitope at the CD40‐CD154 interface that is conserved among primate species

The antagonistic anti‐CD40 antibody, 2C10, and its recombinant primate derivative, 2C10R4, are potent immunosuppressive antibodies whose utility in allo‐ and xenotransplantation have been demonstrated in nonhuman primate studies. In this study, we defined the 2C10 binding epitope and found only slight differences in affinity of 2C10 for CD40 derived from four primate species. Staining of truncation mutants mapped the 2C10 binding epitope to the N‐terminal portion of CD40. Alanine scanning mutagenesis of the first 60 residues in the CD40 ectodomain highlighted key amino acids important for binding of 2C10 and for binding of the noncross‐blocking anti‐CD40 antibodies 3A8 and 5D12. All four 2C10‐binding residues defined by mutagenesis clustered near the membrane‐distal tip of CD40 and partially overlap the CD154 binding surface. In contrast, the overlapping 3A8 and 5D12 epitopes map to an opposing surface away from the CD154 binding domain. This biochemical characterization of 2C10 confirms the validity of nonhuman primate studies in the translation of this therapeutic antibody and provides insight its mechanism of action.     

Repeated Injections of IL‐2 Break Renal Allograft Tolerance Induced via Mixed Hematopoietic Chimerism in Monkeys

Tolerance of allografts achieved in mice via stable mixed hematopoietic chimerism relies essentially on continuous elimination of developing alloreactive T cells in the thymus (central deletion). Conversely, while only transient mixed chimerism is observed in nonhuman primates and patients, it is sufficient to ensure tolerance of kidney allografts. In this setting, it is likely that tolerance depends on peripheral regulatory mechanisms rather than thymic deletion. This implies that, in primates, upsetting the balance between inflammatory and regulatory alloimmunity could abolish tolerance and trigger the rejection of previously accepted renal allografts. In this study, six monkeys that were treated with a mixed chimerism protocol and had accepted a kidney allograft for periods of 1–10 years after withdrawal of immunosuppression received subcutaneous injections of IL‐2 cytokine (0.6–3 × 10⁶ IU/m²). This resulted in rapid rejection of previously tolerated renal transplants and was associated with an expansion and reactivation of alloreactive pro‐inflammatory memory T cells in the host's lymphoid organs and in the graft. This phenomenon was prevented by anti‐CD8 antibody treatment. Finally, this process was reversible in that cessation of IL‐2 administration aborted the rejection process and restored normal kidney graft function.     

Studies Introducing Costimulation Blockade for Vascularized Composite Allografts in Nonhuman Primates

Vascularized composite allografts (VCAs) are technically feasible. Similar to other organ transplants, VCAs are hampered by the toxicity and incomplete efficacy associated with conventional immunosuppression. Complications attributable to calcineurin inhibitors remain prevalent in the clinical cases reported to date, and these loom particularly large given the nonlifesaving nature of VCAs. Additionally, acute rejection remains almost ubiquitous, albeit controllable with current agents. Costimulation blockade offers the potential to provide prophylaxis from rejection without the adverse consequences of calcineurin‐based regimens. In this study, we used a nonhuman‐primate model of VCA in conjunction with immunosuppressive regimens containing combinations of B7‐specific costimulation blockade with and without adhesion blockade with LFA3‐Ig to determine what adjunctive role these agents could play in VCA transplantation when combined with more conventional agents. Compared to tacrolimus, the addition of belatacept improved rejection free allograft survival. The combination with LFA3‐Ig reduced CD2^hi memory T cells, however did not provide additional protection against allograft rejection and hindered protective immunity. Histology paralleled clinical histopathology and Banff grading. These data provide the basis for the study of costimulation blockade in VCA in a relevant preclinical model.     

Spontaneous Resolution of Acute Rejection and Tolerance Induction With IL‐2 Fusion Protein in Vascularized Composite Allotransplantation

Vascularized composite allotransplantation (VCA) has emerged as a treatment option for treating nonlife‐threatening conditions. Therefore, in order to make VCA a safe reconstruction option, there is a need to minimize immunosuppression, develop tolerance‐inducing strategies and elucidate the mechanisms of VCA rejection and tolerance. In this study we explored the effects of hIL‐2/Fc (a long‐lasting human IL‐2 fusion protein), in combination with antilymphocyte serum (ALS) and short‐term cyclosporine A (CsA), on graft survival, regulatory T cell (Treg) proliferation and tolerance induction in a rat hind‐limb transplant model. We demonstrate that hIL‐2/Fc therapy tips the immune balance, increasing Treg proliferation and suppressing effector T cells, and permits VCA tolerance as demonstrated by long‐term allograft survival and donor‐antigen acceptance. Moreover, we observe two distinct types of acute rejection (AR), progressive and reversible, within hIL‐2/Fc plus ALS and CsA treated recipients. Our study shows differential gene expression profiles of FoxP3 versus GzmB, Prf1 or interferon‐γ in these two types of AR, with reversible rejection demonstrating higher Treg to Teff gene expression. This correlation of gene expression profile at the first clinical sign of AR with VCA outcomes can provide the basis for further inquiry into the mechanistic aspects of VCA rejection and future drug targets.     

Desensitization using imlifidase and EndoS enables chimerism induction in allosensitized recipient mice

Mixed hematopoietic chimerism induction as a way to foster tolerance to donor organs in recipients who have been sensitized to donor antigens is challenging. Donor‐specific antibodies (DSA) are a dominant barrier toward successful donor bone marrow engraftment. Although desensitization methods are routinely used in recipients with allosensitization for allogeneic bone marrow transplantation, engraftment is frequently unsuccessful. To overcome the barrier of prior sensitization we tested enzymatic desensitization of donor‐specific IgG using imlifidase and endoglycosidase of Streptococcus pyogenes (EndoS), which both partially block the function of DSA in mice, as a novel approach to improve murine bone marrow engraftment in primed hosts. We found that EndoS was capable of inhibiting antibody‐mediated killing of donor cells in vivo. Furthermore, the effect of EndoS depended on the titer of DSA and the genetic background of the recipients. In combination with imlifidase, EndoS improved the survival of donor bone marrow cells. Together with cyclophosphamide, bortezomib, T cell depletion, and nonlethal irradiation, imlifidase in combination with EndoS allowed allogeneic bone marrow engraftment in sensitized recipients. We conclude that enzymatic inactivation of DSA, using the combination of imlifidase and EndoS, can be used for inducing donor hematopoietic chimerism in allosensitized recipient mice in combination with other desensitization strategies.     

Complement Dependence of Murine Costimulatory Blockade‐Resistant Cellular Cardiac Allograft Rejection

Building on studies showing that ischemia–reperfusion‐(I/R)‐injury is complement dependent, we tested links among complement activation, transplantation‐associated I/R injury, and murine cardiac allograft rejection. We transplanted BALB/c hearts subjected to 8‐h cold ischemic storage (CIS) into cytotoxic T‐lymphocyte associated protein 4 (CTLA4)Ig‐treated wild‐type (WT) or c3^?/? B6 recipients. Whereas allografts subjected to 8‐h CIS rejected in WT recipients with a median survival time (MST) of 37 days, identically treated hearts survived >60 days in c3^?/? mice (p < 0.05, n = 4–6/group). Mechanistic studies showed recipient C3 deficiency prevented induction of intragraft and serum chemokines/cytokines and blunted the priming, expansion, and graft infiltration of interferon‐γ–producing, donor‐reactive T cells. MST of hearts subjected to 8‐h CIS was >60 days in mannose binding lectin (mbl1^?/?mbl2^?/?) recipients and 42 days in factor B (cfb^?/?) recipients (n = 4–6/group, p < 0.05, mbl1^?/?mbl2^?/? vs. cfb^?/?), implicating the MBL (not alternative) pathway. To pharmacologically target MBL‐initiated complement activation, we transplanted BALB/c hearts subjected to 8‐h CIS into CTLA4Ig‐treated WT B6 recipients with or without C1 inhibitor (C1‐INH). Remarkably, peritransplantation administration of C1‐INH prolonged graft survival (MST >60 days, p < 0.05 vs. controls, n = 6) and prevented CI‐induced increases in donor‐reactive, IFNγ‐producing spleen cells (p < 0.05). These new findings link donor I/R injury to T cell–mediated rejection through MBL‐initiated, complement activation and support testing C1‐INH administration to prevent CTLA4Ig‐resistant rejection of deceased donor allografts in human transplant patients.     

Adipose‐derived stromal cell therapy combined with a short course nonmyeloablative conditioning promotes long‐term graft tolerance in vascularized composite allotransplantation

The risks of chronic immunosuppression limit the utility of vascularized composite allotransplantation (VCA) as a reconstructive option in complex tissue defects. We evaluated a novel, clinically translatable, radiation‐free conditioning protocol that combines anti‐lymphocyte serum (ALS), tacrolimus, and cytotoxic T‐lymphocyte‐associated protein 4 immunoglobulin (CTLA4‐Ig) with adipose‐derived stromal cells (ASCs) to allow VCA survival without long‐term systemic immunosuppression. Full‐mismatched rat hind‐limb‐transplant recipients received tacrolimus (0.5 mg/kg) for 14 days and were assigned to 4 groups: controls (CTRL) received no conditioning; ASC‐group received CTLA4‐Ig (10 mg/kg body weight i.p. postoperative day [POD] 2, 4, 7) and donor ASCs (1 × 10⁶ iv, POD 2, 4, 7, 15, 28); the ASC‐cyclophosphamide (CYP)‐group received CTLA4‐Ig, ASC plus cyclophosphamide (50 mg/kg ip, POD 3); the ASC‐ALS‐group received CTLA4‐Ig, ASCs plus ALS (500 µL ip, POD 1, 5). Banff grade III or 120 days were endpoints. ASCs suppressed alloresponse in vitro. Median rejection‐free VCA survival was 28 days in CTRL (n = 7), 34 in ASC (n = 6), and 27.5 in ASC‐CYP (n = 4). In contrast, ASC‐ALS achieved significantly longer, rejection‐free VCA survival in 6/7 animals (86%), with persistent mixed donor‐cell chimerism, and elevated systemic and allograft skin T_regs, with no signs of acute cellular rejection. Taken together, a regimen comprised of short‐course tacrolimus, repeated CTLA4‐Ig and ASC administration, combined with ALS, promotes long‐term VCA survival without chronic immunosuppression.