Taba Kheradmand

Permanent protection of PLG scaffold transplanted allogeneic islet grafts in diabetic mice treated with ECDI-fixed donor splenocyte infusions

Allogeneic islet cell transplantation is a promising treatment for human type 1 diabetes. Currently, human islets are transplanted via intra-portal infusions. While successful, it leads to significant early islet attrition from instant blood-mediated inflammatory reaction. An extra-hepatic site was established by transplanting islet-loaded microporous poly(lactide-co-glycolide) (PLG) scaffolds into the epididymal fat pad in syngeneic islet transplant models. This study examined this technology in allogeneic islet transplantation and determined whether transplant tolerance could be effectively induced to protect PLG scaffold transplanted allogeneic islets. The efficacy of an established tolerance induction strategy using donor splenocytes treated with ethylcarbodiimide(ECDI) was tested. ECDI-fixed donor splenocytes were infused 7 days before and 1 day after islet transplantation. Immediate normoglycemia was restored, and treated mice maintained indefinite normoglycemia whereas untreated mice rejected islet grafts within 20 days of transplantation. Interestingly, efficacy of tolerance induction was superior in PLG scaffold compared with intra-portal transplanted islets. Protection of PLG scaffold islet allografts was associated with several mechanisms of immune regulation. In summary, PLG scaffolds can serve as an alternative delivery system for islet transplantation that does not impair tolerance induction. This approach of combining tolerance induction with scaffold islet transplantation has potential therapeutic implications for human islet transplantation.

Intragraft CD11b(+) IDO(+) cells mediate cardiac allograft tolerance by ECDI-fixed donor splenocyte infusions.

We have previously shown that pre‐ and post‐transplant infusions of donor splenocytes treated with 1‐ethyl‐3‐(3’‐dimethylaminopropyl)‐carbodiimide (ECDI‐SPs) provide permanent donor‐specific protection of islet allografts. The efficacy of donor ECDI‐SPs in protecting vascularized cardiac allografts and mechanism(s) of protection are unknown. In this study, we show that infusions of ECDI‐SPs significantly prolong cardiac allograft survival concomitant with an impressive accumulation of CD11b+IDO+ cells in the cardiac allograft, and that the presence of this population is dependent on Gr1+ cells. Consequently, depletion of Gr1+ cells or inhibition of indoleamine 2,3 dioxygenase (IDO) activity abrogates graft protection by ECDI‐SPs infusions. In addition, T cells from ECDI‐SPs treated recipients secrete high levels of interleukin 10 and interleukin 13 upon in vitro restimulation, which are also dampened in recipients treated with the IDO inhibitor. Furthermore, combination of donor ECDI‐SPs with a short course of rapamycin provides indefinite cardiac allograft survival in 100% of the recipients. These findings reveal a novel mechanism of donor ECDI‐SPs in inducing cardiac transplant tolerance and provide several targets that are amenable to therapeutic manipulations for tolerance induction for cardiac transplantation.

Ethylenecarbodiimide-Fixed Donor Splenocyte Infusions Differentially Target Direct and Indirect Pathways of Allorecognition for Induction of Transplant Tolerance

Strategic exposure to donor Ags prior to transplantation can be an effective way for inducting donor-specific tolerance in allogeneic recipients. We have recently shown that pretransplant infusion of donor splenocytes treated with the chemical cross-linker ethylenecarbodiimide (ECDI-SPs) induces indefinite islet allograft survival in a full MHC-mismatched model without the need for any immunosuppression. Mechanisms of allograft protection by this strategy remain elusive. In this study, we show that the infused donor ECDI-SPs differentially target T cells with indirect versus direct allospecificities. To target indirect allospecific T cells, ECDI-SPs induce upregulation of negative, but not positive, costimulatory molecules on recipient splenic CD11c+ dendritic cells phagocytosing the injected ECDI-SPs. Indirect allospecific T cells activated by such CD11c+ dendritic cells undergo robust initial proliferation followed by rapid clonal depletion. The remaining T cells are sequestered in the spleen without homing to the graft site or the graft draining lymph node. In contrast, direct allospecific T cells interacting with intact donor ECDI-SPs not yet phagocytosed undergo limited proliferation and are subsequently anergized. Furthermore, CD4+CD25+Foxp3+ T cells are induced in lymphoid organs and at the graft site by ECDI-SPs. We conclude that donor ECDI-SP infusions target host allogeneic responses via a multitude of mechanisms, including clonal depletion, anergy, and immunoregulation, which act in a synergistic fashion to induce robust transplant tolerance. This simple form of negative vaccination has significant potential for clinical translation in human transplantation.

Donor‐Specific CD8+Foxp3+ T Cells Protect Skin Allografts and Facilitate Induction of Conventional CD4+Foxp3+ Regulatory T Cells

CD4+ regulatory T cells play a critical role in tolerance induction in transplantation. CD8+ suppressor T cells have also been shown to control alloimmune responses in preclinical and clinical models. However, the exact nature of the CD8+ suppressor T cells, their induction and mechanism of function in allogeneic transplantation remain elusive. In this study, we show that functionally suppressive, alloantigen‐specific CD8+Foxp3+ T cells can be induced and significantly expanded by stimulating naïve CD8+ T cells with donor dendritic cells in the presence of IL‐2, TGF‐β1 and retinoic acid. These CD8+Foxp3+ T cells express enhanced levels of CTLA‐4, CCR4 and CD103, inhibit the up‐regulation of costimulatory molecules on dendritic cells, and suppress CD4 and CD8 T cell proliferation and cytokine production in a donor‐specific and contact‐dependent manner. Importantly, upon adoptive transfer, the induced CD8+Foxp3+ T cells protect full MHC‐mismatched skin allografts. In vivo, the CD8+Foxp3+ T cells preferentially traffic to the graft draining lymph node where they induce conventional CD4+Foxp3+ T cells and concurrently suppress effector T cell expansion. We conclude that donor‐specific CD8+Foxp3+ suppressor T cells can be induced and exploited as an effective form of cell therapy for graft protection in transplantation.

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.

Transient B-Cell Depletion Combined With Apoptotic Donor Splenocytes Induces Xeno-Specific T- and B-Cell Tolerance to Islet Xenografts

Peritransplant infusion of apoptotic donor splenocytes cross-linked with ethylene carbodiimide (ECDI-SPs) has been demonstrated to effectively induce allogeneic donor-specific tolerance. The objective of the current study is to determine the effectiveness and additional requirements for tolerance induction for xenogeneic islet transplantation using donor ECDI-SPs. In a rat-to-mouse xenogeneic islet transplant model, we show that rat ECDI-SPs alone significantly prolonged islet xenograft survival but failed to induce tolerance. In contrast to allogeneic donor ECDI-SPs, xenogeneic donor ECDI-SPs induced production of xenodonor-specific antibodies partially responsible for the eventual islet xenograft rejection. Consequently, depletion of B cells prior to infusions of rat ECDI-SPs effectively prevented such antibody production and led to the indefinite survival of rat islet xenografts. In addition to controlling antibody responses, transient B-cell depletion combined with ECDI-SPs synergistically suppressed xenodonor-specific T-cell priming as well as memory T-cell generation. Reciprocally, after initial depletion, the recovered B cells in long-term tolerized mice exhibited xenodonor-specific hyporesponsiveness. We conclude that transient B-cell depletion combined with donor ECDI-SPs is a robust strategy for induction of xenodonor-specific T- and B-cell tolerance. This combinatorial therapy may be a promising strategy for tolerance induction for clinical xenogeneic islet transplantation.

Preemptive Tolerogenic Delivery of Donor Antigens for Permanent Allogeneic Islet Graft Protection

We have previously developed a robust regimen for tolerance induction in murine models of islet cell transplantation using pre- and posttransplant infusions of donor splenocytes (SPs) treated with a chemical cross-linker ethylcarbodiimide (ECDI). However, the requirement for large numbers of fresh donor SPs for ECDI coupling impairs its clinical feasibility, and additionally, the compatibility of this tolerance regimen with commonly used immunosuppressive drugs is largely unknown. In the current study, we demonstrate that equivalent tolerance efficacy for islet cell transplantation can be successfully achieved not only with a significantly lower dose of ECDI-SPs than originally established but also with culture-expanded donor B-cells or with soluble donor antigens in the form of donor cell lysate, which is ECDI coupled to recipient SPs. We further demonstrate that tolerance induced by donor ECDI-SPs is dependent on a favorable apoptotic-to-necrotic cell ratio post-ECDI coupling and is not affected by a transient course of conventional immunosuppressive drugs including tacrolimus and mycophenolate mofetil. While splenic antigen-presenting cells of the recipient play an important role in mediating the tolerogenic effects of donor ECDI-SPs, splenectomized recipients can be readily tolerized and appear to employ liver Kupffer cells for uptaking and processing of the ECDI-SPs. We conclude that infusion of donor ECDI-SPs is a versatile tolerance strategy that has a high potential for adaptation to clinically feasible regimens for tolerance trials for human islet cell transplantation.

Characterization of a subset of bone marrow-derived natural killer cells that regulates T cell activation in rats

We report that bone marrow‐derived natural killer (BMNK) cells from DA or F344 rats inhibit PMA/ionomycin‐induced T cell proliferation. These NK‐regulatory cells are NKR‐P1Adim, whereas a minor subpopulation is NKR‐P1Abright. Only the NKR‐P1Adim BMNK cells inhibit T cell proliferation. If activated with rat Con A supernatant, the NKR‐P1Adim cells become NKR‐P1Abright and lose the ability to inhibit T cell proliferation. In contrast to BMNK cells, all DA and F344 rat NK cells isolated from the blood, spleen, cervical, or mesenteric lymph nodes or Peyer’s patches are NKR‐P1Abright and lack the ability to inhibit T cell proliferation. Inhibition of T cell proliferation correlates with significant down‐regulation of CD3, suggesting that this may be the mechanism through which the NKR‐P1Adim cells mediate suppression. The nitric oxide synthase inhibitor NG‐monomethyl‐arginine acetate‐abrogated NKR‐P1Adim cell inhibition of T cell proliferation. We conclude that rat bone marrow NKR‐P1Adim cells represent a unique population that may play a role in maintaining immune homeostasis by regulating the clonal expansion of activated T cells.

Antibody-mediated rejection in ABO compatible husband to wife living donor liver transplant and review of the literature.

Role of donor specific antibodies (DSAs) in liver allograft function has not been fully defined. We report an ABO compatible orthotopic liver transplant case with DSAs to donor HLA, where the patient developed immediate antibody-mediated rejection (AMR).The patient, a 43-year-old female with cirrhosis, underwent ABO-compatible living-donor liver transplant from her husband. On post-operative day (POD)1, serum transaminases were sharply elevated. Retrospective testing of pre-transplant serum demonstrated presence of strong class I and class II anti-HLA antibodies and positive T- and B-cell flow-cytometric crossmatches (FCXM). Transaminase levels improved with plasmapheresis and thymoglobulin. On POD7, her liver enzymes became elevated again and allograft biopsy stained positive for C4d. Patient was treated with intravenous immunoglobulin and rituximab and recovered over time. Pre-transplant sera of patient were retrospectively tested by C1q assay to determine the cytotoxic function of DSAs; DSAs were positive for C1q binding. Our results suggest that pre-liver transplant antibody testing may be helpful in identifying patients at risk for development of AMR.

Identification and characterization of novel HLA alleles: Utility of next-generation sequencing methods.

The HLA genes are the most polymorphic of the human genome, and novel HLA alleles are continuously identified, often by clinical Sanger sequencing-based typing (SBT) assays. Introduction of next-generation sequencing (NGS) technologies for clinical HLA typing may significantly improve this process. Here we compare four cases of novel HLA alleles identified and characterized by both SBT and NGS. The tested NGS system sequenced broader regions of the HLA loci, and identified novel polymorphisms undetected by SBT. Subsequent characterization of the novel alleles in isolation of coencoded alleles by SBT required custom-designed primers, while the NGS system was able to sequence both alleles in phase. However, the tested assay was unable to amplify buccal cell DNA for subsequent NGS sequencing, presumably due to the lower quality of these samples. While NGS assays will undoubtedly increase novel allele identification, more stringent DNA sample requirements may be necessary for this new technology.