The natural killer cell activating receptor, NKG2D, is critical to antibody-dependent chronic rejection in heart transplantation.

Chronic rejection is among the most pressing clinical challenges in solid organ transplantation. Interestingly, in a mouse model of heterotopic heart transplantation, antibody-dependent, natural killer (NK) cell-mediated chronic cardiac allograft vasculopathy occurs in some donor-recipient strain combinations, but not others. In this study, we sought to identify the mechanism underlying this unexplained phenomenon. Cardiac allografts from major histocompatibility complex (MHC) mismatched donors were transplanted into immune-deficient C57Bl/6.rag-/- recipients, followed by administration of a monoclonal antibody against the donor MHC class I antigen. We found marked allograft vasculopathy in hearts from C3H donors, but near-complete protection of BALB/c allografts from injury. We found no difference in recipient NK cell phenotype or intrinsic responsiveness to activating signals between recipients of C3H versus BALB/c allografts. However, cardiac endothelial cells from C3H allografts showed an approximately twofold higher expression of Rae-1, an activating ligand of the NK cell receptor natural killer group 2D (NKG2D). Importantly, the administration of a neutralizing antibody against NKG2D abrogated the development of allograft vasculopathy in recipients of C3H allografts, even in the presence of donor-specific antibodies. Therefore, the activating NK cell receptor NKG2D is necessary in this model of chronic cardiac allograft vasculopathy, and strain-dependent expression of NK activating ligands correlates with the development of this disease.

Sensitization in transplantation: Assessment of risk (STAR) 2019 Working Group Meeting Report.

The purpose of the STAR 2019 Working Group was to build on findings from the initial STAR report to further clarify the expectations, limitations, perceptions, and utility of alloimmune assays that are currently in use or in development for risk assessment in the setting of organ transplantation. The goal was to determine the precision and clinical feasibility/utility of such assays in evaluating both memory and primary alloimmune risks. The process included a critical review of biologically driven, state-of-the-art, clinical diagnostics literature by experts in the field and an open public forum in a face-to-face meeting to promote broader engagement of the American Society of Transplantation and American Society of Histocompatibility and Immunogenetics membership. This report summarizes the literature review and the workshop discussions. Specifically, it highlights (1) available assays to evaluate the attributes of HLA antibodies and their utility both as clinical diagnostics and as research tools to evaluate the effector mechanisms driving rejection; (2) potential assays to assess the presence of alloimmune T and B cell memory; and (3) progress in the development of HLA molecular mismatch computational scores as a potential prognostic biomarker for primary alloimmunity and its application in research trial design.

Simultaneous Recognition of Allogeneic MHC and Cognate Autoantigen by Autoreactive T Cells in Transplant Rejection.

The autoimmune condition is a primary obstacle to inducing tolerance in type 1 diabetes patients receiving allogeneic pancreas transplants. It is unknown how autoreactive T cells that recognize self-MHC molecules contribute to MHC-disparate allograft rejection. In this report, we show the presence and accumulation of dual-reactive, that is autoreactive and alloreactive, T cells in C3H islet allografts that were transplanted into autoimmune diabetic NOD mice. Using high-throughput sequencing, we discovered that T cells prevalent in allografts share identical TCRs with autoreactive T cells present in pancreatic islets. T cells expressing TCRs that are enriched in allograft lesions recognized C3H MHC molecules, and five of six cell lines expressing these TCRs were also reactive to NOD islet cells. These results reveal the presence of autoreactive T cells that mediate cross-reactive alloreactivity, and indicate a requirement for regulating such dual-reactive T cells in tissue replacement therapies given to autoimmune individuals.

Linking innate immunity and chronic antibody-mediated allograft rejection.

PURPOSE OF REVIEW: To summarize recent findings linking donor-specific antibodies with innate immunity resulting in chronic allograft rejection. RECENT FINDINGS: Studies in recent years highlight the significance of donor-specific antibodies (DSA) in both acute and chronic allograft rejection. Since chronic rejection is the leading cause of graft failure, this review centers on the contribution of three areas of innate immunity of particular recent focus: complement, NK cells, and macrophages. Recent advances indicate the diverse roles that complement components play both in directly initiating allograft injury and indirectly by contributing to enhanced alloreactivity. NK cells also have emerged as an additional innate response that directly links DSA with chronic graft injury. Finally, recent studies identify alternatively activated macrophages as an additional arm of innate immunity contributing to chronic allograft rejection. SUMMARY: Chronic allograft rejection involves a significant contribution of DSA and differing pathways of the innate immune system. However, key issues remain unresolved. First, it is not always clear which of these varied sources of innate immunity contributing to chronic rejection may be antibody dependent. Moreover, it is not yet clear if these innate pathways represent independent routes that contribute to chronic rejection or rather act in concert to mediate allograft injury.

Sensitization in Transplantation: Assessment of Risk (STAR) 2017 Working Group Meeting Report.

The presence of preexisting (memory) or de novo donor-specific HLA antibodies (DSAs) is a known barrier to successful long-term organ transplantation. Yet, despite the fact that laboratory tools and our understanding of histocompatibility have advanced significantly in recent years, the criteria to define presence of a DSA and assign a level of risk for a given DSA vary markedly between centers. A collaborative effort between the American Society for Histocompatibility and Immunogenetics and the American Society of Transplantation provided the logistical support for generating a dedicated multidisciplinary working group, which included experts in histocompatibility as well as kidney, liver, heart, and lung transplantation. The goals were to perform a critical review of biologically driven, state-of-the-art, clinical diagnostics literature and to provide clinical practice recommendations based on expert assessment of quality and strength of evidence. The results of the Sensitization in Transplantation: Assessment of Risk (STAR) meeting are summarized here, providing recommendations on the definition and utilization of HLA diagnostic testing, and a framework for clinical assessment of risk for a memory or a primary alloimmune response. The definitions, recommendations, risk framework, and highlighted gaps in knowledge are intended to spur research that will inform the next STAR Working Group meeting in 2019.

Anti-LFA-1 induces CD8 T-cell dependent allograft tolerance and augments suppressor phenotype CD8 cells.

The induction of tolerance to transplanted organs is a major objective in transplantation immunology research. Lymphocyte function-associated antigen-1 (LFA-1) interactions have been identified as a key component of the T-cell activation process that may be interrupted to lead to allograft tolerance. In mice, αLFA-1 mAb is a potent monotherapy that leads to the induction of donor-specific transferable tolerance. By interrogating important adaptive and innate immunity pathways, we demonstrate that the induction of tolerance relies on CD8+T-cells. We further demonstrate that αLFA-1 induced tolerance is associated with CD8+CD28-T-cells with a suppressor phenotype, and that while CD8 cells are present, the effector T-cell response is abrogated. A recent publication has shown that CD8+CD28- cells are not diminished by cyclosporine or rapamycin, therefore CD8+CD28- cells represent a clinically relevant population. To our knowledge, this is the first time that a mechanism for αLFA-1 induced tolerance has been described.

Cutting Edge: Roles for Batf3-Dependent APCs in the Rejection of Minor Histocompatibility Antigen-Mismatched Grafts.

In transplantation, a major obstacle for graft acceptance in MHC-matched individuals is the mismatch of minor histocompatibility Ags. Minor histocompatibility Ags are peptides derived from polymorphic proteins that can be presented by APCs on MHC molecules. The APC subtype uniquely responsible for the rejection of minor Ag-mismatched grafts has not yet been identified. In this study, we examined graft rejection in three mouse models: 1) mismatch of male-specific minor Ags, 2) mismatch of minor Ags distinct from male-specific minor Ags, and 3) skin transplant. This study demonstrates that in the absence of pathogen-associated molecular patterns, Batf3-dependent dendritic cells elicit the rejection of cells and grafts expressing mismatched minor Ags. The implication of our findings in clinical transplantation may be significant, as minor Ag reactivity has been implicated in the pathogenesis of multiple allograft tissues.

Direct and indirect allograft recognition: pathways dictating graft rejection mechanisms.

PURPOSE OF REVIEW: The T cell-dependent recognition of allogeneic tissues and organs is complicated by the fact that both donor and host antigen-presenting cells can present donor antigens to host T cells. As such, these pathways result in T cells that can be restricted to either donor ('direct') or host ('indirect') major histocompatibility complex (MHC). These pathways are well recognized, but how these distinct patterns actually dictate allograft recognition is less clear. Thus, the purpose of the review is to summarize results from preclinical animal models in an attempt to clarify the distinct forms of allograft rejection dictated by these recognition pathways. RECENT FINDINGS: CD4 and CD8 donor MHC-restricted T cells are sufficient to reject allografts by a T-cell receptor-mediated direct ('cognate') interaction using a defined array of effector molecules. Conversely, 'noncognate' host MHC-restricted CD4 T cells must interact with intermediate host-type antigen-presenting cells and so greatly amplify the response by triggering antibody and inflammatory responses. SUMMARY: Importantly, 'cognate' CD4 and CD8 T cells have strikingly similar requirements for rejection, suggesting that this effector mechanism is dictated by the nature of allograft recognition rather than by T-cell subset. Conversely, 'noncognate' allograft recognition drives an increasingly appreciated role for inciting innate immunity in mediating allograft injury.

Ectopic expression of Fas Ligand on cardiomyocytes renders cardiac allografts resistant to CD4(+) T-cell mediated rejection.

Fas Ligand limits inflammatory injury and permits allograft survival by inducing apoptosis of Fas-bearing lymphocytes. Previous studies have shown that the CD4(+) T-cell is both sufficient and required for murine cardiac allograft rejection. Here, utilizing a transgenic mouse that over-expresses Fas Ligand specifically on cardiomyocytes as heart donors, we sought to determine if Fas Ligand on graft parenchymal cells could resist CD4(+) T-cell mediated rejection. When transplanted into fully immunocompetent BALB/c recipients Fas Ligand transgenic hearts were acutely rejected. However, when transplanted into CD4(+) T-cell reconstituted BALB/c-rag(-/-) recipients, Fas Ligand hearts demonstrated long-term survival. These results indicate that Fas Ligand over-expression on cardiomyocytes can indeed resist CD4(+) T-cell mediated cardiac rejection and suggests contact dependence between Fas Ligand expressing graft parenchymal cells and the effector CD4(+) T-cells.

Control of in vivo collateral damage generated by T cell immunity.

An ongoing dilemma faced during an immune response is generating an effective, often proinflammatory response to eliminate pathogens and/or infected cells while also minimizing collateral damage to adjacent noninfected tissues. The factors limiting bystander cell injury during an Ag-specific immune response in vivo are largely unknown. In this study, using an in vivo model of islet transplants in TCR transgenic mice, we show that both CD4 and CD8 T cells do have the capacity to inflict adjacent tissue damage and that this injury is greatly enhanced in sensitized hosts. CD4 T cell-mediated killing of specific and bystander cells occurred via different mechanisms. Unlike specific target cell killing, CD4-mediated bystander injury required tissue Fas expression and was inhibited with anti-IFN-γ Ab treatment in vivo. Moreover, bystander cell injury was not entirely nonspecific but rather required, in naive recipients, that the MHC allele expressed by the bystanders was self. Importantly, the coinhibitor programmed death-1 plays an important role in restraining bystander cell injury mediated either by defined TCR transgenic T cells or by polyclonal T cell populations. Thus, the differential requirements for specific versus bystander cell injury suggest that there are opportunities for inhibiting immune pathology without compromising Ag-specific immunity in vivo.

Genetic ablation of arginase 1 in macrophages and neutrophils enhances clearance of an arthritogenic alphavirus.

Chikungunya virus (CHIKV) and Ross River virus (RRV) cause a debilitating, and often chronic, musculoskeletal inflammatory disease in humans. Macrophages constitute the major inflammatory infiltrates in musculoskeletal tissues during these infections. However, the precise macrophage effector functions that affect the pathogenesis of arthritogenic alphaviruses have not been defined. We hypothesized that the severe damage to musculoskeletal tissues observed in RRV- or CHIKV-infected mice would promote a wound-healing response characterized by M2-like macrophages. Indeed, we found that RRV- and CHIKV-induced musculoskeletal inflammatory lesions, and macrophages present in these lesions, have a unique gene-expression pattern characterized by high expression of arginase 1 and Ym1/Chi3l3 in the absence of FIZZ1/Relmα that is consistent with an M2-like activation phenotype. Strikingly, mice specifically deleted for arginase 1 in neutrophils and macrophages had dramatically reduced viral loads and improved pathology in musculoskeletal tissues at late times post-RRV infection. These findings indicate that arthritogenic alphavirus infection drives a unique myeloid cell activation program in inflamed musculoskeletal tissues that inhibits virus clearance and impedes disease resolution in an arginase 1-dependent manner.

NK cells promote islet allograft tolerance via a perforin-dependent mechanism.

Although major histocompatibility complex (MHC) class II-restricted CD4 T cells are well appreciated for their contribution to peripheral tolerance to tissue allografts, little is known regarding MHC class I-dependent reactivity in this process. Here we show a crucial role for host MHC class I-dependent NK cell reactivity for allograft tolerance in mice induced through either costimulation blockade using CD154-specific antibody therapy or by targeting LFA-1 (also known as CD11a). Tolerance induction absolutely required host expression of MHC class I, but was independent of CD8 T cell-dependent immunity. Rather, tolerance required innate immunity involving NK1.1(+) cells, but was independent of CD1d-restricted NKT cells. Therefore, NK cells seem to be generally required for induction of tolerance to islet allografts. Additional studies indicate that CD154-specific antibody-induced allograft tolerance is perforin dependent. Notably, NK cells that are perforin competent are sufficient to restore allograft tolerance in perforin-deficient recipients. Together, these results show an obligatory role for NK cells, through perforin, for induction of tolerance to islet allografts.

Tolerogenic Delivery of a Hybrid Insulin Peptide Markedly Prolongs Islet Graft Survival in the NOD Mouse.

The induction of antigen (Ag)-specific tolerance and replacement of islet ß-cells are major ongoing goals for the treatment of type 1 diabetes (T1D). Our group previously showed that a hybrid insulin peptide (2.5HIP) is a critical autoantigen for diabetogenic CD4+ T cells in the NOD mouse model. In this study, we investigated whether induction of Ag-specific tolerance using 2.5HIP-coupled tolerogenic nanoparticles (NPs) could protect diabetic NOD mice from disease recurrence upon syngeneic islet transplantation. Islet graft survival was significantly prolonged in mice treated with 2.5HIP NPs, but not NPs containing the insulin B chain peptide 9-23. Protection in 2.5HIP NP-treated mice was attributed both to the simultaneous induction of anergy in 2.5HIP-specific effector T cells and the expansion of Foxp3+ regulatory T cells specific for the same Ag. Notably, our results indicate that effector function of graft-infiltrating CD4+ and CD8+ T cells specific for other ß-cell epitopes was significantly impaired, suggesting a novel mechanism of therapeutically induced linked suppression. This work establishes that tolerance induction with an HIP can delay recurrent autoimmunity in NOD mice, which could inform the development of an Ag-specific therapy for T1D.

Deletion of TLR4 reduces apoptosis and improves histology in a murine kidney transplant model.

Acute kidney injury (AKI) after transplantation of human deceased donor kidneys is associated with upregulation of tubular toll like receptor 4 (TLR4), but whether TLR4 is required for AKI is unknown. We hypothesized that TLR4 knockout mice (TLR4KO) subjected to cold ischemia followed by kidney transplant (CI + Txp) would be protected from AKI. C57Bl/6J wild type or TLR4KO kidneys were subjected to CI + Txp into wild type recipients. Tubular cell apoptosis, tubular injury and cast formation were significantly improved in recipients of TLR4KO kidneys. TLR4KO kidneys also demonstrated significantly decreased expression of the effector caspase 8. Brush border injury scores and serum creatinine were not different in recipients of TLR4KO versus wild type kidneys. Phosphorylated RIP3 and MLKL through which TLR4 signals programmed necrosis were expressed in both recipient groups. In addition, TNF-α and TNFR1 expression were significantly increased in recipient serum and TLR4KO kidneys respectively after CI + Txp, suggesting continued activation of programmed necrosis despite TLR4 deletion. Our results suggest that TLR4 deletion decreases apoptosis via inhibition of the death receptor pathway and decreases tubular injury and cast formation.

The impact of Caspase-1 deletion on apoptosis and acute kidney injury in a murine transplant model.

BACKGROUND: Caspase-1 knockout mice (Casp1KO) are protected from Acute Kidney Injury (AKI) after warm ischemia/reperfusion injury in non-transplant models. Since Caspase-1 plays a central role as an inflammatory response initiator, we hypothesized that Casp1KO mice would be protected from AKI following transplant. METHODS: Renal tubular cells (RTECs) were subjected to cold storage and rewarming (CS/REW). C57Bl/6 J wild type or Casp1KO kidneys were subjected to CI for 30 min and then transplanted into wild type recipients (CI + Txp). The recipients underwent bilateral native nephrectomy at the time of transplant. Serum creatinine (sCr) was measured 24 h after native nephrectomy to assess transplant function. RESULTS: We found that RTECs subjected to CS/REW had significantly increased expression of the Caspase-1 and inflammasome protein NLRP1. Wild type kidneys subjected to CI + Txp into wild type recipients also demonstrated significantly increased Caspase-1 and NLRP1 protein expression compared to kidneys transplanted from Casp1KO donors into wild type recipients. Caspase-1 deletion results in significantly decreased RTEC apoptosis in transplanted Casp1KO vs WT kidneys. Surprisingly, however, renal function, ATN scores including brush border injury, cast formation and tubular simplification were similar in both groups and not significantly different. CONCLUSIONS: Our data suggest that other triggers of inflammation and programmed necrosis may need to be inhibited in addition to attenuating Caspase-1 to fully prevent AKI after kidney transplant. Importantly, requirements may be distinct for AKI induced by transplantation as opposed to other transient models such as the clamp model of AKI.

What is a ß cell? - Chapter I in the Human Islet Research Network (HIRN) review series.

BACKGROUND: The pancreatic ß cell, as the sole source of the vital hormone insulin, has been under intensive study for more than a century. Given the potential of newly created insulin-producing cells as a treatment or even cure of type 1 diabetes (T1D) and possibly in severe cases of type 2 diabetes (T2D), multiple academic and commercial laboratories are working to derive surrogate glucose-responsive, insulin-producing cells. SCOPE OF REVIEW: The recent development of advanced phenotyping technologies, including molecular, epigenomic, histological, or functional, have greatly improved our understanding of the critical properties of human ß cells. Using this information, here we summarize the salient features of normal, fully functional adult human ß cells, and propose minimal criteria for what should rightfully be termed 'ß cells' as opposed to insulin-producing but not fully-functional surrogates that we propose should be referred to as 'ß-like' cells or insulin-producing cells. MAJOR CONCLUSIONS: Clear criteria can be established to differentiate fully functional, mature ß cells from 'ß-like' surrogates. In addition, we outline important knowledge gaps that must be addressed to enable a greater understanding of the ß cell.

Priming and effector dependence on insulin B:9-23 peptide in NOD islet autoimmunity.

NOD mice with knockout of both native insulin genes and a mutated proinsulin transgene, alanine at position B16 in preproinsulin (B16:A-dKO mice), do not develop diabetes. Transplantation of NOD islets, but not bone marrow, expressing native insulin sequences (tyrosine at position B16) into B16:A-dKO mice rapidly restored development of insulin autoantibodies (IAAs) and insulitis, despite the recipients' pancreatic islets lacking native insulin sequences. Splenocytes from B16:A-dKO mice that received native insulin-positive islets induced diabetes when transferred into wild-type NOD/SCID or B16:A-dKO NOD/SCID mice. Splenocytes from mice immunized with native insulin B chain amino acids 9-23 (insulin B:9-23) peptide in CFA induced rapid diabetes upon transfer only in recipients expressing the native insulin B:9-23 sequence in their pancreata. Additionally, CD4(+) T cells from B16:A-dKO mice immunized with native insulin B:9-23 peptide promoted IAAs in NOD/SCID mice. These results indicate that the provision of native insulin B:9-23 sequences is sufficient to prime anti-insulin autoimmunity and that subsequent transfer of diabetes following peptide immunization requires native insulin B:9-23 expression in islets. Our findings demonstrate dependence on B16 alanine versus tyrosine of insulin B:9-23 for both the initial priming and the effector phase of NOD anti-islet autoimmunity.

Diverse Routes of Allograft Tolerance Disruption by Memory T Cells.

Memory T lymphocytes constitute a significant problem in tissue and organ transplantation due their contribution to early rejection and their relative resistance to tolerance-promoting therapies. Memory cells generated by environmental antigen exposure, as with T cells in general, harbor a high frequency of T cell receptors (TCR) spontaneously cross-reacting with allogeneic major histocompatibility complex (MHC) molecules. This phenomenon, known as 'heterologous' immunity, is thought to be a key barrier to transplant tolerance induction since such memory cells can potentially react directly with essentially any prospective allograft. In this review, we describe two additional concepts that expand this commonly held view of how memory cells contribute to transplant immunity and tolerance disruption. Firstly, autoimmunity is an additional response that can comprise an endogenously generated form of heterologous alloimmunity. However, unlike heterologous immunity generated as a byproduct of indiscriminate antigen sensitization, autoimmunity can generate T cells that have the unusual potential to interact with the graft either through the recognition of graft-bearing autoantigens or by their cross-reactive (heterologous) alloimmune specificity to MHC molecules. Moreover, we describe an additional pathway, independent of significant heterologous immunity, whereby immune memory to vaccine- or pathogen-induced antigens also may impair tolerance induction. This latter form of immune recognition indirectly disrupts tolerance by the licensing of naïve alloreactive T cells by vaccine/pathogen directed memory cells recognizing the same antigen-presenting cell in vivo. Thus, there appear to be recognition pathways beyond typical heterologous immunity through which memory T cells can directly or indirectly impact allograft immunity and tolerance.