Peripheral Transplantation Tolerance Research Articles

Distinct Graft-Specific TCR Avidity Profiles during Acute Rejection and Tolerance

Mechanisms implicated in robust transplantation tolerance at the cellular level can be broadly categorized into those that inhibit alloreactive Tcells intrinsically (clonal deletion and dysfunction) or extrinsically through regulation. Here, we investigated whether additional population-level mechanisms control Tcells by examining whether therapeutically induced peripheral transplantation tolerance could influence Tcell populations' avidity for alloantigens. Whereas Tcells with high avidity preferentially accumulated during acute rejection of allografts, the alloreactive Tcells in tolerant recipients retained a low-avidity profile, comparable to naive mice despite evidence of activation. These contrasting avidity profiles upon productive versus tolerogenic stimulation were durable and persisted upon alloantigen re-encounter in the absence of any immunosuppression. Thus, peripheral transplantation tolerance involves control of alloreactive Tcells at the population level, in addition to the individual cell level. Controlling expansion or eliminating high-affinity, donor-specific Tcells long term may be desirable to achieve robust transplantation tolerance in the clinic.

Tracking of TCR-Transgenic T Cells Reveals That Multiple Mechanisms Maintain Cardiac Transplant Tolerance in Mice.

Solid organ transplantation tolerance can be achieved following select transient immunosuppressive regimens that result in long-lasting restraint of alloimmunity without affecting responses to other antigens. Transplantation tolerance has been observed in animal models following costimulation or coreceptor blockade therapies, and in a subset of patients through induction protocols that include donor bone marrow transplantation, or following withdrawal of immunosuppression. Previous data from our lab and others have shown that proinflammatory interventions that successfully prevent the induction of transplantation tolerance in mice often fail to break tolerance once it has been stably established. This suggests that established tolerance acquires resilience to proinflammatory insults, and prompted us to investigate the mechanisms that maintain a stable state of robust tolerance. Our results demonstrate that only a triple intervention of depleting CD25+ regulatory T cells (Tregs), blocking programmed death ligand-1 (PD-L1) signals, and transferring low numbers of alloreactive T cells was sufficient to break established tolerance. We infer from these observations that Tregs and PD-1/PD-L1 signals cooperate to preserve a low alloreactive T cell frequency to maintain tolerance. Thus, therapeutic protocols designed to induce multiple parallel mechanisms of peripheral tolerance may be necessary to achieve robust transplantation tolerance capable of maintaining one allograft for life in the clinic.

The Early Induction of PDL1 On Monocytes by Ivig Suppresses T Cell Activation in Allogeneic Mixed Lymphocyte Reactions

AbstractAbstract 4111 Background:Recognition of histocompatibility determinants by allogeneic T cells together with the engagement of co-stimulatory molecules with their ligands expressed on accessory cells (B7:CD28, CD40:CD40L) is essential for the induction of T cell-mediated allograft rejection. In contrast, the PD1:PDL1 negative co-stimulatory pathway plays a critical role in the induction and maintenance of peripheral transplantation tolerance. Despite the use of potent immunosuppressive therapy for the blockade of co-stimulatory pathways, allograft rejection and drug toxicity remain an important problem in transplanted patients. IVIg was first used as a prophylactic agent in these immunocompromised patients to prevent infections, but several studies have suggested that IVIg also improved the rate of graft survival in patients with high risk for rejection. The mechanisms responsible for this anti-inflammatory effect are unclear and remain to be determined. We recently showed, using the allogeneic mixed lymphocyte reaction (MLR) as an in vitro model of allograft rejection and GvHD, that IVIg strongly inhibited T cell activation. In the present study, we sought to determine the mechanisms underlying this inhibition. Methods:For allogeneic MLR, human peripheral blood mononuclear cells (PBMC) from 2 different individuals were mixed together with or without 2 mg/ml of IVIg and incubated for 4 days prior to determination of IL-2 secretion by ELISA as a measure of T cell activation. The expression of TCR, CD3, CD28 and PD1 on T cells and HLA-DR, CD80, CD86 and PDL1 on monocytes was evaluated by flow cytometry. Results:IVIg strongly inhibited IL-2 secretion (>90%; P<0.001) as previously reported. To explain the inhibition of T cell activation in the presence of IVIg, we postulated that the expression of molecules involved in antigen presentation and in different co-stimulatory pathways was modulated by IVIg. We thus evaluated the effect of IVIg on the cell surface expression of various molecules implicated in T cell activation or tolerance, 24 hours after the onset of the MLR. Our results showed no modulation of TCR, CD3, CD28 and PD1 expression on T cells. Similarly, IVIg did not affect the expression of CD86 on monocytes. In contrast, the expression of CD80 was significantly decreased (>30%; P<0.01) on these cells after IVIg treatment. In addition, a strong increase in PDL1 expression (>70%; P<0.05) on monocytes was observed in MLRs done in the presence of IVIg. Finally, the expression of HLA-DR was increased by >60% (P<0.01) and that of CD14 was decreased by >50% (P<0.01) in the presence of IVIg, which is characteristic of the emergence of an anti-inflammatory monocyte population. Conclusion:Altogether, our results suggest that IVIg induces its immunosuppressive effects in allogeneic MLRs by modulating the expression of co-stimulatory molecules on monocytes and by inducing an anti-inflammatory monocyte population. This study contributes to a better understanding of the mechanisms by which IVIg may induce peripheral tolerance and improve graft survival in transplanted patients. Disclosures:No relevant conflicts of interest to declare.

Tolerance-Inducing Immunosuppressive Strategies in Clinical Transplantation

The significant development of immunosuppressive drug therapies within the past 20 years has had a major impact on the outcome of clinical solid organ transplantation, mainly by decreasing the incidence of acute rejection episodes and improving short-term patient and graft survival. However, long-term results remain relatively disappointing because of chronic allograft dysfunction and patient morbidity or mortality, which is often related to the adverse effects of immunosuppressive treatment. Thus, the induction of specific immunological tolerance of the recipient towards the allograft remains an important objective in transplantation. In this article, we first briefly describe the mechanisms of allograft rejection and immune tolerance. We then review in detail current tolerogenic strategies that could promote central or peripheral tolerance, highlighting the promises as well as the remaining challenges in clinical transplantation. The induction of haematopoietic mixed chimerism could be an approach to induce robust central tolerance, and we describe recent encouraging reports of end-stage kidney disease patients, without concomitant malignancy, who have undergone combined bone marrow and kidney transplantation. We discuss current studies suggesting that, while promoting peripheral transplantation tolerance in preclinical models, induction protocols based on lymphocyte depletion (polyclonal antithymocyte globulins, alemtuzumab) or co-stimulatory blockade (belatacept) should, at the current stage, be considered more as drug-minimization rather than tolerance-inducing strategies. Thus, a better understanding of the mechanisms that promote peripheral tolerance has led to newer approaches and the investigation of individualized donor-specific cellular therapies based on manipulated recipient regulatory T cells.

PDL1 Is Required for Peripheral Transplantation Tolerance and Protection from Chronic Allograft Rejection

The PD-1:PDL pathway plays an important role in regulating alloimmune responses but its role in transplantation tolerance is unknown. We investigated the role of PD-1:PDL costimulatory pathway in peripheral and a well established model of central transplantation tolerance. Early as well as delayed blockade of PDL1 but not PDL2 abrogated tolerance induced by CTLA4Ig in a fully MHC-mismatched cardiac allograft model. Accelerated rejection was associated with a significant increase in the frequency of IFN-gamma-producing alloreactive T cells and expansion of effector CD8(+) T cells in the periphery, and a decline in the percentage of Foxp3(+) graft infiltrating cells. Similarly, studies using PDL1/L2-deficient recipients confirmed the results with Ab blockade. Interestingly, while PDL1-deficient donor allografts were accepted by wild-type recipients treated with CTLA4Ig, the grafts developed severe chronic rejection and vasculopathy when compared with wild-type grafts. Finally, in a model of central tolerance induced by mixed allogeneic chimerism, engraftment was not abrogated by PDL1/L2 blockade. These novel data demonstrate the critical role of PDL1 for induction and maintenance of peripheral transplantation tolerance by its ability to alter the balance between pathogenic and regulatory T cells. Expression of PDL1 in donor tissue is critical for prevention of in situ graft pathology and chronic rejection.

Hematopoietic chimerism and central tolerance created by peripheral-tolerance induction without myeloablative conditioning.

Allogeneic hematopoietic chimerism leading to central tolerance has significant therapeutic potential. Realization of that potential has been impeded by the need for myeloablative conditioning of the host and development of graft-versus-host disease (GVHD). To surmount these impediments, we have adapted a costimulation blockade-based protocol developed for solid organ transplantation for use in stem cell transplantation. The protocol combines donor-specific transfusion (DST) with anti-CD154 mAb. When applied to stem cell transplantation, administration of DST, anti-CD154 mAb, and allogeneic bone marrow leads to hematopoietic chimerism and central tolerance with no myeloablation and no GVHD. Tolerance in this system results from deletion of both peripheral host alloreactive CD8+ T cells and nascent intrathymic alloreactive CD8+ T cells. In the absence of large numbers of host alloreactive CD8+ T cells, the transfusion that precedes transplantation need not be of donor origin, suggesting that both allospecific and non-allospecific mechanisms regulate engraftment. Agents that interfere with peripheral transplantation tolerance impair establishment of chimerism. We conclude that robust allogeneic hematopoietic chimerism and central tolerance can be established in the absence of host myeloablative conditioning using a peripheral transplantation tolerance protocol.

Regulatory T cells in the induction and maintenance of peripheral transplantation tolerance

Regulatory T cells in the induction and maintenance of peripheral transplantation tolerance

NOD congenic mice genetically protected from autoimmune diabetes remain resistant to transplantation tolerance induction.

The loss of self-tolerance leading to autoimmune type 1 diabetes in the NOD mouse model involves at least 19 genetic loci. In addition to their genetic defects in self-tolerance, NOD mice resist peripheral transplantation tolerance induced by costimulation blockade using donor-specific transfusion and anti-CD154 antibody. Hypothesizing that these two abnormalities might be related, we investigated whether they could be uncoupled through a genetic approach. Diabetes-resistant NOD and C57BL/6 stocks congenic for various reciprocally introduced Idd loci were assessed for their ability to be tolerized. Surprisingly, in NOD congenic mice that are almost completely protected from diabetes, costimulation blockade failed to prolong skin allograft survival. In reciprocal C57BL/6 congenic mice with NOD-derived Idd loci, skin allograft survival was readily prolonged by costimulation blockade. These data indicate that single or multiple combinations of evaluated Idd loci that dramatically reduce diabetes frequency do not correct resistance to peripheral transplantation tolerance induced by costimulation blockade. We suggest that mechanisms controlling autoimmunity and transplantation tolerance in NOD mice are not completely overlapping and are potentially distinct, or that the genetic threshold for normalizing the transplantation tolerance defect is higher than that for preventing autoimmune diabetes.

The role of peripheral T-cell deletion in transplantation tolerance.

The apoptotic deletion of thymocytes that express self-reactive antigen receptors is the basis of central (thymic) self-tolerance. However, it is clear that some autoreactive T cells escape deletion in the thymus and exist as mature lymphocytes in the periphery. Therefore, peripheral mechanisms of tolerance are also crucial, and failure of these peripheral mechanisms leads to autoimmunity. Clonal deletion, clonal anergy and immunoregulation and/or suppression have been suggested as mechanisms by which 'inappropriate' T-lymphocyte responses may be controlled in the periphery. Peripheral clonal deletion, which involves the apoptotic elimination of lymphocytes, is critical for T-cell homeostasis during normal immune responses, and is recognized as an important process by which self-tolerance is maintained. Transplantation of foreign tissue into an adult host represents a special case of 'inappropriate' T-cell reactivity that is subject to the same central and peripheral tolerance mechanisms that control reactivity against self. In this case, the unusually high frequency of naive T cells able to recognize and respond against non-self-allogeneic major histocompatibility complex (MHC) antigens leads to an exceptionally large pool of pathogenic effector lymphocytes that must be controlled if graft rejection is to be avoided. A great deal of effort has been directed toward understanding the role of clonal anergy and/or active immunoregulation in the induction of peripheral transplantation tolerance but, until recently, relatively little progress had been made towards defining the potential contribution of clonal deletion. Here, we outline recent data that define a clear requirement for deletion in the induction of peripheral transplantation tolerance across MHC barriers, and discuss the potential implications of these results in the context of current treatment modalities used in the clinical transplantation setting.

Requirement for T-cell apoptosis in the induction of peripheral transplantation tolerance.

The mechanisms of allograft tolerance have been classified as deletion, anergy, ignorance and suppression/regulation. Deletion has been implicated in central tolerance, whereas peripheral tolerance has generally been ascribed to clonal anergy and/or active immunoregulatory states. Here, we used two distinct systems to assess the requirement for T-cell deletion in peripheral tolerance induction. In mice transgenic for Bcl-xL, T cells were resistant to passive cell death through cytokine withdrawal, whereas T cells from interleukin-2-deficient mice did not undergo activation-induced cell death. Using either agents that block co-stimulatory pathways or the immunosuppressive drug rapamycin, which we have shown here blocks the proliferative component of interleukin-2 signaling but does not inhibit priming for activation-induced cell death, we found that mice with defective passive or active T-cell apoptotic pathways were resistant to induction of transplantation tolerance. Thus, deletion of activated T cells through activation-induced cell death or growth factor withdrawal seems necessary to achieve peripheral tolerance across major histocompatibility complex barriers.