The emerging role of T cell Ig mucin 1 in alloimmune responses in an experimental mouse transplant model.

T cell Ig mucin 1 (TIM-1) plays an important role in regulating immune responses in autoimmune and asthma models, and it is expressed on both Th1 and Th2 cells. Using an antagonistic TIM-1-specific antibody, we studied the role of TIM-1 in alloimmunity. A short course of TIM-1-specific antibody monotherapy prolonged survival of fully MHC-mismatched vascularized mouse cardiac allografts. This prolongation was associated with inhibition of alloreactive Th1 responses and preservation of Th2 responses. TIM-1-specific antibody treatment was more effective in Th1-type cytokine-deficient Stat4(-/-) recipients as compared with Th2-type cytokine-deficient Stat6(-/-) recipients. Subtherapeutic doses of rapamycin plus TIM-1-specific antibody resulted in allograft acceptance and prevented the development of chronic allograft vasculopathy. Allograft survival via this treatment was accompanied by a Th1- to Th2-type cytokine switch. Depletion of natural Tregs abrogated the graft-protecting effect of the TIM-1-specific antibody. Importantly, CD4(+)CD25(+) Tregs obtained from long-term survivors had enhanced regulatory activity as compared with naive CD4(+)CD25(+) Tregs. Consistent with this, TIM-1-specific antibody treatment both preserved Tregs and prevented the expansion of alloreactive effector Th1 cells in an alloreactive TCR transgenic adoptive transfer model. These studies define previously unknown functions of TIM-1 in regulating alloimmune responses in vivo and may provide a novel approach to promoting transplantation tolerance.

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.

A novel alloantigen-specific CD8+PD1+ regulatory T cell induced by ICOS-B7h blockade in vivo.

Delayed ICOS-B7h signal blockade promotes significant prolongation of cardiac allograft survival in wild-type but not in CD8-deficient C57BL/6 recipients of fully MHC-mismatched BALB/c heart allografts, suggesting the possible generation of CD8(+) regulatory T cells in vivo. We now show that the administration of a blocking anti-ICOS mAb results in the generation of regulatory CD8(+) T cells. These cells can transfer protection and prolong the survival of donor-specific BALB/c, but not third party C3H, heart grafts in CD8-deficient C57BL/6 recipients. This is unique to ICOS-B7h blockade, because B7 blockade by CTLA4-Ig prolongs graft survival in CD8-deficient mice and does not result in the generation of regulatory CD8(+) T cells. Those cells localize to the graft, produce both IFN-gamma and IL-4 after allostimulation in vitro, prohibit the expansion of alloreactive CD4(+) T cells, and appear to mediate a Th2 switch of recipient CD4(+) T cells after adoptive transfer in vivo. Finally, these cells are not confined to the CD28-negative population but express programmed death 1, a molecule required for their regulatory function in vivo. CD8(+)PD1(+) T cells suppress alloreactive CD4(+) T cells but do not inhibit the functions by alloreactive CD8(+) T cells in vitro. These results describe a novel allospecific regulatory CD8(+)PD1(+) T cell induced by ICOS-B7h blockade in vivo.

Differential engagement of Tim-1 during activation can positively or negatively costimulate T cell expansion and effector function.

It has been suggested that T cell immunoglobulin mucin (Tim)-1 expressed on T cells serves to positively costimulate T cell responses. However, crosslinking of Tim-1 by its ligand Tim-4 resulted in either activation or inhibition of T cell responses, thus raising the issue of whether Tim-1 can have a dual function as a costimulator. To resolve this issue, we tested a series of monoclonal antibodies specific for Tim-1 and identified two antibodies that showed opposite functional effects. One anti-Tim-1 antibody increased the frequency of antigen-specific T cells, the production of the proinflammatory cytokines IFN-gamma and IL-17, and the severity of experimental autoimmune encephalomyelitis. In contrast, another anti-Tim-1 antibody inhibited the generation of antigen-specific T cells, production of IFN-gamma and IL-17, and development of autoimmunity, and it caused a strong Th2 response. Both antibodies bound to closely related epitopes in the IgV domain of the Tim-1 molecule, but the activating antibody had an avidity for Tim-1 that was 17 times higher than the inhibitory antibody. Although both anti-Tim-1 antibodies induced CD3 capping, only the activating antibody caused strong cytoskeletal reorganization and motility. These data indicate that Tim-1 regulates T cell responses and that Tim-1 engagement can alter T cell function depending on the affinity/avidity with which it is engaged.

A novel mechanism of action for anti-thymocyte globulin: induction of CD4+CD25+Foxp3+ regulatory T cells.

T cell-depleting agents are being tested as part of clinical tolerance strategies in humans with autoimmunity and transplantation. The immunosuppressive activity of anti-thymocyte globulin (ATG) has been thought to result primarily from depletion of peripheral lymphocytes. Herein is reported for the first time that ATG but not anti-CD52 mAb (alemtuzumab) or the IL-2R antagonists causes rapid and sustained expansion of CD4+CD25+ T cells when cultured with human peripheral blood lymphocytes. These cells display enhanced expression of the regulatory markers glucocorticoid-induced TNF receptor, cytotoxic T lymphocyte-associated antigen-4 (CTLA-4), and forkhead box P3 and efficiently suppress a direct alloimmune response of the original responder lymphocytes. It is interesting that the cells do not suppress memory responses to the recall antigen mumps. Ex vivo expansion of regulatory T cells is due mainly to conversion of CD4+CD25- into CD4+CD25+ T cells and to a lesser degree to proliferation of natural CD4+CD25+ T cells. The induction of regulatory T cells depends on production of Th2 cytokines in the generating cultures. These novel data suggest that ATG not only may promote expansion/generation of regulatory T cells but also may be useful in future ex vivo expansion of these cells for cellular therapy in autoimmunity and clinical transplantation.

How can we measure immunologic tolerance in humans?

Prevention and treatment of allograft rejection in organ transplant recipients relies primarily on non-antigen-specific immunosuppression, with all its associated potential hazards and costs. Currently, the status of the recipient immune response is measured by monitoring pharmacologic drug levels and clinical/pathologic evaluation of graft function. Development of reliable assays that can measure accurately the status of the immune response not only would help clinicians customize the prescription of immunosuppressive drugs in individual patients but also may allow their complete withdrawal in some patients with immunologic tolerance. Furthermore, these assays would facilitate the safe evaluation of novel tolerogenic regimens. Achieving this goal has proved to be very difficult because it requires both a more in-depth understanding of complex mechanisms of tolerance and also identification of transplant patients with acquired tolerance to an allograft that can be studied. This review discusses the current understanding of tolerance mechanisms and outlines the unique and specific challenges in development of tolerance/monitoring assays in the field of transplantation. In addition, several of the most promising candidate assays are discussed in detail.