The Role of Coinhibitory Signaling Pathways in Transplantation and Tolerance

Negative costimulatory molecules, acting through so-called inhibitory pathways, play a crucial role in the control of T cell responses. This negative “second signal” opposes T cell receptor activation and leads to downregulation of T cell proliferation and promotes antigen specific tolerance. Much interest has focused upon these pathways in recent years as a method to control detrimental alloresponses and promote allograft tolerance. However, recent experimental data highlights the complexity of negative costimulatory pathways in alloimmunity. Varying effects are observed from molecules expressed on donor and recipient tissues and also depending upon the activation status of immune cells involved. There appears to be significant overlap and redundancy within these systems, rendering this a challenging area to understand and exploit therapeutically. In this article, we will review the literature at the current time regarding the major negative costimulation pathways including CTLA-4:B7, PD-1:PD-L1/PD-L2 and PD-L1:B7-1, B7-H3, B7-H4, HVEM:BTLA/CD160, and TIM-3:Galectin-9. We aim to outline the role of these pathways in alloimmunity and discuss their potential applications for tolerance induction in transplantation

Tregitope: Immunomodulation Powerhouse

IVIG is frequently used in the ‘pre-conditioning’ regimens for higher risk transplants; its effects are attributed in part to induction of Tregs. We have identified regulatory T cell (Treg) epitopes, now known as Tregitopes, in IgG, the main component of intravenous immunoglobulin therapy (IVIg). Tregitopes provide one explanation for the expansion and activation of Treg cells following IVIg treatment. Tregitopes are peptides that exhibit high affinity binding to multiple human HLA Class II DR; they are conserved across IgG isotypes and mammalian species. In vitro and in vivo, for human PBMC and in animal models, Tregitopes activate Tregs. Studies to delineate the mechanism of action have shown that Tregitopes’ effects are very similar to IVIg in vitro. Here we demonstrate that Tregitopes induce Tregs to produce IL-10, leading to modulation of dendritic cell phenotype (down-regulation of Class II, CD80 and CD86 and up-regulation of ILT3), and describe the effects of Tregitopes in the ABM-TCR-transgenic skin transplantation model. The discovery of Tregitopes in IgG and other autologous proteins may contribute to improved understanding of the mechanism of action of IVIg and lead to the application of these powerful immunomodulators to improve transplantation success and suppress autoimmune disease, in the future

Prolonged, Low-Dose Anti-Thymocyte Globulin, Combined with CTLA4-Ig, Promotes Engraftment in a Stringent Transplant Model

Background: Despite significant nephrotoxicity, calcineurin inhibitors (CNIs) remain the cornerstone of immunosuppression in solid organ transplantation. We, along with others, have reported tolerogenic properties of anti-thymocyte globulin (ATG, Thymoglobulin®), evinced by its ability both to spare Tregs from depletion in vivo and, when administered at low, non-depleting doses, to expand Tregs ex vivo. Clinical trials investigating B7/CD28 blockade (LEA29Y, Belatacept) in kidney transplant recipients have proven that the replacement of toxic CNI use is feasible in selected populations. Methods: Rabbit polyclonal anti-murine thymocyte globulin (mATG) was administered as induction and/or prolonged, low-dose therapy, in combination with CTLA4-Ig, in a stringent, fully MHC-mismatched murine skin transplant model to assess graft survival and mechanisms of action. Results: Prolonged, low-dose mATG, combined with CTLA4-Ig, effectively promotes engraftment in a stringent transplant model. Our data demonstrate that mATG achieves graft acceptance primarily by promoting Tregs, while CTLA4-Ig enhances mATG function by limiting activation of the effector T cell pool in the early stages of treatment, and by inhibiting production of anti-rabbit antibodies in the maintenance phase, thereby promoting regulation of alloreactivity. Conclusion: These data provide the rationale for development of novel, CNI-free clinical protocols in human transplant recipients

Immunophenotyping and Efficacy of Low Dose ATG in Non-Sensitized Kidney Recipients Undergoing Early Steroid Withdrawal: A Randomized Pilot Study

Rabbit antithymocyte globulin (ATG) is commonly used as an induction therapy in renal transplant recipients, but the ideal dosage in tacrolimus-based early steroid withdrawal protocols has not been established. The purpose of this pilot study was to determine the immunophenotyping and efficacy of lower dose ATG in low immunological-risk kidney transplant recipients. In this prospective study, 45 patients were randomized (1∶1) to our standard dose ATG (total dose 3.75 mg/kg)(sATG) vs. lower dose 2.25 mg/kg (lowATG). All patients underwent early steroid withdrawal within 7 days. The primary end point was biopsy-proven acute rejection at 12 months. Prospective immunophenotyping of freshly isolated PBMCs was performed at baseline, 3, 6, 12 months post-transplant. The rate of acute rejection was 17% and 10% in the sATG and lowATG, respectively. Effector memory T cells, Tregs and recent thymic emigrants T cells had similar kinetics post-transplant in both groups. No statistically significant differences were found in graft survival, patient survival or infections between the two groups, though there was a non-significant increase in leukopenia (43%v s. 30%), CMV (8% vs. 0) and BK (4% vs. 0) infections in sATG group vs. lowATG. In sum, in low immunological risk kidney recipients undergoing steroid withdrawal, low dose ATG seems to be efficacious in preventing acute rejection and depleting T cells with potentially lower infectious complications. A larger study is warranted to confirm these findings. Trial Registration ClinicalTrials.gov NCT0054840

OR.107. TIM-1 Plays a Crucial Role in the Expansion of Autopathogneic T-Cells and Regulation of Autoimmunity [abstract only]

T-cell immunoglobulin and mucin (TIM) family Members are differentially expressed on Th1 and Th2 cells. Polymorphisms of TIM-1 have been associated with susceptibility to asthma; however, its role in regulating autoimmunity has not been studied. Here, we have used an agonistic antiTIM-1 antibody (Ab, Clone 3B3) which has previously been shown to costimulate T-cell activation and expansion, to analyze the role of TIM-1 in the development and regulation of experimental autoimmune encephalomyelitis (EAE). Treatment with 3B3 dramatically enhances the severity of EAE as well as the frequency of encephalitogenic CD4+ T-cells and the production of IFN-g and IL-17 by these cells. Furthermore, administration of 3B3 breaks self-tolerance and induces EAE in the disease resistant B10.S strain. We have utilized another anti-TIM-1 Ab (RMT1-10) that does not costimulate T-cell activation in vitro. In contrast to 3B3, treatment with RMT1-10 inhibits the development of EAE and reduces the frequency of encephalitogenic CD4+ T-cells with a commensurate decrease in the production of IFN-g and IL-17. Treatment with RMT1-10 causes CD4+ T-cells to produce more IL-4 and IL-10. We provide evidence that both 3B3 and RMT1-10 bind to the same epitope in the Ig domain of TIM-1, but the binding affinity of 3B3 is much higher than that of RMT1-10. These data suggest that TIM-1 engagement with the agonistic Ab, along with TcR ligation, costimulates T-cell expansion with pro-inflammatory IFN-g and IL-17 production resulting in the breakdown of self-tolerance and development of autoimmunity, whereas blocking anti-TIM-1 Ab causes a decrease in the autopathogenic Th1/ThIL-17 responses. This study demonstrates that TIM-1 is a key cell surface molecule that regulates effector T-cell response and depending on hopw the molecule is engaged, autoimmune responses can be either enhanced or inhibited 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-γ 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-γ 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