Role of Tregs in the acquisition and maintenance of Tconv anergy in transplantation tolerance

Summary Donor-specific transplantation tolerance is an important goal for improving the quality of life of transplant patients, as it eliminates side-effects from chronic immunosuppression. Transient regimens that use a blocking CD154 antibody have been shown to promote donor-specific transplantation tolerance in mice and non-human primates. Although initial clinical trials testing CD154 had been halted due to thromboembolic complications in humans, new humanized Fc-mutated antibodies avoid FcR cross-linking and platelet aggregation and are safe. Three such antibodies are in clinical trials, frexalimab (Sanofi), tegoprubart (AT-1501, Eledon Pharmaceuticals), and TNX-1500, with both TNX-1500 and AT-1501 being slated to start phase 2 trials in kidney transplantation. As such, there is considerable renewed interest in mechanistic experiments to fully understand the consequences of CD154 therapy. We and others have shown that transient administration of CD154, when in combination with injection of donor splenocytes (DST) as a source of systemic alloantigen, results in long- term donor-specific transplantation tolerance of fully mismatched cardiac allografts in mice. This treatment expands Tregs, prevents germinal center formation and therefore development of donor-specific antibodies (DSA), and results in a hypofunctional state of alloreactive conventional T cells (Tconvs) akin to anergy. We have shown that acquisition of this T cell anergic state requires both blockade of CD154/CD40 interactions and persistent expression of the cognate alloantigen in the graft. Our preliminary results also show that CD154+DST-mediated graft acceptance and expansion of Tregs both depend on the presence of classical dendritic cells 1 (cDC1), as they fail to occur in IRF832 mice that lack cDC1s. Although we initially thought that Tregs would function as suppressor cells in this model (reducing the activation of functional Tconvs), our preliminary results reveal an exciting new property of Tregs: their ability to program and maintain anergy in alloreactive Tconvs. Indeed, CD154+DST fails to induce alloreactive Tconv anergy in mice lacking Tregs, and Treg depletion long after establishment of tolerance reinvigorates Tconvs, along with de novo DSA development. We hypothesize that the crosstalk between Tregs, cDC1s and Tconvs during the induction of transplantation tolerance is essential to program alloreactive Tconvs into an anergic state, as well as to maintain this state when established. This hypothesis will be tested in the context of the following Specific Aims. Specific Aim 1. To elucidate the mechanisms by which Tregs program alloreactive T cell anergy during the induction phase of transplantation tolerance. Specific Aim 2. To establish the mechanisms by which elimination of Tregs at the maintenance phase of tolerance triggers alloreactive T cell reinvigoration. This project will define a new property of Tregs (driving Tconv anergy) and establish the role of cDC subsets in this function at the initiation and maintenance of tolerance. This has implications beyond transplantation, in autoimmunity and cancer, for how to manipulate Tconv function. Project Number: 1R01AI194499-01 | Fiscal Year: 2025 | NIH Institute/Center: National Institute of Allergy and Infectious Diseases (NIAID) | Principal Investigator: Maria-Luisa Alegre | Institution: UNIVERSITY OF CHICAGO, CHICAGO, IL | Award Amount: $766,265 | Activity Code: R01 | Study Section: Immunobiology of Transplantation and Alloimmunity Study Section[ITA] View on NIH RePORTER: https://reporter.nih.gov/project-details/1R01AI19449901