Allospecific splenic Tr1 cells drive effector T cell exhaustion through up-regulated Areg-EGFR signaling to promote transplant tolerance

Inducing stable tolerance to transplants remains a challenge in immunology. Previously, we induced tolerance to allogeneic islets in nonhuman primates by preemptive alloantigen delivery to antigen-presenting cells in situ. Here, mass cytometry phenotyping with incorporated donor-derived MHC-I peptide-loaded MHC-II tetramers revealed accumulation of allospecific CD4⁺ T cell clusters in the spleen of tolerant recipients. Areg⁺Tr1 regulatory and terminally exhausted EGFR^hi T (Tex) cells represented the predominant allospecific subsets. Trajectory analysis showed that antigen-experienced effector memory T cells differentiated into suppressive Areg⁺Tr1 and EGFR⁺TOX⁺Nur77⁺TCF-1^- Tex subsets. Cell-cell communication mapping showed that exhausted and effector memory T cells engaged with allospecific Tr1 cells via the Areg-EGFR axis. Gene silencing studies confirmed that Tr1 cells use Areg-EGFR signaling to drive the metabolic suppression and epigenetic reprogramming of CD4⁺ T cells through a Nur77-dependent pathway. These findings point to the splenic Areg⁺Tr1 cell-EGFR⁺Teff cell axis as a critical immunoregulatory pathway in peripheral transplant tolerance.