Comparative ScRNA-Seq Profiling of Antigen-Specific CD4 ⁺ T cells in Semi-Allogeneic Transplantation and Pregnancy Reveals Intersecting Signatures of Rejection and Tolerance

Transplantation tolerance without the need for lifelong immunosuppression is a central goal in transplant immunology yet prior sensitization events remain a major barrier to achieving stable tolerance. In reproductive immunology by contrast, pregnancy represents a spontaneous model of tolerance where the semi-allogeneic fetus evades rejection even in multiparous or previously sensitized mothers. CD8 ⁺ T cell phenotypes of tolerance and rejection have been previously reported in transplant and pregnancy, but the transcriptional states of donor and fetus-specific CD4 ⁺ T cells remain poorly defined. To address this, we performed Single-cell RNA-sequencing (ScRNA-seq) on endogenous, donor-specific CD4 ⁺ T cells across models of naïve or paternally skin sensitized pregnancy as well as in a model of allogeneic heart transplants with or without co-stimulation blockade-induced tolerance. Our systems biology approach allowed us to identify shared and distinct transcriptional clusters of donor-specific CD4 ⁺ Foxp3 ^neg T conventional (Tconvs) and Foxp3 ^pos regulatory (Tregs) T cells from peripheral lymphoid tissue. We expectedly found regulatory populations restricted to tolerance and pregnancy but were surprised to find significant overlap in activated follicular and non-follicular effector phenotypes in rejection and successful pregnancy. We also showed these murine populations were relevant and enriched in human datasets of health and disease respectively. These findings highlight context-dependent differentiation programs of antigen-specific CD4 ⁺ T conventional and regulatory cells and provide new insights into their responses to allogeneic conflict at the intersection of transplant and reproductive immunology.