Single-cell RNAseq analysis of colonic Tregs: native or pTregs driven by different TCRs reactive to food, microbe or self antigens

Antigen-specific regulatory T cells (Tregs) in the gut are essential for maintaining immune tolerance, yet the contributions of different antigen sources and TCR identities to peripheral Treg (pTreg) differentiation remain poorly understood. In this study, we replaced endogenous TCRs in CD4⁺ Tconv cells (CD45.2⁺) with defined TCRs using an AAV-based system. Tconv cells expressing TCRs reactive to food (TF2.2, TF2.7, TF2.6), E. coli Nissle (TM3.5, TM2.3, TM1.2), or self antigens (TS2.3, TS2.1) were transferred into CD45.1⁺ hosts carrying the corresponding antigens. Ten days post-transfer, we performed single-cell RNA sequencing on donor-derived pTreg cells sorted from pooled mesenteric lymph nodes (mLNs), based on Foxp3-GFP expression. In parallel, we profiled TCR-edited transferred cells that did not convert to Foxp3⁺. As negative controls, we included non-edited and TCR-negative donor cells that had undergone the same in vitro culture and transfer process but did not receive differentiation-inducing signals. For comparison with endogenous T cell populations, we also included host-derived CD25hi Tregs as well as GFP⁺ and GFP⁻ cells from age-matched Foxp3-GFP mice.