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

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.