Temporal and Context-Dependent Requirements for the Transcription Factor Foxp3 in Regulatory T Cell Development and Maintenance [transfer organ scRNA-seq]

Regulatory T (Treg) cells, characterized by expression of the lineage-defining transcription factor Foxp3, are critical gatekeepers of immune homeostasis. While Foxp3's essential role in Treg cell development is well established, the precise mechanisms by which Foxp3 governs the Treg-specific transcriptional network remain incompletely understood. Here, we employed a novel chemogenetic model that enables inducible, time-controlled degradation of Foxp3 protein in vivo to dissect its stage-specific functions. We show that Foxp3 is indispensable for the establishment of the Treg transcriptional program and the acquisition of suppressive function during thymic differentiation and in newly generated peripheral Treg cells. In contrast, degradation of Foxp3 in fully differentiated, mature Treg cells resulted in surprisingly minimal transcriptional changes and largely preserved suppressive capacity, affecting only a small subset of genes highly enriched for direct Foxp3 targets. Strikingly, intratumoral Treg cells were uniquely sensitive to Foxp3 loss, leading to impaired suppressive function and enhanced tumor rejection. These findings uncover a differential, context-dependent requirement for Foxp3 across Treg cell ontogeny, highlighting a distinct, largely indirect mechanism by which Foxp3 establishes — and to a more limited extent maintains — Treg identity and function. Overall design: Transfer: For transfer into Tcrbd KO mice, 4 x 106 cells from the Treg + Tconv pool (1 x 106 ROSA26WT, 1 x 106 ROSA26TIR1(F74G), 2 x 106 Tconv) were injected retro-orbitally in 200 µL of sterile PBS (4 mice total). For continuous endogenous Treg depletion, Foxp3DTRCD45.1 were injected i.p. with 1000 ng of DT in 200 µL sterile PBS on day -1 and day 0 followed by 500 ng on day 3 and day 6. Continuous Foxp3 degradation was maintained by daily 5-ph-IAA injections as described above. On day 7, animals were sacrificed. Cross-tissue organ: Foxp3AID/+ROSA26WT and Foxp3AID/+ROSA26TIR1(F74G) mice were treated with 5-ph-IAA for 7 days (4 per group). For each genotype, cells from individual mice and tissue were stained with a unique hashtag antibody. Tregs from SLOs, lung, liver, and LILP were harvested and digested as described in the Methods.