T-bet marks contact-capable regulatory T cells embedded within Th1-dendritic cell networks in skin-draining lymph nodes

Regulatory T cells (Tregs) adapt to inflammatory environments while preserving Foxp3-dependent suppressive identity. In type 1 inflammation, Tregs can upregulate T-bet, but whether T-bet⁺ Tregs represent a stable regulatory subset or exhibit plasticity toward an effector-like state remains incompletely understood. To probe the positioning and interactions of T-bet⁺ Tregs during type 1 inflammation, we performed live lymph node section microscopy in T-bet-ZsGreen Foxp3-RFP dual-reporter mice during a subcutaneous complete Freunds adjuvant (CFA) challenge. T-bet⁺ Tregs accumulated rapidly in the draining lymph nodes within 24 hours, coincident with the appearance of T-bet⁺ dendritic cells (DCs) and Th1-like CD4⁺ T cells. Analysis of lymph node sections revealed that T-bet⁺ Tregs were incorporated into Th1-DC contact networks, displaying increased contact frequency and contact volume compared with T-bet⁻ Tregs. In parallel, flow cytometric analysis of the immune response showed that T-bet⁺ Tregs maintained a stable regulatory profile distinct from T-bet⁺ effector T cells, with retained high expression of Foxp3 and regulatory-associated surface molecules including FR4, CD25, Nrp-1, CD39, and GARP. They also expressed elevated adhesion and activation-associated molecules, including LFA-1, ICOS, PD-1, CD44, and CD103 compared with T-bet⁻ Tregs, and were enriched for CD103⁺ cells across the inflammatory time course. Adoptive transfer studies further showed that pre-existing T-bet expression enhanced recovery of antigen-specific Tregs from CFA-draining lymph nodes in a context-dependent manner, whereas T-bet expression did not confer a unique advantage in canonical in vitro suppression of Th1 proliferation. Together, these findings identify T-bet⁺ Tregs as a contact-capable regulatory subset embedded within early Th1-DC networks during the priming phase of type 1 inflammation rather than a destabilized or effector-converted Treg population.