Regulatory T cells (T reg ) are critical players of immune tolerance that develop in the thymus via two distinct developmental pathways involving CD25 + Foxp3 - and CD25 -Foxp3 lo precursors. However, the mechanisms regulating the recently identified Foxp3 lo precursor pathway remain unclear. Here, we find that the membrane-bound lymphotoxin α 1 β 2 (LTα 1 β 2 ) heterocomplex is upregulated during T reg development upon TCR/CD28 and IL-2 stimulation. We show that Lta expression limits the maturational development of T reg from Foxp3 lo precursors by regulating their proliferation, survival, and metabolic profile. Transgenic reporter mice and transcriptomic analyses further reveal that medullary thymic epithelial cells (mTEC) constitute an unexpected source of IL-4. We demonstrate that LTα 1 β 2 -lymphotoxin β receptor-mediated interactions with mTEC limit T reg development by down-regulating IL-4 expression in mTEC. Collectively, our findings identify the lymphotoxin axis as the first inhibitory checkpoint of thymic T reg development that fine-tunes the Foxp3 lo T reg precursor pathway by limiting IL-4 availability.

Regulatory T cells (T reg ) constitute a subset of CD4 + T cells that specifically express the transcription factor Foxp3 and CD25, also known as IL-2Rα. Naturally occurring CD25 + Foxp3 + T reg critically prevent autoimmune and inflammatory disorders by maintaining selftolerance through the suppression of autoreactive T cells that have escaped thymic selection. The majority of CD25 + Foxp3 + T reg emerge in the thymus from 3 days after birth 1 . In particular, medullary thymic epithelial cells (mTEC) contribute to T reg development by expressing MHC class II and CD80/86 molecules as well as a broad range of selfantigens 2-4 . T reg development follows a "two-step" model in the thymic medulla 5-7 . The first step is driven by the stimulation of the T-cell receptor (TCR) and of CD28 in developing CD4 + single-positive (SP) thymocytes, leading to the generation of CD25 + Foxp3 -precursors (CD25 + T reg P) 5,6,8 . TCR and CD28 signals govern the expression of tumor-necrosis factor receptor (TNFR) members, GITR and OX40, which promote CD25 expression 9 . The second step is driven by γ-chain cytokines, in particular IL-2 and IL-15, that convert CD25 + T reg P into CD25 + Foxp3 + mature T reg 5