Lymphotoxin limits Foxp3⁺ regulatory T cell development from Foxp3^lo precursors via IL-4 signaling

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 α₁β₂ (LTα₁β₂) 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 LTα₁β₂-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.