Integrated T-cell receptor and costimulatory signals determine TGF-β-dependent differentiation and maintenance of Foxp3+ regulatory T cells

Foxp3-expressing Tregs play a non-redundant role in protecting against immune pathologies. Foxp3(+) Tregs can arise intra-and extra-thymically, however, the signals directing their differentiation and maintenance in the periphery are not well understood. We show that stimulation of mouse naive CD4(+) T cells in vitro with optimal doses of anti-CD3/anti-CD28 resulted in high frequencies of Foxp3(+) T cells via a TGF-beta-dependent mechanism. Addition of TGF-beta and retinoic acid overcame the inhibition of Foxp3 expression observed during high-strength anti-CD3/anti-CD28 stimulation. Reducing the strength of TCR or costimulatory signals with inhibitors of mammalian target of rapamycin (mTOR) or MEK/ERK signalling also enhanced expression of Foxp3 in a TGF-beta-dependent manner. Addition of TGF-beta was further required to maintain Foxp3 expression in ex vivo derived Foxp3(+) Tregs upon prolonged anti-CD3/anti-CD28 signalling. Thus, induction/maintenance of Foxp3 expression by TGF-beta is modulated by the integrated strength of TCR/costimulatory signals.