Combined Treatment With TGF-β1, Retinoic Acid, and Lactoferrin Robustly Generate Inducible Tregs (iTregs) Against High Affinity Ligand

Forkhead box P3-positive (Foxp3)-inducible Tregs (iTregs) are readily generated by TGF-β1 at low TCR signaling intensity. TGF-β1–mediated Foxp3 expression is further enhanced by retinoic acid (RA) and lactoferrin (LF). However, the intensity of TCR signaling required for induction of Foxp3 expression by TGF-β1 in combination with RA and LF is unknown. Here, we found that either RA or LF alone decreased TGF-β1–mediated Foxp3 expression at low TCR signaling intensity. In contrast, at high TCR signaling intensity, the addition of either RA or LF strongly increased TGF-β1–mediated Foxp3 expression. Moreover, decreased CD28 stimulation was more favorable for TGF-β1/LF–mediated Foxp3 expression. Lastly, we found that at high signaling intensities of both TCR and CD28, combined treatment with TGF-β1, RA, and LF induced robust expression of Foxp3, in parallel with powerful suppressive activity against responder T cell proliferation. Our findings that TGFβ/RA/LF strongly generate high affinity Ag-specific iTreg population would be useful for the control of unwanted hypersensitive immune reactions such as various autoimmune diseases.