Coordinated IL-2 and TGF-β Signaling Activates Stable Regulatory T Cells and Promotes Immune Tolerance

Abstract Establishing immune tolerance requires the integration of cytokine signals that favor regulatory T cell (Treg) function over effector differentiation, yet how such coordination can be achieved selectively in vivo remains incompletely defined. Regulatory T cells are essential for maintaining immune homeostasis and peripheral tolerance, and both interleukin-2 (IL-2) and transforming growth factor–β (TGF-β) contribute to Treg activation, differentiation, and stability in a highly context-dependent manner. Here, we examine the consequences of coordinated IL-2 and TGF-β3 signaling using an engineered cytokine construct combining attenuated IL-2 with receptor-masked TGF-β3. In vitro, attenuated IL-2/TGF-β3 signaling promoted conversion of naïve CD4⁺ T cells into Foxp3⁺ induced Tregs, expanded endogenous Tregs, and suppressed inflammatory cytokine production by memory and effector CD4⁺ T cells. In vivo, a single administration selectively increased the frequency of CD4⁺Foxp3⁺ regulatory T cells exhibiting an activated and stable phenotype, while minimally activating Foxp3⁻ conventional CD4⁺ T cells relative to IL-2 alone. In a Treg-deficiency–driven adoptive transfer model of autoimmunity, early exposure to coordinated IL-2/TGF-β3 signaling enhanced Treg activation, reduced tissue infiltration by autoreactive T cells, and conferred sustained protection from autoimmune gastritis in the absence of continued treatment. Together, these findings identify a mode of coordinated IL-2 and TGF-β3 signaling sufficient to stabilize regulatory T cell responses and promote immune tolerance while limiting activation of conventional CD4⁺ T cells, highlighting signal integration as a determinant of tolerogenic immune regulation in vivo. One Sentence Summary CUE-401 coordinates IL-2 and TGF-β signaling to expand and activate regulatory T cells and restrain autoimmune responses.