Blockade of TGF-β signaling to enhance the antitumor response is accompanied by dysregulation of the functional activity of CD4⁺CD25⁺Foxp3⁺ and CD4⁺CD25^-Foxp3⁺ T cells

Background: The pleiotropic cytokine, transforming growth factor (TGF)-β, and CD4⁺CD25⁺Foxp3⁺ regulatory T cells (Tregs) play a critical role in actively suppressing antitumor immune responses. Evidence shows that TGF-β produced by tumor cells promotes tolerance via expansion of Tregs. Our group previously demonstrated that blockade of TGF-β signaling with a small molecule TGF-β receptor I antagonist (SM16) inhibited primary and metastatic tumor growth in a T cell dependent fashion. In the current study, we evaluated the effect of SM16 on Treg generation and function. Methods: Using BALB/c, FoxP3eGFP and Rag^-/- mice, we performed FACS analysis to determine if SM16 blocked de novo TGF-β-induced Treg generation in vitro and in vivo. CD4⁺ T cells from lymph node and spleen were isolated from control mice or mice maintained on SM16 diet, and flow cytometry analysis was used to detect the frequency of CD4⁺CD25^-FoxP3⁺ and CD4⁺CD25⁺FoxP3⁺ T cells. In vitro suppression assays were used to determine the ability to suppress naive T cell proliferation in vitro of both CD4⁺CD25⁺FoxP3⁺ and CD4⁺CD25^-FoxP3⁺ T cell sub-populations. We then examined whether SM16 diet exerted an inhibitory effect on primary tumor growth and correlated with changes in FoxP3⁺expression. ELISA analysis was used to measure IFN-γlevels after 72 h co-culture of CD4⁺CD25⁺ T cells from tumor-bearing mice on control or SM16 diet with CD4⁺CD25^- T cells from naive donors. Results: SM16 abrogates TGF-β-induced Treg generation in vitro but does not prevent global homeostatic expansion of CD4⁺ T cell sub-populations in vivo. Instead, SM16 treatment causes expansion of a population of CD4⁺CD25^-Foxp3⁺ Treg-like cells without significantly altering the overall frequency of Treg in lymphoreplete naive and tumor-bearing mice. Importantly, both the CD4⁺CD25^-Foxp3⁺ T cells and the CD4⁺CD25⁺Foxp3⁺ Tregs in mice receiving SM16 diet exhibited diminished ability to suppress naive T cell proliferation in vitro compared to Treg from mice on control diet. Conclusions: These findings suggest that blockade of TGF-β signaling is a potentially useful strategy for blunting Treg function to enhance the anti-tumor response. Our data further suggest that the overall dampening of Treg function may involve the expansion of a quiescent Treg precursor population, which is CD4⁺CD25^-Foxp3⁺.