Blockade of TGF-β signaling to enhance the antitumor response is accompanied by dysregulation of the functional activity of CD4CD25Foxp3 and CD4CD25Foxp3 T cells.

BACKGROUND

The pleiotropic cytokine, transforming growth factor (TGF)-β, and CD4CD25Foxp3 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 CD4CD25FoxP3 and CD4CD25FoxP3 T cells. In vitro suppression assays were used to determine the ability to suppress naive T cell proliferation in vitro of both CD4CD25FoxP3 and CD4CD25FoxP3 T cell sub-populations. We then examined whether SM16 diet exerted an inhibitory effect on primary tumor growth and correlated with changes in FoxP3expression. ELISA analysis was used to measure IFN-γ levels after 72 h co-culture of CD4CD25 T cells from tumor-bearing mice on control or SM16 diet with CD4CD25 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 CD4CD25Foxp3 Treg-like cells without significantly altering the overall frequency of Treg in lymphoreplete naive and tumor-bearing mice. Importantly, both the CD4CD25Foxp3 T cells and the CD4CD25Foxp3 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 CD4CD25Foxp3.