Blockade of surface-bound TGF-β on regulatory T cells abrogates suppression of effector T cell function in the tumor microenvironment

Abstract

Regulatory T cells (T_regs) suppress antitumor immunity by inhibiting the killing of tumor cells by antigen-specific CD8⁺ T cells. To better understand the mechanisms involved, we used ex vivo three-dimensional collagen-fibrin gel cultures of dissociated B16 melanoma tumors. This system recapitulated the in vivo suppression of antimelanoma immunity, rendering the dissociated tumor cells resistant to killing by cocultured activated, antigen-specific T cells. Immunosuppression was not observed when tumors excised from T_reg-depleted mice were cultured in this system. Experiments with neutralizing antibodies showed that blocking transforming growth factor-β (TGF-β) also prevented immunosuppression. Immunosuppression depended on cell-cell contact or cellular proximity because soluble factors from the collagen-fibrin gel cultures did not inhibit tumor cell killing by T cells. Moreover, intravital, two-photon microscopy showed that tumor-specific Pmel-1 effector T cells physically interacted with tumor-resident T_regs in mice. T_regs isolated from B16 tumors alone were sufficient to suppress CD8⁺ T cell-mediated killing, which depended on surface-bound TGF-β on the T_regs Immunosuppression of CD8⁺ T cells correlated with a decrease in the abundance of the cytolytic protein granzyme B and an increase in the cell surface amount of the immune checkpoint receptor programmed cell death protein 1 (PD-1). These findings suggest that contact between T_regs and antitumor T cells in the tumor microenvironment inhibits antimelanoma immunity in a TGF-β-dependent manner and highlight potential ways to inhibit intratumoral T_regs therapeutically.