Cbl-b Deficiency Mediates Resistance to Programed Death-Ligand 1/Programed Death-1 Regulation

Abstract

Casitas B-lineage lymphoma-b (Cbl-b) is an E3 ubiquitin ligase that negatively regulates T cell activation. Cbl-b^-/- T cells are hyper-reactive and co-stimulation independent, and Cbl-b^-/- mice demonstrate robust T cell and NK cell-mediated antitumor immunity. As a result of these murine studies, Cbl-b is considered a potential target for therapeutic manipulation in human cancer immunotherapy. The PD-L1/PD-1 pathway of immune regulation is presently an important therapeutic focus in tumor immunotherapy, and although Cbl-b^-/- mice have been shown to be resistant to several immuno-regulatory mechanisms, the sensitivity of Cbl-b^-/- mice to PD-L1-mediated suppression has not been reported. We now document that Cbl-b^-/- T cells and NK cells are resistant to PD-L1/PD-1-mediated suppression. Using a PD-L1 fusion protein (PD-L1 Ig), this resistance is shown for both <i>in vitro</i> proliferative responses and IFN-γ production and is not associated with decreased PD-1 expression on Cbl-b^-/- cells. In coculture studies, Cbl-b^-/- CD8⁺, but not CD4⁺ T cells, diminish the PD-L1 Ig-mediated suppression of bystander naïve WT CD8⁺ T cells. Using an <i>in vivo</i> model of B16 melanoma in which numerous liver metastases develop in WT mice in a PD-1 dependent manner, Cbl-b^-/- mice develop significantly fewer liver metastases without the administration of anti-PD-1 antibody. Overall, our findings identify a new mode of immuno-regulatory resistance associated with Cbl-b deficiency and suggest that resistance to PD-L1/PD-1-mediated suppression is a novel mechanism by which Cbl-b deficiency leads to enhanced antitumor immunity. Our results suggest that targeting Cbl-b in cancer immunotherapy offers the opportunity to simultaneously override numerous relevant "checkpoints," including sensitivity to regulatory T cells, suppression by TGF-β, and immune regulation by both CTLA-4 and, as we now report, by the PD-L1/PD-1 pathway.