CD96 Is an Immune Checkpoint That Regulates CD8⁺ T-cell Antitumor Function

CD96 is a novel target for cancer immunotherapy shown to regulate NK cell effector function and metastasis. Here, we demonstrated that blocking CD96 suppressed primary tumor growth in a number of experimental mouse tumor models in a CD8⁺ T cell-dependent manner. DNAM-1/CD226, Batf3, IL12p35, and IFNγ were also critical, and CD96-deficient CD8⁺ T cells promoted greater tumor control than CD96-sufficient CD8⁺ T cells. The antitumor activity of anti-CD96 therapy was independent of Fc-mediated effector function and was more effective in dual combination with blockade of a number of immune checkpoints, including PD-1, PD-L1, TIGIT, and CTLA-4. We consistently observed coexpression of PD-1 with CD96 on CD8⁺ T lymphocytes in tumor-infiltrating leukocytes both in mouse and human cancers using mRNA analysis, flow cytometry, and multiplex IHF. The combination of anti-CD96 with anti-PD-1 increased the percentage of IFNγ-expressing CD8⁺ T lymphocytes. Addition of anti-CD96 to anti-PD-1 and anti-TIGIT resulted in superior antitumor responses, regardless of the ability of the anti-TIGIT isotype to engage FcR. The optimal triple combination was also dependent upon CD8⁺ T cells and IFNγ. Overall, these data demonstrate that CD96 is an immune checkpoint on CD8⁺ T cells and that blocking CD96 in combination with other immune-checkpoint inhibitors is a strategy to enhance T-cell activity and suppress tumor growth.