Using bioinformatic and clustering analysis of single cell RNAseq and flow cytometry data, Wang and Zhang et al. deeply analyzed the functional/dysfunctional state of antigen-specific CD8+ T cells in two mouse tumor models following anti-PD-1 and/or agonist-GITR therapy. Combination therapy resulted in durable tumor control, enhanced T cell functionality, and uniquely induced a memory precursor T cell population. Anti-PD-1 prevented dephosphorylation of CD226 and agonist-GITR reduced surface TIGIT presence, tipping the balance of these positive and negative T cell regulators toward stimulation and memory.
Most patients with cancer do not develop durable antitumor responses after programmed cell death protein 1 (PD-1) or programmed cell death ligand 1(PD-L1) checkpoint inhibition monotherapy because of an ephemeral reversal of T cell dysfunction and failure to promote long-lasting immunological T cell memory. Activating costimulatory pathways to induce stronger T cell activation may improve the efficacy of checkpoint inhibition and lead to durable antitumor responses. We performed single-cell RNA sequencing of more than 2000 tumor-infiltrating CD8(+) T cells in mice receiving both PD-1 and GITR (glucocorticoid-induced tumor necrosis factor receptor-related protein) antibodies and found that this combination synergistically enhanced the effector function of expanded CD8(+) T cells by restoring the balance of key homeostatic regulators CD226 and T cell immunoreceptor with Ig and ITIM domains (TIGIT), leading to a robust survival benefit. Combination therapy decreased CD8(+) T cell dysfunction and induced a highly proliferative precursor effector memory T cell phenotype in a CD226-dependent manner. PD-1 inhibition rescued CD226 activity by preventing PD-1-Src homology region 2 (SHP2) dephosphophorylation of the CD226 intracellular domain, whereas GITR agonism decreased TIGIT expression. Unmasking the molecular pathways driving durable antitumor responses will be essential to the development of rational approaches to optimizing cancer immunotherapy.