Single-Cell Analyses Reveal a Functionally Heterogeneous Exhausted CD8⁺ T-cell Subpopulation That Is Correlated with Response to Checkpoint Therapy in Melanoma Available to Purchase

PD-1 pathway inhibitors have revolutionized cancer therapy. However, most patients do not durably benefit, highlighting the need for biomarkers to stratify patients as responders or nonresponders. Although CD8⁺ tumor-infiltrating lymphocytes (TIL) have been associated with immune checkpoint therapy response, there is no consensus on which CD8⁺ TIL subpopulations have the most prognostic value. Preclinical studies have focused on progenitor-like exhausted CD8⁺ T cells (T_PEX) because T_PEX proliferate more in response to PD-1 inhibitors than other exhausted T-cell (T_EX) subpopulations. However, immune checkpoint inhibitor treatment drives T_PEX differentiation into other T_EX populations that can mediate antitumor immunity. These data complicate the ability to identify prognostically important T-cell populations in patients that predict immune checkpoint inhibitor treatment response. In this study, we found that patients with advanced melanoma with ≥20% of CD8⁺ TILs coexpressing PD-1 and CTLA4 (termed CP^Hi TIL) had better objective response rates and survival following PD-1 monotherapy than those below this threshold. Characterization of the CP^Hi TIL subset using bulk and single-cell RNA sequencing showed that although T_PEX-like cells were present within the CP^Hi subset, they were in the minority of these cells. Rather, the CP^Hi population was numerically dominated by other subsets, including cycling, terminally exhausted–like, cytotoxic-like, and/or resident memory-like T_EX populations, and a subset enriched for glycolytic genes. Collectively, these data show that CP^Hi TILs correlate with response in melanoma, but this TIL subset is a heterogeneous mix of different subpopulations that may differentially contribute to antitumor immunity following checkpoint blockade.

Significance: The PD-1⁺ CTLA4⁺ CD8⁺ tumor-infiltrating lymphocyte population correlating with immunotherapy response is a heterogeneous mix of subpopulations, which has important implications for optimizing checkpoint-based immunotherapy.