PD-1 blockade-unresponsive human tumor-infiltrating CD8^(+)T cells are marked by loss of CD28 expression and rescued by IL-15

Blockade of programmed death-1(PD-1)reinvigorates exhausted CD8^(+)T cells,resulting in tumor regression in cancer patients.Recently,reinvigoration of exhausted CD8^(+)T cells following PD-1 blockade was shown to be CD28-dependent in mouse models.Herein,we examined the role of CD28 in anti-PD-1 antibody-induced human T cell reinvigoration using tumor-infiltrating CD8^(+)T cells(CD8^(+)TILs)obtained from non-small-cell lung cancer patients.Single-cell analysis demonstrated a distinct expression pattern of CD28 between mouse and human CD8^(+)TILs.Furthermore,we found that human CD28^(+)CD8^(+)but not CD28^(–)CD8^(+)TILs responded to PD-1 blockade irrespective of B7/CD28 blockade,indicating that CD28 costimulation in human CD8^(+)TILs is dispensable for PD-1 blockade-induced reinvigoration and that loss of CD28 expression serves as a marker of anti-PD-1 antibody-unresponsive CD8^(+)TILs.Transcriptionally and phenotypically,PD-1 blockade-unresponsive human CD28^(–)PD-1^(+)CD8^(+)TILs exhibited characteristics of terminally exhausted CD8^(+)T cells with low TCF1 expression.Notably,CD28^(–)PD-1^(+)CD8^(+)TILs had preserved machinery to respond to IL-15,and IL-15 treatment enhanced the proliferation of CD28^(–)PD-1^(+)CD8^(+)TILs as well as CD28^(+)PD-1^(+)CD8^(+)TILs.Taken together,these results show that loss of CD28 expression is a marker of PD-1 blockade-unresponsive human CD8^(+)TILs with a TCF1–signature and provide mechanistic insights into combining IL-15 with anti-PD-1 antibodies.