TOX-expressing terminally exhausted tumor-infiltrating CD8+ T cells are reinvigorated by co-blockade of PD-1 and TIGIT in bladder cancer

Exhausted T cells in the tumor microenvironment are major targets of immunotherapies. However, the exhaustion status of CD8(+) tumor-infiltrating lymphocytes (TILs) in bladder cancer has not been comprehensively evaluated. Herein, we examined distinct exhaustion status of CD8(+) TILs based on the level of programmed cell death-1 (PD-1) and thymocyte selection-associated high mobility group box protein (TOX) expression in urothelial bladder cancer. We also evaluated the reinvigoration of exhausted CD8(+) TILs upon ex vivo treatment with inhibitory checkpoint blockers. TOX-expressing PD-1(high)CD8(+) TILs had the highest expression of immune checkpoint receptors (ICRs), the most terminally exhausted features, and the highest tumor antigen reactivity among PD-1(+)CD8(+) TILs. Bladder cancer patients with a high percentage of PD-1(high)TOX(+)CD8(+) TILs had more progressed T-cell exhaustion features and higher programmed death-ligand 1 expression in tumor tissues. TIGIT was the most frequent co-expressed ICR on PD-1(+)CD8(+) TILs, and TIGIT blockade enhanced the PD-1 blockade-mediated cytokine production by CD8(+) TILs from bladder cancer patients. Our findings provide an improved understanding of the heterogeneous exhaustion status of CD8(+) TILs and additional immunotherapy strategies to improve outcomes of bladder cancer patients.