PD-1 and TIGIT coexpression enables selective enrichment of clonally expanded tumor-reactive CD8 T cells for melanoma...

Although anti-PD-1 antibodies are approved as first-line treatment for patients with metastatic melanoma (MM), many patients remain resistant. Adoptive cell therapy (ACT) using tumor-infiltrating lymphocytes (TILs) represents a promising alternative or complementary strategy, but heterogeneous clinical response rates indicate that TIL-ACT still requires optimization, particularly through enrichment of tumor-specific T lymphocytes. We recently identified a circulating CD8⁺ T-cell population, termed DPOS, defined by PD-1 and TIGIT coexpression, whose frequency correlates with anti-PD-1 efficacy in MM patients. Because these activated CD8⁺ T cells are enriched in melanoma antigen-specific lymphocytes, we hypothesized that this biomarker combination could enable the selective isolation of TILs with high therapeutic potential.

Deep immunophenotyping, T-cell receptor (TCR) sequencing, in vitro functional assays, and in vivo patient-derived xenograft models were used to characterize and evaluate the antitumor reactivity of the DPOS subset. A clinically compatible workflow for selective isolation and expansion of DPOS TILs was developed using flow cytometry sorting and ex vivo expansion.

DPOS TILs displayed high expression of activation, costimulatory, tissue residency, and pre-exhaustion markers, consistent with an effector-memory phenotype, while maintaining proliferative capacities similar to other CD8⁺ TILs. TCR sequencing and ELISpot assays demonstrated that the DPOS subset is enriched in clonally expanded tumor antigen-specific T cells, with limited overlap between dominant DPOS clonotypes and those from the remaining CD8⁺ TIL compartment. Co-culture assays against autologous tumor cell lines demonstrated that DPOS TILs mediate stronger tumor recognition, characterized by increased cytotoxicity, cytokine secretion, and proliferation following antigen encounter. These functional advantages translated into improved tumor control in two patient-derived xenograft models.

PD-1⁺TIGIT⁺ CD8⁺ TILs define a distinct tumor-reactive population within human melanoma lesions. Our findings support the use of PD-1/TIGIT coexpression as a clinically applicable strategy to enrich functionally tumor-reactive CD8⁺ T cells and provide a rationale for biomarker-guided optimization of TIL-based adoptive cell therapy in melanoma.