CD8⁺ TILs in NSCLC differentiate into TEMRA via a bifurcated trajectory: deciphering immunogenicity of tumor antigens

Abstract

Background CD8 + tumor-infiltrating lymphocytes (TILs) comprise phenotypically and functionally heterogeneous subpopulations. Of these, effector memory CD45RA re-expressing CD8 + T cells (Temra) have been discovered and characterized as the most terminally differentiated subset. However, their exact ontogeny and physiological importance in association with tumor progression remain poorly understood. Methods We analyzed primary tumors and peripheral blood samples from 26 patients with non-small cell lung cancer and analyzed their phenotypes and functional characteristics using flow cytometry, RNA-sequencing, and bioinformatics. Results We found that tumor-infiltrating Temra (tilTemra) cells largely differ from peripheral blood Temra (pTemra), with distinct transcriptomes and functional properties. Notably, although majority of the pTemra was CD27 − CD28 − double-negative (DN), a large fraction of tilTemra population was CD27 + CD28 + double-positive (DP), a characteristic of early-stage, less differentiated effector cells. Trajectory analysis revealed that CD8 + TILs undergo a divergent sequence of events for differentiation into either DP or DN tilTemra. Such a differentiation toward DP tilTemra relied on persistent expression of CD27 and CD28 and was associated with weak T cell receptor engagement. Thus, a higher proportion of DP Temra was correlated with lower immunogenicity of tumor antigens and consequently lower accumulation of CD8 + TILs. Conclusions These data suggest a complex interplay between CD8 + T cells and tumors and define DP Temra as a unique subset of tumor-specific CD8 + TILs that are produced in patients with relatively low immunogenic cancer types, predicting immunogenicity of tumor antigens and CD8 + TIL counts, a reliable biomarker for successful cancer immunotherapy.