Current methods for biomarker discovery and target identification in immuno-oncology rely on static snapshots of tumor immunity.To thoroughly characterize the here temporal nature of antitumor immune responses, we developed a 34-parameter spectral flow cytometry panel and performed high-throughput analyses in critical contexts.We leveraged two distinct preclinical models that recapitulate cancer immunoediting (NPK-C1) and immune checkpoint blockade (ICB) response (MC38), respectively, and profiled multiple relevant tissues at and around key inflection points of immune surveillance and escape and/or ICB response.Machine learning-driven data analysis revealed a pattern of KLRG1 expression that uniquely identified intratumoral effector CD4 T cell populations that constitutively associate with tumor burden across tumor models, and are lost in tumors undergoing regression in response to ICB.Similarly, a Helios-KLRG1+ subset of tumor-infiltrating regulatory metabo 15-gauge finish nailer cordless T cells was associated with tumor progression from immune equilibrium to escape and was also lost in tumors responding to ICB.
Validation studies confirmed KLRG1 signatures in human tumor-infiltrating CD4 T cells associate with disease progression in renal cancer.These findings nominate KLRG1+ CD4 T cell populations as subsets for further investigation in cancer immunity and demonstrate the utility of longitudinal spectral flow profiling as an engine of dynamic biomarker discovery.