Cell surface ADGRG1 and CD86 expression identifies antitumor CD4⁺ T cells in human cancer

Adoptive cell transfer of tumor infiltrating CD4⁺ T cells or T cells genetically modified to express antitumor CD4⁺ T cell receptors (TCRs) has shown clinical efficacy in patients with advanced solid tumors. We previously defined unique transcriptomic states of antitumor, neoantigen-specific CD8⁺ and CD4⁺ tumor infiltrating lymphocytes (TIL) in human cancer. However, optimal cell surface protein markers that identify and would enable viable cell isolation and selective enrichment of antitumor CD4⁺ T cells have yet to be identified. Here, we define transcriptomic and corresponding cell surface protein profiles of antitumor CD4⁺ TIL within metastatic colorectal tumors using high dimensional single cell transcriptomic and proteomic analysis. Comprehensive analysis of 45 antitumor CD4⁺ TCR clonotypes demonstrated uniquely high cell surface protein expression of ADGRG1, CD86, and CD57. In all samples containing tumor neoantigen-reactive CD4⁺ TIL clonotypes, ADGRG1- and CD86-based cell surface enrichment of known reactive TCR clonotypes were 11-fold and 9-fold higher than bulk CD4⁺ TIL, respectively. ADGRG1- and CD86-based cell surface enrichment identified >60 validated antitumor CD4⁺ TCR-clonotypes including tumor organoid-reactive TCRs, as well as neoantigen-specific TCRs targeting private tumor mutations and cancer driver mutations. While both ADGRG1 and CD86 enriched CD4⁺ TIL displayed an exhausted neoantigen-specific gene expression signature, CD86 also enriched for cytotoxic CD4⁺ TIL phenotypes. CD4⁺ TIL isolated based on ADGRG1 expression and expanded in vitro also demonstrated neoantigen reactivity in two samples. Thus, ADGRG1 and CD86 appear to be effective protein markers for isolating human tumor-specific CD4⁺ TCR clonotypes and studying antitumor CD4⁺ TIL.