Cell Surface ADGRG1 and CD86 Expression Identifies Antitumor CD4+ T Cells in Human Cancer

Two cell surface proteins, ADGRG1 and CD86, mark tumor-reactive CD4+ T cells in human solid tumors and can be used to isolate viable cells by flow sorting, according to work published in PNAS. Investigators in the Surgery Branch of the National Cancer Institute paired single cell transcriptomics with surface proteomics across metastatic colorectal, pancreatic, esophageal, rectal, and ovarian tumors. The markers offer a route to antitumor CD4+ TIL for functional study and for TCR-engineered cell therapy.

Tumor-reactive CD8+ T cells can be captured using PD-1, CD39, and CD103, but equivalent markers for CD4+ T cells have been lacking, and PD-1 and ICOS were expressed almost universally on CD4+ TIL in these samples. Because HLA class II-restricted CD4+ responses appear to contribute more in epithelial cancers than in melanoma, the authors profiled 130 immune-related surface proteins by CITE-seq alongside single cell RNA and TCR sequencing in three colorectal tumors, then tested candidate markers by FACS in six further tumors. Sorted TCRs were transduced into healthy donor lymphocytes and screened against 1,326 patient tumor mutations presented by autologous antigen-presenting cells.

Antitumor CD4+ clonotypes showed high surface expression of ADGRG1, CD86, and CD57. In the retrospective samples, ADGRG1 sorting enriched validated neoantigen-reactive clonotypes to 22.1% of sorted cells versus 10.4% for bulk CD4+ TIL, with overall enrichment of 11-fold for ADGRG1 and 9-fold for CD86. In validation samples, ADGRG1 and CD86 sorting recovered 11 of 20 and 9 of 20 reactive clonotypes, while bulk sorting recovered one (P < 0.05). ADGRG1 gave the best ROC performance (AUC, 0.70; PPV, 59.6%; NPV, 76.1%), followed by CD86 (AUC, 0.62). Each marker was present on 94% of neoantigen-reactive populations, yet the two overlapped on only about 1% of CD4+ TIL. Both enriched exhausted and cytotoxic states while selecting against precursor-exhausted cells, and ADGRG1-sorted TIL retained neoantigen reactivity after rapid expansion in two samples. The authors propose evaluating both markers for isolating CD4+ TCR clonotypes for engineered cell therapies.

Reference:

Hitscherich KJ, Dinerman AJ, Hakim AA, et al. Cell surface ADGRG1 and CD86 expression identifies antitumor CD4⁺ T cells in human cancer. Proc Natl Acad Sci U S A. 2026;123(32):e2517883123. https:doi.org/10.1073/pnas.2517883123