Bystander CD4⁺ T cells infiltrate human tumors and are phenotypically distinct

Tumor-specific T cells likely underpin effective immune checkpoint-blockade therapies. Yet, most studies focus on Treg cells and CD8⁺ tumor-infiltrating lymphocytes (TILs). Here, we study CD4⁺ TILs in human lung and colorectal cancers and observe that non-Treg CD4⁺ TILs average more than 70% of total CD4⁺ TILs in both cancer types. Leveraging high dimensional analyses including mass cytometry, we reveal that CD4⁺ TILs are phenotypically heterogeneous, within each tumor and across patients. Consistently, we find different subsets of CD4⁺ TILs showing characteristics of effectors, tissue resident memory (Trm) or exhausted cells (expressing PD-1, CTLA-4 and CD39). In both cancer types, the frequencies of CD39^- non-Treg CD4⁺ TILs strongly correlate with frequencies of CD39^- CD8⁺ TILs, which we and others have previously shown to be enriched for cells specific for cancer-unrelated antigens (bystanders). Ex-vivo, we demonstrate that CD39^- CD4⁺ TILs can be specific for cancer-unrelated antigens, such as HCMV epitopes. Overall, our findings highlight that CD4⁺ TILs can also recognize cancer-unrelated antigens and suggest measuring CD39 expression as a straightforward way to quantify or isolate bystander CD4⁺ T cells.