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 and single-cell sequencing, we reveal that CD4 TILs are heterogeneous at both gene and protein levels, 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). , we demonstrate that CD39 CD4 TILs can be specific for cancer unrelated antigens, such as HCMV epitopes. Overall, our findings highlight that CD4 TILs cells are not necessarily tumor-specific and suggest measuring CD39 expression as a straightforward way to quantify or isolate bystander CD4 T cells.