Background Immunotherapy has demonstrated impressive results in numerous metastatic solid tumors. However, while most studies have focused on immune responses of CD8⁺ tumor-infiltrating lymphocytes (TILs), the functional role of tumor-reacting CD4⁺ TILs remains largely unclear.

Methods To characterize the complete functional repertoire of actively tumor-reacting CD4⁺ TILs, we generated and tested a workflow allowing accurate reproduction of the TIL-tumor cell interaction of eighteen individual patients with three tumor histologies. Via bulk and single-cell RNA sequencing (scRNAseq) of ex vivo-expanded CD4⁺ TILs post-coculture with autologous tumor cells, we obtained a complete transcriptomic characterization of tumor-reacting CD4⁺ TILs following recognition of naturally presented tumor antigens.

Results Recognition of autologous tumor antigens induced detectable transcriptomic and secretome changes in tumor-reacting CD4⁺ TILs, and allowed the generation of a transcriptomic signature of actively tumor-reacting CD4⁺ TILs (TR4S) used to identify multiple subpopulations of functionally distinct tumor-reacting CD4⁺ TILs. The results were then validated on an atlas of public scRNAseq data from fresh tumor tissues of twelve distinct tumor histologies, confirming the presence of multiple tumor-reacting CD4⁺ TIL subpopulations activating distinct functional programs.

Conclusions In conclusion, we characterized the functional landscape of tumor-reacting CD4⁺ TILs on a transcriptomic level. These data highlighted the functional heterogeneity of actively tumor-reacting CD4⁺ TILs, and warrant further investigation into the role of functionally distinct tumor-reacting CD4⁺ TIL subpopulations in the tumor microenvironment.

Ethics Approval All patients were enrolled in clinical trials performed at the National Center for Cancer Immune Therapy (CCIT-DK), Department of Oncology, Copenhagen University Hospital, Herlev, Denmark. All procedures were performed in compliance with clinical protocols approved by the Ethics Committee of the Capital Region of Denmark (Reference H-19076238 and H-20070020) and national regulations for biomedical research, including appropriate data protection of human participants (Reference P-2020-65 and P-2021-303). Written informed consent was provided by all patients before obtaining any samples.