Neoantigen-specific CD4⁺ tumor-infiltrating lymphocytes are potent effectors identified within adoptive cell therapy products for metastatic melanoma patients

Background: Adoptive cell therapy (ACT) with tumor-infiltrating lymphocytes (TILs) is a promising immunotherapeutic approach for patients with advanced solid tumors. While numerous advances have been made, the contribution of neoantigen-specific CD4⁺T cells within TIL infusion products remains underexplored and therefore offers a significant opportunity for progress.

Methods: We analyzed infused TIL products from metastatic melanoma patients previously treated with ACT for the presence of neoantigen-specific T cells. TILs were enriched on reactivity to neoantigen peptides derived and prioritized from patient sample-directed mutanome analysis. Enriched TILs were further investigated to establish the clonal neoantigen response with respect to function, transcriptomics, and persistence following ACT.

Results: We discovered that neoantigen-specific TIL clones were predominantly CD4⁺ T cells and were present in both therapeutic responders and non-responders. CD4⁺ TIL demonstrated an effector T cell response with cytotoxicity toward autologous tumor in a major histocompatibility complex class II-dependent manner. These results were validated by paired TCR and single cell RNA sequencing, which elucidated transcriptomic profiles distinct to neoantigen-specific CD4⁺ TIL.

Conclusions: Despite methods which often focus on CD8+T cells, our study supports the importance of prospective identification of neoantigen-specific CD4⁺ T cells within TIL products as they are a potent source of tumor-specific effectors. We further advocate for the inclusion of neoantigen-specific CD4⁺ TIL in future ACT protocols as a strategy to improve antitumor immunity.