The spontaneous neoantigen-specific CD4⁺ T cell response to a growing tumor is functionally and phenotypically...

CD4⁺ T cells play critical roles in the positive and negative regulation of cellular immunity through the many functional subsets they comprise. The progressive growth of immunogenic tumors which nonetheless generate mutation-specific T cells suggests that effective immune control may be avoided or suppressed at the level of the neoantigen-specific CD4⁺ T cell response. We used a tetramer specific for a validated neoantigen, CTLC_H129>Q/I-E^k, to characterize the ontogeny of natural CD4⁺ T cell responses to an aggressive and poorly immunogenic Major Histocompatibility Complex Class II (MHCII)-deficient tumor, SCC VII, during progressive growth or following therapeutic peptide vaccination. We find that the natural CD4⁺ T cell response to a growing tumor is phenotypically and functionally diverse, with distinct subsets including type 1 helper (T_h1), T follicular helper (T_fh)-like, and regulatory T cell (T_reg) lineages appearing as early as 9 days after tumor implantation. Therapeutic vaccination using the CLTC_H129>Q peptide in adjuvant plus α-PD-1 sharply reduces the frequency of CLTC_H129>Q-specific T_reg frequency in both tumor and tumor-draining lymph node (tdLN). Single cell transcriptomic analysis of CLTC-specific CD4⁺ T cells recapitulated and extended the diversity of the response, with TCRs of varying affinity found within each functional subset. The TCR affinity differences did not strictly correlate with function, however, as even the lowest affinity TCRs isolated from T_reg can mediate therapeutic efficacy against established tumors in the setting of adoptive cellular therapy (ACT). These findings offer unprecedented insight into the functional diversity of a natural neoantigen-specific CD4⁺ T cell response and show how immunotherapeutic intervention influences the phenotype, magnitude, and efficacy of the anti-tumor immune response.