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

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 _h 1), 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.

What is already known on this topic: Little is known about the ontogeny, architecture, development of the CD4 ⁺ NeoAg-specific repertoire induced by progressively-growing tumor. This study was performed to address this topic and contribute new information to aid in its understanding.

What this study adds: This study reveals that the NeoAg-specific CD4 ⁺ T cell response to a growing tumor is phenotypically and functionally diverse, featuring a range of functional T cells subsets including T _H 1, T _FH , and T _reg expressing a range of functional TCR avidities, and demonstrates how an immunotherapeutic NeoAg vaccine can alter their relative composition within the tumor and tumor-draining lymph node.

How this study might affect research practice or policy: This study offers new insights into the diversity of NeoAg-specific CD4 ⁺ T cells and their response to a tumor in the presence or absence of immunotherapeutic intervention. This information could lead to new approaches to immune monitoring in the clinical setting of checkpoint blockade immunotherapy and cancer vaccines. Furthermore, we show that T _reg can be a potent source of TCRs that can mediate therapeutic benefit in the setting of adoptive cell therapy (ACT).