UC San Diego

Although the efficacy of many successful immunotherapies is leveraged by T cells specific for neoantigens (NeoAg), non-self-derived antigens expressed by tumors which typically arise from point mutations, the progressive growth of tumors expressing immunogenic mutations suggests that an effective endogenous immune response is avoided or suppressed. CD4+ T cells play critical roles in the regulation of cellular immunity through the myriad subsets they can comprise but little is known about their development in response to progressive tumor growth, which limits the ability to evaluate therapeutic opportunities and efficacies. I characterized the endogenous NeoAg-specific CD4+ T cell response to a validated clathrin NeoAg (CLTCH129>Q) expressed by SCC VII, an aggressively and poorly immunogenic MHC-II-deficient tumor, using flow cytometry and a CLTCH129>Q/I-Ak tetramer throughout progressive tumor growth. 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 (Th1), T follicular helper (Tfh)-like, and regulatory T cell (Treg) lineages appearing as early as 9 days after tumor inoculation. Therapeutic vaccination substantially alters the relative ratio of these subsets by sharply reducing Treg frequency among CLTCH129>Q-specific CD4+ T cells in both the tumor and tumor-draining lymph node (tdLN). Single cell transcriptomic analysis of CLTCH129>Q/I-Ak tetramer-binding CD4+ T cells recapitulated and extended the diversity of the response while permitting profiling of discovered TCRs following transduction into CD4+ T cells. TCRs of varying affinity are found within each functional subset and while affinity played no role in phenotypic fate, adoptive cellular therapies (ACT) using Th1 cells transduced with a Treg-associated TCR were still capable of mediating therapy equally well as those found on cells expressing anti-tumor phenotypes. These findings offer an 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.