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Therapeutic benefit to immune checkpoint blockade (ICB) is currently limited to the subset of cancers thought to possess a sufficient tumor mutational burden (TMB) to allow for the spontaneous recognition of neoantigens (NeoAg) by autologous T cells. We explored whether the response to ICB of an aggressive low-TMB squamous cell tumor could be improved through combination immunotherapy using functionally defined NeoAg as targets for endogenous CD4+ and CD8+ T cells. We found that, whereas vaccination with CD4+ or CD8+ NeoAg alone did not offer prophylactic or therapeutic immunity, vaccines containing NeoAg recognized by both subsets overcame ICB resistance and led to the eradication of large established tumors that contained a subset of PD-L1+ tumor-initiating cancer stem cells (tCSC), provided the relevant epitopes were physically linked. Therapeutic CD4+/CD8+ T cell NeoAg vaccination produced a modified tumor microenvironment (TME) with increased numbers of NeoAg-specific CD8+ T cells existing in progenitor and intermediate exhausted states enabled by combination ICB-mediated intermolecular epitope spreading. We believe that the concepts explored herein should be exploited for the development of more potent personalized cancer vaccines that can expand the range of tumors treatable with ICB.

CD4 + T cells play key roles in a range of immune responses, either as direct effectors or through accessory cells, including CD8 + T lymphocytes. In cancer, neoantigen (NeoAg)-specific CD8 + T cells capable of direct tumor recognition have been extensively studied, whereas the role of NeoAg-specific CD4 + T cells is less well understood. We have characterized the murine CD4 + T cell response against a validated NeoAg (CLTC H129>Q ) expressed by the MHC-II-deficient squamous cell carcinoma tumor model (SCC VII) at the level of single T cell receptor (TCR) clonotypes and in the setting of adoptive immunotherapy. We find that the natural CLTC H129>Q -specific repertoire is diverse and contains TCRs with distinct avidities as measured by tetramer-binding assays and CD4 dependence. Despite these differences, CD4 + T cells expressing high or moderate avidity TCRs undergo comparable in vivo proliferation to cross-presented antigen from growing tumors and drive similar levels of therapeutic immunity that is dependent on CD8 + T cells and CD40L signaling. Adoptive cellular therapy (ACT) with NeoAg-specific CD4 + T cells is most effective when TCR-engineered cells are differentiated ex vivo with IL-7 and IL-15 rather than IL-2 and this was associated with both increased expansion as well as the acquisition and stable maintenance of a T stem cell memory (T SCM )-like phenotype in tumor-draining lymph nodes (tdLNs). ACT with T SCM -like CD4 + T cells results in lower PD-1 expression by CD8 + T cells in the tumor microenvironment and an increased frequency of PD-1 + CD8 + T cells in tdLNs. These findings illuminate the role of NeoAg-specific CD4 + T cells in mediating antitumor immunity via providing help to CD8 + T cells and highlight their therapeutic potential in ACT., (© 2023. The Author(s).)