Neoantigen-specific stem cell memory-like CD4⁺ T cells mediate CD8⁺ T cell-dependent immunotherapy of MHC class...

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.