CD4+ T cells play a critical role in antitumor immunity via recognition of peptide antigens presented on MHC class II (MHC-II). Although some solid cancers can be induced to express MHC-II, the extent to which this enables direct recognition by tumor-specific CD4+ T cells is unclear. We isolated and characterized T cell antigen receptors (TCRs) from naturally primed CD4+ T cells specific for 2 oncoproteins, HPV-16 E6 and the activating KRASG12V mutation, from patients with head and neck squamous cell carcinoma and pancreatic ductal adenocarcinoma, respectively, and determined their ability to recognize autologous or human leukocyte antigen-matched antigen-expressing tumor cells. We found in both cases that the TCRs were capable of recognizing peptide-loaded target cells expressing the relevant MHC-II or B cell antigen-presenting cells (APCs) when the antigens were endogenously expressed and directed to the endosomal pathway but failed to recognize tumor cells expressing the source protein even after induction of surface MHC-II expression by IFN-γ or transduction with CIITA. These results suggest that priming and functional recognition of both a nuclear (E6) and a membrane-associated (KRAS) oncoprotein are predominantly confined to crosspresenting APCs rather than via direct recognition of tumor cells induced to express MHC-II.

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.