ABSTRACT: 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 of an aggressive low TMB squamous cell tumor to ICB 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. 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.