Understanding resistance to antibody to programmed cell death protein 1 (PD-1; anti-PD-1) is crucial for the development of reversal strategies. In anti-PD-1-resistant models, simultaneous anti-PD-1 and vaccine therapy reversed resistance, while PD-1 blockade before antigen priming abolished therapeutic outcomes. This was due to induction of dysfunctional PD-1⁺CD38^hi CD8⁺ cells by PD-1 blockade in suboptimally primed CD8 cell conditions induced by tumors. This results in erroneous T cell receptor signaling and unresponsiveness to antigenic restimulation. On the other hand, PD-1 blockade of optimally primed CD8 cells prevented the induction of dysfunctional CD8 cells, reversing resistance. Depleting PD-1⁺CD38^hi CD8⁺ cells enhanced therapeutic outcomes. Furthermore, non-responding patients showed more PD-1⁺CD38⁺CD8⁺ cells in tumor and blood than responders. In conclusion, the status of CD8⁺ T cell priming is a major contributor to anti-PD-1 therapeutic resistance. PD-1 blockade in unprimed or suboptimally primed CD8 cells induces resistance through the induction of PD-1⁺CD38^hi CD8⁺ cells that is reversed by optimal priming. PD-1⁺CD38^hi CD8⁺ cells serve as a predictive and therapeutic biomarker for anti-PD-1 treatment. Sequencing of anti-PD-1 and vaccine is crucial for successful therapy.