Characterization of neoantigen-specific T cells in cancer resistant to immune checkpoint therapies

Neoantigen-specific T cells are strongly implicated as being critical for effective immune checkpoint blockade treatment (ICB) (e.g., anti–PD-1 and anti–CTLA-4) and are being targeted for vaccination-based therapies. However, ICB treatments show uneven responses between patients, and neoantigen vaccination efficiency has yet to be established. Here, we characterize neoantigen-specific CD8⁺ T cells in a tumor that is resistant to ICB and neoantigen vaccination. Leveraging the use of mass cytometry combined with multiplex major histocompatibility complex (MHC) class I tetramer staining, we screened and identified tumor neoantigen–specific CD8⁺ T cells in the Lewis Lung carcinoma (LLC) tumor model (mRiok1). We observed an expansion of mRiok1-specific CD8⁺ tumor-infiltrating lymphocytes (TILs) after ICB targeting PD-1 or CTLA-4 with no sign of tumor regression. The expanded neoantigen-specific CD8⁺ TILs remained phenotypically and functionally exhausted but displayed cytotoxic characteristics. When combining both ICB treatments, mRiok1-specific CD8⁺ TILs showed a stem-like phenotype and a higher capacity to produce cytokines, but tumors did not show signs of regression. Furthermore, combining both ICB treatments with neoantigen vaccination did not induce tumor regression either despite neoantigen-specific CD8⁺ TIL expansion. Overall, this work provides a model for studying neoantigens in an immunotherapy nonresponder model. We showed that a robust neoantigen-specific T-cell response in the LLC tumor model could fail in tumor response to ICB, which will have important implications in designing future immunotherapeutic strategies.