Routine detection of reactive Cyfra21-1-specific CD8 T cells and its predictive value for responsiveness to PD-1/PD-L1 inhibitor therapy in NSCLC patients.

Immune checkpoint inhibitors are widely used in non-small cell lung cancer (NSCLC), but with limited overall response rates. Indicators predicting immunotherapy response are hard to routinely detect due to their invasiveness. This study systematically screened for CD8T-cell epitopes in Cytokeratin Fragment Antigen 21-1 (Cyfra21-1), a valuable NSCLC tumor marker, through a combination of in silico prediction, ex vivo co-cultures of peptides with patient-derived peripheral blood mononuclear cells (PBMCs), peptide competition binding assays, and peptide immunization in humanized mice. A total of 37 novel CD8T-cell epitopes were confirmed to exhibit immunogenicity in a real-world lung cancer cohort comprising 150 patients. These epitopes were restricted by 13 HLA-A, 15 HLA-B, and 14 HLA-C prevalent allotypes which cover the majority of Northeast Asian population. Using these epitope peptides, a universal ELISpot assay was established to count reactive Cyfra21-1-specific CD8T cells, and a cohort of 70 NSCLC patients scheduled to receive PD-1/PD-L1 inhibitor therapy was tested. The baseline count of reactive Cyfra21-1-specific CD8T cells in PBMCs was identified as an independent predictor of patient responsiveness to immunotherapy, with an AUC of 0.757 and a cut-off value of 25.5 SFUs/4 × 10⁵ PBMCs that distinguished responders from non-responders. This study is the first to construct a broad-spectrum CD8T-cell epitope library of the Cyfra21-1 antigen in Northeast Asians, establish a universal assay for measuring Cyfra21-1-specific CD8T cell reactivity, and validate the predictive value of Cyfra21-1-specific CD8T cells for immunotherapy response in NSCLC patients.