KIR Expression Defines a Transcriptionally Distinct CD8+ T-cell Population that Confounds Antigen-Specific T-cell Detection

HLA-C-restricted CD8+ T-cell responses remain poorly defined compared with HLA-A and HLA-B responses, despite the central role of HLA-C as both a peptide-presenting molecule and a ligand for killer-cell immunoglobulin-like receptors (KIRs). This dual function creates a major challenge for pHLA multimer-based antigen-specific T-cell analysis, as HLA-C multimers may bind CD8+ T-cells through either antigen-specific TCRs or KIRs. Here, we show that KIR expression on CD8+ T-cells drives TCR-independent binding to pHLA-C multimers, leading to overestimation of HLA-C-restricted antigen-specific T-cell responses. Using HLA-mismatched donor settings and cancer neoantigen pHLA-C multimers, we demonstrate that apparent multimer-positive CD8+ T-cells can arise from KIR-mediated recognition rather than cognate TCR specificity. We further show that KIR blockade before pHLA-C multimer staining removes non-TCR-specific binding, and applying this strategy to large-scale analysis of CMV, EBV, and SARS-CoV-2 antigens, we identify TCR-specific HLA-C-restricted CD8+ T-cell populations. Importantly, KIR-mediated pHLA-C binding is peptide-selective rather than uniform across all peptide-HLA-C complexes. Single-cell transcriptomic, phenotypic, and clonotypic analyses further demonstrate that KIR-mediated and TCR-mediated pHLA-C multimer binding define transcriptionally and clonally distinct CD8+ T-cell populations. Together, these findings establish KIR-mediated pHLA-C recognition as a major confounder in HLA-C multimer-based T-cell analysis. By separating KIR- from TCR-mediated binding, our approach provides a practical framework for accurate discovery and characterization of HLA-C-restricted antigen-specific CD8+ T-cells across viral infection and cancer.