CD96 marks a phenotypically distinct checkpoint-associated HCV-specific CD8+ T-cell subset featuring memory-associated states
Introduction Hepatitis C virus (HCV) infection frequently progresses to chronicity, where persistent antigen exposure drives differentiation of virus-specific CD8+ T cells toward an exhausted phenotype. This state is defined by progressive upregulation of inhibitory receptors and functional impairment of antigen-specific T cells. Because patients span distinct clinical stages from acute infection to spontaneous resolution or therapy induced sustained virological response, HCV provides a unique model to study how T-cell exhaustion is established, maintained, and potentially reversed. While PD1 and TIGIT are well established in this context, CD96 (TACTILE) is a less-characterized receptor that shares its ligand CD155 with TIGIT and DNAM-1 and may modulate CD8+ T-cell differentiation and function. Methods To evaluate the role of CD96 across infection stages, we used HCV-specific MHC class I tetramers combined with high-dimensional flow cytometry to phenotypically profile virus-specific CD8+ T-cells ex vivo in 32 patients spanning acute, subacute, chronic, spontaneously resolved infection, and post-treatment sustained virological response. We assessed CD96 co-expression with PD1/TIGIT, transcription factors (TCF1, TOX, IRF4, T-bet, NR2F6), and relevant differentiation and exhaustion markers. Results CD96+ HCV-specific CD8+ T cells consistently increased relative to bulk across all stages of HCV infection, with approximately four-fold higher frequencies than bulk CD96+ CD8+ T cells (p<0.0001), and highest frequencies of 51,13% observed in the chronic group. Because CD96 expression was predominantly distributed continuously rather than strictly bimodal, we also report CD96 MFI as a continuous measurement alongside CD96+ frequencies. Phenotypically, HCV-specific and bulk CD96+ CD8+ T cells were enriched for IRF4+, TOX+ and TCF1+ subsets, suggesting possible reduced effector features and increased memory-associated characteristics. Notably, the CD96+ PD1+ TIGIT+ subset was about five times higher in HCV-specific than in bulk CD8+ T-cell populations (p<0.0001). In cross-sectional comparison, frequencies of CD96+ PD1+ TIGIT+ HCV-specific CD8+ T cells decreased, while the CD96- PD1- TIGIT- counterparts expanded. Discussion These findings identify CD96 as a marker of a phenotypically distinct checkpoint-associated HCV-specific CD8+ T-cell subset featuring memory-associated rather than terminally exhausted states. Whether this phenotypic association has functional or therapeutic significance for combination immune therapy will require future studies, including direct functional assays of sorted CD96+ versus CD96- HCV-specific CD8+ T cells.