Single-cell RNA and TCR repertoire analysis identify markers of virus-specific T cells

Adoptive cell therapy (ACT) is a promising method for treating cancer and viral infections. Identifying antigen-specific T cells (ASTs) is critical to ACT. We investigated biomarkers for identifying ASTs. Peripheral blood mononuclear cells from healthy donors underwent staining with carboxyfluorescein succinimidyl ester (CFSE) to detect proliferating ASTs. Following exposure to CMV pp65 peptide and interleukin-2 for T-cell expansion, CD3⁺/CD8⁺ T cells were isolated at varying time points, revealing distinct populations. TCR repertoire analysis unveiled nine major clones in CFSE^-/CD3⁺/CD8⁺ T cells on day 7, constituting 93.9 % of total cells. Contrarily, CFSE⁺/CD3⁺/CD8⁺ T cells exhibited minimal overlap with major TCR clones. Combined single-cell RNA-seq analyses highlighted upregulated genes associated with cell cycle proliferation and T-cell cytotoxicity. Engineered T cells expressing dominant clones effectively engaged CMV pp65 peptide, triggering T-cell activation and interferon-γ production. A set of seven upregulated genes in CFSE^-/CD3⁺/CD8⁺ T cells on day 7, indicative of proliferating ASTs, was used to identify antigen-specific CD3⁺/CD8⁺ T cells on days 2-3, exhibiting 93.52 % accuracy. These markers predicted CFSE^-/CD3⁺/CD8⁺ T cells with dominant TCR clones following exposure to EBV peptide with 74.59 % accuracy. In conclusion, we identified new markers facilitating the early isolation of viral antigen-responsive T cells.