Figure 2

Activation-induced markers are upregulated on antigen-specific T cells following antigen-specific activation. T cells from a CMV serum positive, HLA-A*02:01⁺ healthy donor (HD1) were expanded for 10 days in the presence of CMV pp65 peptide (NLVPMVATV) to enable proof-of-concept tests. (A) Pre-expanded T cells were restimulated with cognate CMV pp65 peptide in RAPTER assay culture conditions over a 96-h time course. The percentages of AIM⁺ CD8⁺ T cells was assessed by flow cytometry. (B) Pre-expanded T cells were restimulated with DMSO or CMV pp65 peptide (NLVPMVATV) for 24 h. The percentages of CMV pp65 dextramer⁺ and CD137/4-1BB CD8⁺ T cells was assessed by flow cytometry. (C) The percentage of CD137/4-1BB⁺ T cells within the dextramer⁺ T cell population post-restimulation was assessed by flow cytometry. (D) T cells from HD1 and three additional HDs with varying HLA haplotypes and CMV positivity were expanded for 10 days in the presence of CMV pp65 peptide (NLVPMVATV). The percentages of CMV pp65 dextramer⁺ and CD137/4-1BB CD8⁺ T cells post-restimulation was assessed by flow cytometry. (E) Pre-expanded T cells from HD1 were restimulated with DMSO or CMVpp65 peptide for 24 h and the percentages of dextramer⁺, CD137/4-1BB⁺, and PD-1⁺ CD8⁺ T cells were assessed by flow cytometry. (F) CMV pp65 and MART1 dextramer⁺ (Dex⁺), and CMV pp65 CD137/4-1BB⁺, and PD-1⁺ CD8⁺ T cells were isolated by FACS then analyzed by scTCR-SEQ. Correlation map shows the overlap between TCR sequences across conditions. All dextramer⁺ TCRs were compared to CD137/4-1BB⁺, and PD-1⁺ TCRs with clone sizes >  = 10 cells.