Fig. 1: EBV-specific CD8⁺ T cells differ in their preferred, phenotypically defined differentiation states dependent on the recognized antigen.

A Schematic of experimental design: CD8⁺ T cells from peripheral blood of young adults (< 40 years) were stained with the panel of antibodies shown in Supplementary Data 3 and HLA-A*02:01 tetramers loaded with indicated EBV peptides and then subjected to spectral flow cytometry. B UMAP reference plots and feature plots of indicated cell surface marker showing bulk CD8⁺ T cell subsets. Functional subset assignment was based on classical marker profiles including CD45RA/RO, CCR7, and CD28. C Representative flow cytometry scatter plots of tetramer staining and box plot of tetramer-positive cell frequencies (n = 7). Fluorophores for tetramers were PE-Cy5 (BMLF1), BV480 (EBNA3C), APC (BRLF1), and PE (LMP2). D Reference mapping of tetramer-positive cells projected on UMAP plots from bulk CD8⁺ T cells. E Bubble chart of tetramer-positive cell distributions among bulk CD8⁺ T cell-defined clusters defined in (B). The bubble size and color reflect the median proportion of antigen-specific T cells in a given cluster. F Comparison of probability vectors reflecting the subset distributions of tetramer-positive cells. Significance levels between the indicated pairwise comparisons are shown as bubble size and color. G Principal component analysis (PCA) of cell frequency distributions. H Protein levels of indicated transcription factors or cytotoxic molecules in tetramer-positive cells as assessed by spectral flow cytometry (n = 8). Data show the median with 95% confidence intervals (C), median (E), or mean ± SEM in (H). All datapoints represent distinct biological replicates. Data were compared by two-sided permutation tests (F), or two-tailed, unpaired t-tests comparing all lytic (BMLF1, BRLF1) versus all latent (LMP2, EBNA3C) antigen-specific T cell populations (H). Source data are provided as a Source Data file.