Fig 2: Antigen-specific CD8+ T cell subset differentiation and phenotype are based on virus specificity independent of age(A) T cell subset distribution of flu-specific (red) and CMV-specific(blue) CD8+ T cells based on CD45RA and CCR7 expression (CD45RA+/CCR7+; CD45RA−/CCR7+, TCM; CD45RA−/CCR7−, TEM; CD45RA+/CCR7−, TEMRA) shown in representative flow cytometry plots. Gray contour plots depict CD45RA and CCR7 expression of total CD8+ T cells within the same sample. Numbers indicate frequency of multimer+ virus-specific cells within each subset.(B) Subset distribution of flu-specific (shades of red) and CMV-specific (shades of blue) CD8+ T cells compiled from 3–21 donors in blood and indicated tissue sites.(C) Frequencies of flu-multimer+ (red) and CMV-multimer+ (blue) CD8+ T cells maintained as CD69− TEM cells (top) and TEMRAs (bottom) from 3–21 donors for each tissue site.(D and E) Frequencies of flu-specific (red, left) and CMV-specific (blue, right) CD8+CD69− TEM cells (D) and CD8+ TEMRAs (E) in indicated tissue sites as a function of age for each individual donor. The line of best fit was determined by Pearson correlation; “pearsonr” refers to correlation coefficient, p values are indicated for each comparison, and red font indicates significant correlation for BM.Statistical significance for comparison of means was calculated by unpaired t test and indicated by **p ≤ 0.01; *p ≤ 0.05. Subset frequencies shown from donors with ≥10 multimer+ T cells.

Fig 3: Distinct frequencies and subset distribution of flu-specific memory CD8+ T cells between adult and pediatric donors(A) Distribution of flu-specific CD8+ T cells in lung, LLN, and MLN of pediatric and adult donors shown in representative flow cytometry plots. Numbers indicate frequency of multimer+ cells within total CD8+ T cell population.(B) Frequencies of flu-multimer+ CD8+ T cells in pediatric (pink; n = 15) and adult (gray; n = 31) donors from 5–21 donors for each tissue site. Inset shows frequencies of flu-multimer+ cells in pediatric tissues with y axis spanning 0%–1%.(C) Memory T cell subset distribution of flu-specific CD8+ T cells in indicated tissues of pediatric donors shown in representative flow cytometry plots (left) and compiled in graph (right) showing frequencies of flu-multimer+ CD8+ T cells in pediatric (pink) and adult (gray) donors maintained as TCM (left) and TEM (right) in lung (top) and LLN (bottom) tissue sites. Subset frequencies shown from donors with ≥10 multimer+ T cells.(D) Expression of tissue residency markers CD69 and CD103 by flu-specific CD8+ T cells in indicated tissues of pediatric donors shown in representative flow cytometry plots (left) and compiled in graph (right) showing frequencies of flu-multimer+ CD69+CD103+CD8+ TRM cells (gated on TEM) in pediatric (pink) and adult (gray) donors in lung (top) and LLN (bottom) tissue sites. TRM frequencies shown from donors with ≥5 multimer+ TEM cells.Statistical significance for comparison of means was calculated by unpaired t test and indicated by *p ≤ 0.05; ns, not significant.

Fig 4: Sex-related differences in tissue maintenance of virus-specific CD8+ T cells(A) Age range of male and female donors in this study (male: green, n = 19; female: yellow, n = 20).(B) Frequencies of flu-multimer+ (left) and CMV-multimer+ (right) CD8+ T cells from 3–15 donors for each site stratified by sex.(C and D) Frequencies of flu-multimer+ (left) and CMV-multimer+ (right) CD8+ TEM cells (C) and CD8+ TEMRAs (D) from 2–13 donors for each site stratified by sex.(E) Frequencies of total CD8+ TEM cells (top) and TEMRA (bottom) from 7–16 donors for each site stratified by sex.(F) Frequencies of flu-multimer+ (left) and CMV-multimer+ (right) CD69+ CD103+ CD8+ TRM cells gated on multimer+ cells from 2–10 donors for each site stratified by sex. Subset frequencies shown from donors with ≥10 multimer+ T cells, and TRM frequencies shown from donors with ≥5 multimer+ TEM cells.Statistical significance for comparison of means was calculated by unpaired t test and indicated by **p ≤ 0.01; *p ≤ 0.05; ns, not significant.

Fig 5: Tissue segregation of the anti-viral T cell response revealed by single-cell transcriptome and functional profiling(A) Antigen-responding CD69+IFN-γ+CD8+ T cells in the spleen and LLN of donor D481 following stimulation by DMSO negative control, flu antigen-specific peptide pool, CMV antigen-specific peptide pool, and anti-CD3/CD28 bead positive control, shown in representative flow cytometry plots. Colored gates (red: flu; blue: CMV) indicate populations that were subsequently sorted for sequencing.(B) Scatterplot showing 2,000 variable genes (red) as determined by calculating average expression and dispersion of each gene. The top 10 most highly variable genes are labeled on the graph.(C) UMAP embedding of virus-reactive CD8+ T cells colored by cluster as identified via unsupervised hierarchical clustering (see STAR Methods), tissue, or virus specificity.(D) Heatmap showing top 10 most differentially expressed genes in each cluster. See Table S4 for complete list of differentially expressed genes (padj < 0.1).(E) Pairwise comparisons of log(x+1) normalized cytokine levels in supernatants from in vitro stimulation of single-cell suspensions from blood (n = 6), BM (n = 7), spleen (n = 9), lung (n = 8), and LLN (n = 10) with flu (red) or CMV (blue) peptide pools. Statistical significance between flu- and CMV-stimulated conditions was calculated by paired t test and indicated by **p ≤ 0.01; *p ≤ 0.05.(F) Cytokine levels in blood, BM, spleen, lung, and LLN supernatant samples.Statistical significance was calculated using one-way ANOVA followed by Tukey’s multiple comparisons test indicated by ***p ≤ 0.001; **p ≤ 0.01; *p ≤ 0.05.