Fig 1.
Alterations in T-cell pool in CMV infection are not solely explained by the presence of CMV-specific CD8+ T-cells.
(A) Absolute T-cell numbers in CMV- (in blue) and CMV+ (in red) individuals for CD4+ (left panel) and CD8+ (right panel) TTN, TCM, TEM, and TEMRA cells. (B) Expression of CD57, KLRG-1, and CD28 by CD4+ and CD8+ T-cells, measured per memory subpopulation (TCM, TEM, and TEMRA) and compared between CMV+ and CMV- individuals. (C) CMV-specific CD8+ T-cells analyzed using 7 different HLA-class I dextramers for immunodominant CMV epitopes (yielding N = 38 CMV-specific CD8+ T-cell populations in N = 32 CMV+ individuals–see S1 Table for epitopes used). Upper panel: CMV-specific CD8+ T-cells in absolute numbers and frequency of total CD8+ T-cells. Middle panel: phenotype of CMV-specific CD8+ T-cells based on division in TN (naïve, CD27+CD45RO-), TCM, TEM, and TEMRA cells. Lower panel: association of percentage TEM/EMRA phenotype of CMV-specific CD8+ T-cells and the frequency of CMV-specific cells within the CD8+ T-cell pool. Error bars in upper and middle panel represent the range. (D) Comparison of expression of CD57, KLRG-1, and CD28 by CMV-specific CD8+ T-cells and total memory CD8+ T-cells of CMV+ individuals, analyzed per memory subpopulation (TCM, TEM, and TEMRA). (E) Left panels: CD8+ T-cells in CMV- and CMV+ individuals, clustered by t-SNE analysis based on the expression of CD57, KLRG-1, CD127, CD27, CD45RO, CCR7, and CD95. Numbers 1–9 represent identified clusters of CD8+ T-cells based on cell density (with red representing high density of cells, and blue low density of cells). Cluster 5–8 (CD57high, KLRG-1high, CD27low) are outlined in black in all t-SNE graphs. Right panels: Percentage of cluster 5–8 within the total CD8+ T-cell pool of CMV- and CMV+ individuals. (F) Phenotype of cluster 5–8 is presented in a heatmap by the mean fluorescence intensity of CD57, KLRG-1, CD127, CD27, CD45RO, CCR7, and CD95 as used in the t-SNE analysis. (G) Left panel: CMV-specific CD8+ T-cells clustered by the same t-SNE analysis as total CD8+ T-cells. Middle panel: Percentage of clusters 5–8 among CMV-specific T-cells. Right panel: Percentage of cluster 9 among CMV-specific T-cells. (H) Phenotype of cluster 9 is presented in a heatmap by the mean fluorescence intensity of CD57, KLRG-1, CD127, CD27, CD45RO, CCR7, and CD95 as used in the t-SNE analysis. Expression levels are given in arbitrary units ranging from zero (black) to high (white). Bars and horizontal lines in all figures represent medians. Differences between CMV- and CMV+ individuals were assessed by Mann-Whitney U test. Correlation was tested by Spearman’s correlation. Stars indicate significant differences as follows: * P-value <0.05, ** P-value <0.01, *** P-value <0.001, *** P-value <0.0001.
Fig 2.
Altered expression of lifespan-associated markers by CD4+ TEM/EMRA cells in CMV infection.
(A) Representative flow cytometric plots for gating of Ki-67 and Bcl-2 expression. Graphs represent percentage of positive cells for Ki-67 and Bcl-2 in CMV- and CMV+ individuals by CD4+ and CD8+ T-cells, measured per memory subpopulation. (B) and (C) Principal component analysis (PCA) of CD4+ (B) and CD8+ (C) T-cells based on lifespan-associated markers KLRG-1, CD57, CD28, Ki-67, and Bcl-2 per memory subpopulation. Blue dots represent CMV- individuals, red dots represent CMV+ individuals. (D) Comparison of expression of Ki-67 and Bcl-2 by the total memory CD8+ T-cell pool of CMV+ individuals and by CMV-specific CD8+ T-cells, measured per memory subpopulation. (E) PC analysis of CD8+ T-cells based on lifespan-associated markers KLRG-1, CD57, CD28, Ki-67 and Bcl-2 per memory subpopulation. Bars in (A) and (D) represent medians. Differences between CMV- and CMV+ individuals were tested by Mann-Whitney U test. Stars indicate significant differences as follows: * P-value <0.05, ** P-value <0.01, *** P-value <0.001, *** P-value <0.0001.
Fig 3.
Dynamics of CD4+ and CD8+ T-cells in CMV- and CMV+ individuals.
(A) Graphical representation of the study design for the deuterated water labelling protocol. Created with BioRender.com. (B) Deuterium labelling enrichment (%DNA labelled) of different T-cell subpopulations in CMV- and CMV+ individuals. Label enrichment was scaled between 0 and 100% by normalizing for the estimated maximum enrichment of granulocytes (see Materials and methods). Symbols depict the mean of duplicate measurements. (C) Summary of estimated production rates of TTN, TCM, and TEM/EMRA CD4+ and CD8+ T-cells in CMV- (blue symbols) and CMV+ (red symbols) individuals. All estimates were obtained by fitting a single-exponential model to the data sets per individual (see Materials and methods and S6 and S7 Figs). It was not possible to reliably estimate the production rate for TCM cells from E23 (CMV-) and TEMRA cells from E34 (CMV+). E30 only received 2H2O for 3.5 weeks. Horizontal lines represent median values. Differences between groups were assessed by Kruskal-Wallis test. Data from each individual are represented by unique symbols.
Table 1.
Selected individuals for heavy water labelling study.
Table 2.
Median (range) of production rates (p) of the different T-cell subsets.
Fig 4.
Dynamics of CMV-specific CD8+ T-cells and CD8+ TEM/EMRA cells in CMV+ and CMV- individuals.
(A) Frequency within the total CD8+ T-cell pool (upper panel) and absolute count (lower panel) of CMV-specific CD8+ T-cells over time in the CMV+ individuals selected for the deuterium labelling study. (B) Deuterium labelling enrichment (%DNA labelled) of CMV-specific CD8+ T-cells compared to CD8+ TEM/EMRA cells of CMV+ (upper panel) or CMV- (lower panel) individuals. Label enrichment was scaled between 0 and 100% by normalizing for the estimated maximum enrichment of granulocytes (see Materials and methods). Symbols depict the mean of duplicate measurements. It should be noted that for one individual (E34) it was not possible to determine the production rate of CD8+ TEM/EMRA cells because too many samples were out of range of the standard lines for deuterium enrichment measurement. E30 only received 2H2O for 3.5 weeks. (C) Estimated average production rates of CMV-specific CD8+ T-cells compared to CD8+ TEM/EMRA cells of CMV+ (upper panel) or CMV- (lower panel) individuals. All estimates were obtained by fitting a single-exponential model to the data sets per individual (see Materials and methods and S7 Fig). (D) Association between the absolute cell number and the average production rate of CMV-specific CD8+ T-cells. Horizontal lines represent median values. Differences between groups were assessed by Mann-Whitney U test, or Wilcoxon matched-pairs signed rank test for comparisons within the same individuals. Correlation was tested by Spearman’s correlation. P-values < 0.1 are presented in the figure, ns = not significant. Data from each individual are represented by unique symbols.
Fig 5.
Association between T-cell production rates and expression of senescence, proliferation, and apoptosis markers.
The estimated average production rates of different CD4+ and CD8+ T-cell subsets as estimated by heavy water labelling were plotted against their expression (% positive) of CD57, KLRG-1, CD28, Ki-67, and Bcl-2. Data points of donor E04 are circled in the Ki-67 graph. Correlation was tested by Spearman’s correlation, P-values and r-values are indicated in the graphs. Different symbols represent different memory subpopulations.
Fig 6.
CMV infection affects the phenotype but not the lifespan of T-cells in humans.
Both CD4+ and CD8+ T-cells in CMV-infected individuals upregulate the expression of senescence markers (e.g. CD57, KLRG-1) and downregulate the expression of co-stimulatory and proliferation markers (e.g. CD28, Ki-67). By separately analyzing the numerically large immunodominant CD8+ T-cell populations, we found that these do not explain the differences in T-cell phenotype between CMV+ and CMV- individuals. Though the increased late-differentiated state and size of the immunodominant CMV-specific CD8+ T-cell pool might suggest an altered lifespan, we find that these cells have similar lifespans as non-CMV-specific CD8+ T-cells. Created with BioRender.com.