Sara van den Berg

146 Chapter 6 Figure 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- and CMV+ individuals for CD4 + (left panel) and CD8 + (right panel) T TN , T CM , T EM , and T EMRA cells. B. Expression of CD57, KLRG-1, and CD28 by CD4 + and CD8 + T-cells, mea- sured per memory subpopulation (T CM , T EM , and T EMRA ) and compared between CMV+ and CMV- individuals. C. CMV-specific CD8 + T-cells analyzed using 7 different HLA-class I tetramers for 8 immunodominant CMV epitopes (yielding N = 38 CMV-specific CD8 + T-cell populations in N = 32 CMV+ individuals – see Supplementary Table 1 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 T N (naïve, CD27 + CD45RO ‑ ), T CM , T EM , and T EMRA cells. Lower panel: association of percentage T EM/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 rep- resent 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 (T CM , T EM , and T EMRA ). 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 (CD57 high , KLRG-1 high , CD27 low ) are outlined in black in all t-SNE graphs. Cluster 9 represents a cluster that was only observed in CMV+ individuals. Right panels: Percentage of cluster 5-8 within the total CD8 + T-cell pool of CMV- and CMV+ individuals . F. Phenotype of clyster 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. Right panel: Percentage of clusters 5-8 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. Alterations in the CD8+ T-cell pool in CMV infection are not solely explained by the presence of CMV-specific T-cells Although the impact of latent CMV infection on the T-cell pool is well known [2, 4, 5], the underlying mechanism for these changes remains unclear. Given that the above described changes in the phenotype of the CD8 + T-cell pool of CMV+ individuals resembles the phenotype of CMV-specific T-cells, we investigated whether these changes could be explained by the sheer presence of large numbers of CMV-specific CD8 + T-cells. We used an MFI-based t-SNE cluster analysis, including the markers CD57, KLRG-1, CD127, CD27, CD45RO, CCR7, and CD95. T-SNE analysis of CMV- and CMV+ individuals ( N = 22 and N = 32, respectively) again revealed large differences in T-cell phenotype ( Figure 1E , F ). CMV+ individuals had significantly more cells in clusters 5-8 ( Figure 1E , outlined in black, p < 0.0001) ( Supplementary Figure 2 ). These clusters represent CD57 high KLRG-1 high CD27 low T-cells ( Figure 1F ), whereas clusters 1-4 in CMV- individuals is characterised by either low KLRG-1 (cluster 1) or low CD57 (clusters 2-4) expression. Subsequently, CMV-specific CD8 + T-cells specific for different CMV-epitopes were plotted on the same t-SNE cluster-axes ( Figure 1G ). Only 10.0% (median) of CMV-specific CD8 + T-cells were part of the CD57 high KLRG-1 high CD27 low cluster ( Figure 1E right panel ). A proportion of CMV-specific CD8 + T-cells (15% on average per donor) contributed to a relatively small cluster (cluster 9) of T-cells in CMV+ individuals ( Figure 1G right panel ). This cluster was hardly observed in CMV- individuals. The unique combination of T-cell markers in cluster 9 seems to be exclusive for CMV-specific CD8 + T-cells. It can be described as a T CM or T EM like population (CD27 medium CD45RO high ) with high