Cytomegalovirus and Epstein-Barr virus co-infected young and middle-aged adults can have an aging-related T-cell phenotype

Abstract

Cytomegalovirus (CMV) is known to alter circulating effector memory or re-expressing CD45RA⁺ (TemRA) T-cell numbers, but whether Epstein-Barr virus (EBV) does the same or this is amplified during a CMV and EBV co-infection is unclear. Immune cell numbers in blood of children and young, middle-aged, and senior adults (n = 336) were determined with flow cytometry, and additional multivariate linear regression, intra-group correlation, and cluster analyses were performed. Compared to non-infected controls, CMV-seropositive individuals from all age groups had more immune cell variance, and CMV⁺ EBV^- senior adults had more late-differentiated CD4⁺ and CD8⁺ TemRA and CD4⁺ effector memory T-cells. EBV-seropositive children and young adults had a more equal immune cell composition than non-infected controls, and CMV^- EBV⁺ senior adults had more intermediate/late-differentiated CD4⁺ TemRA and effector memory T-cells than non-infected controls. CMV and EBV co-infected young and middle-aged adults with an elevated BMI and anti-CMV antibody levels had a similar immune cell composition as senior adults, and CMV⁺ EBV⁺ middle-aged adults had more late-differentiated CD8⁺ TemRA, effector memory, and HLA-DR⁺ CD38^- T-cells than CMV⁺ EBV^- controls. This study identified changes in T-cell numbers in CMV- or EBV-seropositive individuals and that some CMV and EBV co-infected young and middle-aged adults had an aging-related T-cell phenotype.

Figure 1

CD4⁺ T-cell numbers in blood of CMV⁻ EBV⁻, CMV⁻ EBV⁺, CMV⁺ EBV⁻, or CMV⁺ EBV⁺ children and young, middle-aged, and senior adults. For these four CMV and EBV seropositivity depending groups, (a) numbers of CD4⁺ naïve (CCR7⁺ CD45RA⁺), central memory (CCR7⁺ CD45RA⁻), effector memory (CCR7⁻ CD45RA⁻), or TemRA (CCR7⁻ CD45RA⁺) T-cells are shown, together with numbers of early- (CD45RA⁺ CD28⁺ CD27⁺), intermediate- (CD45RA⁺ CD28⁺ CD27⁻), or late-differentiated (CD45RA⁺ CD28⁻ CD27⁻) (b) CD4⁺ TemRA T-cells or (c) CD4⁺ effector memory T-cells. (d) Differences in numbers of HLA-DR^+/− and CD38^+/− CD4⁺ T-cell numbers between CMV⁻ EBV⁻, CMV⁻ EBV⁺, CMV⁺ EBV⁻, or CMV⁺ EBV⁺ individuals. Cell numbers are per µl blood and data are geomeans with 95% confidence intervals. A two-way ANOVA with a Dunnett’s multiple comparison test was used as statistical test. *p < 0.05 and ** ^{or $$}p < 0.01. An asterisk was used to indicate significant differences compared to the CMV⁻ EBV⁻, non-infected group and a dollar sign indicates a comparison with the CMV⁺ EBV⁻ group. n = 59 for children, n = 88 for young adults, n = 92 for middle-aged adults, and n = 90 for senior adults. Number of CMV⁻ EBV⁻, CMV⁻ EBV⁺, CMV⁺ EBV⁻, or CMV⁺ EBV⁺ individuals per age group are indicated within the figure.

Figure 2

CD8⁺ T-cell numbers in blood of CMV⁻ EBV⁻, CMV⁻ EBV⁺, CMV⁺ EBV⁻, or CMV⁺ EBV⁺ children and young, middle-aged, and senior adults. (a) Numbers of CD8⁺ naïve (CCR7⁺ CD45RA⁺), central memory (CCR7⁺ CD45RA⁻), effector memory (CCR7⁻ CD45RA⁻), or TemRA (CCR7⁻ CD45RA⁺) T-cells in CMV⁻ EBV⁻, CMV⁻ EBV⁺, CMV⁺ EBV⁻, or CMV⁺ EBV⁺ individuals. For these four CMV and EBV seropositivity depending groups, numbers of early- (CD45RA⁺ CD28⁺ CD27⁺), intermediate- (CD45RA⁺ CD28⁻ CD27⁺), or late-differentiated (CD45RA⁺ CD28⁻ CD27⁻) (b) CD8⁺ TemRA T-cells or (c) CD8⁺ effector memory T-cells and (d) HLA-DR^+/− and CD38^+/− CD8⁺ T-cell numbers were determined. Cell numbers are per µl blood and data are geomeans with 95% confidence intervals. * ^{or $}p < 0.05, ** ^{or $$}p < 0.01 and ^***p < 0.001. A two-way ANOVA with a Dunnett’s multiple comparison test was used as statistical test. An asterisk was used to indicate significant differences compared to the CMV⁻ EBV-, non-infected group and a dollar sign indicates a comparison with the CMV⁺ EBV⁻ group. n = 59 for children, n = 88 for young adults, n = 92 for middle-aged adults, and n = 90 for senior adults. Number of CMV⁻ EBV⁻, CMV⁻ EBV⁺, CMV⁺ EBV⁻, or CMV⁺ EBV⁺ individuals per age group are indicated within the figure.

Figure 3

The immune variance between study subjects of the same age category was assessed with an intra-group Spearman correlation analysis. The intra-group correlation coefficients of (a) CMV⁻ EBV⁻ and CMV⁺ EBV⁻, (b) CMV⁻ EBV⁻ and CMV⁻ EBV⁺, or (c) CMV⁺ EBV⁻ and CMV⁺ EBV⁺ individuals are plotted. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. The number of study participants per group are mentioned within the graph.

Figure 4

Multivariate regression analysis with absolute immune cell numbers using age, sex, CMV- and EBV-antibody levels as potential predicting factors. (a) A heatmap depicting correlation coefficients from the multivariate regression analysis for T-cells, NK-cells, and B-cells per independent predictor being age, sex, CMV- and EBV-antibody levels, or a combination of these factors. Stripes are used to indicate which factor correlated the strongest with quantities of an immune cell and correlation coefficients ranged from 1 to − 1. (b) A table summarizing the factor(s) that significantly correlated with the analyzed immune cells. n = 336. Correlation coefficients and combined correlation coefficients are given in parentheses. Scatter plots with log transformed anti-CMV antibody levels (RU/ml) and (c) late-differentiated CD4⁺ TemRA T-cells or CD4⁺ effector memory T-cells (CCR7⁻ CD45RA^+/− CD28⁻ CD27⁻, respectively) or (d) late-differentiated CD8⁺ TemRA T-cells or CD8⁺ effector memory T-cells (CCR7⁻ CD45RA^+/− CD28⁻ CD27⁻, respectively). n = 335, but only data for CMV-seropositive individuals is plotted. Correlation coefficients and linear regression lines with only CMV-antibody levels as predicting factor are shown in black and those with CMV-antibody levels and age as predicting factors are shown in red.

Figure 5

Cluster analysis with percentages of 44 immune cell subpopulations quantified from blood samples of all study participants. (a) A principal component analysis plot showing the five clusters the study participants were grouped in. (b) Information on age, CMV- and EBV-antibody levels per cluster. Percentages of (c) CD4⁺ or (d) CD8⁺ naïve (CCR7⁺ CD45RA⁺), central memory (CCR7⁺ CD45RA⁻), late-differentiated TemRA (CD45RA⁺ CD28⁻ CD27⁻), late-differentiated effector memory (CD45RA⁻ CD28⁻ CD27⁻), HLA-DR⁺ and CD38⁺, and HLA-DR⁺ and CD38⁻ T-cells per cluster. n = 68 for cluster-1, n = 80 for cluster-2, n = 40 for cluster-3, n = 65 for cluster-4, and n = 82 for cluster-5.

Figure 6

Schematic study design with an overview of the performed analyses and the main findings of this study. This image was partially created with BioRender.com.