Intensity of the humoral response to cytomegalovirus is associated with the phenotypic and functional status of the immune system

Abstract

Cytomegalovirus (CMV) infection exerts an enormous effect on human immunity, as it is associated with an immune-impaired response, a variety of chronic diseases, and overall survival in elderly individuals. Levels of anti-CMV antibodies may be associated with the differentiation degree of T cell subsets. Titers are significantly higher in the elderly and positively correlated with specific CD4(+) T cell responses to CMV. In the elderly, antibody titers are associated with the degree of differentiation and the T cell receptor excision circle (TREC) content in CD4(+) T cells, with other features of the immune risk profile, and with a reduced ability to respond to immunization in vivo. Associations may be absent in young subjects because their anti-CMV antibody titers are lower than those of the elderly. However, comparing young and elderly individuals with similar antibody levels reveals differences in their highly differentiated and naïve T cells. These are more marked in individuals with high titers. In parallel with the increase in anti-CMV antibodies, the elderly experience a significant reduction in absolute counts of naïve CD4(+) T cells, which may be a strategy to compensate for the expansion of differentiated cells and to avoid an increase in total T cells. In summary, our results show that titers of anti-CMV antibodies, and not only CMV seropositivity, are related to differentiation status and immunocompetence in the elderly, making this as an important prognostic marker of the status of immune system function.

Fig 1

Frequencies of CMV infection and titers of anti-CMV antibodies in young and elderly subjects and response of CD4⁺ T cells from elderly subjects to CMV and anti-CD3. Immunoglobulin G levels of CMV-specific antibodies were determined by ELISA and a semiquantitative diagram was used to calculate the CMV titer. (A) Histograms show the percentages of CMV-seropositive (black bars) and CMV-seronegative (white bars) young (n = 70) and elderly (n = 92) subjects. The calculated titers of the patient samples are indicated as VIRO units (VU). Percentages of CMV-infected individuals were compared using the χ² test. (B) Titers of anti-CMV antibodies in infected young (n = 37) and elderly (n = 82) subjects are illustrated in the box plots. Significant group differences, assessed with the Mann-Whitney U test, are indicated. (C and D) CD69 expression and proliferative capacity of CD4⁺ T cells from elderly subjects in response to CMV in vitro stimulation were analyzed. (C) Correlation between anti-CMV antibody titers and the frequency of CD69 expression in CD4⁺ subsets from elderly individuals in response to a CMV supernatant (10⁴ PFU/ml) (n = 37) and to anti-CD3 (10 ng/ml) (n = 22). Whole blood from CMV-seropositive elderly people was stimulated for 18 h. (D) Proliferative capacity of CD4⁺ T cell subsets in response to CMV stimulation (n = 28) and to anti-CD3 (n = 27). PBMCs were labeled with CFSE (1.5 μM) and cultured in the presence of CMV supernatant or anti-CD3 for 5 days. The percentage of dividing CD4⁺ T cells is represented. Spearman correlation coefficients and corresponding P values are presented in the upper left-hand corner. n.s., not significant.

Fig 2

Distribution of CD4⁺ T cells into naïve (CD45RA⁺ CCR7⁺), central memory (CD45RA⁻ CCR7⁺), effector memory (CD45RA⁻ CCR7⁻), and effector memory RA (CD45RA⁺ CCR7⁻) related to CMV seropositivity and anti-CMV antibody titers in elderly subjects. CD45RA and CCR7 expression was analyzed by flow cytometry in gated CD3⁺ CD4⁺ T cells. (A) Individual segments of the pie charts show the proportions of each subset in CMV-seronegative and CMV-seropositive elderly subjects. Representative dot plots of the subsets defined by CD45RA and CCR7 expression for individuals in each group show the percentages of positive cells. Student's t test or the Mann-Whitney U test was used to examine differences between the groups, and P values are represented. (B) Relationship between anti-CMV antibody titers and frequency of the CD4⁺ T cell populations defined by CD45RA and CCR7 expression. (C) TREC content was measured in CD4⁺ T cells from young (n = 25) and elderly (n = 30) CMV-seropositive subjects. The CD4⁺ population was isolated by magnetic bead separation and the TREC copy number was determined by real-time PCR. Experiments were conducted in duplicate, and the results are presented relative to anti-CMV antibody titers in each subject. Spearman correlation coefficients and corresponding P values are shown in the upper left-hand corner.

Fig 3

Relationship between CMV seropositivity and anti-CMV antibody titer with CD4/CD8 ratio and percentages of CD8⁺ CD28^null and CD4⁺ CD28^null T cells in young and elderly subjects. CD4/CD8 ratio and percentages of CD8⁺ CD28^null and CD4⁺ CD28^null T cells were compared between CMV-seropositive and CMV-seronegative young (A) and elderly (B) subjects. Significant differences are indicated (Student's t test or Mann-Whitney U test). (C) Correlation of anti-CMV antibody titers and CD4/CD8 ratio and percentages of CD8⁺ CD28^null and CD4⁺ CD28^null T cells in young (black squares) and elderly (white circles) subjects are represented in the dot plots. Spearman correlation coefficients and corresponding P values are listed on the right-hand side.

Fig 4

Grouping of young and elderly subjects by anti-CMV antibody titer. (A) Distribution of frequencies of anti-CMV antibody titers in young and elderly subjects and classification into groups defined according to the antibody levels in young people (50th and 95th percentiles). (B) Comparisons of naïve and CD28^null T cells (CD4⁺ and CD8⁺) between young and elderly individuals with similar anti-CMV antibody titers. Differences in the median frequencies between the young and the elderly in each group are represented by lines. Significant differences between subsets, assessed by Student's t test or the Mann-Whitney U test, are indicated. (C) Absolute counts of naïve and CD28^null T cells (CD4⁺ and CD8⁺) in elderly subjects. The Kruskal-Wallis test was used to identify significant differences in frequencies between groups. “*p” indicates a significant difference in comparisons of all groups; “**p” indicates a significant difference when only groups of CMV-seropositive subjects were considered. (D) Absolute counts of CD3⁺ T cells, distributed into CD4⁺ (white bars) and CD8⁺ (black bars), belonging to each group in elderly subjects.

Fig 5

Levels of TNF-α and IL-15 related to anti-CMV antibody titers. Spearman correlation coefficients and corresponding P values are shown in the upper left-hand corner.

Fig 6

Response to influenza virus vaccination and its correlation with anti-CMV antibody (Ab) titer. Anti-influenza virus antibody titers were quantified by ELISA in the sera of elderly individuals after vaccination. The Kruskal-Wallis test was used to compare the influenza antibody titer between CMV groups. “*p” indicates a significant difference in comparisons between all groups; “**p” indicates a significant difference when only groups of CMV-seropositive subjects were considered.