Marked skewing of entire T-cell memory compartment occurs only in a minority of CMV-infected individuals and is unrelated to the degree of memory subset skewing among CMV-specific T-cells.

Background: Chronic CMV infection drives the clonal expansion and accumulation of terminally differentiated, dysfunctional CMV-specific T-cells. CMV infection also appears to accelerate the differentiation of non-CMV-specific T-cells; however, the extent of this phenomenon is unclear. Methods: The distribution of CD4 and CD8 T-cells into four memory subsets determined by CD45RA and CCR7 expression was analyzed in 96 CMV-infected (CMV+) and 81 CMV-uninfected (CMV-) older individuals. In CMV+ individuals, the distribution of IFN-γ producing CMV-specific T-cells into the same subsets was analyzed following stimulation with 16 different CMV antigens using flowcytometry (intracellular cytokine staining). We used previously published results to extrapolate the relative size of the entire CMV-specific CD4 and CD8 T-cell response from the summated response to selected antigens. The T-cell memory subset distribution across all CMV antigen-induced responses (weighted mean) was then used to calculate memory subset proportions (in % of CD4 or CD8 T-cells) of CMV-specific and non-CMV-specific T-cells. These were compared to the corresponding proportions in CMV- individuals. Results: Only a minority (20%-30%) of CMV+ individuals displayed overall proportions of terminally differentiated T-cell memory subsets above an upper outlier boundary defined in CMV- individuals. The calculated proportions of these subsets among non-CMV-specific T-cells in CMV+ individuals also exceeded the corresponding proportions in CMV- people, suggesting that their differentiation could be CMV-driven. In CMV+ people showing overall subset distributions within the outlier limits, the memory subset distributions of non-CMV-specific T-cells were more like those in CMV- people. Logistic regression revealed that CMV infection, age, and sex all had significant effects on one or more of the non-CMV-specific CD4 or CD8 T-cell memory subsets in CMV+ individuals, with CMV infection showing the strongest effect overall. Surprisingly, except for the CD45RA-/CCR7- CD4 T-cell subset, we only found weak correlations between corresponding memory subset proportions among all T-cells and CMV-specific T-cells. Conclusion: Our analysis supports an effect of CMV infection on non-CMV-specific T-cells; however, it is limited to a minority of individuals and not closely related to the degree of memory subset differentiation of CMV-specific T-cells. We propose that unknown predisposing factors might determine to what extent CMV infection affects non-CMV-specific T-cell differentiation.

In-Depth Profiling of T-Cell Responsiveness to Commonly Recognized CMV Antigens in Older People Reveals Important Sex Differences.

The impact of biological sex on T-cell immunity to Cytomegalovirus (CMV) has not been investigated in detail with only one published study comparing CMV-specific T-cell responses in men and women. Many studies, however, have shown an association between CMV infection and immunosenescence, with broad effects on peripheral blood lymphocyte subsets as well as the T and B-cell repertoires. Here, we provide a detailed analysis of CMV-specific T-cell responses in (n=94) CMV+ older people, including 47 women and 47 men aged between 60 and 93 years. We explore sex differences with respect to 16 different CMV proteins arranged in 14 peptide pools (overlapping peptides). Following ex vivo stimulation, CD4 and CD8 T-cells producing IFN-γ, TNF, and IL-2 were enumerated by flow-cytometry (intracellular cytokine staining). T-cell responses were evaluated in terms of each cytokine separately or in terms of cytokines produced simultaneously (polyfunctionality). Surface memory phenotype and CD3 downmodulation were assessed in parallel. The polyfunctionality index and a memory subset differentiation score were used to identify associations between response size, cytokine production, polyfunctionality, and memory subset distribution. While no significant sex differences were found with respect to overall CMV target protein selection, the T-cell response in men appeared more focused and accompanied by a more prominent accumulation of CMV-specific memory CD4 and CD8 T-cells. T-cell polyfunctionality and differentiation were similar in the sexes, however, CMV-specific T-cells in men produced more pro-inflammatory cytokines. Particularly, TNF production by CD4 T-cells was stronger in men than in women. Also, compared with women, men had larger responses to CMV proteins with immediate-early/early kinetics than women, which might have been driven by CMV reactivation. In conclusion, the CMV-specific T-cell response in men was larger and more pro-inflammatory than in women. Our findings may help explain sex differences in CMV-associated pathologies.

Cytomegalovirus infection is associated with an increase in aortic stiffness in older men which may be mediated in part by CD4 memory T-cells.

Human Cytomegalovirus (CMV) infection is associated with atherosclerosis, higher cardiovascular disease (CVD) risk, and an increase in memory T-cells (Tmem). T-cells have also been implicated in CVD, independently of CMV infection. To better understand the CMV-associated CVD risk, we examined the association between CMV (IgG) serostatus and central aortic (carotid-to-femoral) pulse wave velocity (cfPWV), an early, independent predictor of CVD. We also investigated if such an association might be reflected by the distribution of Tmem and/or other T-cell subsets. Methods: Healthy older volunteers (60-93 years) underwent routine clinical and laboratory evaluation, including assessment of cfPWV in eligible participants. Flow-cytometry was used to assess proportions of memory T-cells, CD28null T-cells, and CMV-specific T-cells. The following associations were examined; CMV serostatus/cfPWV, CMV serostatus/proportion of Tmem, proportion of Tmem/cfPWV, CD28null T-cells/cfPWV, and CMV-specific T-cells/cfPWV. Linear regression models were used to adjust for age, sex, socioeconomic status, smoking, waist-to-hip ratio, cholesterol, and blood pressure as required. Results: Statistically significant positive associations were found (P-values for the fully adjusted models are given); CMV serostatus/cfPWV in men (P ≤ 0.01) but not in women, CMV serostatus/proportions of CD4 Tmem in men (P ≤ 0.05) but not in women; proportions of CD4 Tmem/cfPWV among CMV seropositive (CMV+) people (P ≤ 0.05) but not CMV seronegative (CMV-) people. Conclusion: CMV infection increases the CVD risk of older men by increasing cfPWV. This may be mediated in part by increased proportions of CD4 Tmem, higher numbers of which are found in CMV+ older people and more so among men than women. Given the high prevalence of CMV worldwide, our findings point to a significant global health issue. Novel strategies to mitigate the increased CVD risk associated with CMV may be required.

CD28null pro-atherogenic CD4 T-cells explain the link between CMV infection and an increased risk of cardiovascular death.

An increased risk of cardiovascular death in Cytomegalovirus (CMV)-infected individuals remains unexplained, although it might partly result from the fact that CMV infection is closely associated with the accumulation of CD28null T-cells, in particular CD28null CD4 T-cells. These cells can directly damage endothelium and precipitate cardiovascular events. However, the current paradigm holds that the accumulation of CD28null T-cells is a normal consequence of aging, whereas the link between these T-cell populations and CMV infection is explained by the increased prevalence of this infection in older people. Resolving whether CMV infection or aging triggers CD28null T-cell expansions is of critical importance because, unlike aging, CMV infection can be treated. Methods: We used multi-color flow-cytometry, antigen-specific activation assays, and HLA-typing to dissect the contributions of CMV infection and aging to the accumulation of CD28null CD4 and CD8 T-cells in CMV+ and CMV- individuals aged 19 to 94 years. Linear/logistic regression was used to test the effect of sex, age, CMV infection, and HLA-type on CD28null T-cell frequencies. Results: The median frequencies of CD28null CD4 T-cells and CD28null CD8 T-cells were >12-fold (p=0.000) but only approximately 2-fold higher (p=0.000), respectively, in CMV+ (n=136) compared with CMV- individuals (n=106). The effect of CMV infection on these T-cell subsets was confirmed by linear regression. Unexpectedly, aging contributed only marginally to an increase in CD28null T-cell frequencies, and only in CMV+ individuals. Interestingly, the presence of HLA-DRB1*0301 led to an approximately 9-fold reduction of the risk of having CD28null CD4 T-cell expansions (OR=0.108, p=0.003). Over 75% of CMV-reactive CD4 T-cells were CD28null. Conclusion: CMV infection and HLA type are major risk factors for CD28null CD4 T-cell-associated cardiovascular pathology. Increased numbers of CD28null CD8 T-cells are also associated with CMV infection, but to a lesser extent. Aging, however, makes only a negligible contribution to the expansion of these T-cell subsets, and only in the presence of CMV infection. Our results open up new avenues for risk assessment, prevention, and treatment.