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

Abstract

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 CD28^null T-cells, in particular CD28^null CD4 T-cells. These cells can directly damage endothelium and precipitate cardiovascular events. However, the current paradigm holds that the accumulation of CD28^null 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 CD28^null 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 CD28^null 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 CD28^null T-cell frequencies. Results: The median frequencies of CD28^null CD4 T-cells and CD28^null 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 CD28^null 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 CD28^null CD4 T-cell expansions (OR=0.108, p=0.003). Over 75% of CMV-reactive CD4 T-cells were CD28^null. Conclusion: CMV infection and HLA type are major risk factors for CD28^null CD4 T-cell-associated cardiovascular pathology. Increased numbers of CD28^null 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.

Keywords: CD28null CD4 T-cells; CD28null CD8 T-cells; Cytomegalovirus; aging; cardiovascular disease; coronary complications.

Figure 1

CD28^null CD4 T-cell numbers are by an order of magnitude higher in CMV+ compared to CMV- people. (A) Dotplots showing CD27/CD28 expression on CD4 T-cells in CMV- and CMV+ individuals. CD28^null cells are indicated (circle). (B) The CD28^null CD4 T-cell frequency distribution (log10-transformed CD4 T-cell fractions) is shown for the whole cohort (top, n=242), CMV- (middle, n=106) and CMV+ individuals (bottom, n=136). (C) Scatterplots show log10-transformed fractions of CD28^null CD4 T-cells. The UQ+1.5×IQR for CMV- individuals is indicated (dotted line). (D) Scatterplots show the log10-transformed fractions of the CD27+ and CD27- subsets of CD28^null CD4 T-cells. Error bars in scatterplots show median, upper, and lower quartiles. (E) The ROC curve shows the separation of CMV- and CMV+ populations by CD28^null CD4 T-cell frequencies.

Figure 2

In CMV+ people, CD28^null CD8 T-cell numbers are about twice as high as in CMV- people. (A) Dotplots show CD27 versus CD28 expression on CD8 T-cells in CMV- and CMV+ individuals. CD28^null cells are indicated (circle). (B) The CD28^null CD8 T-cell frequency distribution (log10-transformed CD8 T-cell fractions) is shown in the whole cohort (top, n=242), CMV- (middle, n=106) and CMV+ individuals (bottom, n=136). (C) Scatterplots show log10-transformed fractions of CD28^null CD8 T-cells. (D) Scatterplots show log10-transformed fractions of the CD27+ and CD27- subsets of CD28^null CD8 T-cells. Error bars in scatterplots show median, upper, and lower quartiles. (E) The ROC curve shows the separation of CMV- and CMV+ populations by CD28^null CD8 T-cell frequencies.

Figure 3

Effect of age, HLA and CRP on CD28^null CD4 T-cells. (A-B) Scatterplots show differences in CD28^null CD4 (A) and CD8 (B) T-cell frequencies between young and older CMV- and CMV+ people (n.s.= not significant). CD28^null T-cell fractions were log10-transformed for better visualization. (C) Box-plots show the levels of CD28^null CD4 T-cell frequencies in four subgroups based on CMV infection status (CMV- green, CMV+ red) and CRP levels (≤ 5 mg/L or >5mg/L). (D) Bar-charts show significant differences in the frequencies of arginine (R), lysine (K), and alanine (A) at positions 71 and 74 of exon 2 of HLA-DRB1 in individuals with and without expansions. Significance levels are indicated.

Figure 4

Expression of CD28 by CMV-specific cells. PBMCs from a representative CMV+ individual stimulated with CMV-peptide (UL83). Plots show unstimulated (left) and stimulated CD4 T-cells (middle). CD28 expression is shown versus IFN-ɣ (top), TNF (middle), and IL-2 (bottom), each identifying responding cells. SEB stimulation (positive control) strongly induced all three cytokines (not shown). Individual scatter graphs (right) show CD28 expression in responding cells (proteins UL55, UL83, or UL86, n=7). At least 40% and up to almost 90% (median 70%) of IFN-ɣ and TNF-producing CMV-specific T-cells are CD28^null. Error bars show median and interquartile range.