A novel cytomegalovirus-induced regulatory-type T-cell subset increases in size during older life and links virus-specific immunity to vascular pathology

Abstract

Background: Cytomegalovirus (CMV) infection directly targets vascular endothelium and smooth muscle and at older ages is associated with accelerated vascular pathology and mortality. CMV-specific cellular immunity might directly contribute to this process.

Methods: Conventional ex vivo activation-induced T-cell responses to 19 dominant CMV antigens, along with CMV-specific inducible regulatory-type CD4+ T cells (iTregs), were measured in healthy older people, using a novel protocol that included classic Treg markers alongside the activation marker CD134. Measurements were correlated with diastolic, systolic, and mean arterial blood pressure, a surrogate marker for arterial stiffness.

Results: CMV-specific iTregs recognized the same antigens as conventional CD4+ T cells and were significantly more frequent at older ages. They suppressed antigen-specific and nonspecific proliferation and in large part expressed Foxp3. Frequencies of CMV-specific iTregs and CD8+ T cells (summated response) were significantly associated with diastolic and mean arterial pressures. Confounders, including age, body mass index, smoking, antihypertensive medication use, or C-reactive protein levels, did not explain these observations.

Conclusions: A novel CMV-induced regulatory-type CD4+ T-cell subset is readily detectable in CMV-infected people and, like the aggregate CD8+ T-cell response to the most dominant CMV antigens, is quantitatively associated with arterial stiffness in older life. Whereas CD8+ effector T cells might directly cause vascular injury, iTregs may attenuate this response.

Keywords: Arteriosclerosis; Regulatory T-Cells; hypertension; iTreg; immunology; infection; inflammation.

Figure 1.

CD25⁺CD39⁺CD134⁺ CD4⁺ T cells result from short term ex vivo activation with specific antigens and coexpress the regulatory T-cell (Treg) marker Foxp3. Panels show experiments in a cytomegalovirus (CMV)–positive donor (A) and a CMV-negative donor (B). Whole blood was stimulated with medium alone (negative control; unstimulated), CMV lysate, the CMV UL83 peptide pool, and phytohemagglutinin (PHA; positive control). CD39⁺ cells are not significantly increased by CMV lysate or UL83 stimulation but are strongly increased by PHA stimulation. Note that CD134 is upregulated in response to CMV antigens in the CMV-positive participant but not the CMV-negative participant (second row in A and B). C, A representative experiment in an older CMV-positive participant using CMV-lysate stimulation and negative control is shown. Plots show CD4/CD39 staining (left), CD25/Foxp3 staining (middle), and CD134/Foxp3 staining (right). Percentages of relevant subsets as the percentage of CD4⁺ T cells are shown in bold in unstimulated (middle, top), and CMV lysate–stimulated replicates (middle, bottom). The increase of 0.89% of Foxp3⁺ cells in terms of CD4⁺ T cells is the maximum contribution that short-term activation could have made to the Foxp3⁺CD134⁺ subset seen after stimulation (right), accounting for a maximum of 48% of these cells. The results shown are representative of 3 independent experiments.

Figure 2.

CD39⁺CD25⁺CD134⁺CD4⁺ inducible regulatory T cells (iTregs) are able to suppress autologous CD4⁺ T-cell proliferation. The ability of UL83-stimulated CD39⁺CD25⁺CD134⁺CD4⁺ iTregs to suppress proliferation was tested after sorting them from peripheral blood mononuclear cells by a fluorescence-activated cell sorter and resting them in high-dose interleukin 2 for 2 days. For the suppressor assay iTregs (Tr) were cocultured with PKH67-stained CD4⁺CD25⁻ responder cells (Te) and stimulated with soluble anti-CD3 antibody or UL83 (pp65) peptide pool in the presence of autologous CD3⁻ T cells as antigen-presenting cells. iTregs were added at Tr:Te ratios of 1:1 and 1:10. Responder cell proliferation in the absence of Tregs or in the absence of stimulation were used as positive and negative controls, respectively. Histograms show increasing lipid dye dilution (PKH67) from right to left. Responder cell proliferation was measured as the percentage of cells with decreased PKH67 staining.

Figure 3.

CD39⁺CD25⁺CD134⁺CD4⁺ inducible regulatory T cells (iTregs) are induced by the same antigens that commonly induce effector responses. The induction of iTregs was studied in whole-blood assays of cytomegalovirus (CMV)–positive young donors (A) and older donors (B) in response to CMV peptide pools including the 6 most dominant CMV CD4⁺ T-cell target antigens (UL55, UL86, UL83, UL32, UL99, and UL153). CMV UL83 (pp65) appeared to elicit the biggest iTreg responses in both young and older participants. C, The dot plot shows the relationship between the sum (Σ) of the CMV peptide pool–induced iTreg responses and the CMV lysate–induced iTreg response. D, A significant correlation between the CD8⁺ effector T-cell response to CMV (ie, the summated CD8⁺ effector T-cell responses to 19 different CMV proteins) and CMV lysate–induced CD4⁺ iTregs was observed across the whole CMV-positive population sample.

Figure 4.

Both CD25⁺CD39⁺CD4⁺ regulatory T cells (Tregs) and cytomegalovirus (CMV)–specific inducible Tregs (iTregs) are significantly expanded in older participants, and their frequencies expose a sex-based difference in older age. Tregs, as well as CMV-specific or tuberculin-specific iTregs, were analyzed by flow cytometry in young and older individuals following ex vivo antigen-specific stimulation; medium alone was used as negative control. A, A significant difference in CD4⁺CD25⁺CD39⁺ Treg frequencies between young donors (n = 39) and older donors (n = 74) was observed in the absence of stimulation. B, CD25⁺CD39⁺CD134⁺ CMV-specific iTregs were significantly higher in older CMV-positive participants, compared with young CMV-positive participants, following ex vivo antigen-specific stimulation with CMV lysate. C, Background levels in CMV-negative participants. D, There was no such difference in regard to tuberculin-specific iTregs (including CMV-positive and CMV-negative donors). E, When the same analysis was done looking separately at men and women, frequencies were clearly higher in older women than in older men, whereas no significant increase with age-based or sex-based differences was seen with respect to tuberculin-specific iTregs. F, The difference in CMV-induced iTregs between CMV-positive older women and men is statistically significant. Abbreviations: Stim, stimulated; unstim, unstimulated.