Cytomegalovirus-specific T-cells are associated with immune senescence, but not with systemic inflammation, in people living with HIV

Abstract

In people living with HIV (PLWHIV), coinfection with cytomegalovirus (CMV) has been associated with inflammation, immunological ageing, and increased risk of severe non-AIDS related comorbidity. The effect of CMV-specific immune responses on systemic inflammation, immune activation and T-cell senescence was evaluated in 53 PLWHIV treated with combination antiretroviral therapy (cART). Activated-, terminally differentiated-, naïve-, and senescent T-cells were assessed by flow cytometry, and plasma levels of CMV IgG, interleukin-6, tumor necrosis factor-α, high-sensitivity C-reactive protein and soluble-CD14 were measured. In PLWHIV, expression of interleukin-2, tumor necrosis factor-α and interferon-γ was measured by intracellular-cytokine-staining after stimulation of T-cells with CMV-pp65, CMV-IE1, and CMV-gB. Increased CMV-specific T-cell responses were associated with a higher ratio of terminally differentiated/naïve CD8+ T-cells and with increased proportions of senescent CD8+ T-cells, but not with systemic inflammation or sCD14. Increased CMV-specific CD4+ T-cell responses were associated with increased proportions of activated CD8+ T-cells. In PLWHIV with expansion of CMV-specific T-cells or increased T-cell senescence, CMV-specific polyfunctionality was maintained. That the magnitude of the CMV-specific T-cell response was associated with a senescent immune phenotype, suggests that a dysregulated immune response against CMV may contribute to the immunological ageing often described in PLWHIV despite stable cART.

Figure 1

CMV-specific immune responses in PLWHIV. In a cohort of PLWHIV, CMV IgG antibody levels were measured, and CMV-specific CD8+ and CD4+ T-cell responses were examined by measuring intracellular expression of IFN-γ, TNF-α, and IL-2 after stimulation with CMV-pp65, CMV-IE1, and CMV-gB. By summing up the frequency of CD4+ or CD8+ T-cells within each unique combination of functions (IFN-γ, TNF-α or IL-2), the magnitude of the total CMV-specific response (%CD8+ or %CD4+) was analysed. (A) CMV IgG antibody levels in PLWHIV and HIV-negative age-matched controls (190 U/mL (118–528) versus 86 U/mL (69–109). (B) Total CMV-pp65-, CMV-IE1-, and CMV-gB-specific CD8+ and CD4+ T-cell responses. (C) Frequency of CMV-specific CD8+ and CD4+ T-cells expressing IFN-γ, TNF-α, and IL-2. In (B) and (C), blue circles represent CMV-pp65, red circles represent CMV-IE1, and green circles represent CMV-gB. For each variable, median and interquartile range are shown. Mann-Whitney test was performed to compare PLWHIV with uninfected controls in (A). Wilcoxon paired signed-rank test was used to compare frequency of CMV-specific CD8+ and CD4+ T-cells in (B) and (C). *p < 0.01, **p < 0.001, ***p < 0.0001. Bonferroni corrected significance level for (B) and (C) with 3 end points evaluated; p < 0.017.

Figure 2

Relationship between CMV IgG levels and CMV-specific T-cell responses in PLWHIV. Associations between total CMV-specific T-cell responses and CMV IgG levels for total CMV-pp65-specific CD8+ T-cell responses (A), total CMV-gB-specific CD8+ T-cell responses (B), and total CMV-IE1-specific CD8+ T-cell responses were characterized.

Figure 3

Polyfunctionality profiles of CD8+ and CD4+ CMV-pp65-specific T-cells with increasing total response size. Total CMV-pp65-specific CD8+ and CD4+ T-cell response sizes were separated into quartiles. Pie charts demonstrating the relative contribution of each functional cell subset for each quartile in total CD8+ (A) and total CD4+ (B) T-cell responses in PLWHIV. Polyfunctionality visualization and analysis was performed using Spice Version 4.2.3. Bar graph illustrates the median frequency of cells in each functional subset for each quartile. Differences between pie charts were tested with permutation tests set at 10.000 repetitions. The difference between quartiles of total CMV-pp65-specific T-cell responses and proportions of polyfunctional T-cell subsets were first tested with the Kruskal-Wallis test and if significant quartile 1 to 2, 2 to 3 and 3 to 4 was compared with the Mann-Whitney U test. *p < 0.01 **p < 0.001. ***p < 0.0001. Bonferroni corrected significance level with 4 end points evaluated; p < 0.013.