Impaired polyfunctionality of CD8+ T cells in severe sepsis patients with human cytomegalovirus reactivation

Abstract

Human cytomegalovirus (HCMV) establishes a lifelong chronic latent infection and often reactivates in immunocompromised patients. In addition, HCMV reactivates in patients with sepsis or other critical illnesses, particularly in patients with poor prognoses. However, the immunological characteristics of sepsis patients with HCMV reactivation have not been elucidated. In the present study, we examined T-cell responses in severe sepsis patients with and without HCMV reactivation. First, HCMV pp65-specific T-cell functions were assessed by intracellular cytokine staining (ICS) for IFN-γ, TNF-α, and MIP-1β and by CD107a staining. We analyzed the ICS data for each function individually and found no difference between the patient groups. However, the relative frequency of polyfunctional CD8⁺ T cells was significantly decreased in sepsis patients with HCMV reactivation. Next, we examined programmed cell death protein 1 (PD-1) expression. It was significantly increased in the CD8⁺ T-cell population in severe sepsis patients with HCMV reactivation, indicating CD8⁺ T-cell exhaustion. Interestingly, the frequency of PD-1⁺ cells in the CD8⁺ T-cell population was inversely correlated with the relative frequency of polyfunctional CD8⁺ T cells. Herein, we demonstrate that HCMV reactivation in severe sepsis patients is associated with PD-1 expression and impaired polyfunctionality of CD8⁺ T cells.

Figure 1

The relative frequency of single cytokine-producing T cells in severe sepsis patients. Peripheral blood mononuclear cells (PBMCs) from severe sepsis patients without (n=27) and with (n=13) human cytomegalovirus (HCMV) reactivation were stimulated with HCMV pp65 overlapping peptides, and intracellular cytokine staining for IFN-γ, TNF-α and MIP-1β was performed. Fluorochrome-conjugated anti-CD107a was also added to the culture when the PBMCs were stimulated to assess the antigen-specific degranulating activity of the T cells. The data were analyzed for single cytokine⁺ cells or CD107a⁺ cells in the total CD8⁺ T-cell population (a) and for single cytokine⁺ cells in the total CD4⁺ T-cell population (b). The data were also analyzed for the percentage of the total CD8⁺ or CD4⁺ T-cell population (c) or the absolute counts (d) of T cells with any type of function (total responding cells). The horizontal lines represent the median values.

Figure 2

Polyfunctionality of human cytomegalovirus (HCMV) pp65-specific T cells in severe sepsis patients. The data from Figure 1 were analyzed for the polyfunctionality of T cells according to the number of T-cell functions. CD8⁺ T cells were analyzed for IFN-γ, TNF-α, MIP-1β and CD107a (a–d), and CD4⁺ T cells were analyzed for IFN-γ, TNF-α and MIP-1β (e–g). The pie graphs show the fraction of T cells positive for a given number of functions among T cells with any type of function (total responding cells) (a, e). The percentage of T cells positive for a given number of functions was compared between severe sepsis patients without (n=27) and with (n=13) CMV reactivation (b, f). Detailed analyses of polyfunctionality were presented with every possible combination of functions, and the data were compared between the groups (c, g). Representative polychromatic dot plots for IFN-γ and TNF-α are shown. T cells positive for a given number of functions are marked by different colors to show their distribution in the dot plots for IFN-γ and TNF-α (d). The horizontal lines represent the median values. *P<0.05, **P<0.01.

Figure 3

Geometric mean fluorescence intensity (gMFI) of cytokine-producing T cells in severe sepsis patients. The data from Figure 1 were analyzed for the gMFI of each function. The gMFI of the cytokine-producing cell population was divided by the gMFI of the cytokine-negative cell population to normalize the data. The data were analyzed for single cytokine⁺ cells or CD107a⁺ cells in the CD8⁺ T cells (a) and for single cytokine⁺ cells in the CD4⁺ T cells (b). The horizontal lines represent the median values. *P<0.05.

Figure 4

Correlation between PD-1 expression and the polyfunctionality of CD8⁺ T cells in severe sepsis patients. (a) The percentage of PD-1⁺ cells and gMFI of PD-1 in the CD8⁺ T-cell population were compared between severe sepsis patients without (n=13) and with human cytomegalovirus (HCMV) reactivation (n=6). (b) Correlational analysis was performed between the percentage of PD-1⁺ cells in the total CD8⁺ T-cell population and the percentage of highly polyfunctional (quadruple-positive) cells among the CD8⁺ T cells with any type of function (total responding CD8⁺ T cells). (c) Correlational analysis was performed between the percentage of PD-1⁺ cells in the total CD8⁺ T-cell population and HCMV DNA titers. The horizontal lines represent the median values. *P<0.05, **P<0.01.