Human Cytomegalovirus Infection Increases Both Antibody- and Non-Antibody-Dependent Cellular Reactivity by Natural Killer Cells

Abstract

Background: Antibody-mediated rejection in solid organ transplantation is an important immunological barrier to successful long-term graft survival. Next to complement activation, natural killer (NK) cells have been implicated in the process. Human cytomegalovirus (CMV), independently associated with decreased graft survival, has a strong imprint on the immune response. Here, we assessed the effect of CMV status on alloreactive NK cell reactivity.

Methods: We compared antibody-mediated NK cell cytolytic activity (CD107a expression) and IFNγ production between healthy CMV-seropositive (n = 8) and CMV-seronegative (n = 11) individuals, in cocultures of NK cells with anti-HLA class I or rituximab (control) antibody-coated Raji cells.

Results: First, we showed that within the NKG2C+ NK cells, it is specifically the NKG2C+/A- subset that is enriched in CMV+ individuals. We then observed that in particular the NK cell antibody-dependent cell mediated cytotoxicity (ADCC), but also non-ADCC alloreactivity toward HLA-positive target cells was increased in CMV+ individuals as compared to CMV- ones. This enhanced ADCC as well as non-ADCC NK cell reactivity in CMV+ individuals was particularly characterized by a significantly higher number of ILT2+ and NKG2C+ NK cells that possessed cytolytic activity and/or produced IFNγ in response to HLA-positive target cells.

Conclusions: With regard to organ transplantation, these data suggest that CMV infection enhances NK cell alloreactivity, which may pose an additional adverse effect on graft survival, especially in the presence of donor specific antibodies.

FIGURE 1

Flow cytometric analysis of NK receptor expression on isolated NK cells from CMV+ (n = 8) and CMV− (n = 11) individuals. A, Gating strategy and example dot plots from a single representative CMV+ individual. CD56+/CD3− cells NK cells gated from the FSc/SSc plot, were further subdivided into CD56^dim and `CD56^bright cells. Both subsets were further analyzed for the expression of selected markers (for data on CD56^bright NK cells see Figure S1, SDC, http://links.lww.com/TXD/A55). B-C, Scatter plots showing cumulative data for the percentage of NK receptor-positive/negative cells (as indicated on the x-axis) within the CD56^dim NK population in CMV+ and CMV− individuals; the solid lines indicate median value. Statistical differences were calculated using the nonparametric Mann-Whitney test and differences were considered significant at *P < 0.05, **P < 0.01.

FIGURE 2

Percentage of IFNγ-producing and degranulating (CD107a expressing) NK cells in response to allogeneic Raji cells coated with (anti-HLA) antibodies in CMV+ (n = 8) and CMV− (n = 11) individuals. 2 × 10⁵ NK cells from CMV+ and CMV− individuals were cultured for 4 hours, either alone, or with uncoated or anti–HLA-A3 IgG, anti–HLA-A3 IgM (negative control) or RTX (positive control) antibody-coated Raji cells. Coculture was done at a 1:1 ratio for 4 hours in 200-μL medium, in the presence of anti-CD107a BV510 antibody, 10 μg/mL brefeldin A and 10 μg/mL monensin. After incubation, the cells were additionally stained for IFNγ and selected NK cell markers and analysed using flow cytometry. The upper left panel shows the gating strategy, whereby NK cells were gated out based on the CD56/CD3 plot, using software settings that included the co-cultured Raji cells (indicated by arrow). The upper right panel shows a representative example of a CMV+ individual. The lower panel shows scatter plots of cumulative data on IFNγ expression (left panel) and CD107a expression (right panel); the solid line indicates median value. Statistical analysis was done using the nonparametric Mann-Whitney test for unpaired analysis, and the nonparametric Wilcoxon matched-pair signed rank test where appropriate and differences were considered significant at *P < 0.05, **P < 0.01, or ***P < 0.001.

FIGURE 3

Phenotypic characteristics of the reactive NK cell population (CD107a+ or IFNγ+) in CMV+ (n = 8) and CMV− (n = 11) individuals upon coculture with uncoated or (allo) antibody-coated Raji cells (from the experiments in Figure 2). The expression of selected NK cells markers (NKG2C/A, ILT2, KIR2DL1/S1, KIR2DL2/L3/S2, and KIR3DL1/S1) was examined on IFNγ+ and CD107a+ cells. Scatter plots of cumulative data on % positive cells are shown; the solid line indicates median value. Statistical differences were calculated using the nonparametric Mann-Whitney test and differences were considered significant at *P < 0.05, **P < 0.01.

FIGURE 4

Contribution of the NKG2C+ and ILT2+ populations to overall NK cell reactivity. Data are derived from the experiments in Figure 2. From total NK cells, the proportions of reactive (IFNγ+ and/or CD107a+) NKG2C and ILT2-positive and -negative cells were calculated and compared between CMV+ and CMV− individuals. Statistical differences were calculated using the nonparametric Mann-Whitney test and differences were considered significant at *P < 0.05, **P < 0.01.