NK cells, displaying early activation, cytotoxicity and adhesion molecules, are associated with mild dengue disease

Abstract

During the innate immune response against infections, Natural Killer (NK) cells are as important effector cells as are Cytotoxic T lymphocytes (CTL) generated after antigenic stimulation in the adaptative response. NK cells increase in numbers, after viral infection or vaccination. We investigated the NK cell and CD8 T lymphocyte status in 55 dengue infected patients. The NK (CD56+CD3-) and CD56+ T cell (CD56+CD3+) rates rise during the acute phase of disease. The majority of NK cells from dengue patients display early markers for activation (CD69, HLA-DR, and CD38) and cell adhesion molecules (CD44, CD11a) during the acute phase of disease. The intracellular cytotoxic granule, TIA-1, is also up-regulated early in NK cells. Most of these markers appear also on CD8+ T lymphocytes but during the late acute phase. Circulating IL-15 is elevated in a significant number of patients during early acute infection and its values were statistically correlated with NK frequencies and cytotoxic markers on NKs. We have therefore shown that dengue virus infection is very likely stimulating a cytotoxic response that may be efficient in controlling the virus in synergism with CD8+ T lymphocytes. Interestingly, the heightened CD56+CD3-, CD56+CD3+, CD56+TIA-1+ and CD56+CD11a+ cell rates are associated with mild dengue clinical manifestations and might indicate a good prognosis of the disease.

Figures

Fig. 1

NK CD56+CD3−, CD56+CD3+ and CD56+CD16+ cell frequencies during dengue. Representative dot plots for NK CD56+CD3−, CD56+CD3+ (a) and CD56+CD16+ (b) cell labellings from a healthy donor and a dengue patient at 5 days after the disease onset (early acute phase) are shown. Numbers in each quadrant indicate the percentage for NK CD56+CD3−, CD56+CD3+ or CD56+CD16+ within the lymphocyte gate. PBMCs from healthy subjects (n = 13) or from dengue patients in early (days 1–5), late acute (days 6–10) and convalescent (after 11 days) phases were labelled as described in Materials and methods and analysed by flow cytometry within lymphocyte gate. Individual and mean percentages for (c) CD56+CD3− (d) CD56+CD3+ or (e) CD56+CD16+ cells among lymphocytes are shown in all groups. Statistical significance was assessed by the Mann–Whitney U-test. ***P < 0·001, **P < 0·01 and *P < 0·05.

Fig. 2

Representative contour plots for increased expression of activation cell markers during dengue. PBMC from healthy subjects or from patients with acute dengue were labelled as described in Materials and methods and analysed by flow cytometry within lymphocyte gate. Ex vivo (a) CD69, (b) HLA-DR, (c) CD38 and (d) CD44 coexpression on CD56+ NK cells from a healthy control and a dengue patient with early acute infection was illustrated. Numbers in quadrants indicate the percentage of cells within the corresponding cell subset.

Fig. 3

CD69 and Tia-1 expression on NK cells and CD8+ T lymphocytes during dengue. PBMC from healthy subjects samples or from dengue patients in early (days 1–5), late acute (days 6–10) and convalescent (11 days after disease onset) phase were labelled as described in Materials and methods and analysed by double-colour flow cytometry for particular activation marker expression in CD56+ and CD8+ T lymphocytes. Mean percentages of (a,b) CD69 and (c,d) Tia-1+ among (a,c) CD56+ and (b,d) CD8+ T lymphocytes are shown in all groups. Statistical significance was assessed by the Mann–Whitney U-test. ***P < 0·001, **P < 0·01, *P < 0·05.

Fig. 4

CD44 and CD11a expression on CD56+ NK cells during acute dengue.PBMC from healthy phases subjects or from dengue patients in early (days 1–5), late acute (days 6–10) and convalescent (after 11 days after disease onset) phases were labelled as described in Materials and methods and analysed by double-colour flow cytometry for particular adhesion molecule expression in CD56+. Mean percentages for (a) CD44 and (b) CD11a expression among gated CD56+ NK cells are shown in all groups. Statistical significance was assessed by the Mann–Whitney U-test. ***P < 0·001, **P < 0·01 and *P < 0·05.

Fig. 5

Representative contour plots for increased expression of cytotoxic cell markers during dengue. PBMC from patients with acute dengue were labelled as described in Materials and methods and analysed by flow cytometry within lymphocyte gate. Ex vivo (a) Isotype labelling for (b) TIA-1 in double stained CD56+, CD8+ or CD4+ cells (c)Ex vivo CD11a and CD16 coexpression on gated CD56+ cells. Numbers in quadrants indicate the percentage of cells within the corresponding cell subset.

Fig. 6

(a) Alteration in IL-15 plasma levels during the dengue time course and association with NK cells. IL-15 levels in dengue patients increase in early acute phase returning to normal on convalescence. Statistical significance was assessed by the Mann–Whitney U-test with P = 0·0001. Positive correlation between IL-15 and (b) CD56+CD3− or (c) CD56+Tia-1+ cells was performed by Spearman regression analysis. PBMCs from dengue patients were analysed by flow cytometry as described in Figs 1 and 2. IL-15 levels were measured by ELISA. ***P < 0·001, **P < 0·01, *P < 0·05.