Intracellular cytokine production by dengue virus-specific T cells correlates with subclinical secondary infection

Abstract

The pathophysiology of dengue virus infection remains poorly understood, although secondary infection is strongly associated with more severe disease. In the present study, we performed a nested, case-control study comparing the responses of pre-illness peripheral blood mononuclear cells between children who would subsequently develop either subclinical or symptomatic secondary infection 6-11 months after the baseline blood samples were obtained and frozen. We analyzed intracellular cytokine production by CD4(+) and CD8(+) cells in response to stimulation with dengue antigen. We found higher frequencies of dengue virus-specific TNFα, IFNγ-, and IL-2-producing T cells among schoolchildren who subsequently developed subclinical infection, compared with those who developed symptomatic secondary dengue virus infection. Although other studies have correlated immune responses during secondary infection with severity of disease, to our knowledge this is the first study to demonstrate a pre-infection dengue-specific immune response that correlates specifically with a subclinical secondary infection.

Figure 1.

Gating strategy. Shown in the top row are gated lymphocytes in forward versus side scatter (left); CD3⁺, live/dead negative gated cells (center); and CD4⁺ or CD8⁺ gated cells (right). In the middle row are CD4⁺ cells producing TNFα (left), IFNγ (center), or IL-2 (right); the bottom row shows the same arrangement for CD8⁺ cells. The frequencies of cytokine-producing cells were determined by subtraction of the background percentage of cytokine-producing antigen cells in the control antigen tube. This figure represents the PBMC responses from a patient with subclinical secondary infection; the antigen used to stimulate PBMCs here were derived from DENV-3.

Figure 2.

CD4⁺ intracellular cytokine production. TNFα, IFNγ, and IL-2 production was measured in response to antigen stimulation with 4 dengue serotypes of presecondary infection PBMC from 27 Thai schoolchildren. Comparison groups are 10 children (triangles) who developed symptomatic infection and 17 children (circles) who had subclinical infection established retrospectively through dengue IgG ELISA and HI titers. Median percentage of cytokine-producing cells and P values to detect significant median differences using the Wilcoxon rank sum test are shown.

Figure 3.

CD8⁺ intracellular cytokine production. TNFα, IFNγ, and IL-2 production was measured in response to antigen stimulation with 4 dengue serotypes of presecondary infection PBMCs from 27 Thai schoolchildren. Comparison groups are 10 children (triangles) who developed symptomatic infection and 17 children (circles) who had subclinical infection established retrospectively through dengue IgG ELISA and HI titers. Median percentage of cytokine-producing cells and P values to detect significant median differences using the Wilcoxon rank sum test are shown.

Figure 4.

Lack of polyfunctionality in cytokine production following antigen stimulation. Twenty-five of the 27 donors had T cells that produced only 1 cytokine (upper left and lower right quadrants) but did not have cells that simultaneously produced 2 cytokines (upper right quadrants). The top row represents CD4+ cells, and the bottom row represents CD8+ cells. At left is TNFα (y-axis) versus IFNγ (x-axis); the center shows TNFα versus IL-2; the right shows IFNγ versus IL-2.