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Published on: April 10, 2018
Cytokine production by dengue virus antigen-responsive human T lymphocytes in vitro examined using a double
1Department of Medicine, University of Massachusetts Medical School, Worcester 01655, USA.
Insights
This study developed a double immunocytochemistry method to identify cytokine-producing cells in dengue virus infection. Antigen-stimulated CD4+ and CD8+ T cells were found to produce cytokines, supporting their role in dengue pathogenesis.
Area of Science:
- Immunology
- Virology
- Cell Biology
Background:
- Cytokines are implicated in the pathogenesis of viral infections like dengue.
- Identifying specific cytokine-producing cells is crucial for understanding disease mechanisms.
Purpose of the Study:
- To develop and validate a double immunocytochemistry method for characterizing cytokine-producing cells in dengue virus-immune individuals.
- To identify the specific immune cells responsible for cytokine production upon dengue antigen stimulation.
Main Methods:
- Developed a double immunocytochemistry technique using immunoalkaline phosphatase and immunoperoxidase staining.
- Analyzed peripheral blood mononuclear cells (PBMC) from dengue virus-immune donors stimulated with dengue antigens.
- Quantified cytokine production (IFN-gamma, IL-2, IL-4, TNF-beta) and identified cell surface markers (CD4+, CD8+ T cells).
Main Results:
- The double immunocytochemistry method effectively distinguished cytokine-producing cells.
- Antigen stimulation led to increased numbers of IFN-gamma, IL-2, IL-4, and TNF-beta-producing cells, primarily CD4+ and CD8+ T cells.
- Results were consistent with enzyme-linked immunosorbent assay (ELISA) findings.
Conclusions:
- The developed double immunocytochemistry technique is suitable for detecting and characterizing cytokine-producing cells in PBMC.
- Antigen-stimulated CD4+ and CD8+ T cells produce cytokines that may contribute to dengue virus infection pathogenesis.
Abstract:
A number of studies suggest that cytokines may contribute to the pathogenesis of viral infections, including dengue. In this study, we developed a double immunocytochemical method and characterized cytokine-producing cells in the peripheral blood mononuclear cells (PBMC) of dengue virus-immune donors after in vitro stimulation with specific dengue antigens. We found that double immunostaining using immunoalkaline phosphatase (Vector blue) for cytokines [interferon-gamma (IFN-gamma), interleukin (IL) -2, -4, -1alpha, -1beta, and -6, tumor necrosis factor beta (TNF-beta), and TNF-alpha] and immunoperoxidase [diaminobenzidine (DAB)] for cell surface markers (CD3, CD4, CD8, CD20, and CD68) provided the best distinction of double-positive cells from single-positive or -negative cells. The number of IFN-gamma, IL-2, IL-4, and TNF-beta-positive cells increased 2 or 3 days after stimulation with specific dengue antigens. No or very few cytokine-producing cells were detected in the PBMC of non-immune donors stimulated with dengue antigens and the PBMC of immune donors stimulated with a control antigen. The analysis of cell surface markers showed that mainly CD4+ and CD8+ T cells produced these cytokines. The results obtained by immunocytochemistry were consistent with cytokine levels detected in the culture medium assayed by enzyme-linked immunosorbent assay. In conclusion, this double immunocytochemistry technique is suitable for the detection and characterization of cytokine-producing cells in PBMC. Furthermore, the results support the hypothesis that antigen-stimulated CD4+ and CD8+ T cells produce cytokines that may play a role in the pathogenesis of dengue virus infection.
