Patterns of intracellular cytokines in CD4 and CD8 T cells from patients with mycobacterial infections.
Using a short-term bulk culture protocol designed for an intracellular-staining method based on a flow cytometry approach to the frequencies of cytokine-producing cells from tuberculosis and leprosy patients, we found distinct patterns of T cell subset expression. The method also reveals the profile of peak cytokine production and can provide simultaneous information about the phenotype of cytokine-producing cells, providing a reliable assay for monitoring the immunity of these patients. The immune response of Mycobacterium leprae and purified protein derivative (PPD) in vitro to a panel of mycobacteria-infected patients from an endemic area was assessed in primary mononuclear cell cultures. The kinetics and source of the cytokine pattern were measured at the single-cell level. IFN-gamma-, TNF-alpha-, IL-4- and IL-10-secreting T cells were intracytoplasmic evaluated in an attempt to identify M. leprae- and PPD-specific cells directly from the peripheral blood. The analysis by this approach indicated that TNF-alpha was the first (8 h) to be produced, followed by IFN-gamma (16 h), IL-10 (20 h) and IL-4 (24 h), and double-staining experiments confirmed that CD4+ were a greater source of TNF-alpha than of CD8+ T cells (P < 0.05). Both T cell subsets secreted similar amounts of IFN-gamma. We conclude that the protocol permits rapid evaluation of cytokine production by different T cell populations. The method can also be used to define immune status in non-infected and contact individuals.
Laboratório de Hanseníase, Departamento de Medicina Tropical, Instituto Oswaldo Cruz, FIOCRUZ, Rio de Janeiro, RJ, Brazil. pzuquim@ioc.fiocruz.br
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D015496:CD4-Positive T-Lymphocytes / Q000276:immunology*; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology*; D016207:Cytokines / Q000096:biosynthesis*; D003593:Cytoplasm / Q000276:immunology; D005434:Flow Cytometry; D006801:Humans; D007371:Interferon-gamma / Q000096:biosynthesis; D016753:Interleukin-10 / Q000096:biosynthesis; D015847:Interleukin-4 / Q000096:biosynthesis; D007918:Leprosy / Q000276:immunology*; D009166:Mycobacterium leprae / Q000276:immunology*; D014373:Tuberculin / Q000276:immunology; D014397:Tuberculosis, Pulmonary / Q000276:immunology*; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis
D016207:Cytokines; D014373:Tuberculin; D014409:Tumor Necrosis Factor-alpha; D016753:Interleukin-10; D015847:Interleukin-4; D007371:Interferon-gamma