Patterns of intracellular cytokines in CD4 and CD8 T cells from patients with mycobacterial infections

Abstract

Using a short-term bulk culture protocol designed for an intracellularstaining 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 cytokineproducing 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-γ-, TNF-α-, 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-α was the first (8 h) to be produced, followed by IFN-γ (16 h), IL-10 (20 h) and IL-4 (24 h), and double-staining experiments confirmed that CD4+ were a greater source of TNF-α than of CD8+ T cells (P < 0.05). Both T cell subsets secreted similar amounts of IFN-γ. We conclude that the protocol permits rapid evaluation of cytokine production by different T cell populations.

Flow Cytometric Detection of Gamma Interferon Can Effectively DiscriminateMycobacterium bovisBCG-Vaccinated Cattle fromM. bovis-Infected Cattle

“…These results are in contrast to those assessing the potential of CFC for the diagnosis of M. tuberculosis infection. In a number of such studies where cells were stimulated with PPD, it was not possible to distinguish BCG-vaccinated patients from TB patients (1,16,33,34). Accurate diagnosis was observed only where ESAT-6 was used as the stimulating antigen, and an overall sensitivity of between 90 and 95% was achieved.…”

“…Accurate diagnosis was observed only where ESAT-6 was used as the stimulating antigen, and an overall sensitivity of between 90 and 95% was achieved. The number of responding CD4 ϩ T cells ranged from 0.1 to approximately 1%, although overall responses to ESAT-6 were very low (1,16,33,34). Although ESAT-6 has been suggested as a potential diagnostic tool for distinguishing M. bovis-infected cattle from BCG-vaccinated animals, it is clear that only a proportion of infected animals respond to this antigen (15).…”

Immunologic Homeostasis during Infection: Coexistence of Strong Pulmonary Cell-Mediated Immunity to SecondaryCryptococcus neoformansInfection While the Primary Infection Still Persists at Low Levels in the Lungs

“…Additionally, TNF-␣ plays a critical role in immunity to C. neoformans (35,36). However, similar functional distinctions in T cells have been reported in other infections (37,38). In the CD8 ϩ T cell response to Listeria monocytogenes, intracellular cytokine staining for IFN-␥ detects higher frequencies of tetramer positive (Ag specific) cells than TNF-␣ (39).…”

Oral coinfection can stress peripheral lymphocyte to inflammatory activity in leprosy

“…Usually the peripheral profile of cytokines in leprosy (serum and plasma) reflects the measure of many sources of cytokine production 17,18 without identifying which specific cell is really compromised 19,20 . It has been observed that PBMCs present different levels of proliferation index of cytokines when they are stimulated in vitro with Mycobacterium leprae extracts [21][22][23] .…”