Direct demonstration of cytokine synthesis heterogeneity among human memory/effector T cells by flow cytometry

Abstract

The array of cytokines produced by T cells in effector sites is a primary means by which these cells mediate host defense. It is well recognized that cloned T cells are heterogeneous with regard to cytokine synthesis and, thus, in their ability to mediate specific immune responses, but the extent to which the patterns of cytokine secretion observed in cloned cells reflect actual populations of memory/effector T cells existing in vivo is largely unknown. Here, we report our findings using a multiparameter flow cytometric assay that allows simultaneous determination of an individual T-cell' ability to produce multiple cytokines and its phenotype after only short (4 to 8 hours) in vitro incubation with an activating stimulus and the secretion inhibitor Brefeldin A. This assay shows a rapid accumulation of interleukin-2 (IL-2), IL-4, and gamma-interferon (gamma-IFN) in the cytoplasm of CD4+ cells after stimulation with either accessory cell- independent (phorbol 12-myristate 13-acetate [PMA] + ionomycin [I]) or accessory cell-dependent (staphylococcal enterotoxins [SE] A and B) T- cell-activating stimuli. Further analysis showed that production of gamma-IFN and IL-4 is predominantly, if not exclusively, restricted to the CD45ROhigh memory/effector T-cell subset, whereas IL-2 may be produced by both the CD45ROhigh and CD45ROlow subsets. Simultaneous determination of IL-2 and gamma-IFN production among CD45ROhigh/CD4+ T cells showed distinct subsets that produce each of these cytokines alone (an average of 30% for IL-2 alone, 8% for gamma-IFN alone), both (16%), or neither (46%). Similar analyses with the small IL-4-producing memory/effector T-cell subset (only 4.3% of total CD4+/CD45ROhigh T cells) showed that an average of 51% of these IL-4-producing cells also synthesize average of 51% of these IL-4-producing cells also synthesize IL-2, 23% synthesize only IL-4, 16% synthesize all three cytokines, and 9.6% synthesize IL-4 and gamma-IFN. These patterns of cytokine synthesis were found to be similar with both PMA + I and SEA/SEB stimulation and were observed in both peripheral blood memory/effector CD4+ T cells and in T cells of similar phenotype obtained from cutaneous delayed-type hypersensitivity sites. Taken together, these data strongly support the in vivo existence of human memory/effector T- cell subsets with “preprogrammed” cytokine synthesis potential, although they suggest that these subsets may be more complex than originally proposed in the TH1/TH2 hypothesis.

Longitudinal study of intracellular T cell cytokine production in infants compared to adults

“…Jung et al [17] also showed that freshly isolated unstimulated blood lymphocytes do not produce IFN-g, IL2 or IL4 cytokines as measured by intracellular staining or mRNA analysis. Similar to data from Picker et al [2], a 4-h incubation with PMA/I at 37°C was optimal for intracellular cytokine expression (data not shown). Furthermore, while PMA/I does reduce the fluorescence intensity of anti-CD4 staining [2], the percentage of CD4 positive helper T cells was not significantly different between unstimulated and stimulated samples (n = 26, mean ± s.e.m.…”

“…Validation criteria are reported on PMA/I stimulated blood samples because cytokine levels in unstimulated T cells were below the limit of sensitivity of the assay (≥1 cell in 10 000) as reported previously [2,9]. Jung et al [17] also showed that freshly isolated unstimulated blood lymphocytes do not produce IFN-g, IL2 or IL4 cytokines as measured by intracellular staining or mRNA analysis.…”

“…However, these methodologies do not identify specific, cytokine-secreting cell types. Flow cytometric detection of intracellular cytokine production by individual T lymphocytes [1][2][3][4][5][6] minimizes the influence of extracellular cytokine inhibitors and cytokine depletion by receptor binding. This method permits identification of the cell type synthesizing a given cytokine, and is less time-consuming and labour intensive than ELISPOT assays [7].…”

“…Flow cytometric analysis of intracellular cytokines has been tested on whole blood [4,[9][10][11] and purified peripheral blood mononuclear cells (PBMC) [2]. Suni et al [6] have shown that intracellular T cell cytokine expression in response to soluble cytomegalovirus antigen is similar in whole blood cultures compared to expression in PBMC cultures.…”

Analysis of a Chronic Myelogenous Leukemia Patient Vaccinated with Leukemic Dendritic Cells Following Autologous Peripheral Blood Stem Cell Transplantation

“…CD4 + or CD8 + T cells was performed as described. 20) Briefly, the patient's CD4 + or CD8 + T cells were continuously treated with FACS lysing and permeabilization solutions (Becton Dickinson Immunocytometry System; San Jose, CA). The cells were subsequently incubated with FITC-anti-IFN-γ (Becton Dickinson) and PE-anti-IL-4 (Becton Dickinson) in 0.1% BSA-PBS; FITC-mouse IgG2a and PE-mouse IgG1 (Becton Dickinson) were used as controls.…”