Brefeldin A, but not monensin, enables flow cytometric detection of interleukin-4 within peripheral T cells responding to ex vivo stimulation with Chlamydia trachomatis

Abstract

Intracellular cytokine staining (ICS) assay optimization should include selection of suitable cytokine secretion inhibitors. Here, peripheral blood mononuclear cells (PBMC) from women with proven history of C. trachomatis genital tract infection were used to compare the ability of brefeldin A (BFA) and monensin (MN) to concurrently trap interferon-γ (IFN-γ), tumor necrosis factor (TNF), interleukin (IL)-4, and IL-17 within T cells responding to ex vivo stimulation with chlamydial antigen. While flow cytometric analyses showed similar intracellular levels of TNF, IFN-γ, and IL-17 among T cells treated with BFA or both BFA and MN, markedly more IL-4 was found inside T cells treated with BFA compared to those that received MN or BFA and MN. The latter findings oppose current ICS recommendations informing that ICS results are unaffected by concomitant use of BFA and MN, and also suggests that MN may be an unsuitable cytokine secretion inhibitor for ICS assays designed to measure intracellular IL-4 accumulation.

Copyright © 2012 Elsevier B.V. All rights reserved.

Figures

Fig. 1

BFA and MN have comparable effects on T cell viability. PBMC were isolated from non-pregnant women with history of NAAT-confirmed genital tract C. trachomatis infection and stimulated 96 h with inactivated C. trachomatis EB. During the last 7 h, cultures were treated with BFA, MN, or BFA/MN as indicated, and then stained with a live/dead cell marker, followed by staining as indicated in Materials and methods. A) Contour plots depict the gating strategy used to define CD4+ T cells that proliferated in response to stimulation with chlamydial antigen. Plots show sequentially (left to right) the gating hierarchy used to interrogate for live non-B cell non-monocyte cells (Live/DeadlowCD14−CD19−CD20−), singlets, CD3ε+CD4+ cells, and finally to define proliferating cells as events that diluted the cell proliferation dye. B) Representative contour plots show percentages of Live/DeadlowCD3ε+ cells (i.e., live T cells) treated with BFA, MN, or BFA/MN that responded to chlamydial antigen. C) Graph depicts percentages of live T cells stratified by treatment group (n=11 samples per group). For B and C, Live/Dead staining was performed, with no addition of any fluorescently labeled antibodies in the same channel.

Fig. 2

MN alters intracellular cytokine levels detected within CD4+ T cells that responded to chlamydial antigen. PBMC cultures were stimulated 48, 72, and 96 h with inactivated C. trachomatis EB. During the last 7 h, cultures were treated with BFA, MN, or BFA/MN as indicated, and processed for flow cytometric analyses as described in the Materials and methods. A) PBMC stimulated for 48, 72, and 96 h, and treated with BFA during the last 7 h of the respective incubation times are shown. Representative contour plots depict increased accumulation of IFN-γ-producing CD4+ T cells among proliferating cells with longer stimulation periods. B) PBMC stimulated for 96 h were treated during the last 7 h of incubation with BFA, MN, or BFA/MN as indicated, and then processed for flow cytometric analyses. Representative contour plots show gates used to identify proliferating CD4+ T cells that produced cytokines in cultures treated with BFA, MN, or BFA/MN. Numbers indicate percentages within each gate (data shown are from one representative experiment of three independent experiments performed).

Fig. 3

MN severely decreases levels of IL-4 detected within T cells stimulated by chlamydial antigen. Graph depicts the integrated median fluorescence intensity (iMFI) levels of IFN-γ, IL-4, TNF and IL-17 in CD4+ T cells that proliferated in response to inactivated EB. iMFI was calculated as (percentage of positive cells)×(MFI for each indicated cytokine), and treatment groups were compared using repeated measures one-way ANOVA and Tukey’s test for multiple comparisons (* P<0.05, ** P<0.01; *** P<0.001). Each symbol represent individual samples (n=11 samples per treatment group), and horizontal bars indicate mean values. Data shown are pooled from three independent experiments.