Interleukin-4 suppresses the cytotoxic potential of in vitro generated, adaptive regulatory CD4 T cells by down-regulation of granzyme B

Abstract

Regulatory CD4+ T cells (Tregs) control immune responses using secretion of anti-inflammatory cytokines and/or cytotoxic mechanisms and play a central role in the outcomes of several immune pathologies. Previous studies suggest an impaired function of Tregs in allergy, especially during allergen seasons, but the underlying mechanism is not known. Therefore, we analysed the impact of the T helper type 2 cytokine interleukin (IL)-4 on in vitro generated adaptive Tregs (aTregs), which have been reported to use the granzyme B (GrB)/perforin pathway to kill autologous immune cells. aTregs were generated by co-ligation of CD3 and CD46 on CD4+ T lymphocytes and granzyme expression was analysed using flow cytometry. To quantify GrB and perforin expression as well as IL-10 secretion in response to IL-4, specific enzyme-linked immunosorbent assays were performed in cell lysates and/or culture supernatants. Using a flow cytometry-based cytotoxicity assay the impact of IL-4 on the cytotoxic potential of aTregs was investigated. While IL-4 did not affect IL-10 secretion and perforin expression in aTregs, a significant suppression of GrB synthesis was detected in the presence of IL-4. In addition, IL-4-mediated suppression of GrB led to impaired cytotoxicity of aTregs against K562 target cells. In conclusion, our data suggest that IL-4 might play a role in impaired aTreg function in allergy.

Figure 2

Interleukin (IL)-4 suppresses granzyme B synthesis in adaptive regulatory T cells (aTregs). aTregs were generated by co-ligation of CD3 and CD46 in the presence of IL-2 and indicated IL-4 concentrations for 2 days. (a) Granzyme B (GrB) concentrations in cell lysates and supernatants of aTregs were determined by enzyme-linked immunosorbent assay (ELISA). The relative change compared with the control sample (no IL-4) is displayed. Mean values ± standard deviation (SD) for five different subjects are shown. Asterisks indicate significant differences between control samples (no IL-4) and IL-4-stimulated samples. P < 0·05 was regarded as significant. (b) The specificity of the IL-4 effect was confirmed using a neutralizing IL-4 antibody (10 μg/ml) or an isotype control antibody (10 μg/ml). Experiments for three different subjects are shown. (c) Median fluorescence intensity (MFI) values of GrB staining in aTregs generated in the absence or presence of 100 ng/ml IL-4 as determined by flow cytometry. Data for 10 different donors are shown and median values are indicated (−). *P values < 0·05 were regarded as significant. (d) Perforin concentrations in cell lysates of aTregs from five subjects were determined by ELISA.

Figure 1

Expression of granzymes in CD4⁺ T cells. CD4⁺ T cells were cultured with (i) interleukin (IL)-2; (ii) IL-2, anti-CD3 and anti-CD28; or (iii) IL-2, anti-CD3 and anti-CD46 for 3 days. Cell size and granularity are shown in forward-scatter/side-scatter plots and the activation state in CD4/CD25 plots, respectively. Granzyme expression is displayed in histograms with percentages or median fluorescence intensity (MFI) values indicated. One representative experiment of three is shown. FITC, fluorescein isothiocyanate; PE, phycoerythrin.

Figure 3

Granzyme B (GrB)-dependent cytotoxicity of adaptive regulatory T cells (aTregs) is suppressed by interleukin (IL)-4. (a) Adaptive CD4⁺ Tregs kill K562 cells in a GrB/perforin-dependent manner. Ethyleneglycoltetraacetic acid (EGTA) (4 mm) and the GrB inhibitor z-AAD-cmk (50 μm) were added to samples as indicated. Mean values ± standard deviation (SD) of seven independent experiments are shown. (b) aTregs were generated in the absence or presence of IL-4 (100 ng/ml). Killing of K562 cells by aTregs from eight subjects is shown. Mean values are indicated (−). *P values < 0·05 were regarded as significant.

Figure 4

Impact of interleukin (IL)-4 on cytokine release by adaptive regulatory T cells (aTregs). aTregs were generated with concentrations of IL-4 for 2 days as indicated. IL-10 and interferon (IFN)-γ concentrations were determined in the culture supernatants of five subjects. The relative change compared with the control sample (no IL-4) is displayed. Mean values ± standard deviation are shown.