Fig. 5
Resistance to glucocorticoid and regulatory T cell (Treg)-mediated immunosuppression in circulatory CD4+CD28null T cells. A flow cytometry-based in vitro immunosuppression assay using activation-induced CD69 upregulation was used to evaluate suppressive effects of glucocorticoid (GC) and Tregs on T-cell subsets. Representative flow cytometry histograms depicting glucocorticoid-mediated suppression of CD4+CD28+ and CD4+CD28null T cell subsets in myositis patients are shown in (a) and (b), respectively. Grey tinted histograms depict CD69 levels on unstimulated control T cells, black line depicts CD69 upregulation on stimulated control T cells and the bold black line depicts glucocorticoid-induced suppression of CD69 upregulation. The histograms show lower suppression of CD28null T cells compared to CD28+ T cells. At group level also, CD4+CD28null T cells were more resistant to glucocorticoid-mediated suppression compared to CD28+ both in myositis patients (n = 6) (c) and in healthy donors (n = 6) (d). Viable Tregs were isolated based on a CD3+CD4+CD25high gating strategy, as shown in the representative flow cytometry dot plot (e). CD4+CD28null T cells were more resistant to Treg-mediated suppression compared with CD28+ T cells (n = 6) (f). *p < 0.05. MFI mean fluorescence intensity