Figure 3

Inhibiting function of Breg cells in primary Sjögren’s syndrome (pSS) patients. Magnetic-bead purified CD4 + CD25- effector T cells were cultured alone or 1:1 with flow-cytometry sorted CD19 + CD24^hiCD38^hi cells, stimulated for 72 h with 0.5 μg/mL plate-bound CD3 monocolonal antibodies (mAb), 100 ng/mL CD40L and 0.1 μg/mL CpG ODN 2006. GolgiPlug was added during the final 5 h along with PMA + Iono. Cells were surface-stained for the expression of CD4 and intracellular-stained IFN-γ and TNF-α. Expression of IFN-γ and TNF-α were assessed by flow cytometry. Isotype-matched mAbs were set to be negative controls (A, H). Flow cytometry plots of IFN-γ and TNF-α expression by effector T cells (Teff) alone (B, E, I, L) or in the presences of CD19 + CD24^hiCD38^hi B cells are shown (C, F, J, M). Heterologous Teff/Breg cells from HC and Breg/Teff cells from pSS patients were cross-cultured at 1:1 in the same condition. The results are shown in D, G, K, N. Data are representative of six independent experiments (six different donors in each group).