Figure 1.
Expansion of circulating Breg cells in SLE patients.
(A) Human PBMCs were labeled with lymphocyte-specific antibodies (CD19, CD5, and CD1d). The percentage of CD5+CD1dhigh cells among a CD19 gate was determined by flow cytometry. (B) Results of flow cytometric analysis of Breg cells in patients with active SLE (n = 16), patients with inactive SLE (n = 14), and control subject (n = 15). (C) A positive correlation between the proportion of CD5+CD1dhigh cells among CD19+ B cells and the clinical severity of the flare as scored using the SLEDAI (n = 30) was observed.
Figure 2.
Infiltration of IL-10+ B cells in involved skin of SLE patients.
(A) Consecutive levels obtained by serially section showed typical pathological changes of lupus (upper panel), lymphocytes infiltration confirmed by CD20 (middle panel) and IL-10 (lower panel) immunohistochemical staining (× 100 magnification). Further magnification of the black-bordered box shows typical IL-10+ lymphocytes (×400 magnification). (B) The counts of IL-10+ lymphocytes in skins were showed in right (SLE patients n = 10, healthy controls n = 4).
Figure 3.
IL-10 production in Breg cells of SLE patients.
(A) Real-time RT-PCR analysis of IL-10 mRNA expression in PBMCs from patients with active SLE (n = 16), patients with inactive SLE (n = 14), and control subject (n = 15). (B) Serum levels of IL-10 were detected in patients with active SLE (n = 16), patients with inactive SLE (n = 14), and control subject (n = 15) by ELISA. (C) PBMCs were isolated and stimulated with LPS for 24 hours and PIB for the final 5 hours. CD19+IL-10+ cells were detected by flow cytometry analysis in a CD19 gate (left). Results of flow cytometric analysis of CD19+IL-10+ cells (right, n = 6 for each group). (D) Sorted CD19+CD5+CD1dhigh B cells from SLE patients and healthy controls were stimulated with LPS for 24 hours and PIB for the last 5 hours. IL-10 mRNA expression was detected by real-time RT-PCR. Results shown are representative of at least three independent experiments. (E) Sorted CD19+CD5+CD1dhigh B cells from SLE patients and healthy controls were stimulated with LPS for 24 hours and PI for the last 5 hours. IL-10 in supernatants was detected by ELISA. Results shown are representative of at least three independent experiments.
Figure 4.
Tfh cells are associated with expansion of Breg cells in SLE patients.
(A) Human PBMCs were labeled with lymphocyte-specific antibodies (CD4, CXCR5 and PD-1). The percentage of CXCR5+PD-1+ cells among CD4+ T cells was analyzed by flow cytometry (left). Results of flow cytometric analysis of Tfh cells (right) in patients with active SLE (n = 16), patients with inactive SLE (n = 14), and control subject (n = 15). (B) A positive correlation between the proportion of CXCR5+PD-1+ cells among CD4+ T cells and the clinical severity of the flare as scored using the SLEDAI (n = 30) was noted. (C) A positive correlation between the proportion of CD4+CXCR5+PD-1+ T cells and CD19+CD5+CD1dhigh B cells in PBMCs from SLE patients (n = 30) was observed. (D) Serum IL-21 levels were detected in patients with active SLE (n = 16), patients with inactive SLE (n = 14), and control subject (n = 15) by ELISA. (E) A positive correlation between serum IL-10 levels and IL-21 levels in SLE patients (n = 30) was observed.
Figure 5.
Tfh cell-derived IL-21 promotes IL-10 production during the differentiation of Breg cells.
(A) Sorted B cells (CD43 depletion) from healthy controls were cultured in the presence of LPS and the indicated concentrations of IL-21 for 48 hours followed by stimulation with PI for the final 5 hours (Control: LPS+PIB). IL-10 in supernatants was detected by ELISA. Results shown are representative of at least three independent experiments. (B) Sorted B cells (CD43 depletion) from healthy controls were cultured in the presence of LPS with or without 40 ng/ml IL-21 for 48 hours followed by stimulation with PIB for the last 5 hours (Control: LPS+PIB). IL-10+ cells were analyzed in a CD19 gate by flow cytometry. Results shown are representative of at least three independent experiments. (C) Sorted CD4+CXCR5+PD-1+ T cells were cultured with anti-CD3 and anti-CD28 for 48 hours, IL-21 was detected in the supernatants by ELISA. Results shown are representative of at least three independent experiments (C: healthy controls; S: SLE patients). (D) Sorted B cells (CD43 depletion) were stimulated for 48 hours with LPS plus supernatants from cultures of Tfh cells of SLE patients with or without neutralization of IL-21. The cells were also stimulated with PI for the last 5 hours. Levels of IL-10 in the supernatants were determined by ELISA (Control: LPS+PI; Tfh (S): LPS+supernatants from Tfh cells of SLE patients+PI; Tfh (C): LPS+supernatants from Tfh cells of healthy controls+PI). Results shown are representative of at least three independent experiments. (E) Sorted B cells (CD43 depletion) were cultured for 48 hours in the presence of LPS plus supernatants from Tfh cells of SLE patients. These cells were cultured with or without neutralization of IL-21 and were stimulated with PIB for the last 5 hours (Control: LPS+PIB; Tfh (S): LPS+supernatants from Tfh cells of SLE patients+PIB). IL-10+ cells were analyzed by flow cytometry with a CD19 gate. Results shown are representative of at least three independent experiments.
Table 1.
Patient characteristics.