B cell subsets and dysfunction of regulatory B cells in IgG4-related diseases and primary Sjögren's syndrome: the similarities and differences

Abstract

Introduction: IgG4-related disease (IgG4-RD) is a multisystem-involved autoimmune disease. Abnormally activated and differentiated B cells may play important roles. Regulatory B cells (Breg) are newly defined B cell subgroups with immunosuppressive functions. In this study, we investigated the differences of B cell subsets, the expressions of co-stimulatory molecules on B cells, and the function of Breg cells in patients with IgG4-RD, primary Sjögren's syndrome (pSS) as well as in healthy controls (HC).

Methods: Newly diagnosed IgG4-RD patients (n = 48) were enrolled, 38 untreated pSS patients and 30 healthy volunteers were recruited as disease and healthy controls. To analyze B cell subsets and B cell activity, PBMCs were surface stained and detected by flow cytometry. The function of Breg cells was tested by coculturing isolated CD19 + CD24(hi)CD38(hi) Breg cells with purified CD4 + CD25- T cells. Serum cytokines were measured by ELISA and cytometric bead array. Relationship between clinical data and laboratory findings were analyzed as well.

Results: Compared with pSS patients and HC, IgG4-RD patients had a lower frequency of peripheral Breg cells. Interestingly, CD19 + CD24-CD38(hi) B cell subsets were significantly higher in peripheral B cells from IgG4-RD patients than in pSS patients and HC, which correlated with serum IgG4 levels. The expression of BAFF-R and CD40 on B cells was significantly lower in IgG4-RD patients compared with those in pSS patients and HC. Unlike HC, Breg cells from pSS patients lacked suppressive functions.

Conclusions: B cells in patients with IgG4-RD and pSS display a variety of abnormalities, including disturbed B cell subpopulations, abnormal expression of key signaling molecules, co-stimulatory molecules, and inflammatory cytokines. In addition, a significantly increased B cell subset, CD19 + CD24-CD38(hi) B cells, may play an important role in the pathogenesis of IgG4-RD.

Figure 1

Expression of B-cell subsets in IgG4-related disease (RD), primary Sjögren’s syndrome (pSS), and healthy controls (HC). Representative flow cytometry pictures of different B-cell subsets from HC, IgG4-RD, and pSS patients (A). The percentages of CD19+ B cells out of total lymphocytes in each group (B). Percentages of Breg cells, mature B cells, and memory B cells out of total B cells in each group (C, D, E). Summary of different B-cell subsets in different populations (F). Percentages of CD19 + CD24-CD38^hi B cells out of total B cells in each group (G). Values are shown as mean ± standard error of the mean, *P <0.05, **P <0.01, ***P <0.001.

Figure 2

Expression of BAFF-R, CD40, CD80, and CD86 on peripheral B cells in IgG4-related disease (RD), primary Sjögren’s syndrome (pSS), and healthy controls (HC). Representative flow cytometry pictures of BAFF-R expression on CD19+ B cells of HC, IgG4-RD, and pSS patients (A). Scatter plots showing the percentages of CD19 + BAFF-R + cells in CD19+ cells (B). Scatter plots of the percentages of BAFF-R + expression on different B cell subsets (C-F). The changes in mean fluorescence intensity (MFI) of BAFF-R from healthy subjects after stimulation with recombinant human BAFF (rhBAFF) for 48 h (G). One representative example of decreased BAFF-R MFI on B cells by flow cytometry after 48 h culture in the presence of rhBAFF (2 ng/mL) (H). Representative flow cytometry of CD40 expression on CD19+ B cells of HC, IgG4-RD, and pSS patients (I). Scatter plots showing the percentages of CD19 + CD40+ cells in CD19+ cells (J-K). Percentages of CD80 and CD86 expression on CD19+ B cells of HC, IgG4-RD, and pSS patients (L,M). Values are shown as mean ± standard error of the mean, *P <0.05; **P <0.01; ***P <0.001.

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).

Figure 4

IgG4 secreting by CD19 + CD24 + CD38- memory B cells from IgG4-related disease patients. Isolated CD19 + CD24 + CD38- memory B cells from IgG4-RD, primary Sjögren’s syndrome (pSS) patients and healthy controls (HC) were cultured in vitro, stimulated with CD40L and CpG ODN 2006. At day 7, the supernatants were collected and IgG4 level was evaluated by CBA. The results are shown in A-C.

Figure 5

Serum levels of cytokines and BAFF in IgG4-related disease (RD), primary Sjögren’s syndrome (pSS), and healthy controls (HC). Serum cytokines of IL-4 (A), IL-6 (B), IL-5 (C), IL-13 (D), IL-10 (E), IFN-γ (F), IL-17A (H), TGF-β (I) and BAFF (G) levels from serum samples of IgG4-RD (n = 28), pSS patients (n = 28), and HC (n = 24) were tested by ELISA. Values are shown as mean ± standard error of the mean, *P <0.05; **P <0.01; ***P <0.001.

Figure 6

Correlation of different B-cell subsets with laboratory findings in IgG4-related disease (RD) patients. Correlations between the percentages of CD19 + CD24^hiCD38^hi Breg cells and levels of IgG, IgM, IgA and IgG4, IgG4/IgG ratio, as well as erythrocyte sedimentation rate (ESR) in IgG4-RD patients (n = 28) were analyzed by Pearson’s rank test (A-F). Correlations between the percentages of CD19 + CD24-CD38^hi B cells and levels of IgG, IgM, IgA and IgG4, IgG4/IgG ratio, as well as ESR in IgG4-RD patients were analyzed by Pearson’s rank test (G-L).