Abstract T follicular helper (Tfh) cells aid effector B cells, and augment autoimmunity, whereas the role of Tfh cells on regulatory B (Breg) cells in systemic lupus erythematosus (SLE) is not known. The aim of this study is to investigate the percentage of Breg cells in SLE, and the role of Tfh cells on Breg cells. First, we demonstrated the presence of Breg cells in SLE peripheral blood mononuclear cells and in involved skins. Both the percentage of circulating Breg cells and the ability to produce interleukin-10 (IL-10) were elevated in SLE patients. The percentage of Breg cells increased during SLE flares and decreased following disease remission. Second, Tfh cell expansion was not only related to autoantibody production but also correlated with the increased percentage of Breg cells. Third, in vitro studies revealed that Tfh cell-derived IL-21 could promote IL-10 production and Breg cell differentiation. In conclusions, these data imply that SLE flares may be linked to the expansion of Tfh cells and that Breg cells are increased in a regulatory feedback manner. Thus, SLE development may be associated with the complex regulation of Tfh cells and diverse B cell subsets.

Citation: Yang X, Yang J, Chu Y, Xue Y, Xuan D, Zheng S, et al. (2014) T Follicular Helper Cells and Regulatory B Cells Dynamics in Systemic Lupus Erythematosus. PLoS ONE 9(2): e88441. https://doi.org/10.1371/journal.pone.0088441 Editor: Frederic Rieux-Laucat, Pavillon Kirmisson, France Received: August 14, 2013; Accepted: January 10, 2014; Published: February 14, 2014 Copyright: © 2014 Yang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by grants from National Natural Science Foundation of China (No. 81373213, 81072463, 81000693), Program of Shanghai Subject Chief Scientist (No. 11XD1401100), and Medical Guide Project from Shanghai Science and Technology Committee (No. 134119a8400). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist.

Introduction Systemic lupus erythematosus (SLE) is an autoimmune disease that involves multiple organ systems [1]. The pathogenic mechanisms that cause lupus are unclear; however, the immune balance between regulatory T or B lymphocytes and effector T and B lymphocytes may be disturbed, contributing to the autoimmune injuries in SLE [2], [3], [4], [5]. Interleukin (IL)-10-producing regulatory B (Breg) cells have recently been identified. These cells, which represent 1∼3% of adult mouse spleen B cells, predominantly represent a subset of CD19+CD5+CD1dhigh B cells and function to negatively regulate immune responses [3], [6], [7]. The absence or loss of Breg cells exacerbates disease symptoms in contact hypersensitivity, experimental autoimmune encephalomyelitis, chronic colitis, and collagen-induced arthritis models [8], [9], [10], [11]. IL-10 is a key cytokine produced by Breg cells, and diminished disease severity was observed following administration of IL-10 in the NZM2410 mouse model of lupus [12], whereas more severe disease occurred in both MRL/lpr mice on a IL-10 KO background and in Breg cell-deficient NZB/W mice [13], [14]. The finding that transfer of IL-10-secreting CD21hiCD23hi B cells mitigates disease in MRL/lpr mice [15] further suggests that B cell-derived IL-10 limits disease activity. Although several studies showed that Breg cells were present in lupus-prone mice, including MRL/lpr and NZW mice [6], [13], [16], the dynamic change of Breg cells in SLE patients is not clear, and the mechanism of Breg cell differentiation in SLE patients is unknown. T follicular helper (Tfh) cells, a subset of CD4+ T cells found in germinal centers (GCs), express high levels of C-X-C chemokine receptor type 5 (CXCR5), programmed death-1 (PD-1), and inducible costimulatory molecule (ICOS) [17], [18], [19]. Recently, expanded circulating Tfh cells were characterized as CD4+CXCR5+ICOShighPD-1high in peripheral blood mononuclear cells (PBMCs) from SLE patients [20]. In addition, production of the CXCR5 ligand CXCL13 was also found to be elevated in SLE patients [21]. IL-21 is a key cytokine produced by Tfh cells [18], [19]. Our previous study demonstrated that the genotype and allele frequencies for copy number amplifications of IL-21 are significantly higher in SLE patients than in healthy controls [22]. Tfh cell-derived IL-21 is thought to drive the differentiation of B cells to produce antibodies, a process that serves as an important regulator of humoral immune responses [19], [23]. Recent studies showed that IL-21 is a pleiotropic cytokine, at least under specific circumstances, IL-21 can also exert anti-inflammatory actions due to its ability to inhibit dendritic cell maturation and stimulate IL-10 production in T cells [24], [25]. Our recent study proved that Tfh cell-derived IL-21 could promote the differentiation of Breg cells in lupus-prone MRL/lpr mice [16], however the relationship between Tfh and Breg cells in SLE patients is not known. Whether Tfh cell-derived IL-21 may also play a key role in the differentiation of Breg cells in SLE patients need be clarified. Here, we provided evidence that Breg cells were present among PBMCs and involved skins in SLE patients. In detailed studies of Breg and Tfh cells from 30 SLE patients, we showed that Breg cells exhibited expansion rather than redistribution in vivo, and this expansion of Breg cells was related to disease activity. Further study demonstrated that expansion of Breg cells was related to Tfh cells in SLE. Tfh cell-derived IL-21 could promote IL-10 production and the differentiation of Breg cells. These data suggest that Tfh cell-derived IL-21 may induce the production of the anti-inflammatory cytokine IL-10 and result in expansion of Breg cells in SLE. Thus, the pathophysiology of SLE may be linked to a complex immune relationship between Tfh cells and diverse B subsets.

Discussion The ability of B cells to negatively regulate cellular immune responses and inflammation has been described previously [7]. Most recently, CD19+CD5+CD1dhigh B cells with the capacity to produce IL-10 have been named Breg cells (B10) in mice [3], [6], [7]. Remarkably, Breg cells are potent negative regulators of inflammation and autoimmunity in mouse models of disease in vivo [10], [11], [33]. Recently, IL-10-producing CD1dhigh, or CD5+IL-10+ Breg cells were identified in human [26], [27], [34], however little is known the dynamic changes of Breg cells in active or inactive SLE patient. The balance between Breg cell negative regulation and B-cell positive contributions to immune responses are likely to vary in different diseases as well as during the course of disease. Breg cell numbers increase during some autoimmunity animal models like NZB/W mice [6], [13], our recent data proved that Breg cells were expanded in MRL/lpr mice [16]. Here, we demonstrated that the percentage of peripheral blood CD19+CD5+CD1dhigh Breg cells was significantly increased in active SLE patients and was positively correlated with disease activity, Breg cells decreased during disease relief. Breg cells produced more IL-10 in active SLE patients than healthy control. In addition, more IL-10+ B cells were detected in involved skin of SLE patients when compared with controls. In addition, the percentage of CD19+CD24+CD38+ Breg cells was also expanded in SLE patients than heanlty control, which was consistent with previous results [29]. The absolute numbers of CD19+CD5+CD1dhigh cells, CD19+CD24+CD38+ cells, and CD19+IL-10+ cells increased but not significantly in SLE patients when compared with healthy controls, which might be attributed to peripheral lymphopenia in SLE patients during flares. The percentage of Breg cells was expanded in SLE patients and decreased following remission than in healthy controls, these data suggested that Breg cells are dynamic during the development of autoimmunity. Maintaining immunological balance involves the capacity of the immune system to upregulate immunosuppressive responses, which may limit deterioration by the autoimmune response. The upregulation of Breg cells in active SLE patients may reflect a regulatory feedback mechanism to restore cellular tolerance and ameliorate harmful autoimmune responses. B10 cells were functionally identified by their ability to express cytoplasmic IL-10 after 5 hours of ex vivo stimulation, whereas progenitor B10 (B10pro) cells required 48 hours of in vitro stimulation before they acquired the ability to express IL-10. Recent study showed that the percentages of B10 cells in SLE patients were not significantly different from controls, but the percentages of B10+Bpro cells in SLE patients were significantly different from controls [28], these data implied that B cells in SLE have more potential to produce IL-10. In our study, modified methods were taken, the B cells were stimulated with LPS 24 hours and the last 5 hours of PIB stimulation, which was based on the previous reported methods [6]. Consistent with previous results [28], [35], our study confirmed that both IL-10 production and the percentage of CD19+IL-10+ B cells were increased in SLE patients; however, the reason behind this expansion of Breg cells in SLE was not addressed in the previous studies [28]. Our data showed that the absolute numbers of CD4+CXCR5+PD-1+ Tfh cells were not significantly increased in SLE patients than in healthy controls, however the percentage of CD4+CXCR5+PD-1+ Tfh cells were expanded in active SLE patients and that Tfh cell-derived IL-21 contributed to autoantibody production. Further analysis showed that the percentage of Tfh cells was positively related to disease activity in SLE, which suggested that Tfh cells may contribute to autoimmunity by helping B effector cells and inducing humoral immunity [19], [36]. Secondly, we unexpectedly identified a strong positive correlation between Tfh cells and Breg cells in SLE patients, suggesting that Tfh cells may contribute to the expansion of Breg cells in SLE. Our in vitro data further revealed that SLE patient Tfh cell-derived IL-21 in synergy with LPS and PI promoted IL-10 production and the differentiation of Breg cells. This finding was verified as treatment of these cultures with an IL-21-neutralizing antibody inhibited IL-10 production and the generation of CD19+IL-10+ cells. IL-21 is a pleiotropic cytokine, and at least under certain circumstances, IL-21 can stimulate anti-inflammatory IL-10 production in T and B cells [24], [25], [32], [37]. The generation of T and B subsets during autoimmune disease requires complex and reciprocal regulation; thus, micro-environmental cytokines or other factors may be involved in the development of pro-inflammatory or anti-inflammatory lymphocyte subsets. Our data suggest that Tfh cells facilitate immune homeostasis by increasing the number of regulatory B cells and the production of IL-10 via the stimulation of IL-21 in SLE patients. All together, we define a novel role of Tfh cells in immune regulatory actions to promote production of the immunosuppressive cytokine IL-10, which extends the existing recognization that Tfh cells merely induce humoral responses and augment autoimmunity. Furthermore, IL-21 may serve as a potential upstream promoter for Breg cell differentiation and IL-10 production in SLE. These findings suggest that particular emphasis should be given to the regulatory function of Tfh cells and IL-21 in the treatment of SLE.

Materials and Methods SLE Patients and Healthy Controls This study was approved by the Ethical Committee of Huashan Hospital and Zhongshan Hospital, Fudan University (Shanghai, People’s Republic of China). Thirty consecutive adult patients (28 women and 2 men, mean age 37.6±12.3 years) with a diagnosis of SLE, based on the American College of Rheumatology 1997 revised criteria [38], were included in the study. All patients enrolled in the study after giving informed and written consent. All SLE patients were referred to the Division of Rheumatology, Huashan Hospital or to the Department of Dermatology, Zhongshan Hospital, Fudan University, Shanghai, China. Disease activity was assessed by the SLE Disease Activity Index (SLEDAI). One group comprised subjects with active SLE (SLEDAI ≥6, n = 16, mean age 35.9±12.0 years, 15 women and 1 man), while the second group comprised subjects with inactive SLE (SLEDAI <6, n = 14, mean age 39.6±12.7 years, 13 women and 1 man) [2]. The following treatment was provided for the SLE group: prednisone, hydroxychloroquine+prednisone, or hydroxychloroquine +prednisone+cyclophosphamide. For the control group, 15 age and sex matched healthy individuals (mean age 36.2±12.7 years; 14 women and 1 man) were enrolled after giving informed consent. The ages, sex, and treatments of the patients are presented in Table 1. PPT PowerPoint slide

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larger image TIFF original image Download: Table 1. Patient characteristics. https://doi.org/10.1371/journal.pone.0088441.t001 B and T Cell Isolation, Culture Conditions, and Differentiation Human B cells were purified from PBMCs of healthy donors (CD43 depletion) by negative selection following the manufacturer’s instructions (Miltenyi Biotec, Bergisch Gladbach, Germany). For the differentiation of Breg cells, purified B cells (2×106 cells/ml) were cultured in 10 µg/ml lipopolysaccharide (LPS; Sigma-Aldrich, St. Louis, MO) for 24 or 48 hours and stimulated with PIB (50 ng/ml phorbol 12-myristate 13-acetate [PMA], Sigma-Aldrich; 500 ng/ml ionomycin [Sigma-Aldrich]; and 20 µg/ml brefeldin A [BFA], eBioscience, San Diego, CA) for the last 5 hours, as previously described [6], [28]. Where indicated, cultures were supplemented with indicated doses of IL-21 (PeproTech, RockyHill, NJ) and LPS for 48 hours, and stimulated with PIB for the last 5 hours. In experiments to detect IL-10 in culture supernatants, BFA was not added. For some experiments, CD20+CD27− naïve B cells (eBioscience) were sorted from PBMCs of healthy donors by flow cytometry, and cultured in certain conditions. To determine the effects of Tfh cell-derived IL-21 on the activation of Breg cells, CD4+CXCR5+PD-1+ Tfh cells from active SLE patients were first sorted by flow cytometry. The resultant Tfh cells (2×106 cells/ml) were stimulated with 2 µg/ml plate-bound anti-CD3 and 2 µg/ml soluble anti-CD28 (eBioscience) for 48 hours. Supernatants were collected for later use. Purified B cells or naïve B cells (2×106 cells/ml) from healthy donors were cultured with 10 µg/ml LPS (Sigma-Aldrich) in the presence or absence of 20% supernatants from the above-stimulated Tfh cells or 20 µg/ml anti-IL-21 neutralizing antibody (eBioscience) for 48 hours. Culture media with the same doses of anti-CD3 and anti-CD28 was used as a vehicle control. Cultures were stimulated with PIB for the last 5 hours. IL-10+ cells were analyzed by flow cytometry with a CD19 gate. In experiments to detect IL-10 in culture supernatants by enzyme-linked immunosorbent assay (ELISA), BFA was not added. For some experiments, CD19+CD5+CD1dhigh Breg cells (4×105 cells) were obtained via cell sorting from PBMCs of SLE patients and healthy controls and were then cultured in the presence of LPS for 24 hours and PIB for the last 5 hours for the detection of IL-10 mRNA expression. For detecting IL-10 in culture supernatants, BFA was not added. ELISA Sera from SLE patients and healthy controls were collected and frozen at −80°C until needed. Concentrations of anti-double-stranded DNA (anti-dsDNA) were determined by ELISA (R&D, Minneapolis, MN). Serum levels of IL-21 and IL-10 in SLE patients were also detected by commercial ELISA (eBioscience). In some experiments, isolated B cells (5×105 cells) were cultured and stimulated with PMA and ionomycin (PI, Sigma-Aldrich) for the last 5 hours. IL-10 was detected in the supernatants by ELISA (eBioscience). Sorted CD4+CXCR5+PD-1+ Tfh cells (5×105 cells) were stimulated with 2 µg/ml plate-bound anti-CD3 and 2 µg/ml soluble anti-CD28 (eBioscience) for 48 hours. IL-21 in supernatants was detected by ELISA (eBioscience). Flow Cytometry For detection of Tfh cells, human PBMCs were stained with Alexa Fluor 647-conjugated anti-CD4, Alexa Fluor 488-conjugated anti-CXCR5, and phycoerythrin (PE)-conjugated anti-PD-1 (all from BD Pharmingen, San Jose, CA). Cells were gated for CD4+ T cells first and then for CXCR5+PD-1+ Tfh cells. For detection of Breg cells, PBMCs were stained with PerCP/Cy5.5-conjugated anti-CD19, fluorescein isothiocyanate (FITC)-conjugated anti-CD5, and PE-conjugated anti-CD1d (eBioscience) for 15 minutes. CD5+CD1dhigh cells were analyzed with a CD19+ gate. For intracellular IL-10 staining, PBMCs were incubated for 24 hours with 10 µg/ml LPS and stimulated with PIB for the last 5 hours. Surface staining with PerCP/Cy5.5-conjugated CD19 or FITC-conjugated anti-CD5 was first performed for 15 min, and cells were re-suspended in Fixation/Permeabilization solution (Invitrogen). Intracellular staining of PE-conjugated anti-IL-10 was performed according to the manufacturer’s protocol (eBioscience). After staining, IL-10+ cells were analyzed with a CD19+ gate by flow cytometry. For some experiments, cells were stained with FITC-conjugated CD19 and PE-conjugated anti-IL-10 (eBioscience) and detected by immunofluorescence microscopy. Immunohistochemistry Skin biopsies from 10 SLE patients were obtained after informed consent, 4 normal skin biopsies (Three skin biopsies were from healthy donors after informed consent, one tissue was obtained from orthopedic surgery after informed consent) were used as controls. Tissues were processed and embedded in paraffin using routine methods. Tissue blocks were serially sectioned to obtain consecutive levels. Sections were stained with hematoxylin and eosin, and immunohistochemistry with the following antibodies was performed as previously described [2]. Antibodies to CD20 and IL-10 (Abcam, Cambridge, MA) were used. Immunohistochemical staining was assessed by two independent pathologists without knowledge of patient characteristics. The positive cells in per surface were counted under ×400 magnification, and five randomly selected independent microscopic fields were counted for each sample to ensure that the data were representative and homogeneous. Analyses of Cytokine and Transcription Factor mRNA Expression Total RNA was purified with the Trizol reagent (Invitrogen). cDNAs were synthesized using Primescript RT Master Mix Perfect Real-time Kit (TaKaRa, Tokyo, Japan), and mRNA expression was determined with the Bio-Rad iCycler 7500 Optical System (Bio-Rad, Richmond, CA) using a SYBR Premix EX Taq Real-time PCR Master Mix (TaKaRa). The 2−ΔΔCt method was used to normalize transcription to β-actin and to calculate the fold induction relative to controls. The following primer pairs were used: Hum β-actin, forward ATCATGTTTGAGACCTTCAACA and reverse CATCTCTTGCTCGAAGTCCA and Hum IL-10, forward GAAGTGAAAACGAGACCAAGGT and reverse CTGCAAGTTAGATCCTCAGG. Statistical Analyses Results were expressed as means ± standard deviation. The statistical significance was determined by analysis of variance for comparisons of multiple means followed by the Bonferroni post hoc test, or the Student’s t-test, and the Mann-Whitney U-test. Correlations were determined by Spearman’s ranking.

Supporting Information Figure S1. The absolute numbers of Breg cells in SLE patients. (A) The results of flow cytometric analysis of absolute numbers of CD19+CD5+CD1dhigh cells in patients with SLE (n = 30) and healthy controls (n = 15). (B) A positive correlation between the absolute numbers of CD19+ CD5+CD1dhigh cells and the clinical severity of the flare as scored using the SLEDAI (n = 30) was observed. (C) Human PBMCs were labeled with lymphocyte-specific antibodies (CD19, CD24, and CD38). The percentage of CD24+CD38+ cells among a CD19 gate was determined by flow cytometry (left). Results of flow cytometric analysis of percentage of CD24+CD38+ cells among a CD19 gate cells in patients with SLE and control subject (right, n = 7 for each group). (D) The results of flow cytometric analysis of absolute numbers of CD19+CD24+CD38+ cells in patients with SLE and healthy controls (n = 7 for each group). https://doi.org/10.1371/journal.pone.0088441.s001 (TIF) Figure S2. IL-10+ cells in skins of SLE patients. (A) The skin tissues from SLE patient were serially sectioned to obtain consecutive levels. The sections were stained with antibodies to IL-10 and isotype control. (B) The skin tissues from SLE patient were stained with CD20 and IL-10, the CD20+IL-10+ cells were analyzed by immunofluorescence microscopy. https://doi.org/10.1371/journal.pone.0088441.s002 (TIF) Figure S3. IL-10+ cells in PBMCs of SLE patients. (A) PBMCs were isolated and stimulated with LPS for 24 hours and PIB for the final 5 hours. The presence of CD19+IL-10+ cells in PBMCs from active SLE patients was detected by immunofluorescence microscopy. The arrow indicates typical positive cells. (B) CD19+IL-10+ cells were detected by flow cytometry analysis in a CD19 gate (n = 6 for each group). https://doi.org/10.1371/journal.pone.0088441.s003 (TIF) Figure S4. Tfh cells in PBMCs of SLE patients. (A) The presence of CXCR5+PD-1+ cells in PBMCs of active SLE patients was detected by immunofluorescence microscopy. The arrow indicates the typical positive cells. (B) The results of flow cytometric analysis of absolute numbers of CD4+CXCR5+PD-1+ cells in patients with SLE (n = 30) and healthy controls (n = 15). https://doi.org/10.1371/journal.pone.0088441.s004 (TIF) Figure S5. Tfh cell-derived IL-21 promotes the differentiation of Breg cells. Sorted CD20+CD27− naïve B cells 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 among the sorted B cells were analyzed by flow cytometry. Results shown are representative of at least three independent experiments. https://doi.org/10.1371/journal.pone.0088441.s005 (TIF)

Acknowledgments We thank Prof. Xiao Kang Li, Prof. Liwei Lv and Song Guo Zheng for their precious suggestions and comments. We thank Huiming Ren, Weizhe Ma, Xiaoye Gu, Xiaoxia Zhu, Xue Xu, Minrui Liang, Haiyan Chen and Ning Kong for helpful discussions and experimental technique helps. We thank the patients, the healthy volunteer donors, and the doctors for their participation in this study.

Author Contributions Conceived and designed the experiments: XY HZ. Performed the experiments: XY JY HZ. Analyzed the data: XY JY YC YX DX SZ HZ. Contributed reagents/materials/analysis tools: XY JY YC YX DX SZ HZ. Wrote the paper: XY JY HZ.