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CD70-mediated CD27 expression downregulation contributed to the regulatory B10 cell impairment in rheumatoid arthritis
Molecular Immunology 119 (2020) 92–100
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CD70-mediated CD27 expression downregulation contributed to the regulatory B10 cell impairment in rheumatoid arthritis
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Lianjie Shia,b,c,1, Fanlei Hub,1, Lei Zhub, Chuanhui Xud, Huaqun Zhub, Yingni Lib, Hongjiang Liue, Chun Lib, Na Liub, Liling Xub, Rong Mub,*, Zhanguo Lib,* a
Department of Rheumatology and Immunology, Peking University International Hospital, Beijing, China Department of Rheumatology and Immunology, Peking University People’s Hospital & Beijing Key Laboratory for Rheumatism Mechanism and Immune Diagnosis (BZ0135), Beijing, China c State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, China d Department of Rheumatology, Allergy and Immunology, Tan Tock Seng Hospital, Singapore e The First People’s Hospital of Yichang, China Three Gorges University, Yichang, Hubei, China b
ARTICLE INFO
ABSTRACT
Keywords: Rheumatoid arthritis Regulatory CD70 CD27 interleukin-10
Regulatory B10 cells have been shown to exhibit impaired functions in autoimmune diseases. However, the underlying mechanism is still obscure. In the present study, we aimed to understand the regulatory characteristics of regulatory B10 cells and how these cells are involved in the development of rheumatoid arthritis (RA). Here, we chose CD19+CD24hiCD27+ as the phenotype of regulatory B10 cells. We found that the frequencies of CD19+CD24hiCD27+ regulatory B10 cells were decreased and that their IL-10-producing function was impaired in patients with RA compared with healthy controls (HCs). The impairment in CD19+CD24hiCD27+ B10 cells was partially attributed to the decreased expression of CD27 induced by the upregulated CD70 expression on CD19 + B cells and CD4 + T cells. The proportion of CD19+CD24hiCD27+ regulatory B10 cells could be restored by blocking the CD70-CD27 interaction with an anti-CD70 antibody. Furthermore, the CD70-CD27 interaction significantly elevated IL-10 expression and might compensate for the decreased number of CD19+CD24hiCD27+ B cells. Hence, the CD70-CD27 interaction might play a critical role in the numerical and functional impairments of regulatory B10 cells, thus contributing to RA pathogenesis. In conclusion, the change in CD19+CD24hiCD27+ regulatory B10 cells in RA was only a consequence, not the cause, of RA development, but the increased expression of CD70 might be the culprit.
1. Introduction
The IL-10-producing regulatory B cells, B10 cells, are widely studied and are broadly recognized in collagen-induced arthritis (CIA) in many studies, including studies on phenotype and function (Yanaba et al., 2008; Yang et al., 2012). Compelling evidence in mice suggests that regulatory B10 cells potentially prevent pathogenic autoimmunity predominantly via the suppressive cytokine IL-10 (Bouaziz et al., 2008). The absence of regulatory B10 cells exacerbates disease activity in CIA (Mauri et al., 2003). However, knowledge about the IL-10-producing regulatory B10 cell subset in humans is limited. IL-10 expression remains the best hallmark of regulatory B cells, namely, IL-10+ B cells. Very recently, studies have demonstrated that IL-10+ B cells in blood represented subsets of CD19+CD24hiCD27+ or CD19+CD24hiCD38hi subpopulations (Flores-Borja et al., 2013; Iwata et al., 2011). Both subsets contain a similar total number of IL-10-producing B cells.
Rheumatoid arthritis (RA) is a chronic, systemic inflammatory disease affecting approximately 0.2–1.0 % (Scott et al., 2010) of the population worldwide and 0.2-0.93 % of the population in China (Li et al., 2012; Xiang and Dai, 2009; Zeng et al., 2008). B cells play an important role in the development of RA through autoantibodies, such as rheumatoid factor (RF) and anti-cyclic-citrullinated peptide antibodies (ACPA). Recently, several studies have demonstrated that the role of B cells in RA extends beyond the production of antibodies (Blair et al., 2010; Iwata et al., 2011). A subset of B cells named regulatory B cells negatively regulates the cellular immune response via cytokines such as interleukin (IL)-10, IL-35, and transforming growth factor (TGF)-β (Rosser and Mauri, 2015; Tedder, 2015).
Corresponding authors at: Department of Rheumatology and Immunology, Peking University People’s Hospital & Beijing Key Laboratory for Rheumatism Mechanism and Immune Diagnosis (BZ0135), 11 South Xi Zhi Men Avenue, Xi Cheng District, Beijing, China. E-mail addresses: [email protected] (R. Mu), [email protected] (Z. Li). 1 These authors contributed equally to this work. ⁎
https://doi.org/10.1016/j.molimm.2020.01.016 Received 19 March 2019; Received in revised form 10 January 2020; Accepted 23 January 2020 0161-5890/ © 2020 Published by Elsevier Ltd.
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CD19+CD24hiCD27+ B cells include not only B10 cells but also B10pro cells. Much effort has been given to the study of CD19+CD24hiCD27+ regulatory B10 cells in autoimmune diseases (Iwata et al., 2011; Jin et al., 2013; Zha et al., 2012), but several questions remain unresolved, including whether the impaired CD19+CD24hiCD27+ B cells are a cause or just a consequence of RA development. In the present study, we validated that IL-10 is predominately expressed by the CD19+CD24hiCD27+ B cell subset of CD19+ B cells, which is consistent with a previous study (Iwata et al., 2011). Indeed, CD19+CD24hiCD38hi B cells were previously identified as immature transitional B cells with regulatory capacity via IL-10 secretion. However, CD19+CD24hiCD27+ B cells include not only IL-10-competent B10 cells but also B10pro cells (Iwata et al., 2011). Hence, we investigated CD19+CD24hiCD27+ B cells to delineate the pathogenic role of regulatory B10 cells.
and IL-1β levels were determined using ELISA (Neobioscience Technology Co, Ltd, Beijing, China). The ELISA procedure was performed according to the manufacturer’s instructions. 3.1. Real-time quantitative PCR (RT-qPCR) assays Total RNA was extracted from cells using an RNeasy mini kit (QIAGEN, USA). Reverse transcription was performed with a Revert Aid First Strand cDNA synthesis kit (Fermentas, Glen Burnie, MD, USA) according to the manufacturer’s instructions. The resulting cDNA was subjected to PCR and real-time PCR analyses. RT-qPCR was performed with SYBR Green Master mix (Applied Biosystems, Foster City, CA, USA) using primers for GAPDH, IL-10, IL-1β, TNF-α, CD27 and CD70. Primer sequences were as follows: GAPDH forward, 5′-AAGGTGAAGGTCGG AGTCAA-3′; GAPDH reverse, 5′-AATGAAGGGGTCATTGATGG-3′; IL-10 forward, 5′-CTTCGAGATC TCCGAGATGC CTTC-3′; IL-10 reverse, 5′-ATTCTTCACC TGCTCCACGG CCTT-3′; hCD27 forward, 5′-ACCCTCAGCCCACCCACTTA-3′; hCD27 reverse, 5′-CAGGGTGAAAACAAGGAACATT-3; hCD70 forward, 5′-TGCTTTGGTCCCATTGGTCG-3′; and hCD70 reverse, 5′-TCCTGCTGAGGTCCTGTGTGATTC-3′. For qPCR, gene expression was quantified relative to the expression of the housekeeping gene GAPDH, and normalized to control by standard 2−ΔΔCT calculation.
2. Materials and methods 2.1. Patients and healthy controls (HCs) A total of 120 RA patients (109 women and 11 men) were enrolled in this study, and the patients had a mean age of 46.6 years (ranging from 21 to 60 years). All the patients met the 1987 revised classification criteria of the American College of Rheumatology (ACR). All the patients manifested active disease, which was defined as a Disease Activity Score involving 28 joints with a C-reactive protein (DAS 28CRP) value greater than 3.2. Twenty RA patients received conventional synthetic disease-modifying anti-rheumatic drug (DMARD) treatment (including methotrexate, leflunomide or hydroxychloroquine) for at least 3 months, and they had no response to the treatment. Then another DMARD, iguratimod, was added and the patients were followd up for 24 weeks. Disease activity was evaluated before and after treatment. Seventy-three age-matched HCs (64 women and 9 men) were recruited. All the participants provided informed consent, and the study and consent forms were approved by the Institutional and Medical Ethics Review Board of Peking University People’s Hospital. All RA patients underwent clinical assessments at baseline, which consisted of determining the numbers of swollen joints (28 joints evaluated) and tender joints (28 joints evaluated). Laboratory assessments included complete blood counts, and RF, ACPA, and C-reactive protein (CRP) measurements.
3.2. Flow cytometry and cell sorting For B cell staining, 100 μl of whole blood from the RA patients and HCs was collected and stained with antibodies for the markers CD19 (APC-Cy7, Biolegend), CD24 (PE, eBioscience), and CD27 (APC, eBioscience). For each sample, isotype controls were also used. The cells were incubated with the antibodies at 4 °C for 30 min and then with 2 ml of BD FACS Lysing Solution (BD Biosciences) at room temperature for 10 min. Labelled cells were washed with PBS and analysed with BD FACS Aria II flow cytometers (BD Biosciences). For intracellular staining, PBMCs from healthy individuals or RA patients were stimulated with PIB (50 ng/ml PMA, 1000 ng/ml ionomycin, and 10 mg/ml Brefeldin A, Becton Dickinson, San Diego, CA, USA) for 5 h. Then the cells were stained with APC-CY7 conjugated anti-CD19, fixed and permeabilized, followed by intracellular staining using PE-conjugated anti-IL-10. Next, the cells were analysed with a FACS Arial II flow cytometer. Data were analysed using FlowJo software. To isolate B cell subsets, PBMCs were stained with anti-CD19-APC/ Cy7, CD24-PE, and CD27-APC, and then the targeted B cell subpopulations, including CD19+CD24hiCD27+ and CD19+CD24loCD27B cells were sorted by flow cytometry (> 99 % purity). These sorted cells were subsequently subjected to RT-PCR, RT-qPCR and ELISPOT.
2.2. Cell isolation and treatment Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Paque Plus gradient centrifugation, and 106 cells/mL were resuspended in RPMI 1640 medium (HyClone, Logan, UT, USA) supplemented with 10 % foetal bovine serum (FBS) and 1 % antibiotic and cultured in a 5 % CO2 incubator. A cytokine stimulation assay was conducted with 1 × 106 PBMCs per well in 6-well plates stimulated with tumour necrosis factor (TNF)-α (10 ng/mL), IL-1β (2 ng/mL), IL-6 (10 ng/mL), interferon (IFN)-γ (10 ng/mL), and IL-17 (50 ng/mL) for 72 h. The cells were analysed by flow cytometry to determine the number of CD19+CD24hiCD27+ cells after the treatment. During the study, PBMCs from healthy individuals were treated with anti-CD3 and anti-CD28 antibodies for 72 h with or without anti-CD70 antibody (1 μg/mL) pretreatment for 1 h. Subsequently, the cells and the supernatants were prepared for flow cytometry and enzyme-linked immunosorbent assay (ELISA) analysis as described.
3.3. Statistical analysis Statistical analyses were performed using SPSS 17.0. A two-tailed Student’s t test was used to analyse the differences between the RA patients and HCs. The correlations between clinical and laboratory parameters were evaluated by Spearman correlation coefficients. A paired t test was used to compare paired data before and after DMARD treatment. A P-value < 0.05 was considered statistically significant. 4. Results
3. ELISA
4.1. IL-10+ B cells were enriched in the CD19+CD24hiCD27+ regulatory B10 cell subset
Serum samples and cell culture supernatants were obtained and stored at -70 °C until analysis. The soluble CD27 level was determined by using the human sCD27 instant ELISA Kit (eBioscience, USA). TNF-α
To validate CD19+CD24hiCD27+ as the phenotype of IL-10+ B cells, we compared the expression of IL-10 in the CD19+CD24hiCD27+ and CD19+CD24loCD27− subsets by PCR, ELISPOT (data not shown 93
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Fig. 1. Frequencies of IL-10+ B cells and proportion of IL-10-competent CD19+CD24hiCD27+ regulatory B10 cells are decreased in rheumatoid arthritis. (A) Representative flow cytometry depicting the gating strategies for CD19+CD24hi CD27+ B cells and CD19+CD24loCD27− B cells. The bar chart shows the IL-10 transcript levels in sorted CD19+CD24hiCD27+ B cells and CD19+CD24loCD27− B cells quantified by qPCR, and the cumulative data is represented as the mean ± SEM of five independent experiments. (B) Flow cytometric analysis of the proportion of CD19+CD24hiCD27+ B cells in the peripheral blood of 50 RA patients and 30 healthy donors. Representative flow charts and statistical results are shown (independent samples t test). (C) Representative flow cytometry plots showing the proportion of CD19+IL-10+ B cells in a healthy donor and a patient with RA. The dot plot shows the cumulative results for the proportion (independent t test). (D) Bar graph showing decreased IL-10 mRNA expression in CD19+CD24hiCD27+ B cells isolated from patients with RA compared with those from healthy individuals. * P < 0.05, **P < 0.01. Data are representative of at least three independent experiments.
here) and real-time PCR, as in our previous study (Hu et al., 2017). The mRNA expression of IL-10 in the CD19+CD24hiCD27+ B cells was approximately ten-fold of that in the CD19+CD24loCD27− B cells from the HCs (Fig. 1A).
decreased in the RA patients (n = 50) compared with the HCs (n = 30, 14.19 ± 8.13 % vs. 31.79 ± 12.64 %, respectively, P < 0.01) (Fig. 1B). To confirm our findings, we used intracellular staining and flow cytometry analysis to determine the number of B10 cells (IL-10+ B cells) in another 9 healthy individuals and 8 RA patients. The IL-10+ B cells constituted 3.4 ± 0.75 % of the total number of B cells in the RA patients and 4.4 ± 0.7 % in the healthy individuals (P < 0.01) (Fig. 1C). We used RT-qPCR to detect the IL-10 expression of CD19+CD24hiCD27+ B cells from the RA patients and HCs. Interestingly, the CD19+CD24hiCD27+ B cells from the RA patients expressed less IL-10 than those from the healthy individuals (P = 0.02) (Fig. 1D).
4.2. Decreased proportion and impaired IL-10 production of CD19+ B cells and downregulated IL-10 expression of CD19+CD24hiCD27+ regulatory B10 cells in RA The proportion of CD19+CD24hiCD27+ regulatory B10 cells was 94
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4.3. The proportion of CD19+CD24hiCD27+ regulatory B10 cells was associated with treatment response, and a good response to TNF inhibitor treatment was observed, but was not correlated with disease activity in RA
in both the CD4+ T cells and CD19+ B cells from the RA patients compared with those from the healthy donors. The expression of CD70 on CD4+ T cells was approximately three-fold higher in the cells from the RA patients than in those from the HCs (Fig. 4A, B). CD27 can be expressed in a membrane-bound form and a soluble form. T cell activation leads to the shedding of CD27 from the cell surface, resulting in soluble CD27 (sCD27), which might signal via the CD27-CD70 pathway. We detected the sCD27 level in the serum from both the RA patients and HCs and showed that the level of sCD27 was higher in the RA patients (Fig. 4C). However, we cannot distinguish whether the sCD27 originated from T cells, B cells or another cell type. To further ascertain the proportion of sCD27 in the serum derived from B cells and the shedding of sCD27 attributed to the CD70-CD27 interaction, we cultured PBMCs from the HCs in vitro. The cells were divided into three groups and treated differently. An anti-CD70 antibody was used to block the CD70-CD27 interaction. Anti-CD3 and antiCD28 antibodies were used to stimulate T cell activity. We detected the expression of membrane-bound CD27 on both CD4+ T cells and CD19+ B cells as well as the expression of sCD27 in the cell supernatant and found that CD27 expression decreased significantly on CD19+ B cells, while the sCD27 and IL-10 levels were increased in the cell culture supernatants (the latter data are not shown). However, the higher membrane-bound CD27 level and lower sCD27 level in the supernatants were found after B cells were pre-treated with the anti-CD70 antibody (Fig. 4D–G). These results suggest that the upregulation of CD70 expression might decrease the proportion of CD19+CD24hiCD27+ regulatory B10 cells in RA via the CD70-CD27 interaction.
To elucidate the association of CD19+CD24hiCD27+ regulatory B10 cells with disease activity, we assessed the proportion of CD19+CD24hiCD27+ regulatory B10 cells in 20 RA patients with high disease activity (DAS 28-CRP > 5.1) and followed these patients for 24 weeks. Ten of the 20 patients showed satisfactory responses to conventional synthetic DMARD treatment, resulting in disease remission (2.6 < DAS 28-CRP < 3.2). The proportion of CD19+CD24hiCD27+ regulatory B10 cells in the peripheral blood was elevated to a mean level of 18 % in most patients at week 24, suggesting an association between the regulatory B10 cell level and RA pathophysiology (Fig. 2A). We evaluated the CD19+CD24hiCD27+ regulatory B10 cell subset in another 10 patients before and after treatment with TNF inhibitors (TNFis). All patients achieved low disease activity or remission. Interestingly, we found that the proportion of CD19+CD24hiCD27+ regulatory B10 cells in the TNFi treatment group was higher than that in in the csDMARD-treated group and comparable to that in the HC group (Fig. 2B). CD19+CD24hiCD27+ regulatory B10 cells were restored after TNFi treatment, suggesting that TNF-α might be involved in the mechanism that leads to the decreased number and impaired function of CD19+CD24hiCD27+ regulatory B10 cells in RA. We also performed correlation analysis between the proportion of CD19+CD24hiCD27+ regulatory B10 cells and clinical characteristics of 27 RA patients with complete clinical information. Interestingly, no correlations were found between DAS28-CRP, RF, and ACPA and the proportion of CD19+CD24hiCD27+ B cells. However, the absolute number of CD19+CD24hiCD27+ B cells was correlated with the titre of ACPA (Fig. 2C).
4.7. TNF-α and IL-1β decreased the proportion of CD19+CD24hiCD27+ B cells likely by upregulating CD70 expression in PBMCs PBMCs derived from healthy donors were cultured and treated with TNF-α or IL-1β. CD70 expression was analysed by flow cytometry and PCR. We found that both TNF-α and IL-1β upregulated CD70 expression on CD4+ T cells and CD19+ B cells (Fig. 5A, B), and these results were consistent with the decrease in the proportion of CD19+CD24hiCD27+ regulatory B10 cells after TNF-α and IL-1β stimulation but not IL-6, IFN-γ or IL-17 stimulation (Fig. 5C).
4.4. TNF-α and IL-1β decreased the proportion of CD19+CD24hiCD27+ regulatory B10 cells To determine the role of TNF-α in the decreased proportion of CD19+CD24hiCD27+ regulatory B10 cells, PBMCs from the RA patients were either left untreated or treated with TNF-α, IL-1β, IL-6, IFN-γ and IL-17. The results demonstrated that TNF-α and IL-1β significantly decreased the proportion of CD19+CD24hiCD27+ cells in vitro, but no differences were observed in the IL-6, IFN-γ and IL-17 groups (Fig. 2D, 2E). These findings suggested that TNF-α and IL-1β mediated the decrease in the CD19+CD24hiCD27+ B cell subset.
5. Discussion The present study demonstrates decreases in both the proportion and IL-10 expression of CD19+CD24hiCD27+ regulatory B10 cells in RA patients, which may contribute to the immunologic abnormalities of this disease. The mechanism of the downregulation of this regulatory B cell subset could be the shedding of sCD27 from the cell surface through the CD70-CD27 interaction mediated by the high CD70 expression induced by proinflammatory cytokines, notably TNF-α and IL-1β. An antiCD70 antibody could restore the proportion of CD19+CD24hiCD27+ regulatory B10 cells and thus might be a potential therapeutic target for RA treatment. Our results showed that the proportion of CD19+CD24hiCD27+ regulatory B10 cells was decreased in RA, which is consistent with results for CD19+IL-10+ B cells. This finding confirms that the proportion of regulatory B cells is decreased in RA. We also found that CD19+CD24hiCD27+ regulatory B10 cells derived from RA patients exhibit impaired IL-10 expression. Our findings were not consistent with the reported elevated serum IL-10 levels in RA and the findings reported in previous studies (Cush et al., 1995; Llorente et al., 1994), probably because the elevated serum IL-10 levels in RA compensate for the numerical deficit and impaired IL-10 secretion observed by RTqPCR. Another highlight of this study is the remarkable recovery of CD19+CD24hiCD27+ regulatory B10 cell frequencies after TNFi treatment in RA, which prompted us to explore the mechanisms underlying the changes in this B cell subset in RA. TNF-α plays a key role in the
4.5. The decreased expression of CD27might be responsible for the decreased proportion of CD19+CD24hiCD27+ regulatory B10 cells No difference was found in the proportion of CD19+ B cells (data not shown) or the CD24 expression on CD19+ B cells (Fig. 3A) between the HCs and RA patients. However, the RA patients showed remarkably lower CD27 expression, which was reduced by 24 % (Fig. 3B). This result suggests that the decreasing proportion of the CD19+CD24hiCD27+ regulatory B10 cells might be due to the downregulation of CD27 expression on the cell surface. We also detected the relative CD27 expression in CD19+CD27+ B cells by RT-qPCR and the median fluorescence intensity (MFI) of CD24 and CD27 in CD19+CD24hiCD27+ regulatory B10 cells by flow cytometry and confirmed that the CD27 expression in these subsets was lower in the RA patients than the HCs (Fig. 3C, D). 4.6. Upregulated CD70 expression may decrease the proportion of CD19+CD24hiCD27+ regulatory B10 cells in RA We analysed the expression of CD70, the ligand for CD27, on CD4+ T and CD19+ B cells. We found that CD70 expression was upregulated 95
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Fig. 2. The proportion of CD19+CD24hi CD27+ regulatory B10 cells is associated with the treatment, especially for TNFi treatment. (A) The graph shows the variation in the proportion of CD19+CD24hi CD27+ regulatory B10 cells in patients who had a good response to treatment (paired t test). (B) The decreased proportion of CD19+CD24hiCD27+ regulatory B10 cells in patients recovered after TNFi treatment (independent t test). (C) The correlation of CD19+CD24hiCD27+ regulatory B10 cells and clinical characteristic. (D) Peripheral blood mononuclear cells (PBMCs) from healthy individuals (n = 3) were stimulated with TNF-α, IL-1β, IL-6, IFN-γ and IL-17 for 72 h. The proportion of CD19+CD24hiCD27+ regulatory B10 cells was analysed by flow cytometry. The bar chart shows the variation of CD19+CD24hiCD27+ regulatory B10 cells after stimulation. (E) Representative flow cytometry for one healthy individual shows the proportion of CD19+CD24hi CD27+ regulatory B10 cells after TNF-α and IL-1β stimulation. * P < 0.05, **P < 0.01.
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Fig. 3. The decreased proportion of CD19+CD24hiCD27+ regulatory B10 cells in RA is attributed to the decreased expression of CD27. (A) Representative flow cytometry plots for one healthy individual and one RA patient show the proportion of CD24+ B cells in the CD19+ B cell population. (B) Representative flow cytometry plots show the proportion of CD27+ cells in the CD19+ B cell population. (C) CD19+CD24hiCD27+ regulatory B10 cells from 5 healthy donors and 10 patients with RA were sorted by flow cytometry and subjected to real-time PCR analysis of CD27 expression. The relative CD27 expression is shown. (D) The bar graph shows the cumulative data for the CD27 median fluorescence intensity (MFI) in CD19+CD24hiCD27+ regulatory B10 cells from 29 healthy individuals and 41 RA patients, as measured by flow cytometry. The results are presented as the mean ± SD.
CD19+ B cells and found that the CD19+CD24hiCD27+ regulatory B10 cells from the RA patients showed a lower CD27 MFI than those from the HCs. We found that CD70 expression was upregulated on both CD4 + T cells and CD19 + B cells and accompanied by decreased CD19+CD24hiCD27+ regulatory B10 cell numbers and increased sCD27 and total IL-10 levels in the supernatants. Generally, CD27, a member of the tumour necrosis factor receptor (TNFR) superfamily, is a wellcharacterized marker of memory B cells (Agematsu, 2000; Agematsu et al., 1997). The interaction of CD27 with its unique ligand CD70 has been reported to release a truncated form of CD27 (Loenen et al., 1992; Prasad et al., 1997). We hypothesize that the decreased CD27 expression on B cells might be attributed to CD70-CD27 interactions, as CD70 expression is upregulated by TNF-α and IL-1β. However, the CD27
pathogenesis of RA (McInnes and Schett, 2011). Thus, we assume that the changes involving CD19+CD24hiCD27+ regulatory B10 cells in RA are attributed to TNF-α. Consistent with our hypothesis, the proportion of CD19+CD24hiCD27+ regulatory B10 cells was remarkably decreased by TNF-α and IL-1β but not by IL-6, IL-8, IL-17 or IFN-γ. Nevertheless, the roles of TNF-α in the proportion and IL-10-secreting functions of CD19+CD24hiCD27+ regulatory B10 cells remain to be elucidated. We analysed the expression of CD24 and CD27 by the B cells. In the RA patients, the expression of CD27 on CD19+ B cells was downregulated. However, no differences in CD24 expression on CD19+ B cells were found between the RA patients and HCs. Our findings suggested that TNF-α might decrease the proportion of CD19+CD24hiCD27+ regulatory B10 cells by lowering the expression of CD27. Furthermore, we compared the MFI of CD24 and CD27 on 97
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Fig. 4. CD70 expression is upregulated in lymphocytes in RA patients, and the upregulated CD70 expression could decrease the proportion of CD19+CD24hiCD27+ regulatory B10 cells in vitro. PBMCs from healthy individuals were cultured with anti-CD3 and anti-CD28 antibodies for 72 h with or without anti-CD70 antibody (1 μg/mL) pretreatment for 1 h. (A, B) Representative flow cytometry plots for one healthy individual and one RA patient show the proportion of CD70+ B cells in the CD19+ B or CD4+ T cell population. (C) Serum was collected from 12 healthy controls and 20 patients with RA, and the soluble CD27 level was detected by ELISA. (D) Representative flow cytometry plots for one healthy individual show the proportion of CD70+ T cells in the CD4+ T cell population. The bar chart shows the cumulative data for the proportion of CD4+CD70+ T cells blocked or not blocked by the anti-CD70 antibody. (E) Representative flow cytometry plots for one healthy individual show the proportion of CD70+ B cells in the CD19+ B cell population. The bar graph shows the cumulative data for the CD19+CD70+ B cell proportion in 3 healthy individuals treated or not treated with the antiCD70 antibody. (F) Representative contour plots and the cumulative data for three healthy individuals show the proportion of CD27+ B cells in the CD19+ B cell population before and after anti-CD70 antibody treatment. (G) The supernatants were collected, and soluble CD27 expression was assessed by ELISA. The bar chart shows the cumulative data for three independent experiments. The data are presented as the mean ± SD. * P < 0.05, **P < 0.01.
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Fig. 5. TNF-α increases CD70 expression on lymphocytes in vitro. (A, B) Representative flow cytometry plots for one healthy individual show the proportion of CD4+CD70 + T cells or CD19+CD70 + B cells after treatment with TNF-α or IL-1β. The graph shows the cumulative data for the proportion of CD4+CD70 + T cells or CD19+CD70 + B cells in different groups. (C) The bar chart shows the cumulative data as the mean ± SD of three independent experiments. * P < 0.05, **P < 0.01.
expression on CD19+ B cells was generally persistent, and the level of sCD27 in the supernatants was decreased following pretreatment with the anti-CD70 antibody (to block the CD70-CD27 interactions). We also assessed CD70 mRNA expression in PBMCs stimulated with different cytokines, and the result was consistent with the upregulation of CD70 expression by TNF-α and IL-1β. In addition, flow cytometry results showed that TNF-α and IL-1β slightly upregulated CD70 expression on both CD4+ T cells and CD19+ B cells. Previous studies demonstrated that TNF-α and IL-1α enhanced CD70 expression on human lymphocytes (Lens et al., 1997; Tesselaar et al., 2003). Our results suggested that TNF-α decreased CD27 expression on B cells likely via the upregulation of CD70 expression. The upregulated CD70 expression on CD4+ T cells has been reported in a previous study (Lee et al., 2007). In another study, blocking CD27-CD70 interactions with an anti-CD70 antibody ameliorated joint
disease in murine CIA (Oflazoglu et al., 2009). The protection of regulatory B cells conferred by blocking CD70-CD27 interactions might help to explain the amelioration of joint disease in CIA with an antiCD70 antibody. Recently, Park et al. (Park et al., 2014) also found that CD70-expressing CD4+ T cells produce IFN-γ and IL-17 in RA, which indicated that CD70 was involved in RA pathogenesis. Consistent with previous studies, our study demonstrated that CD70 might mediate RA pathogenesis. Our results further showed that in addition to the CD70 expression on CD4+ T cells, CD70 expression on CD19+ B cells was also upregulated in RA, resulting in elevated serum levels of sCD27 and a decreased proportion of CD19+CD24hiCD27+ regulatory B10 cells in the PBMC population. These results also agreed with those of our in vitro study. Our present study has several limitations. First, we did not perform functional studies of CD19+CD24hiCD27+ regulatory B10 cells and 99
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only assessed these cells by evaluating IL-10 expression because a functional study of this subset was recently published by our team (Hu et al., 2017). Secondly, we only detected CD70 expression on CD4+ T and CD19+ B cells, we did not include CD8+ T cells. In conclusion, the current findings demonstrate that the decreased proportion and IL-10 expression of impaired CD19+CD24hiCD27+ regulatory B10 cells in RA might be attributed to the upregulated CD70 expression on CD4+ T and CD19+ B lymphocytes. Our data might provide a molecular basis to support the rationale for anti-CD70 therapy for RA.
Hu, F., Liu, H., Liu, X., Zhang, X., Xu, L., Zhu, H., Li, Y., Shi, L., Ren, L., Zhang, J., Li, Z., Jia, Y., 2017. Pathogenic conversion of regulatory B10 cells into osteoclast-priming cells in rheumatoid arthritis. J. Autoimmun. 76, 53–62. Iwata, Y., Matsushita, T., Horikawa, M., Dilillo, D.J., Yanaba, K., Venturi, G.M., Szabolcs, P.M., Bernstein, S.H., Magro, C.M., Williams, A.D., Hall, R.P., St Clair, E.W., Tedder, T.F., 2011. Characterization of a rare IL-10-competent B-cell subset in humans that parallels mouse regulatory B10 cells. Blood 117, 530–541. Jin, L., Weiqian, C., Lihuan, Y., 2013. Peripheral CD24hi CD27+ CD19+ B cells subset as a potential biomarker in naive systemic lupus erythematosus. Int. J. Rheum. Dis. 16, 698–708. Lee, W.W., Yang, Z.Z., Li, G., Weyand, C.M., Goronzy, J.J., 2007. Unchecked CD70 expression on T cells lowers threshold for T cell activation in rheumatoid arthritis. J. Immunol. 179, 2609–2615. Lens, S.M., Baars, P.A., Hooibrink, B., van Oers, M.H., van Lier, R.A., 1997. Antigenpresenting cell-derived signals determine expression levels of CD70 on primed T cells. Immunology 90, 38–45. Li, R., Sun, J., Ren, L.M., Wang, H.Y., Liu, W.H., Zhang, X.W., Chen, S., Mu, R., He, J., Zhao, Y., Long, L., Liu, Y.Y., Liu, X., Lu, X.L., Li, Y.H., Wang, S.Y., Pan, S.S., Li, C., Li, Z.G., 2012. Epidemiology of eight common rheumatic diseases in China: a large-scale cross-sectional survey in Beijing. Rheumatology Oxford (Oxford) 51, 721–729. Llorente, L., Richaud-Patin, Y., Fior, R., Alcocer-Varela, J., Wijdenes, J., Fourrier, B.M., Galanaud, P., Emilie, D., 1994. In vivo production of interleukin-10 by non-T cells in rheumatoid arthritis, Sjogren’s syndrome, and systemic lupus erythematosus. A potential mechanism of B lymphocyte hyperactivity and autoimmunity. Arthritis Rheum. 37, 1647–1655. Loenen, W.A., De Vries, E., Gravestein, L.A., Hintzen, R.Q., Van Lier, R.A., Borst, J., 1992. The CD27 membrane receptor, a lymphocyte-specific member of the nerve growth factor receptor family, gives rise to a soluble form by protein processing that does not involve receptor endocytosis. Eur. J. Immunol. 22, 447–455. Mauri, C., Gray, D., Mushtaq, N., Londei, M., 2003. Prevention of arthritis by interleukin 10-producing B cells. J. Exp. Med. 197, 489–501. McInnes, I.B., Schett, G., 2011. The pathogenesis of rheumatoid arthritis. N. Engl. J. Med. 365, 2205–2219. Oflazoglu, E., Boursalian, T.E., Zeng, W., Edwards, A.C., Duniho, S., McEarchern, J.A., Law, C.L., Gerber, H.P., Grewal, I.S., 2009. Blocking of CD27-CD70 pathway by antiCD70 antibody ameliorates joint disease in murine collagen-induced arthritis. J. Immunol. 183, 3770–3777. Park, J.K., Han, B.K., Park, J.A., Woo, Y.J., Kim, S.Y., Lee, E.Y., Lee, E.B., Chalan, P., Boots, A.M., Song, Y.W., 2014. CD70-expressing CD4 T cells produce IFN-gamma and IL-17 in rheumatoid arthritis. Rheumatology 53, 1896–1900. Prasad, K.V., Ao, Z., Yoon, Y., Wu, M.X., Rizk, M., Jacquot, S., Schlossman, S.F., 1997. CD27, a member of the tumor necrosis factor receptor family, induces apoptosis and binds to Siva, a proapoptotic protein. Proc Natl Acad Sci U S A 94, 6346–6351. Rosser, E.C., Mauri, C., 2015. Regulatory B cells: origin, phenotype, and function. Immunity 42, 607–612. Scott, D.L., Wolfe, F., Huizinga, T.W., 2010. Rheumatoid arthritis. Lancet 376, 1094–1108. Tedder, T.F., 2015. B10 cells: a functionally defined regulatory B cell subset. J. Immunol. 194, 1395–1401. Tesselaar, K., Xiao, Y., Arens, R., van Schijndel, G.M., Schuurhuis, D.H., Mebius, R.E., Borst, J., van Lier, R.A., 2003. Expression of the murine CD27 ligand CD70 in vitro and in vivo. J. Immunol. 170, 33–40. Xiang, Y.J., Dai, S.M., 2009. Prevalence of rheumatic diseases and disability in China. Rheumatol. Int. 29, 481–490. Yanaba, K., Bouaziz, J.D., Haas, K.M., Poe, J.C., Fujimoto, M., Tedder, T.F., 2008. A regulatory B cell subset with a unique CD1dhiCD5+ phenotype controls T cell-dependent inflammatory responses. Immunity 28, 639–650. Yang, M., Deng, J., Liu, Y., Ko, K.H., Wang, X., Jiao, Z., Wang, S., Hua, Z., Sun, L., Srivastava, G., Lau, C.S., Cao, X., Lu, L., 2012. IL-10-producing regulatory B10 cells ameliorate collagen-induced arthritis via suppressing Th17 cell generation. Am. J. Pathol. 180, 2375–2385. Zeng, Q.Y., Chen, R., Darmawan, J., Xiao, Z.Y., Chen, S.B., Wigley, R., Le Chen, S., Zhang, N.Z., 2008. Rheumatic diseases in China. Arthritis Res. Ther. 10, R17. Zha, B., Wang, L., Liu, X., Liu, J., Chen, Z., Xu, J., Sheng, L., Li, Y., Chu, Y., 2012. Decrease in proportion of CD19+ CD24(hi) CD27+ B cells and impairment of their suppressive function in Graves’ disease. PLoS One 7, e49835.
Ethics Statement This study was carried out in accordance with the recommendations of the “Institutional Medical Ethics Review Board of Peking University People’s Hospital” with written informed consent from all subjects. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by the “Institutional Medical Ethics Review Board of Peking University People’s Hospital.” Funding This work was supported by grants from the National Natural Science Foundation of China (81501396 to Dr. Lianjie Shi, 81771706 to Dr. Rong Mu, 81701614 to Dr. Hongjiang Liu, and 81671604 to Dr. Fanlei Hu), China Postdoctoral Science Foundation (2016 M600874), and Peking University International Hospital Research Funds (YN2017QX01 and YN2016QN01). Declaration of Competing Interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. References Agematsu, K., 2000. Memory B cells and CD27. Histol. Histopathol. 15, 573–576. Agematsu, K., Nagumo, H., Yang, F.C., Nakazawa, T., Fukushima, K., Ito, S., Sugita, K., Mori, T., Kobata, T., Morimoto, C., Komiyama, A., 1997. B cell subpopulations separated by CD27 and crucial collaboration of CD27+ B cells and helper T cells in immunoglobulin production. Eur. J. Immunol. 27, 2073–2079. Blair, P.A., Norena, L.Y., Flores-Borja, F., Rawlings, D.J., Isenberg, D.A., Ehrenstein, M.R., Mauri, C., 2010. CD19(+)CD24(hi)CD38(hi) B cells exhibit regulatory capacity in healthy individuals but are functionally impaired in systemic Lupus Erythematosus patients. Immunity 32, 129–140. Bouaziz, J.D., Yanaba, K., Tedder, T.F., 2008. Regulatory B cells as inhibitors of immune responses and inflammation. Immunol. Rev. 224, 201–214. Cush, J.J., Splawski, J.B., Thomas, R., McFarlin, J.E., Schulze-Koops, H., Davis, L.S., Fujita, K., Lipsky, P.E., 1995. Elevated interleukin-10 levels in patients with rheumatoid arthritis. Arthritis Rheum. 38, 96–104. Flores-Borja, F., Bosma, A., Ng, D., Reddy, V., Ehrenstein, M.R., Isenberg, D.A., Mauri, C., 2013. CD19+CD24hiCD38hi B cells maintain regulatory T cells while limiting TH1 and TH17 differentiation. Sci. Transl. Med. 5, 173ra123.
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CD70-mediated CD27 expression downregulation contributed to the regulatory B10 cell impairment in rheumatoid arthritis
T
Lianjie Shia,b,c,1, Fanlei Hub,1, Lei Zhub, Chuanhui Xud, Huaqun Zhub, Yingni Lib, Hongjiang Liue, Chun Lib, Na Liub, Liling Xub, Rong Mub,*, Zhanguo Lib,* a
Department of Rheumatology and Immunology, Peking University International Hospital, Beijing, China Department of Rheumatology and Immunology, Peking University People’s Hospital & Beijing Key Laboratory for Rheumatism Mechanism and Immune Diagnosis (BZ0135), Beijing, China c State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, China d Department of Rheumatology, Allergy and Immunology, Tan Tock Seng Hospital, Singapore e The First People’s Hospital of Yichang, China Three Gorges University, Yichang, Hubei, China b
ARTICLE INFO
ABSTRACT
Keywords: Rheumatoid arthritis Regulatory CD70 CD27 interleukin-10
Regulatory B10 cells have been shown to exhibit impaired functions in autoimmune diseases. However, the underlying mechanism is still obscure. In the present study, we aimed to understand the regulatory characteristics of regulatory B10 cells and how these cells are involved in the development of rheumatoid arthritis (RA). Here, we chose CD19+CD24hiCD27+ as the phenotype of regulatory B10 cells. We found that the frequencies of CD19+CD24hiCD27+ regulatory B10 cells were decreased and that their IL-10-producing function was impaired in patients with RA compared with healthy controls (HCs). The impairment in CD19+CD24hiCD27+ B10 cells was partially attributed to the decreased expression of CD27 induced by the upregulated CD70 expression on CD19 + B cells and CD4 + T cells. The proportion of CD19+CD24hiCD27+ regulatory B10 cells could be restored by blocking the CD70-CD27 interaction with an anti-CD70 antibody. Furthermore, the CD70-CD27 interaction significantly elevated IL-10 expression and might compensate for the decreased number of CD19+CD24hiCD27+ B cells. Hence, the CD70-CD27 interaction might play a critical role in the numerical and functional impairments of regulatory B10 cells, thus contributing to RA pathogenesis. In conclusion, the change in CD19+CD24hiCD27+ regulatory B10 cells in RA was only a consequence, not the cause, of RA development, but the increased expression of CD70 might be the culprit.
1. Introduction
The IL-10-producing regulatory B cells, B10 cells, are widely studied and are broadly recognized in collagen-induced arthritis (CIA) in many studies, including studies on phenotype and function (Yanaba et al., 2008; Yang et al., 2012). Compelling evidence in mice suggests that regulatory B10 cells potentially prevent pathogenic autoimmunity predominantly via the suppressive cytokine IL-10 (Bouaziz et al., 2008). The absence of regulatory B10 cells exacerbates disease activity in CIA (Mauri et al., 2003). However, knowledge about the IL-10-producing regulatory B10 cell subset in humans is limited. IL-10 expression remains the best hallmark of regulatory B cells, namely, IL-10+ B cells. Very recently, studies have demonstrated that IL-10+ B cells in blood represented subsets of CD19+CD24hiCD27+ or CD19+CD24hiCD38hi subpopulations (Flores-Borja et al., 2013; Iwata et al., 2011). Both subsets contain a similar total number of IL-10-producing B cells.
Rheumatoid arthritis (RA) is a chronic, systemic inflammatory disease affecting approximately 0.2–1.0 % (Scott et al., 2010) of the population worldwide and 0.2-0.93 % of the population in China (Li et al., 2012; Xiang and Dai, 2009; Zeng et al., 2008). B cells play an important role in the development of RA through autoantibodies, such as rheumatoid factor (RF) and anti-cyclic-citrullinated peptide antibodies (ACPA). Recently, several studies have demonstrated that the role of B cells in RA extends beyond the production of antibodies (Blair et al., 2010; Iwata et al., 2011). A subset of B cells named regulatory B cells negatively regulates the cellular immune response via cytokines such as interleukin (IL)-10, IL-35, and transforming growth factor (TGF)-β (Rosser and Mauri, 2015; Tedder, 2015).
Corresponding authors at: Department of Rheumatology and Immunology, Peking University People’s Hospital & Beijing Key Laboratory for Rheumatism Mechanism and Immune Diagnosis (BZ0135), 11 South Xi Zhi Men Avenue, Xi Cheng District, Beijing, China. E-mail addresses: [email protected] (R. Mu), [email protected] (Z. Li). 1 These authors contributed equally to this work. ⁎
https://doi.org/10.1016/j.molimm.2020.01.016 Received 19 March 2019; Received in revised form 10 January 2020; Accepted 23 January 2020 0161-5890/ © 2020 Published by Elsevier Ltd.
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CD19+CD24hiCD27+ B cells include not only B10 cells but also B10pro cells. Much effort has been given to the study of CD19+CD24hiCD27+ regulatory B10 cells in autoimmune diseases (Iwata et al., 2011; Jin et al., 2013; Zha et al., 2012), but several questions remain unresolved, including whether the impaired CD19+CD24hiCD27+ B cells are a cause or just a consequence of RA development. In the present study, we validated that IL-10 is predominately expressed by the CD19+CD24hiCD27+ B cell subset of CD19+ B cells, which is consistent with a previous study (Iwata et al., 2011). Indeed, CD19+CD24hiCD38hi B cells were previously identified as immature transitional B cells with regulatory capacity via IL-10 secretion. However, CD19+CD24hiCD27+ B cells include not only IL-10-competent B10 cells but also B10pro cells (Iwata et al., 2011). Hence, we investigated CD19+CD24hiCD27+ B cells to delineate the pathogenic role of regulatory B10 cells.
and IL-1β levels were determined using ELISA (Neobioscience Technology Co, Ltd, Beijing, China). The ELISA procedure was performed according to the manufacturer’s instructions. 3.1. Real-time quantitative PCR (RT-qPCR) assays Total RNA was extracted from cells using an RNeasy mini kit (QIAGEN, USA). Reverse transcription was performed with a Revert Aid First Strand cDNA synthesis kit (Fermentas, Glen Burnie, MD, USA) according to the manufacturer’s instructions. The resulting cDNA was subjected to PCR and real-time PCR analyses. RT-qPCR was performed with SYBR Green Master mix (Applied Biosystems, Foster City, CA, USA) using primers for GAPDH, IL-10, IL-1β, TNF-α, CD27 and CD70. Primer sequences were as follows: GAPDH forward, 5′-AAGGTGAAGGTCGG AGTCAA-3′; GAPDH reverse, 5′-AATGAAGGGGTCATTGATGG-3′; IL-10 forward, 5′-CTTCGAGATC TCCGAGATGC CTTC-3′; IL-10 reverse, 5′-ATTCTTCACC TGCTCCACGG CCTT-3′; hCD27 forward, 5′-ACCCTCAGCCCACCCACTTA-3′; hCD27 reverse, 5′-CAGGGTGAAAACAAGGAACATT-3; hCD70 forward, 5′-TGCTTTGGTCCCATTGGTCG-3′; and hCD70 reverse, 5′-TCCTGCTGAGGTCCTGTGTGATTC-3′. For qPCR, gene expression was quantified relative to the expression of the housekeeping gene GAPDH, and normalized to control by standard 2−ΔΔCT calculation.
2. Materials and methods 2.1. Patients and healthy controls (HCs) A total of 120 RA patients (109 women and 11 men) were enrolled in this study, and the patients had a mean age of 46.6 years (ranging from 21 to 60 years). All the patients met the 1987 revised classification criteria of the American College of Rheumatology (ACR). All the patients manifested active disease, which was defined as a Disease Activity Score involving 28 joints with a C-reactive protein (DAS 28CRP) value greater than 3.2. Twenty RA patients received conventional synthetic disease-modifying anti-rheumatic drug (DMARD) treatment (including methotrexate, leflunomide or hydroxychloroquine) for at least 3 months, and they had no response to the treatment. Then another DMARD, iguratimod, was added and the patients were followd up for 24 weeks. Disease activity was evaluated before and after treatment. Seventy-three age-matched HCs (64 women and 9 men) were recruited. All the participants provided informed consent, and the study and consent forms were approved by the Institutional and Medical Ethics Review Board of Peking University People’s Hospital. All RA patients underwent clinical assessments at baseline, which consisted of determining the numbers of swollen joints (28 joints evaluated) and tender joints (28 joints evaluated). Laboratory assessments included complete blood counts, and RF, ACPA, and C-reactive protein (CRP) measurements.
3.2. Flow cytometry and cell sorting For B cell staining, 100 μl of whole blood from the RA patients and HCs was collected and stained with antibodies for the markers CD19 (APC-Cy7, Biolegend), CD24 (PE, eBioscience), and CD27 (APC, eBioscience). For each sample, isotype controls were also used. The cells were incubated with the antibodies at 4 °C for 30 min and then with 2 ml of BD FACS Lysing Solution (BD Biosciences) at room temperature for 10 min. Labelled cells were washed with PBS and analysed with BD FACS Aria II flow cytometers (BD Biosciences). For intracellular staining, PBMCs from healthy individuals or RA patients were stimulated with PIB (50 ng/ml PMA, 1000 ng/ml ionomycin, and 10 mg/ml Brefeldin A, Becton Dickinson, San Diego, CA, USA) for 5 h. Then the cells were stained with APC-CY7 conjugated anti-CD19, fixed and permeabilized, followed by intracellular staining using PE-conjugated anti-IL-10. Next, the cells were analysed with a FACS Arial II flow cytometer. Data were analysed using FlowJo software. To isolate B cell subsets, PBMCs were stained with anti-CD19-APC/ Cy7, CD24-PE, and CD27-APC, and then the targeted B cell subpopulations, including CD19+CD24hiCD27+ and CD19+CD24loCD27B cells were sorted by flow cytometry (> 99 % purity). These sorted cells were subsequently subjected to RT-PCR, RT-qPCR and ELISPOT.
2.2. Cell isolation and treatment Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Paque Plus gradient centrifugation, and 106 cells/mL were resuspended in RPMI 1640 medium (HyClone, Logan, UT, USA) supplemented with 10 % foetal bovine serum (FBS) and 1 % antibiotic and cultured in a 5 % CO2 incubator. A cytokine stimulation assay was conducted with 1 × 106 PBMCs per well in 6-well plates stimulated with tumour necrosis factor (TNF)-α (10 ng/mL), IL-1β (2 ng/mL), IL-6 (10 ng/mL), interferon (IFN)-γ (10 ng/mL), and IL-17 (50 ng/mL) for 72 h. The cells were analysed by flow cytometry to determine the number of CD19+CD24hiCD27+ cells after the treatment. During the study, PBMCs from healthy individuals were treated with anti-CD3 and anti-CD28 antibodies for 72 h with or without anti-CD70 antibody (1 μg/mL) pretreatment for 1 h. Subsequently, the cells and the supernatants were prepared for flow cytometry and enzyme-linked immunosorbent assay (ELISA) analysis as described.
3.3. Statistical analysis Statistical analyses were performed using SPSS 17.0. A two-tailed Student’s t test was used to analyse the differences between the RA patients and HCs. The correlations between clinical and laboratory parameters were evaluated by Spearman correlation coefficients. A paired t test was used to compare paired data before and after DMARD treatment. A P-value < 0.05 was considered statistically significant. 4. Results
3. ELISA
4.1. IL-10+ B cells were enriched in the CD19+CD24hiCD27+ regulatory B10 cell subset
Serum samples and cell culture supernatants were obtained and stored at -70 °C until analysis. The soluble CD27 level was determined by using the human sCD27 instant ELISA Kit (eBioscience, USA). TNF-α
To validate CD19+CD24hiCD27+ as the phenotype of IL-10+ B cells, we compared the expression of IL-10 in the CD19+CD24hiCD27+ and CD19+CD24loCD27− subsets by PCR, ELISPOT (data not shown 93
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Fig. 1. Frequencies of IL-10+ B cells and proportion of IL-10-competent CD19+CD24hiCD27+ regulatory B10 cells are decreased in rheumatoid arthritis. (A) Representative flow cytometry depicting the gating strategies for CD19+CD24hi CD27+ B cells and CD19+CD24loCD27− B cells. The bar chart shows the IL-10 transcript levels in sorted CD19+CD24hiCD27+ B cells and CD19+CD24loCD27− B cells quantified by qPCR, and the cumulative data is represented as the mean ± SEM of five independent experiments. (B) Flow cytometric analysis of the proportion of CD19+CD24hiCD27+ B cells in the peripheral blood of 50 RA patients and 30 healthy donors. Representative flow charts and statistical results are shown (independent samples t test). (C) Representative flow cytometry plots showing the proportion of CD19+IL-10+ B cells in a healthy donor and a patient with RA. The dot plot shows the cumulative results for the proportion (independent t test). (D) Bar graph showing decreased IL-10 mRNA expression in CD19+CD24hiCD27+ B cells isolated from patients with RA compared with those from healthy individuals. * P < 0.05, **P < 0.01. Data are representative of at least three independent experiments.
here) and real-time PCR, as in our previous study (Hu et al., 2017). The mRNA expression of IL-10 in the CD19+CD24hiCD27+ B cells was approximately ten-fold of that in the CD19+CD24loCD27− B cells from the HCs (Fig. 1A).
decreased in the RA patients (n = 50) compared with the HCs (n = 30, 14.19 ± 8.13 % vs. 31.79 ± 12.64 %, respectively, P < 0.01) (Fig. 1B). To confirm our findings, we used intracellular staining and flow cytometry analysis to determine the number of B10 cells (IL-10+ B cells) in another 9 healthy individuals and 8 RA patients. The IL-10+ B cells constituted 3.4 ± 0.75 % of the total number of B cells in the RA patients and 4.4 ± 0.7 % in the healthy individuals (P < 0.01) (Fig. 1C). We used RT-qPCR to detect the IL-10 expression of CD19+CD24hiCD27+ B cells from the RA patients and HCs. Interestingly, the CD19+CD24hiCD27+ B cells from the RA patients expressed less IL-10 than those from the healthy individuals (P = 0.02) (Fig. 1D).
4.2. Decreased proportion and impaired IL-10 production of CD19+ B cells and downregulated IL-10 expression of CD19+CD24hiCD27+ regulatory B10 cells in RA The proportion of CD19+CD24hiCD27+ regulatory B10 cells was 94
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4.3. The proportion of CD19+CD24hiCD27+ regulatory B10 cells was associated with treatment response, and a good response to TNF inhibitor treatment was observed, but was not correlated with disease activity in RA
in both the CD4+ T cells and CD19+ B cells from the RA patients compared with those from the healthy donors. The expression of CD70 on CD4+ T cells was approximately three-fold higher in the cells from the RA patients than in those from the HCs (Fig. 4A, B). CD27 can be expressed in a membrane-bound form and a soluble form. T cell activation leads to the shedding of CD27 from the cell surface, resulting in soluble CD27 (sCD27), which might signal via the CD27-CD70 pathway. We detected the sCD27 level in the serum from both the RA patients and HCs and showed that the level of sCD27 was higher in the RA patients (Fig. 4C). However, we cannot distinguish whether the sCD27 originated from T cells, B cells or another cell type. To further ascertain the proportion of sCD27 in the serum derived from B cells and the shedding of sCD27 attributed to the CD70-CD27 interaction, we cultured PBMCs from the HCs in vitro. The cells were divided into three groups and treated differently. An anti-CD70 antibody was used to block the CD70-CD27 interaction. Anti-CD3 and antiCD28 antibodies were used to stimulate T cell activity. We detected the expression of membrane-bound CD27 on both CD4+ T cells and CD19+ B cells as well as the expression of sCD27 in the cell supernatant and found that CD27 expression decreased significantly on CD19+ B cells, while the sCD27 and IL-10 levels were increased in the cell culture supernatants (the latter data are not shown). However, the higher membrane-bound CD27 level and lower sCD27 level in the supernatants were found after B cells were pre-treated with the anti-CD70 antibody (Fig. 4D–G). These results suggest that the upregulation of CD70 expression might decrease the proportion of CD19+CD24hiCD27+ regulatory B10 cells in RA via the CD70-CD27 interaction.
To elucidate the association of CD19+CD24hiCD27+ regulatory B10 cells with disease activity, we assessed the proportion of CD19+CD24hiCD27+ regulatory B10 cells in 20 RA patients with high disease activity (DAS 28-CRP > 5.1) and followed these patients for 24 weeks. Ten of the 20 patients showed satisfactory responses to conventional synthetic DMARD treatment, resulting in disease remission (2.6 < DAS 28-CRP < 3.2). The proportion of CD19+CD24hiCD27+ regulatory B10 cells in the peripheral blood was elevated to a mean level of 18 % in most patients at week 24, suggesting an association between the regulatory B10 cell level and RA pathophysiology (Fig. 2A). We evaluated the CD19+CD24hiCD27+ regulatory B10 cell subset in another 10 patients before and after treatment with TNF inhibitors (TNFis). All patients achieved low disease activity or remission. Interestingly, we found that the proportion of CD19+CD24hiCD27+ regulatory B10 cells in the TNFi treatment group was higher than that in in the csDMARD-treated group and comparable to that in the HC group (Fig. 2B). CD19+CD24hiCD27+ regulatory B10 cells were restored after TNFi treatment, suggesting that TNF-α might be involved in the mechanism that leads to the decreased number and impaired function of CD19+CD24hiCD27+ regulatory B10 cells in RA. We also performed correlation analysis between the proportion of CD19+CD24hiCD27+ regulatory B10 cells and clinical characteristics of 27 RA patients with complete clinical information. Interestingly, no correlations were found between DAS28-CRP, RF, and ACPA and the proportion of CD19+CD24hiCD27+ B cells. However, the absolute number of CD19+CD24hiCD27+ B cells was correlated with the titre of ACPA (Fig. 2C).
4.7. TNF-α and IL-1β decreased the proportion of CD19+CD24hiCD27+ B cells likely by upregulating CD70 expression in PBMCs PBMCs derived from healthy donors were cultured and treated with TNF-α or IL-1β. CD70 expression was analysed by flow cytometry and PCR. We found that both TNF-α and IL-1β upregulated CD70 expression on CD4+ T cells and CD19+ B cells (Fig. 5A, B), and these results were consistent with the decrease in the proportion of CD19+CD24hiCD27+ regulatory B10 cells after TNF-α and IL-1β stimulation but not IL-6, IFN-γ or IL-17 stimulation (Fig. 5C).
4.4. TNF-α and IL-1β decreased the proportion of CD19+CD24hiCD27+ regulatory B10 cells To determine the role of TNF-α in the decreased proportion of CD19+CD24hiCD27+ regulatory B10 cells, PBMCs from the RA patients were either left untreated or treated with TNF-α, IL-1β, IL-6, IFN-γ and IL-17. The results demonstrated that TNF-α and IL-1β significantly decreased the proportion of CD19+CD24hiCD27+ cells in vitro, but no differences were observed in the IL-6, IFN-γ and IL-17 groups (Fig. 2D, 2E). These findings suggested that TNF-α and IL-1β mediated the decrease in the CD19+CD24hiCD27+ B cell subset.
5. Discussion The present study demonstrates decreases in both the proportion and IL-10 expression of CD19+CD24hiCD27+ regulatory B10 cells in RA patients, which may contribute to the immunologic abnormalities of this disease. The mechanism of the downregulation of this regulatory B cell subset could be the shedding of sCD27 from the cell surface through the CD70-CD27 interaction mediated by the high CD70 expression induced by proinflammatory cytokines, notably TNF-α and IL-1β. An antiCD70 antibody could restore the proportion of CD19+CD24hiCD27+ regulatory B10 cells and thus might be a potential therapeutic target for RA treatment. Our results showed that the proportion of CD19+CD24hiCD27+ regulatory B10 cells was decreased in RA, which is consistent with results for CD19+IL-10+ B cells. This finding confirms that the proportion of regulatory B cells is decreased in RA. We also found that CD19+CD24hiCD27+ regulatory B10 cells derived from RA patients exhibit impaired IL-10 expression. Our findings were not consistent with the reported elevated serum IL-10 levels in RA and the findings reported in previous studies (Cush et al., 1995; Llorente et al., 1994), probably because the elevated serum IL-10 levels in RA compensate for the numerical deficit and impaired IL-10 secretion observed by RTqPCR. Another highlight of this study is the remarkable recovery of CD19+CD24hiCD27+ regulatory B10 cell frequencies after TNFi treatment in RA, which prompted us to explore the mechanisms underlying the changes in this B cell subset in RA. TNF-α plays a key role in the
4.5. The decreased expression of CD27might be responsible for the decreased proportion of CD19+CD24hiCD27+ regulatory B10 cells No difference was found in the proportion of CD19+ B cells (data not shown) or the CD24 expression on CD19+ B cells (Fig. 3A) between the HCs and RA patients. However, the RA patients showed remarkably lower CD27 expression, which was reduced by 24 % (Fig. 3B). This result suggests that the decreasing proportion of the CD19+CD24hiCD27+ regulatory B10 cells might be due to the downregulation of CD27 expression on the cell surface. We also detected the relative CD27 expression in CD19+CD27+ B cells by RT-qPCR and the median fluorescence intensity (MFI) of CD24 and CD27 in CD19+CD24hiCD27+ regulatory B10 cells by flow cytometry and confirmed that the CD27 expression in these subsets was lower in the RA patients than the HCs (Fig. 3C, D). 4.6. Upregulated CD70 expression may decrease the proportion of CD19+CD24hiCD27+ regulatory B10 cells in RA We analysed the expression of CD70, the ligand for CD27, on CD4+ T and CD19+ B cells. We found that CD70 expression was upregulated 95
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Fig. 2. The proportion of CD19+CD24hi CD27+ regulatory B10 cells is associated with the treatment, especially for TNFi treatment. (A) The graph shows the variation in the proportion of CD19+CD24hi CD27+ regulatory B10 cells in patients who had a good response to treatment (paired t test). (B) The decreased proportion of CD19+CD24hiCD27+ regulatory B10 cells in patients recovered after TNFi treatment (independent t test). (C) The correlation of CD19+CD24hiCD27+ regulatory B10 cells and clinical characteristic. (D) Peripheral blood mononuclear cells (PBMCs) from healthy individuals (n = 3) were stimulated with TNF-α, IL-1β, IL-6, IFN-γ and IL-17 for 72 h. The proportion of CD19+CD24hiCD27+ regulatory B10 cells was analysed by flow cytometry. The bar chart shows the variation of CD19+CD24hiCD27+ regulatory B10 cells after stimulation. (E) Representative flow cytometry for one healthy individual shows the proportion of CD19+CD24hi CD27+ regulatory B10 cells after TNF-α and IL-1β stimulation. * P < 0.05, **P < 0.01.
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Fig. 3. The decreased proportion of CD19+CD24hiCD27+ regulatory B10 cells in RA is attributed to the decreased expression of CD27. (A) Representative flow cytometry plots for one healthy individual and one RA patient show the proportion of CD24+ B cells in the CD19+ B cell population. (B) Representative flow cytometry plots show the proportion of CD27+ cells in the CD19+ B cell population. (C) CD19+CD24hiCD27+ regulatory B10 cells from 5 healthy donors and 10 patients with RA were sorted by flow cytometry and subjected to real-time PCR analysis of CD27 expression. The relative CD27 expression is shown. (D) The bar graph shows the cumulative data for the CD27 median fluorescence intensity (MFI) in CD19+CD24hiCD27+ regulatory B10 cells from 29 healthy individuals and 41 RA patients, as measured by flow cytometry. The results are presented as the mean ± SD.
CD19+ B cells and found that the CD19+CD24hiCD27+ regulatory B10 cells from the RA patients showed a lower CD27 MFI than those from the HCs. We found that CD70 expression was upregulated on both CD4 + T cells and CD19 + B cells and accompanied by decreased CD19+CD24hiCD27+ regulatory B10 cell numbers and increased sCD27 and total IL-10 levels in the supernatants. Generally, CD27, a member of the tumour necrosis factor receptor (TNFR) superfamily, is a wellcharacterized marker of memory B cells (Agematsu, 2000; Agematsu et al., 1997). The interaction of CD27 with its unique ligand CD70 has been reported to release a truncated form of CD27 (Loenen et al., 1992; Prasad et al., 1997). We hypothesize that the decreased CD27 expression on B cells might be attributed to CD70-CD27 interactions, as CD70 expression is upregulated by TNF-α and IL-1β. However, the CD27
pathogenesis of RA (McInnes and Schett, 2011). Thus, we assume that the changes involving CD19+CD24hiCD27+ regulatory B10 cells in RA are attributed to TNF-α. Consistent with our hypothesis, the proportion of CD19+CD24hiCD27+ regulatory B10 cells was remarkably decreased by TNF-α and IL-1β but not by IL-6, IL-8, IL-17 or IFN-γ. Nevertheless, the roles of TNF-α in the proportion and IL-10-secreting functions of CD19+CD24hiCD27+ regulatory B10 cells remain to be elucidated. We analysed the expression of CD24 and CD27 by the B cells. In the RA patients, the expression of CD27 on CD19+ B cells was downregulated. However, no differences in CD24 expression on CD19+ B cells were found between the RA patients and HCs. Our findings suggested that TNF-α might decrease the proportion of CD19+CD24hiCD27+ regulatory B10 cells by lowering the expression of CD27. Furthermore, we compared the MFI of CD24 and CD27 on 97
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Fig. 4. CD70 expression is upregulated in lymphocytes in RA patients, and the upregulated CD70 expression could decrease the proportion of CD19+CD24hiCD27+ regulatory B10 cells in vitro. PBMCs from healthy individuals were cultured with anti-CD3 and anti-CD28 antibodies for 72 h with or without anti-CD70 antibody (1 μg/mL) pretreatment for 1 h. (A, B) Representative flow cytometry plots for one healthy individual and one RA patient show the proportion of CD70+ B cells in the CD19+ B or CD4+ T cell population. (C) Serum was collected from 12 healthy controls and 20 patients with RA, and the soluble CD27 level was detected by ELISA. (D) Representative flow cytometry plots for one healthy individual show the proportion of CD70+ T cells in the CD4+ T cell population. The bar chart shows the cumulative data for the proportion of CD4+CD70+ T cells blocked or not blocked by the anti-CD70 antibody. (E) Representative flow cytometry plots for one healthy individual show the proportion of CD70+ B cells in the CD19+ B cell population. The bar graph shows the cumulative data for the CD19+CD70+ B cell proportion in 3 healthy individuals treated or not treated with the antiCD70 antibody. (F) Representative contour plots and the cumulative data for three healthy individuals show the proportion of CD27+ B cells in the CD19+ B cell population before and after anti-CD70 antibody treatment. (G) The supernatants were collected, and soluble CD27 expression was assessed by ELISA. The bar chart shows the cumulative data for three independent experiments. The data are presented as the mean ± SD. * P < 0.05, **P < 0.01.
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Fig. 5. TNF-α increases CD70 expression on lymphocytes in vitro. (A, B) Representative flow cytometry plots for one healthy individual show the proportion of CD4+CD70 + T cells or CD19+CD70 + B cells after treatment with TNF-α or IL-1β. The graph shows the cumulative data for the proportion of CD4+CD70 + T cells or CD19+CD70 + B cells in different groups. (C) The bar chart shows the cumulative data as the mean ± SD of three independent experiments. * P < 0.05, **P < 0.01.
expression on CD19+ B cells was generally persistent, and the level of sCD27 in the supernatants was decreased following pretreatment with the anti-CD70 antibody (to block the CD70-CD27 interactions). We also assessed CD70 mRNA expression in PBMCs stimulated with different cytokines, and the result was consistent with the upregulation of CD70 expression by TNF-α and IL-1β. In addition, flow cytometry results showed that TNF-α and IL-1β slightly upregulated CD70 expression on both CD4+ T cells and CD19+ B cells. Previous studies demonstrated that TNF-α and IL-1α enhanced CD70 expression on human lymphocytes (Lens et al., 1997; Tesselaar et al., 2003). Our results suggested that TNF-α decreased CD27 expression on B cells likely via the upregulation of CD70 expression. The upregulated CD70 expression on CD4+ T cells has been reported in a previous study (Lee et al., 2007). In another study, blocking CD27-CD70 interactions with an anti-CD70 antibody ameliorated joint
disease in murine CIA (Oflazoglu et al., 2009). The protection of regulatory B cells conferred by blocking CD70-CD27 interactions might help to explain the amelioration of joint disease in CIA with an antiCD70 antibody. Recently, Park et al. (Park et al., 2014) also found that CD70-expressing CD4+ T cells produce IFN-γ and IL-17 in RA, which indicated that CD70 was involved in RA pathogenesis. Consistent with previous studies, our study demonstrated that CD70 might mediate RA pathogenesis. Our results further showed that in addition to the CD70 expression on CD4+ T cells, CD70 expression on CD19+ B cells was also upregulated in RA, resulting in elevated serum levels of sCD27 and a decreased proportion of CD19+CD24hiCD27+ regulatory B10 cells in the PBMC population. These results also agreed with those of our in vitro study. Our present study has several limitations. First, we did not perform functional studies of CD19+CD24hiCD27+ regulatory B10 cells and 99
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only assessed these cells by evaluating IL-10 expression because a functional study of this subset was recently published by our team (Hu et al., 2017). Secondly, we only detected CD70 expression on CD4+ T and CD19+ B cells, we did not include CD8+ T cells. In conclusion, the current findings demonstrate that the decreased proportion and IL-10 expression of impaired CD19+CD24hiCD27+ regulatory B10 cells in RA might be attributed to the upregulated CD70 expression on CD4+ T and CD19+ B lymphocytes. Our data might provide a molecular basis to support the rationale for anti-CD70 therapy for RA.
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Ethics Statement This study was carried out in accordance with the recommendations of the “Institutional Medical Ethics Review Board of Peking University People’s Hospital” with written informed consent from all subjects. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by the “Institutional Medical Ethics Review Board of Peking University People’s Hospital.” Funding This work was supported by grants from the National Natural Science Foundation of China (81501396 to Dr. Lianjie Shi, 81771706 to Dr. Rong Mu, 81701614 to Dr. Hongjiang Liu, and 81671604 to Dr. Fanlei Hu), China Postdoctoral Science Foundation (2016 M600874), and Peking University International Hospital Research Funds (YN2017QX01 and YN2016QN01). Declaration of Competing Interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. References Agematsu, K., 2000. Memory B cells and CD27. Histol. Histopathol. 15, 573–576. Agematsu, K., Nagumo, H., Yang, F.C., Nakazawa, T., Fukushima, K., Ito, S., Sugita, K., Mori, T., Kobata, T., Morimoto, C., Komiyama, A., 1997. B cell subpopulations separated by CD27 and crucial collaboration of CD27+ B cells and helper T cells in immunoglobulin production. Eur. J. Immunol. 27, 2073–2079. Blair, P.A., Norena, L.Y., Flores-Borja, F., Rawlings, D.J., Isenberg, D.A., Ehrenstein, M.R., Mauri, C., 2010. CD19(+)CD24(hi)CD38(hi) B cells exhibit regulatory capacity in healthy individuals but are functionally impaired in systemic Lupus Erythematosus patients. Immunity 32, 129–140. Bouaziz, J.D., Yanaba, K., Tedder, T.F., 2008. Regulatory B cells as inhibitors of immune responses and inflammation. Immunol. Rev. 224, 201–214. Cush, J.J., Splawski, J.B., Thomas, R., McFarlin, J.E., Schulze-Koops, H., Davis, L.S., Fujita, K., Lipsky, P.E., 1995. Elevated interleukin-10 levels in patients with rheumatoid arthritis. Arthritis Rheum. 38, 96–104. Flores-Borja, F., Bosma, A., Ng, D., Reddy, V., Ehrenstein, M.R., Isenberg, D.A., Mauri, C., 2013. CD19+CD24hiCD38hi B cells maintain regulatory T cells while limiting TH1 and TH17 differentiation. Sci. Transl. Med. 5, 173ra123.
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