Characteristics Of B Cell Subsets In Primary Sjogren 's Syndrome And Their B Cell Function

Part 1 The characteristics of B cell subsets and the function of regulatory B cells in primary Sjogren’s syndromeBackgroundPrimary Sjogren’s syndrome (pSS) is a chronic autoimmune disorder that typically affects exocrine glands-mainly labial and lacrimal, however, a broad spectrum of systemic manifestations may characterize the disease. B cells play a central role in the immunopathogenesis and exhibit sign of hyperactivity. Regulatory B cells (Breg) are newly defined B cell subgroups. In this study, we investigated the differences of B cell subsets, the expressions of co-stimulatory molecules on B cell subsets, and the function of Breg cells in patients with pSS patients and healthy controls (HC). We also analyzed the relationship between Breg cells and clinical manifestations of pSS patients.Methods84 newly diagnosed pSS patients and 69 HC were recruited, and 16 labial gland specimens were also collected. To analyze B cell subsets and B cell activity, PBMCs were surface stained and detected by flow cytometry. IL-10 levels in Breg cells were detected by intracellular cytokine measurement. The expressions of CD20 and IL-10 in labial gland specimens were detected by Immunohistochemistry and immunofluorescence. The function of Breg cells was tested by co- culturing isolated Breg cells with purified CD4+CD25- T cells, and supernatant cytokines were measured by cytometric bead array (CBA). T follicular helper cells (Tfh) and IL-21R on B cells were detected by flow cytometry. We also analyzed the relationship between Breg cells and clinical manifestations of pSS patients.Results1. The frequency of Breg cells in pSS patients was much higher compared to HC(9.23±0.45%, and 2.55±0.16%, respectively; p<0.001). The frequency of mature B cells in pSS patients was much higher compared to HC(63.72±1.08%, and 56.50±1.14%, respectively; p<0.001). However, there were decreased percentages of memory B cells in pSS patients and HC(6.34±0.46%,19.36±1.03%, respectively;p<0.001). And there were decreased percentages of CD19+CD24hi CD27+ cells in pSS patients and HC (6.05±0.53%, 16.39±1.15%, respectively;p<0.001). There was no difference in morphology among B cell subsets.2. The percentages of CD8、CD44、CD95、TACI on Breg cells were significantly lower compared with those on memory B cells in patients (p<0.05).3. The level of IL-10 in CD19+B cells and Breg cells was much lower in pSS patients compared with HC (8.23±1.91%, and 25.55±0.63%, respectively,p<0.001; 28.43±5.49%, and 53.13±7.93%, respectively, p=0.030).4. Breg cells from pSS patients did not show effects of suppressing proinflammatroy cytokines by CD4+CD25-T cells, such as IFN-y, TNF-a, IL-4, IL-17, IL-21 and IL-9 (p<0.05).5. There were abundant CD20+ B cells and sporadic IL-10+ cells in labial glands of pSS patients compared with HC (p<0.05).6. The frequency of Tfh cells and the levels of IL-21 which they secreted were much higher in pSS patients compared with HC (21.88±0.82%,17.91±0.88%, respectively, p=0.002; 27.43±2.94%,9.97±1.91%, respectively, p=0.018). The percentages of IL-21 R on Breg cells were much lower in pSS patients compared with HC (62.27±2.43%, 75.25±2.18%, respectively;p<0.001).7. In vitro, the level of IL-10 in Breg cells could be significant increased when B cells among pSS patients or HC were stimulated with CD40L, CpG, and rh IL-21.8. The frequency of Breg cells was significantly increased in pSS patients with high levels of IgG or ESR, positive of anti-SSA antibody or anti-SSB antibody, and high disease activity index compared with the patients without these characteristics. The frequency of Breg cells were positive correlated with IgG or ESR among pSS patients.ConclusionsB cells in pSS patients displayed a variety of abnormalities. The expression of key molecules on Breg cells was much different from memory B cells in pSS patients. The IL-10 levels in Breg cells were decreased in peripheral blood and salivary gland of pSS patients. The function of Breg cells in pSS patients was impaired, and they could not suppress proinflammatroy cytokines secreted by effector T cells efficiently. There were some extent relationships between the frenquency of Breg cells and clinical characteristics of pSS patients.Part 2 Analysis of differentially expressed genes and microRNAs of B cells in primary Sjogren’s syndrome by RNA sequencingBackgroundPrimary Sjogren’s syndrome (pSS) is a chronic autimmune disease mainly characterized by the inflammation of exocrine glands. There two key insights into pSS pathogenesis, which included "IFN signature" and hyperactivity of B cells. mRNA and microRNA (miRNA) are very important to control the gene expression. In this research, we analyzed the differentially expressed genes (DEG) and miRNA of B cells in pSS patients by RNA-sequencing.MethodsPeripheral blood samples from 3 pSS patients and 3 age-matched healthy controls (HC) were collected. CD19+ B cells were sorted by Magnetic cell sorting method. Total RNA was extracted and cDNA of transcriptome or miRNA analysis were prepared and RNA-sequencing was performed to screen the DEG and miRNA. The GO Terms was used to uncover the biological function of DEGs, and the KEGG pathway enrichment was used to find out the related signal pathway. The mRNA-miRNA conjoint analysis was also performed.Results1. There were a total of 73 significantly DEGs in B cells of pSS patients compared to HC, including 51 upregulated DEGs (such as IFI44L、IFI44、IFIT1、IFITM1、IFIT3、 IFIT2、IRF7、IFI6 and ISG15) and 22 downregulated DEGs (such as ESR2 and EGR1). GO Terms and KEGG pathway analyses showed that most of the upregulated DEGs were enriched in IFN signaling and IFN regulatory pathway, and also showed the relationship with microbial infection, such as influenza A virus, hepatitis C virus, measles and herpes simplex virus.2. There were five significantly differentially expressed miRNAs, including hsa-miR-4485-3p、hsa-miR-144-5p、hsa-miR-144-3p、hsa-miR-451a、hsa-miR-4732-3p. GO Terms and KEGG pathway analyses showed that most of the target genes which regulated by those miRNAs were enrichment on herpes simplex virus and TGF-β signaling pathway.3. DEGs and differentially expressed miRNAs conjoint analysis showed that the target DEGs which regulated by those miRNAs participated in cytoskeleton formation and modification of DNA or RNA. such as RASD2、CKAP4、SPARS2L和METTL.ConclusionsThere were 51 upregulated DEGs and 22 downregulated DEGs in B cells of pSS patients. GO Terms and KEGG pathway analyses showed that most of the upregulated DEGs were enriched in IFN related signaling pathway, and also showed the significant relationship with microbial infection.Conjoint analysis showed that the target DEGs which regulated by differentially expressed miRNAs participated in cytoskeleton formation and modification of DNA or RNA. There maybe more than one regulatory methods lead to DEGs in B cells of pSS patients.Part 3 Abnormality of the B cell receptor repertoire in primary Sjogren’s syndromeBackgroundPrimary Sjogren’s syndrome (pSS) is characterized by mononuclear inflammatory infiltrates and IgG plasma cells in exocrine glands. B cells play a central role in the immunopathogenesis and exhibit signs of hyperactivity. B cell receptor (BCR) work as important part for the antigen epitope recognition in adaptive immune, especially on the complementary-determining region 3 (CDR3). Based on the next-generarion sequencing, quantitative data on the degree of expansion of individual clones within the BCR repertoire could be obtained, and analyze the differences between BCR CDR3 repertoire in pSS patients and healthy controls (HC) preliminarily.MethodsPeripheral blood samples from 20 pSS patients and 19 age-matched HC were collected. CD19+B cells were sorted by Magnetic cell sorting method. Genome DNA of CD19+B cells was extracted. After two rounds of polymerase chain reaction (PCR) and PCR production purification, BCR H chain CDR3 sequencing was performed. Quantitative evaluation and global comparison were analyzed.Results1. The CDR3 lengths of BCR in pSS patients were much shorter compared with HC (53.03±1.68bp,55.47±1.52, respectively; p<0.001).2. The D50 index in pSS patients was (0.217±0.014), which was much lower than that in HC (0.229±0.020) respectively (p=0.04). This indicated that the diversity of BCR CDR3 in pSS patients was much reduced.3. There were significant differences of V/J gene usages between pSS patients and HC. The frequencies of usage of IGHV1, IGHV4, IGHV5, IGHV6, IGHJ1, IGHJ2 and IGHJ3 were much higher among pSS patient compared with HC (p<0.05). While The frequency of usage of IGHV3, was much lower among pSS patient compared with HC (p<0.001).4. The share clone analysis showed that there were three dominant clonotypes among the pSS patients with anti-SSB antibody, including CARAGMDVW, CARGRYYYYGMDVW,CAKDNKPLNYDILTGQMNYGMDVW.ConclusionsThe CDR3 lengths and diversity of BCR was significantly decreased in pSS patients. There was a certain degree preference of V/J gene usages in BCR of pSS patients. The share clone analysis showed three dominant clonotypes among the pSS patients with anti-SSB antibody. The BCR CDR3in pSS patients was abnormal. The analysis of BCR CDR3 might help to explore the function of B cells in the pathogenesis of pSS and to find some latent antigents.