Enrichment and Clinical Relevance of FcRL5⁺CD27⁺ B Cells in Autoimmune Hepatitis

Enrichment and Clinical Relevance of FcRL5⁺CD27⁺ B Cells in Autoimmune Hepatitis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Enrichment and Clinical Relevance of FcRL5⁺CD27⁺ B Cells in Autoimmune Hepatitis Yuyang Liu, Xiting Pu, Chengcen Guo, Haoyu Wen, Yudong Zhao, Qiyun Xia, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6509177/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background: Dysregulation of B cells has been implicated in the development of autoimmune hepatitis (AIH), yet identification of potentially pathogenic subpopulations remains elusive. Fc receptor-like 5 (FcRL5), a B cell surface marker, represents a potential identifier of pathogenic subsets. This study aimed to investigate the function and immunophenotypic profile of FcRL5⁺B cells in AIH and evaluates their therapeutic targetability. Methods: Immunohistochemistry was performed on liver biopsies from AIH, nonalcoholic steatohepatitis (NASH), chronic hepatitis B (CHB), and healthy controls. Flow cytometry was conducted on liver grafts from AIH patients, healthy donor livers, and peripheral blood samples. FcRL5⁺CD27⁺ B cells were induced in vitro using CpG and IL-15 stimulation. Phenotypic analysis was performed by flow cytometry, and RNA sequencing was conducted on total B cells after induction. STAT3 and STAT5 inhibitors were added at the start of induction to evaluate their effects on cell expansion. Results: FcRL5⁺ cells were significantly enriched in the livers of AIH patients, predominantly within the CD19⁺CD27⁺ memory B cell compartment. These cells exhibited a pro-inflammatory phenotype, characterized by high expression of CXCR3 and CD11c, along with robust secretion of GM-CSF. Longitudinal analysis revealed a marked decrease in intrahepatic FcRL5⁺ cell frequency following clinical remission in AIH patients. RNA sequencing and functional assays indicated that the JAK-STAT pathway regulates the proliferation and function of FcRL5⁺CD27⁺ B cells, with STAT3 and STAT5 phosphorylation playing critical roles. Conclusion: This study highlights the importance of FcRL5⁺CD27⁺ memory B cells in the pathogenesis of autoimmune hepatitis and identifies them as promising targets for therapeutic intervention. Autoimmune hepatitis FcRL5 memory B cells JAK-STAT pathway inflammation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Background Autoimmune hepatitis (AIH) is a chronic inflammatory liver disease characterized by an aberrant immune response in which the immune system erroneously recognizes hepatocytes as foreign antigens, leading to persistent hepatic injury. Key clinical hallmarks of AIH include the presence of circulating autoantibodies, elevated serum IgG levels, and distinctive histopathological features( 1 ). Current investigations into the hepatic immune microenvironment of AIH have predominantly focused on CD8⁺ tissue-resident memory T (T RM ) cells, while the role of B lymphocytes remains underexplored ( 2 – 5 ). However, accumulating evidence underscores the pivotal role of B cells in the pathogenesis of AIH, as they orchestrate complex immunoregulatory networks ( 6 ). Notably, recent studies have demonstrated that SepSecS-specific memory B cells in soluble liver antigen (SLA)-positive AIH patients can activate cognate T cell populations via antigen presentation, initiating and sustaining autoimmune responses ( 7 ). Clinically, B cell-targeted therapies such as rituximab (an anti-CD20 monoclonal antibody) ( 8 , 9 )and belimumab (an anti-BAFF monoclonal antibody) ( 10 , 11 )have shown therapeutic efficacy in refractory AIH cases, further supporting the central involvement of B cells in disease progression. Fc receptor-like 5 (FcRL5), a member of the immunoglobulin (Ig) superfamily, has attracted attention for its ability to selectively bind intact IgG molecules via multiple Ig-like domains, while discriminating against structurally compromised IgG. This selective recognition suggests that FcRL5 may serve as a receptor for newly secreted, functional IgG ( 12 ). In CHB, atypical memory B cells (atMBCs) exhibit a distinct distribution pattern compared to circulating B cells, characterized by preferential accumulation within the liver and elevated expression of FcRL5 ( 13 ). Additionally, in systemic lupus erythematosus (SLE), TLR7 and IL-21 signaling pathways promote the accumulation of FcRL5⁺ memory B cells, which subsequently differentiate into autoreactive plasmablasts ( 15 ). In chronic infection models in mice, FcRL5 is primarily expressed on germinal center-derived memory B cells( 16 ). Furthermore, studies on human tonsillar memory B cells have revealed that atypical FCRL4⁻FcRL5⁺ subsets express transcription factors and immunoglobulin genes indicative of plasmacytic differentiation potential, while FCRL4⁺FcRL5⁺ subsets display tissue-resident features( 17 ). In malaria, FcRL5 is enriched in atMBCs independently of FCRL4, highlighting its functional diversity ( 18 ). In summary, FcRL5 exhibits functional diversity across various immunopathological contexts, involving the differentiation, localization, and tissue residency of B cells. Despite these advances, the role of FcRL5 in AIH remains uncharacterized. To address this gap, our study identifies a distinct population of FcRL5⁺CD27⁺ memory B cells enriched in the liver tissue of AIH patients, revealing both its clinical relevance and functional distinction from other B cell subsets. Moreover, we propose a potential regulatory mechanism mediated by the downstream STAT3/5 signaling pathway of IL-15. These findings not only enhance our understanding of AIH pathogenesis but also offer new avenues for targeted therapeutic intervention. Methods Liver Tissue Human liver tissue samples were provided by the Department of Gastroenterology at Renji Hospital. The cohort comprised paraffin-embedded liver sections from 51 cases of AIH, 16 cases of CHB, 7 cases of NASH, and 6 healthy controls (HC) for immunohistochemical analysis. Among the healthy controls, 6 sections were derived from liver transplant donors, while the remaining samples were obtained via liver biopsy. Notably, 15 AIH patients underwent a second liver biopsy, and complete clinical data were collected for all cases. All patients fulfilled the clinical diagnostic criteria for AIH( 19 ), CHB( 20 ), or NASH( 21 ). The histological grading of inflammation and fibrosis in AIH patients was evaluated using the Scheuer scoring system( 22 ), and patients were further stratified into remission and non-remission groups according to the Histological Activity Index ( 23 ). In addition, liver tissues from 3 AIH transplant patients and 4 healthy donors were collected for flow cytometry analysis. All sample collections were conducted in strict compliance with ethical principles and met the ethical review standards of Renji Hospital, with approval from the ethics committee (approval number: KY2021-063-B). All procedures adhered to the principles set forth in the Declaration of Helsinki, ensuring that the rights, safety, and privacy of the subjects were fully protected. Immunohistochemistry Liver tissue sections were paraffin-embedded and subsequently deparaffinized and rehydrated. Antigen retrieval was performed using a microwave-based method with EDTA buffer (pH 9.0). Endogenous peroxidase activity was blocked using 3% hydrogen peroxide solution. Sections were then blocked with 50% goat serum at room temperature for 30 minutes, followed by incubation with primary anti-FcRL5 antibody (Servicebio, GB111823-100, 1:500 dilution) at 4°C overnight. After washing with PBS, sections were incubated with HRP-conjugated secondary antibody (1:500 dilution) at room temperature for 20 minutes. DAB was applied for 30 seconds for color development, followed by hematoxylin counterstaining for 10 seconds. Slides were sealed with neutral resin. FcRL5 + cells were counted in five randomly selected portal areas under a 400× high-power field by three independent observers, and the average value was used for analysis. Immunofluorescence Liver tissue sections were paraffin-embedded, deparaffinized, and rehydrated. Antigen retrieval was performed using a microwave-based method with EDTA buffer (pH 9.0). Endogenous peroxidase activity was blocked using 3% hydrogen peroxide solution. Sections were then blocked with 50% donkey serum at room temperature for 30 minutes, followed by incubation with primary antibodies—anti-FcRL5 (Servicebio, GB111823-100, 1:500 dilution) and anti-CD27 (Abcam, ab268268, 1:200 dilution)—at 4°C overnight. After washing with PBS, fluorophore-conjugated secondary antibodies (Goat Anti-Rabbit IgG, Abcam, ab150078, 1:200 dilution; Goat Anti-Mouse IgG, Abcam, ab6785, 1:200 dilution) were applied and incubated at room temperature for 30 minutes. Nuclei were counterstained with DAPI, and sections were mounted. Co-localization of target proteins was observed under a laser confocal microscope at 400× magnification. Multiplex Immunofluorescence We use Absin four-color Multiplex Immunofluorescence kit (abs50012). Liver tissue sections were paraffin-embedded, deparaffinized, and rehydrated. Antigen retrieval was performed using a microwave-based method with EDTA buffer (pH 9.0). Endogenous peroxidase activity was blocked using 3% hydrogen peroxide solution. Sections were then blocked with 50% goat serum at room temperature for 30 minutes, followed by incubation with the primary antibody against FcRL5 (Servicebio, GB111823-100, 1:500 dilution) at 4°C overnight. After washing with TBST, HRP-conjugated secondary antibody was applied and incubated at room temperature for 20 minutes. Following another TBST wash, 50 µL of TSA amplification reagent (dye 520, dye 570 or dye 650) was added and incubated in the dark at room temperature for 10 minutes. Antigen retrieval was then repeated, followed by the same blocking and incubation procedures as described above for the application of additional primary antibodies, including anti-CD27 (Abcam, ab268268, 1:200 dilution), anti-CD19 (Abcam, ab134114, 1:100 dilution), and anti-CD138 (Abcam, ab128936, 1:4000 dilution). A final round of microwave-based antigen retrieval was performed, and nuclei were counterstained with DAPI. Co-localization of target proteins was visualized using a laser confocal microscope under 400× magnification. Isolation of Human Liver Immune Cells Immune cells from human liver tissue were isolated using a density gradient centrifugation method ( 21 ). Briefly, approximately 30 g of fresh liver tissue was collected and digested in a digestion solution at 37°C in a water bath for 30 minutes. The digested tissue was then homogenized using a tissue dissociator, filtered sequentially through a 70 µm cell strainer, and further ground using a sterile syringe plunger. The resulting cell suspension was resuspended in RPMI 1640 medium (Corning, Manassas, USA) and centrifuged at 50 ×g for 5 minutes at 4°C to remove liver tissue debris. The supernatant was then centrifuged at 750 ×g for 10 minutes at 4°C, and the supernatant was discarded. The pellet was resuspended in 30 mL of 33% Percoll solution and carefully overlaid onto 10 mL of 70% Percoll solution (Percoll, Cytiva, Cat# 17-0891-01). The mixture was centrifuged at 900 ×g for 30 minutes at 4°C with acceleration set to 3 and deceleration set to 0. After centrifugation, the upper layer was discarded, and immune cells were carefully collected from the interphase. Finally, the isolated cells were washed with 1× PBS to obtain purified intrahepatic immune cells. Isolation of Peripheral Blood Mononuclear Cells (PBMCs) PBMCs were isolated using a density gradient centrifugation method. Briefly, fresh peripheral blood from healthy donors was diluted with 1× PBS at a ratio of 1:3 and carefully layered over an equal volume of Ficoll solution (Cytiva, Cat# 17144003). The mixture was centrifuged at 400 ×g for 30 minutes at 20°C with acceleration set to 3 and deceleration set to 0. After centrifugation, the white buffy coat layer containing mononuclear cells was carefully collected. The isolated cells were then washed with 1× PBS to obtain purified PBMCs. In Vitro Induction and Inhibition Assay of FcRL5⁺CD27⁺ B Cells PBMCs were seeded in 48-well plates at a density of 3 × 10⁵ cells/mL in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 U/mL penicillin, 100 µg/mL streptomycin, and 50 mM 2-mercaptoethanol. To induce FcRL5⁺CD27⁺ B cells, recombinant human IL-15 (50 ng/mL, R&D Systems) and CpG ODN 2006 (1 µg/mL, InvivoGen) were added to the cultures. Simultaneously, cells were treated with either the STAT3 inhibitor C188-9 (Sigma-Aldrich; stock 100 mg/mL, working concentrations: 0, 5, 10, 15, and 20 µM) or the STAT5 inhibitor STAT5-IN-1 (MedChemExpress; stock 20 mg/mL, working concentrations: 50, 100, 150, and 200 µM) to assess their effects on B cell proliferation. A DMSO vehicle control was included only in the STAT5-IN-1 group due to its higher stock concentration in DMSO. All cultures were maintained at 37°C in a humidified incubator with 5% CO₂ for 4 days. After incubation, the frequency of FcRL5⁺CD27⁺ B cells were evaluated by flow cytometry. Flow Cytometry Analysis Cells were collected and transferred to flow cytometry tubes. PBS containing 3% FBS was used as the staining buffer. For surface marker staining, 100 µL of antibody cocktail at working concentrations was added to each tube, and cells were incubated for 30 minutes at 4°C in the dark. The antibodies used included: Live/Dead (FVS780, BD, 565388), CD3 (BD, 555916), CD19 (BD, 740968, 566396), FcRL5 (Invitrogen, 50-3078-42; Miltenyi, 130-127-304), CD27 (BD, 566450, 563327), CD38 (BD, 563151), CD21 (BD, 563163), CD138 (BD, 566050), IgG (BD, 561298), IgD (BioLegend, 987902), CCR6 (BD, 565173), CXCR3 (BD, 562452), CXCR5 (BD, 562747), CD62L (BD, 563808), HLA-DR (BD, 555558), CD69 (BD, 557745), and CD11c (BD, 563930). For intracellular cytokine staining, cells were stimulated with Leukocyte Activation Cocktail (BD, 550583) at 37°C with 5% CO₂ for 5 hours. After stimulation, cells were centrifuged at 1500 rpm for 5 minutes at 4°C, resuspended in staining buffer, and subjected to surface marker staining as described above. Cells were then fixed and permeabilized using BD Cytofix/Cytoperm solution, incubated at 4°C for 20 minutes in the dark, and washed with Perm/Wash buffer. Intracellular cytokine antibodies were then added and incubated at 4°C in the dark for 30 minutes. The antibodies included: GM-CSF (BD, 554507), GZMB (BD, 560213), IL-4 (BD, 560672), IFN-γ (BD, 564039), IL-17A (BD, 562933), and TNF-α (BD, 340512). For phospho-protein detection, the BD Phospho-Flow staining kit was used. After surface marker staining, cells were fixed with 1× TFP Fix/Perm Buffer and incubated at 4°C for 50 minutes in the dark. Cells were then washed with TFP Perm/Wash Buffer, incubated with pre-chilled Perm Buffer III on ice for 20 minutes in the dark, and stained with phospho-specific antibodies including pSTAT3 (BioLegend, 698907) and pSTAT5 (BD, 612567) for 50 minutes at 4°C in the dark. After all staining procedures, cells were washed once with 1× PBS and resuspended in 200 µL of 1× PBS. Flow cytometric analysis was performed using a BD LSRFortessa™ X-20 or Cytek® Northern Lights™ cytometer. Data were analyzed using FlowJo software (version 10.8.1). RNA Sequencing (RNA-Seq) To evaluate the changes in total B cells before and after stimulation with CpG in combination with IL-15, CD19⁺ B cells were isolated using CD19 magnetic microbeads (Miltenyi Biotec, Cat# 130-050-301). Total RNA was extracted from the sorted B cells using the TRIzol method, and RNA-Seq libraries were constructed. After PCR amplification, library quality was assessed using the Agilent 2100 Bioanalyzer. Libraries that passed quality control were subjected to paired-end sequencing (PE150) on the Illumina NovaSeq 6000 platform, with a target sequencing depth of 30–50 million reads per sample. All library construction, sequencing, and bioinformatics analysis were performed by OBiO Technology (Shanghai, China). Bioinformatics analysis of raw sequencing data included quality control, alignment to the reference genome, and identification of differentially expressed genes (DEGs). DEGs were defined as those with an adjusted P value (padj) 0.07. Subsequent analyses included Gene Ontology (GO) enrichment analysis and Gene Set Enrichment Analysis (GSEA). Statistical Analysis Statistical analysis was conducted using GraphPad Prism version 10.1.1. Data distribution and variance homogeneity were assessed by Shapiro–Wilk and Levene’s tests. Parametric or non-parametric tests were selected accordingly. Unpaired t tests or Mann–Whitney tests were used for two-group comparisons, while one-way ANOVA with Dunnett’s or Kruskal–Wallis with Dunn’s post-hoc tests were applied for multiple groups. Paired data were analyzed using paired t tests or Wilcoxon signed-rank tests, as appropriate. For multifactorial comparisons, two-way ANOVA followed by suitable corrections was used. Statistical significance was defined as p < 0.05. Results Hepatic Expression of FcRL5 in AIH Patients and Its Clinical Correlation with AIH To investigate the expression of FcRL5 in the liver, we performed immunohistochemical staining for FcRL5 in liver tissues from patients with AIH (n = 51), CHB (n = 16), NASH (n = 7), and HC (n = 6) (Table 1 ). We found that FcRL5 + cells were significantly enriched in the portal areas and lobular inflammatory regions of AIH patients ( p < 0.0001) (Fig. 1 A). Notably, although FcRL5 + cells were also detected in the NASH ( p = 0.0011), CHB ( p = 0.0005), and healthy control groups, their numbers were markedly lower compared to those in the AIH group (Fig. 1 B). Table 1 Clinical characteristics of patients with AIH, NASH, CHB, and healthy controls. Autoimmune hepatitis (n = 51) Nonalcoholic steatohepatitis (n = 7) Chronic hepatitis B (n = 16) Healthy control (n = 6) female/male 42/9 4/3 6/10 5/1 age(year) 52.24 ± 1.64 40.29 ± 5.05 41.38 ± 2.8 39.17 ± 3.32 TB(µmol/L) 17.69 ± 2.26 11.47 ± 1.63 13.59 ± 1.74 12.87 ± 1.14 DB(µmol/L) 9.09 ± 1.80 3.49 ± 0.40 4.56 ± 0.50 4.02 ± 0.27 AKP(U/L) 83.02 ± 3.82 65.57 ± 5.80 90.25 ± 5.90 77.67 ± 11.15 GGT(U/L) 64.72 ± 10.07 73.00 ± 24.89 59.64 ± 22.30 89.00 ± 25.59 ALT(U/L) 119.4 ± 49.82 98.43 ± 22.20 63.25 ± 20.12 21.00 ± 3.92 AST(U/L) 63.05 ± 14.23 53.14 ± 10.80 41.54 ± 2.52 18.17 ± 2.95 Albumin(g/L) 40.29 ± 0.63 44.93 ± 0.93 41.30 ± 0.83 45.10 ± 1.69 IgA(g/L) 2.51 ± 0.13 2.09 ± 0.52 2.44 ± 0.64 NA IgM(g/L) 1.52 ± 0.10 2.07 ± 0.50 1.97 ± 0.40 NA IgG(g/L) 13.96 ± 0.42 14.08 ± 1.82 12.24 ± 1.15 NA Data are shown as mean ± SEM. To further explore the potential relationship between FcRL5 expression and disease progression in AIH, we analyzed the correlation between the number of FcRL5 + cells and the histological grades of hepatic inflammation and fibrosis stage, as well as various clinical biochemical parameters. The results demonstrated a significant positive correlation between the number of FcRL5 + cells and both the degree of hepatic inflammation (r = 0.4546, p = 0.0008) and fibrosis stage (r = 0.4592, p = 0.0007) in AIH patients (Fig. 1 C). Additionally, analysis of clinical biochemical indicators showed that FcRL5 expression positively correlated with serum levels of albumin (ALB) (r = -0.3676, p = 0.008), total bilirubin (TB) (r = 0.2814 p = 0.0455), direct bilirubin (DB) (r = 0.3217, p = 0.0213), and immunoglobulin G (IgG) (r = 0.3131, p = 0.0253) (Fig. 1 D). Given that FcRL5 is primarily expressed on B cells, we further characterized the phenotype of FcRL5 + cells through immunofluorescence co-localization analysis. The results showed that the majority of FcRL5 + cells co-localized with the B cell marker CD19 in AIH liver tissues (Fig. 2 A), and a subset of these cells also co-localized with the classical memory B cell marker CD27 (Fig. 2 B). However, co-localization with the plasma cell-specific marker CD138 was not observed (Fig. 2 C). Multiplex immunofluorescence further demonstrated that FcRL5 was predominantly expressed in CD19⁺CD27⁺ memory B cells (Fig. 2 D). Phenotypic and Functional Analysis of FcRL5⁺CD27⁺ Memory B Cells in the Liver of AIH Patients To confirm that FcRL5 is primarily expressed in CD19⁺CD27⁺ memory B cells, we analyzed hepatic immune cells from AIH patients. Within the B cell gate, a distinct cluster of FcRL5⁺ cells were observed (Fig. 3 A). Cytometric t-distributed stochastic neighbour embedding (t-SNE) flow cytometry data analysis identified four B cell subsets based on CD27 and CD38 expression: memory B cells (CD27⁺CD38⁻), naïve B cells (CD27⁻CD38⁻), pre-germinal center B cells (CD27⁻CD38⁺), and plasma cells/plasmablasts (CD27⁺CD38⁺). Notably, the region with high FcRL5 expression was predominantly located within the memory B cell cluster (Fig. 3 B). Consistent with this finding, FcRL5⁺ cells exhibited high levels of CD21, CD27, and IgG, while expressing low levels of CD38, CD138, and IgD (Fig. 3 B). When comparing AIH patients to HCs, the proportion of FcRL5⁺CD27⁺ cells among B cells was significantly increased in the AIH group ( p = 0.0233) (Fig. 3 C). Furthermore, a comparison between peripheral blood and liver immune cells from AIH patients revealed a marked enrichment of FcRL5⁺CD27⁺ B cells in the liver ( p = 0.0167) (Fig. 3 D). To further investigate the phenotypic features of hepatic FcRL5⁺CD27⁺ B cells in AIH patients, we analyzed the expression of several functional markers. These cells showed significantly elevated expression of CXCR3 ( p = 0.0110) and CD11c ( p = 0.0037). Conversely, CD69 and CD62L expression was relatively lower but without statistical significance. Importantly, FcRL5⁺CD27⁺ B cells demonstrated robust secretion of GM-CSF ( p = 0.0218), and also showed a trend toward increased production of pro-inflammatory cytokines IFN-γ and TNF-α (Fig. 3 E). Taken together, these findings suggest that FcRL5⁺CD27⁺ B cells possess liver-homing potential and represent a pro-inflammatory subset characterized by active cytokine secretion. Dynamic Changes in FcRL5⁺ Cells During Repeat Liver Biopsies in AIH Patients To further investigate the dynamic changes of FcRL5⁺ cells during disease progression and their potential clinical relevance, we collected paired liver biopsy samples from AIH patients(n = 15) who underwent a second liver biopsy. Compared to the first biopsy, both hepatic inflammation grade (Fig. 4 A) and fibrosis stage (Fig. 4 B) were generally reduced at the time of the second biopsy, indicating clinical remission following treatment with glucocorticoid. Immunohistochemical analysis revealed that both the staining intensity and number of FcRL5⁺ cells were higher in the first biopsy samples than in the second ones ( p = 0.0012) (Fig. 4 D). Quantitative analysis confirmed a significant decrease in FcRL5⁺ cell numbers in the second biopsies (Fig. 4 C). To determine whether the change in FcRL5⁺ cell abundance is associated with treatment outcomes, we further compared the number of FcRL5⁺ cells in the initial biopsies between patients who achieved histological remission and those who did not. Although the non-remission group showed a higher average number of FcRL5⁺ cells than the remission group, the difference did not reach statistical significance ( p = 0.9451) (Fig. 4 E). In Vitro Induction and Phenotypic Characterization of FcRL5⁺CD27⁺ B Cells Interleukin-15 (IL-15), a crucial homeostatic cytokine, plays a pivotal role in sustaining the survival and functional integrity of tissue-resident immune cells. To further explore the immunophenotypic signature of FcRL5⁺CD27⁺ B cells, we cultured peripheral B cells in vitro using CpG combined with or without IL-15 stimulation (Fig. 5 A). After four days of stimulation, combined stimulation with CpG and IL-15 doubled the proportion of FcRL5⁺CD27⁺ B cells (approximately 29%) compared to CpG stimulation alone ( p < 0.0001) (Fig. 5 B), and the mean fluorescence intensity (MFI) of FcRL5 in total B cells was significantly increased ( p < 0.0001) (Fig. 5 C). Phenotypic analysis by flow cytometry showed that FcRL5⁺CD27⁺ B cells expressed significantly higher levels of CD11c ( p = 0.0349) and CD62L ( p = 0.0118) compared to other B cell subsets, with a downward trend in CXCR5. Cytokine profiling further demonstrated elevated levels of granzyme B (GZMB) ( p = 0.0459), with increasing trends in IFN-γ and GM-CSF, and a significant decrease in IL-4 ( p = 0.0217) (Fig. 5 D). These findings indicate that in vitro–induced FcRL5⁺CD27⁺ B cells display a pronounced pro-inflammatory phenotype. To assess molecular changes pre- and post-induction, we performed RNA sequencing (RNA-Seq) on sorted CD19⁺ B cells. The volcano plot illustrated the distribution of differentially expressed genes following induction (Fig. 5 E). Heatmap analysis revealed notable upregulation of the following gene categories: memory-related genes (CD27, CD38); B cell activation markers (FAS, CD80, CD86); chemokines and chemokine receptors (CXCR3, CCR5, ITGAE, CXCL9, CXCL10); cytokine secretion genes (CSF2 [GM-CSF], GZMB); and components of the JAK-STAT signaling pathway (JAK3, STAT3), along with the transcription factor TBX21 (Fig. 5 F). Functional Characteristics of FcRL5⁺CD27⁺ B Cells and Regulation via the JAK-STAT Signaling Pathway To elucidate the functional characteristics and regulatory mechanisms of FcRL5⁺CD27⁺ B cells, we conducted GO enrichment and GSEA. GO analysis indicated significant enrichment in biological processes such as “response to cytokine” and “immune system process” after induction (Fig. 6 A). Consistently, GSEA showed significant upregulation of gene sets related to B cell activation, cytokine regulation, lymphocyte migration, and the JAK-STAT signaling pathway in the induced group (Fig. 6 B). These findings are in line with our earlier gene expression analysis (heatmap), which showed marked upregulation of JAK3 and STAT3. To confirm activation of the JAK-STAT pathway, we assessed phosphorylation levels of STAT3 and STAT5 via flow cytometry. Compared to the control group, phosphorylated STAT5 was significantly increased in total B cells following induction ( p = 0.0005) (Fig. 6 C). Subset analysis revealed that FcRL5⁺CD27⁺ B cells exhibited significantly higher phosphorylation of both STAT3 ( p = 0.0007) and STAT5 ( p = 0.0084) compared to other B cell subsets (Fig. 6 D). To further clarify the regulatory role of the JAK-STAT pathway in FcRL5⁺CD27⁺ B cells, we treated PBMCs with the STAT3-specific inhibitor C188-9 and the STAT5-specific inhibitor STAT5-IN-1. Under both culture systems the frequency of FcRL5⁺CD27⁺ B cells was significantly reduced in a dose-dependent manner ( p < 0.0001) (Figs. 6 E, 6 F). Discussion Memory B cells serve as direct precursors to antibody-secreting plasma cells and play pivotal roles in various autoimmune diseases ( 24 ). For instance, in systemic lupus erythematosus (SLE), memory B cells enhance autoimmune responses via a positive feedback loop involving Toll-like receptor signaling and the CD40L pathway ( 25 ). Similarly, in immune thrombocytopenic purpura, the activation of CD95⁺ memory B cells exacerbates autoimmune responses ( 26 ). Notably, FcRL5⁺ T-bet⁺ memory B cells exhibit transcriptional profiles reminiscent of effector CD8⁺ T cells, and FcRL5⁺ memory B cells derived from tonsils are more prone to differentiate into antibody-secreting cells in vitro ( 27 ). In healthy donors, FcRL5⁺ cells are primarily enriched in the peripheral blood within atypical CD21⁻/ lo CD27⁻ tissue-like memory B cell populations ( 28 ). Our study revealed a significant intrahepatic enrichment of FcRL5⁺ cells in patients with AIH, primarily localized to CD19⁺CD27⁺ memory B cells. The frequencies of intrahepatic FcRL5⁺ cells were substantially higher than those observed in peripheral blood, suggestive of a tissue-resident phenotype. Furthermore, FcRL5⁺ cell abundance was positively correlated with hepatic inflammation, fibrosis, and clinical biochemical markers such as IgG, DB, and ALB, indicating a close association with disease severity. Research on tissue-resident memory B (B RM ) cells. provides valuable insights into the biological features of FcRL5⁺ cells. For example, lung-resident B RM cells established post-influenza infection highly express CD69, CCR6, and CXCR3 ( 29 ), while studies on local respiratory immunity highlight the essential role of CXCR3 in the localization and functional maintenance of B RM cells in the lung mucosa ( 30 ). In the gut, the co-expression of CD45RB and CD69 has been identified as a hallmark of tissue-resident memory B cells ( 31 ). Similarly, our analysis revealed that intrahepatic FcRL5⁺CD27⁺ B cells in AIH patients display a distinct phenotype characterized by high expression of CXCR3 and CD11c, along with robust secretion of GM-CSF. Longitudinal analysis of paired liver biopsy samples from AIH patients before and after treatment revealed a significant reduction in FcRL5⁺ cells following disease remission. Although the mean abundance of FcRL5⁺ cells was higher in the non-remission group at the time of initial biopsy, the difference did not reach statistical significance, suggesting that FcRL5⁺ cells may reflect disease activity to some extent, but their value as a prognostic marker requires further validation. At the molecular level, Toll-like receptor 9 (TLR9) signaling activated by CpG is a potent stimulator of B cell proliferation and differentiation ( 32 , 33 ). Previous studies have shown that stimulation with FCRL3, FCRL4, or FCRL5 alone does not induce B cell responses; instead, their function relies on co-engagement with the B cell receptor or TLR9 ( 34 ). IL-15 may play a dual role in AIH. Clinically, serum IL-15 levels and the number of IL-15⁺ B cells are positively correlated with ALT levels in patients ( 35 ). Conversely, in a ConA-induced murine model of AIH, IL-15 protects against liver injury by reducing IL-4, IL-5, and TNF-α production by NKT cells ( 36 ). Our previous work demonstrated that an IL-15 plus TGF-β protocol could successfully induce CD8⁺ T RM cells. in vitro, resembling those found in AIH livers ( 3 ). In the current study, we used in vitro stimulation with CpG and IL-15, combined with RNA-Seq analysis, to investigate the properties of FcRL5⁺CD27⁺ B cells. The induced cells exhibited pronounced pro-inflammatory characteristics, including high secretion of IL-4 and GZMB, as well as elevated CD11c expression. These features partially mirrored those of liver-derived FcRL5⁺CD27⁺ B cells, suggesting that the in vitro model may, to some extent, recapitulate their functional state in vivo. Nevertheless, discrepancies in the expression of CXCR5 and CD62L between in vitro-induced and liver-derived cells may be attributable to differences in culture conditions or the surrounding microenvironment. RNA-Seq further revealed that the induced B cells exhibited significant upregulation of genes involved in B cell activation, cytokine regulation, lymphocyte migration, and the JAK-STAT signaling pathway, including marked increases in JAK3 and STAT3 expression. The JAK-STAT pathway, a downstream effector of IL-15 signaling ( 37 ), plays a crucial role in regulating B cell function. Specifically, STAT3 is essential for the activation of human memory B cells ( 38 ), while STAT5 is implicated in the activation of germinal center B cells, governing proliferation and differentiation into memory B cells ( 39 ). These findings align with our flow cytometry data showing high levels of phosphorylated STAT3 and STAT5. Importantly, the proliferation of FcRL5⁺CD27⁺ B cells was significantly inhibited by selective STAT3 and STAT5 inhibitors, suggesting that the JAK-STAT pathway may contribute to AIH pathogenesis by regulating the proliferation and function of this B cell subset. This study has several limitations. We did not evaluate whether FcRL5⁺CD27⁺ B cells directly contribute to liver injury through the secretion of pro-inflammatory mediators. Additionally, the effects of JAK-STAT inhibition on these cells were not validated in animal models. Differences between the in vitro induction system and the in vivo environment also limit the interpretation of some findings. Future studies employing AIH animal models or single-cell sequencing approaches are warranted to further elucidate the precise roles and mechanisms of FcRL5⁺CD27⁺ B cells in AIH pathogenesis. Conclusions This study identifies FcRL5⁺CD27⁺ memory B cells as key players in the pathogenesis of AIH. We demonstrated that FcRL5⁺ cells are significantly enriched in the livers of AIH patients and display a pro-inflammatory phenotype, characterized by high expression of CXCR3 and CD11c, as well as robust secretion of GM-CSF. Longitudinal analysis showed that the frequency of FcRL5⁺ cells declined notably following clinical remission, suggesting their potential as biomarkers of disease activity. Furthermore, in vitro experiments and RNA sequencing revealed that STAT3 and STAT5 signaling plays a pivotal role in regulating the proliferation of FcRL5⁺CD27⁺ B cells. These findings not only deepen our understanding of B cell–mediated mechanisms in AIH but also provide a potential foundation for the development of FcRL5-targeted immunotherapies. Abbreviations AIH: Autoimmune Hepatitis ALB: Albumin atMBCs: Atypical Memory B Cells CHB: Chronic Hepatitis B DAB: 3,3'-Diaminobenzidine DB: Direct Bilirubin DEGs: Differentially Expressed Genes EDTA: Ethylenediaminetetraacetic Acid FBS: Fetal Bovine Serum FcRL5: Fc Receptor-Like 5 GO: Gene Ontology GPA: Granulomatosis with Polyangiitis GSEA: Gene Set Enrichment Analysis HRP: Horseradish Peroxidase Ig: Immunoglobulin IgG: Immunoglobulin G IL-15: Interleukin-15 MBCs: Memory B Cells MFI: Mean Fluorescence Intensity MPA: Microscopic Polyangiitis NASH: Non-Alcoholic Steatohepatitis PBMCs: Peripheral Blood Mononuclear Cells PBS: Phosphate-Buffered Saline PE150: Paired-End 150 RNA-seq: RNA Sequencing SLA: Soluble Liver Antigen SLE: Systemic Lupus Erythematosus TB: Total Bilirubin TBST: Tris-buffered Saline with Tween TRMs: Tissue-Resident Memory T Cells padj: Adjusted P Value Declarations Ethics approval and consent to participate. This study strictly adheres to the principles outlined in the and the ethical guidelines of China. It has been approved by the relevant ethics committee. All patients and healthy donors participating in this study provided written informed consent, explicitly acknowledging that their peripheral blood and liver tissues would be used for scientific research. They were fully informed about the nature, purpose, potential risks, and benefits of the study. Consent for publication Not applicable Availability of data and materials The original data applied in this research are accessible from the corresponding author. Competing interests The authors declare that they have no competing interests in this section. Funding This work was supported by the National Natural Science Foundation of China grants (#82070581 and 81770564 to Qixia Wang) and Hospital-pharma Integration Project on Innovation Coordination (NO. SHDC2022CRT002 to Xiong Ma). Authors' contributions QW, ZY, and XM were responsible for project design, research supervision, and manuscript review and revision; YL and XP were responsible for experimental design, execution, and data analysis; CG contributed to manuscript writing, RNA sequencing data analysis, and data verification; HW, YZ, and QX provided technical support, performed data analysis, and contributed to result validation, respectively. All authors made significant contributions to the conduct of the study and the completion of the manuscript. Acknowledgements We thank OBiO Technology (Shanghai, China) for providing technical support in RNA library construction, sequencing, and bioinformatics analysis. References Muratori L, Lohse AW, Lenzi M. Diagnosis and management of autoimmune hepatitis. BMJ (clin Res Ed,). 2023 Feb 6;380:e070201. Taylor SA, Assis DN, Mack CL. The Contribution of B Cells in Autoimmune Liver Diseases. Semin Liver Dis. 2019 Nov;39(4):422–31. 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Clin Immunol (orlando Fla,). 2009 Feb;130(2):199–212. Nellore A, Zumaquero E, Scharer CD, Fucile CF, Tipton CM, King RG, et al. A transcriptionally distinct subset of influenza-specific effector memory B cells predicts long-lived antibody responses to vaccination in humans. Immunity. 2023 Apr;56(4):847-863.e8. Li H, Borrego F, Nagata S, Tolnay M. Fc receptor-like 5 expression distinguishes two distinct subsets of human circulating tissue-like memory B cells. J Immunol (baltim Md,: 1950). 2016 May 15;196(10):4064–74. Tan HX, Juno JA, Esterbauer R, Kelly HG, Wragg KM, Konstandopoulos P, et al. Lung-resident memory B cells established after pulmonary influenza infection display distinct transcriptional and phenotypic profiles. Sci Immunol. 2022 Jan 28;7(67):eabf5314. Oh JE, Song E, Moriyama M, Wong P, Zhang S, Jiang R, et al. Intranasal priming induces local lung-resident B cell populations that secrete protective mucosal antiviral IgA. Sci Immunol. 2021 Dec 10;6(66):eabj5129. Weisel NM, Weisel FJ, Farber DL, Borghesi LA, Shen Y, Ma W, et al. Comprehensive analyses of B-cell compartments across the human body reveal novel subsets and a gut-resident memory phenotype. Blood. 2020 Dec 10;136(24):2774–85. Wen L, Zhang B, Wu X, Liu R, Fan H, Han L, et al. Toll-like receptors 7 and 9 regulate the proliferation and differentiation of B cells in systemic lupus erythematosus. Front Immunol. 2023;14:1093208. Kumar V. Toll-like receptors in adaptive immunity. In: Kumar V, editor. Toll-like Receptors in Health and Disease [Internet]. Cham: Springer International Publishing; 2022 [cited 2025 Apr 11]. p. 95–131. Available from: https://doi.org/10.1007/164_2021_543 Tolnay M. Lymphocytes sense antibodies through human FCRL proteins: Emerging roles in mucosal immunity. J Leukoc Biol. 2022 Feb;111(2):477–87. Fujimori S, Chu PS, Teratani T, Harada Y, Suzuki T, Amiya T, et al. IL-15-producing splenic B cells play pathogenic roles in the development of autoimmune hepatitis. JHEP Rep. 2023 Jul;5(7):100757. Li B, Sun R, Wei H, Gao B, Tian Z. Interleukin-15 prevents concanavalin A-induced liver injury in mice via NKT cell-dependent mechanism. Hepatology. 2006 Jun;43(6):1211–9. Hu X, Li J, Fu M, Zhao X, Wang W. The JAK/STAT signaling pathway: from bench to clinic. Signal Transduct Target Ther. 2021 Nov 26;6(1):402. Huang W, de Vries C, Sharma RK, Wangriatisak K, Chatzidionysiou K, Malmström V, et al. JAK inhibitors and B cell function: a comparative study of their impact on plasma cell differentiation, cytokine production, and naïve B cell activation. Eur J Immunol. 2025 Mar;55(3):e202451437. Scheeren FA, Naspetti M, Diehl S, Schotte R, Nagasawa M, Wijnands E, et al. STAT5 regulates the self-renewal capacity and differentiation of human memory B cells and controls bcl-6 expression. Nat Immunol. 2005 Mar;6(3):303–13. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6509177","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":452326896,"identity":"308b5061-eb64-4df0-9036-70d4454523fd","order_by":0,"name":"Yuyang Liu","email":"","orcid":"","institution":"Shanghai Jiao Tong University School of Medicine Affiliated Renji Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yuyang","middleName":"","lastName":"Liu","suffix":""},{"id":452326897,"identity":"ff106b5c-4e76-49c8-a959-4b5544c86ed3","order_by":1,"name":"Xiting Pu","email":"","orcid":"","institution":"Shanghai Jiao Tong University School of Medicine Affiliated Renji Hospital","correspondingAuthor":false,"prefix":"","firstName":"Xiting","middleName":"","lastName":"Pu","suffix":""},{"id":452326898,"identity":"a1fa582b-80df-4956-b742-8789bdefcf24","order_by":2,"name":"Chengcen Guo","email":"","orcid":"","institution":"Shanghai Jiao Tong University School of Medicine Affiliated Renji Hospital","correspondingAuthor":false,"prefix":"","firstName":"Chengcen","middleName":"","lastName":"Guo","suffix":""},{"id":452326899,"identity":"f6e29f10-e877-41fd-84e7-ec942b8126ca","order_by":3,"name":"Haoyu Wen","email":"","orcid":"","institution":"Shanghai Jiao Tong University School of Medicine Affiliated Renji Hospital","correspondingAuthor":false,"prefix":"","firstName":"Haoyu","middleName":"","lastName":"Wen","suffix":""},{"id":452326900,"identity":"5e6f501a-085e-4fdb-b66d-778a551e7dd7","order_by":4,"name":"Yudong Zhao","email":"","orcid":"","institution":"Shanghai Jiao Tong University School of Medicine Affiliated 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Hospital","correspondingAuthor":false,"prefix":"","firstName":"Xiong","middleName":"","lastName":"Ma","suffix":""},{"id":452326904,"identity":"c8e94fc3-8e24-4f87-bc04-085c1c8005ad","order_by":8,"name":"Zhengrui You","email":"","orcid":"","institution":"Shanghai Jiao Tong University School of Medicine Affiliated Renji Hospital","correspondingAuthor":false,"prefix":"","firstName":"Zhengrui","middleName":"","lastName":"You","suffix":""},{"id":452326905,"identity":"90d47be4-0d52-446c-b4a9-766a912c1bb2","order_by":9,"name":"Qixia Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3ElEQVRIie3PPQrCQBCG4RFBmw3pZIKCVxgJ+ANCrpIgWMU+lQSU2HgARcErWFpOCGiz9oKNIlgLtipqKYK7pcW+1RTfUwyAyfSn8SVC4S0yfh16InecyGaFsBCkE6lH8q6VRG1C4WbWUGNPm7COooCiUR5f2IqhapdYQeS5iyhQtObbJTsrqE1n/m9S33XWSC8E3FtyTYJPeyUJEvTpTcIDB4kW6WTEPgrahcCpDvHkOTjGjMIZrymNJap/cUYhZfdH37OLg9P1FrWrdllBAD4HqJp/E5PJZDJ99wSi3UuywPn3hQAAAABJRU5ErkJggg==","orcid":"","institution":"Shanghai Jiao Tong University School of Medicine Affiliated Renji Hospital","correspondingAuthor":true,"prefix":"","firstName":"Qixia","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2025-04-23 05:50:49","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6509177/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6509177/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":82562097,"identity":"2e6e3e02-a615-4695-9e9d-5326b1f5dae2","added_by":"auto","created_at":"2025-05-13 01:42:05","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2850600,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFcRL5 is highly expressed in AIH livers and correlates with disease severity.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A–B) Representative IHC images and quantification per high-power field (HPF) of FcRL5⁺ cells (brown) in liver samples from AIH (n=51), NASH (n=7), CHB (n=16), and HC (n=6) groups. (C) Correlation of intrahepatic FcRL5⁺ cell counts with inflammation grade and fibrosis stage in AIH patients. (D) Associations between FcRL5 expression and serum ALB, TB, DB, and IgG levels. Data are shown as mean ± SEM. *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ****p \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"FIGURE1.png","url":"https://assets-eu.researchsquare.com/files/rs-6509177/v1/e18acecaad9b91de3bd3ed67.png"},{"id":82560338,"identity":"4c9bf292-8bbf-49cc-96c6-bfbb83803ad8","added_by":"auto","created_at":"2025-05-13 01:34:05","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1806334,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFcRL5 localizes predominantly to CD27⁺ memory B cells in AIH livers.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A–C) Immunofluorescence staining showing FcRL5 co-localized separately with CD19, CD27, or CD138 in serial liver sections from AIH patients. (D) Multicolor staining demonstrating co-expression of FcRL5, CD19, and CD27 within the same tissue context.\u003c/p\u003e","description":"","filename":"FIGURE2.png","url":"https://assets-eu.researchsquare.com/files/rs-6509177/v1/16010ef7cd853b5235a1ad78.png"},{"id":82560339,"identity":"d5dcfdbe-b953-4a2a-9f4e-685b678aa64c","added_by":"auto","created_at":"2025-05-13 01:34:05","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":843280,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhenotypic and functional features of intrahepatic FcRL5⁺CD27⁺B cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Flow cytometry gating strategy identifying FcRL5⁺ B cell subsets in AIH livers. (B) Automated optimized parameters for T-distributed stochastic neighbour embedding (opt-SNE) graphs from flow cytometric analysis (n=3) highlights FcRL5 enrichment in CD27⁺CD38⁻memory B cells. (C) Comparison of FcRL5⁺CD27⁺B cell frequencies in AIH (n=3) and HC (n=4). (D) Enrichment of FcRL5⁺CD27⁺B cells in liver (n=3) versus blood of AIH (n=7) patients. (E) Expression of CXCR3, CD11c, and GM-CSF secretion analyzed by flow cytometry. Data are shown as mean ± SEM. *p \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"FIGURE3.png","url":"https://assets-eu.researchsquare.com/files/rs-6509177/v1/f8b57691abe0b2c0a0530d5b.png"},{"id":82560342,"identity":"5dd21f75-d7ca-4de0-8875-97ebe1349cb1","added_by":"auto","created_at":"2025-05-13 01:34:05","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":3245887,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDynamic changes in FcRL5⁺ cells during AIH progression.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A–B) Inflammation grades and fibrosis stages at baseline (n=15) and follow-up (n=15) liver biopsy in AIH patients. (C) Representative IHC images and quantification of FcRL5⁺ cells at both time points. (D) Significant reduction in FcRL5⁺ cells after remission. (E) Higher FcRL5⁺cell counts at baseline in non-remission (n=3) vs. remission (n=12) group. Data are shown as mean ± SEM. *p \u0026lt; 0.05, **p \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"FIGURE4.png","url":"https://assets-eu.researchsquare.com/files/rs-6509177/v1/075fdcea62c58a9ca7fa509c.png"},{"id":82562096,"identity":"2b2be5df-6b3e-435d-8f64-ab3cc5c3ce98","added_by":"auto","created_at":"2025-05-13 01:42:05","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":576546,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIn vitro induction and molecular features of FcRL5⁺CD27⁺B cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Schematic of the induction protocol using CpG and IL-15. (B) Flow cytometry showing expansion of FcRL5⁺CD27⁺B cells post-induction (n=3 per group). (C) Mean fluorescence intensity of FcRL5 in total B cells (n=6 per group). (D) Analysis of surface markers and cytokine secretion reveals increased CD11c and CXCR3 expression, elevated GZMB, and reduced IL-4 levels (n=3 per group). (E) Volcano plot of differentially expressed genes in total B cells before and after induction. (F) Heatmap showing induction-associated changes in genes related to memory formation, activation, chemotaxis, cytokines, and the JAK-STAT pathway. Data are shown as mean ± SEM. *p \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"FIGURE5.png","url":"https://assets-eu.researchsquare.com/files/rs-6509177/v1/b1309d8d57c55bc4e832b2f8.png"},{"id":82560341,"identity":"6f957aaa-1e79-44d6-a5bf-808f0a354094","added_by":"auto","created_at":"2025-05-13 01:34:05","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":679964,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFunctional features and STAT3/5 regulation of induced FcRL5⁺CD27⁺B cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) GO enrichment analysis reveals pathways related to cytokine response and immune activation. (B) GSEA identifies upregulated gene sets linked to B cell activation, cytokine regulation, migration, and JAK-STAT signaling. (C) STAT3/5 phosphorylation in total B cells pre- and post-induction (n=6 per group). (D) Comparison of STAT3/5 phosphorylation in FcRL5⁺CD27⁺vs. other B cell subsets (n=6 per group). (E–F) Dose-dependent suppression of FcRL5⁺CD27⁺ B cells by STAT3 inhibitor C188-9 (E) and STAT5 inhibitor STAT5-IN-1 (F). DMSO vehicle was included only in STAT5-IN-1 conditions (n=3 per group). Data are shown as mean ± SEM. *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ****p \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"FIGURE6.png","url":"https://assets-eu.researchsquare.com/files/rs-6509177/v1/38234ce01efe97d3ac3363e8.png"},{"id":84582370,"identity":"b0849bbb-3e6b-474f-9fa4-2c9f391eef56","added_by":"auto","created_at":"2025-06-13 19:18:56","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":12387492,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6509177/v1/4040dfb9-25aa-4443-a875-54b13706cc71.pdf"}],"financialInterests":"","formattedTitle":"Enrichment and Clinical Relevance of FcRL5⁺CD27⁺ B Cells in Autoimmune Hepatitis","fulltext":[{"header":"Background","content":"\u003cp\u003eAutoimmune hepatitis (AIH) is a chronic inflammatory liver disease characterized by an aberrant immune response in which the immune system erroneously recognizes hepatocytes as foreign antigens, leading to persistent hepatic injury. Key clinical hallmarks of AIH include the presence of circulating autoantibodies, elevated serum IgG levels, and distinctive histopathological features(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Current investigations into the hepatic immune microenvironment of AIH have predominantly focused on CD8⁺ tissue-resident memory T (T\u003csub\u003eRM\u003c/sub\u003e) cells, while the role of B lymphocytes remains underexplored (\u003cspan additionalcitationids=\"CR3 CR4\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). However, accumulating evidence underscores the pivotal role of B cells in the pathogenesis of AIH, as they orchestrate complex immunoregulatory networks (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). Notably, recent studies have demonstrated that SepSecS-specific memory B cells in soluble liver antigen (SLA)-positive AIH patients can activate cognate T cell populations via antigen presentation, initiating and sustaining autoimmune responses (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Clinically, B cell-targeted therapies such as rituximab (an anti-CD20 monoclonal antibody) (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e)and belimumab (an anti-BAFF monoclonal antibody) (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e)have shown therapeutic efficacy in refractory AIH cases, further supporting the central involvement of B cells in disease progression.\u003c/p\u003e \u003cp\u003eFc receptor-like 5 (FcRL5), a member of the immunoglobulin (Ig) superfamily, has attracted attention for its ability to selectively bind intact IgG molecules via multiple Ig-like domains, while discriminating against structurally compromised IgG. This selective recognition suggests that FcRL5 may serve as a receptor for newly secreted, functional IgG (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). In CHB, atypical memory B cells (atMBCs) exhibit a distinct distribution pattern compared to circulating B cells, characterized by preferential accumulation within the liver and elevated expression of FcRL5 (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). Additionally, in systemic lupus erythematosus (SLE), TLR7 and IL-21 signaling pathways promote the accumulation of FcRL5⁺ memory B cells, which subsequently differentiate into autoreactive plasmablasts (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). In chronic infection models in mice, FcRL5 is primarily expressed on germinal center-derived memory B cells(\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). Furthermore, studies on human tonsillar memory B cells have revealed that atypical FCRL4⁻FcRL5⁺ subsets express transcription factors and immunoglobulin genes indicative of plasmacytic differentiation potential, while FCRL4⁺FcRL5⁺ subsets display tissue-resident features(\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). In malaria, FcRL5 is enriched in atMBCs independently of FCRL4, highlighting its functional diversity (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). In summary, FcRL5 exhibits functional diversity across various immunopathological contexts, involving the differentiation, localization, and tissue residency of B cells.\u003c/p\u003e \u003cp\u003eDespite these advances, the role of FcRL5 in AIH remains uncharacterized. To address this gap, our study identifies a distinct population of FcRL5⁺CD27⁺ memory B cells enriched in the liver tissue of AIH patients, revealing both its clinical relevance and functional distinction from other B cell subsets. Moreover, we propose a potential regulatory mechanism mediated by the downstream STAT3/5 signaling pathway of IL-15. These findings not only enhance our understanding of AIH pathogenesis but also offer new avenues for targeted therapeutic intervention.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eLiver Tissue\u003c/h2\u003e \u003cp\u003eHuman liver tissue samples were provided by the Department of Gastroenterology at Renji Hospital. The cohort comprised paraffin-embedded liver sections from 51 cases of AIH, 16 cases of CHB, 7 cases of NASH, and 6 healthy controls (HC) for immunohistochemical analysis. Among the healthy controls, 6 sections were derived from liver transplant donors, while the remaining samples were obtained via liver biopsy. Notably, 15 AIH patients underwent a second liver biopsy, and complete clinical data were collected for all cases. All patients fulfilled the clinical diagnostic criteria for AIH(\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e), CHB(\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e), or NASH(\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). The histological grading of inflammation and fibrosis in AIH patients was evaluated using the Scheuer scoring system(\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e), and patients were further stratified into remission and non-remission groups according to the Histological Activity Index (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). In addition, liver tissues from 3 AIH transplant patients and 4 healthy donors were collected for flow cytometry analysis. All sample collections were conducted in strict compliance with ethical principles and met the ethical review standards of Renji Hospital, with approval from the ethics committee (approval number: KY2021-063-B). All procedures adhered to the principles set forth in the Declaration of Helsinki, ensuring that the rights, safety, and privacy of the subjects were fully protected.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eImmunohistochemistry\u003c/h3\u003e\n\u003cp\u003eLiver tissue sections were paraffin-embedded and subsequently deparaffinized and rehydrated. Antigen retrieval was performed using a microwave-based method with EDTA buffer (pH 9.0). Endogenous peroxidase activity was blocked using 3% hydrogen peroxide solution. Sections were then blocked with 50% goat serum at room temperature for 30 minutes, followed by incubation with primary anti-FcRL5 antibody (Servicebio, GB111823-100, 1:500 dilution) at 4\u0026deg;C overnight. After washing with PBS, sections were incubated with HRP-conjugated secondary antibody (1:500 dilution) at room temperature for 20 minutes. DAB was applied for 30 seconds for color development, followed by hematoxylin counterstaining for 10 seconds. Slides were sealed with neutral resin. FcRL5\u003csup\u003e+\u003c/sup\u003e cells were counted in five randomly selected portal areas under a 400\u0026times; high-power field by three independent observers, and the average value was used for analysis.\u003c/p\u003e\n\u003ch3\u003eImmunofluorescence\u003c/h3\u003e\n\u003cp\u003eLiver tissue sections were paraffin-embedded, deparaffinized, and rehydrated. Antigen retrieval was performed using a microwave-based method with EDTA buffer (pH 9.0). Endogenous peroxidase activity was blocked using 3% hydrogen peroxide solution. Sections were then blocked with 50% donkey serum at room temperature for 30 minutes, followed by incubation with primary antibodies\u0026mdash;anti-FcRL5 (Servicebio, GB111823-100, 1:500 dilution) and anti-CD27 (Abcam, ab268268, 1:200 dilution)\u0026mdash;at 4\u0026deg;C overnight. After washing with PBS, fluorophore-conjugated secondary antibodies (Goat Anti-Rabbit IgG, Abcam, ab150078, 1:200 dilution; Goat Anti-Mouse IgG, Abcam, ab6785, 1:200 dilution) were applied and incubated at room temperature for 30 minutes. Nuclei were counterstained with DAPI, and sections were mounted. Co-localization of target proteins was observed under a laser confocal microscope at 400\u0026times; magnification.\u003c/p\u003e\n\u003ch3\u003eMultiplex Immunofluorescence\u003c/h3\u003e\n\u003cp\u003eWe use Absin four-color Multiplex Immunofluorescence kit (abs50012). Liver tissue sections were paraffin-embedded, deparaffinized, and rehydrated. Antigen retrieval was performed using a microwave-based method with EDTA buffer (pH 9.0). Endogenous peroxidase activity was blocked using 3% hydrogen peroxide solution. Sections were then blocked with 50% goat serum at room temperature for 30 minutes, followed by incubation with the primary antibody against FcRL5 (Servicebio, GB111823-100, 1:500 dilution) at 4\u0026deg;C overnight. After washing with TBST, HRP-conjugated secondary antibody was applied and incubated at room temperature for 20 minutes. Following another TBST wash, 50 \u0026micro;L of TSA amplification reagent (dye 520, dye 570 or dye 650) was added and incubated in the dark at room temperature for 10 minutes. Antigen retrieval was then repeated, followed by the same blocking and incubation procedures as described above for the application of additional primary antibodies, including anti-CD27 (Abcam, ab268268, 1:200 dilution), anti-CD19 (Abcam, ab134114, 1:100 dilution), and anti-CD138 (Abcam, ab128936, 1:4000 dilution). A final round of microwave-based antigen retrieval was performed, and nuclei were counterstained with DAPI. Co-localization of target proteins was visualized using a laser confocal microscope under 400\u0026times; magnification.\u003c/p\u003e\n\u003ch3\u003eIsolation of Human Liver Immune Cells\u003c/h3\u003e\n\u003cp\u003eImmune cells from human liver tissue were isolated using a density gradient centrifugation method (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). Briefly, approximately 30 g of fresh liver tissue was collected and digested in a digestion solution at 37\u0026deg;C in a water bath for 30 minutes. The digested tissue was then homogenized using a tissue dissociator, filtered sequentially through a 70 \u0026micro;m cell strainer, and further ground using a sterile syringe plunger. The resulting cell suspension was resuspended in RPMI 1640 medium (Corning, Manassas, USA) and centrifuged at 50 \u0026times;g for 5 minutes at 4\u0026deg;C to remove liver tissue debris. The supernatant was then centrifuged at 750 \u0026times;g for 10 minutes at 4\u0026deg;C, and the supernatant was discarded. The pellet was resuspended in 30 mL of 33% Percoll solution and carefully overlaid onto 10 mL of 70% Percoll solution (Percoll, Cytiva, Cat# 17-0891-01). The mixture was centrifuged at 900 \u0026times;g for 30 minutes at 4\u0026deg;C with acceleration set to 3 and deceleration set to 0. After centrifugation, the upper layer was discarded, and immune cells were carefully collected from the interphase. Finally, the isolated cells were washed with 1\u0026times; PBS to obtain purified intrahepatic immune cells.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eIsolation of Peripheral Blood Mononuclear Cells (PBMCs)\u003c/h2\u003e \u003cp\u003ePBMCs were isolated using a density gradient centrifugation method. Briefly, fresh peripheral blood from healthy donors was diluted with 1\u0026times; PBS at a ratio of 1:3 and carefully layered over an equal volume of Ficoll solution (Cytiva, Cat# 17144003). The mixture was centrifuged at 400 \u0026times;g for 30 minutes at 20\u0026deg;C with acceleration set to 3 and deceleration set to 0. After centrifugation, the white buffy coat layer containing mononuclear cells was carefully collected. The isolated cells were then washed with 1\u0026times; PBS to obtain purified PBMCs.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eIn Vitro Induction and Inhibition Assay of FcRL5⁺CD27⁺ B Cells\u003c/h3\u003e\n\u003cp\u003ePBMCs were seeded in 48-well plates at a density of 3 \u0026times; 10⁵ cells/mL in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 U/mL penicillin, 100 \u0026micro;g/mL streptomycin, and 50 mM 2-mercaptoethanol. To induce FcRL5⁺CD27⁺ B cells, recombinant human IL-15 (50 ng/mL, R\u0026amp;D Systems) and CpG ODN 2006 (1 \u0026micro;g/mL, InvivoGen) were added to the cultures. Simultaneously, cells were treated with either the STAT3 inhibitor C188-9 (Sigma-Aldrich; stock 100 mg/mL, working concentrations: 0, 5, 10, 15, and 20 \u0026micro;M) or the STAT5 inhibitor STAT5-IN-1 (MedChemExpress; stock 20 mg/mL, working concentrations: 50, 100, 150, and 200 \u0026micro;M) to assess their effects on B cell proliferation. A DMSO vehicle control was included only in the STAT5-IN-1 group due to its higher stock concentration in DMSO. All cultures were maintained at 37\u0026deg;C in a humidified incubator with 5% CO₂ for 4 days. After incubation, the frequency of FcRL5⁺CD27⁺ B cells were evaluated by flow cytometry.\u003c/p\u003e\n\u003ch3\u003eFlow Cytometry Analysis\u003c/h3\u003e\n\u003cp\u003eCells were collected and transferred to flow cytometry tubes. PBS containing 3% FBS was used as the staining buffer. For surface marker staining, 100 \u0026micro;L of antibody cocktail at working concentrations was added to each tube, and cells were incubated for 30 minutes at 4\u0026deg;C in the dark. The antibodies used included: Live/Dead (FVS780, BD, 565388), CD3 (BD, 555916), CD19 (BD, 740968, 566396), FcRL5 (Invitrogen, 50-3078-42; Miltenyi, 130-127-304), CD27 (BD, 566450, 563327), CD38 (BD, 563151), CD21 (BD, 563163), CD138 (BD, 566050), IgG (BD, 561298), IgD (BioLegend, 987902), CCR6 (BD, 565173), CXCR3 (BD, 562452), CXCR5 (BD, 562747), CD62L (BD, 563808), HLA-DR (BD, 555558), CD69 (BD, 557745), and CD11c (BD, 563930).\u003c/p\u003e \u003cp\u003eFor intracellular cytokine staining, cells were stimulated with Leukocyte Activation Cocktail (BD, 550583) at 37\u0026deg;C with 5% CO₂ for 5 hours. After stimulation, cells were centrifuged at 1500 rpm for 5 minutes at 4\u0026deg;C, resuspended in staining buffer, and subjected to surface marker staining as described above. Cells were then fixed and permeabilized using BD Cytofix/Cytoperm solution, incubated at 4\u0026deg;C for 20 minutes in the dark, and washed with Perm/Wash buffer. Intracellular cytokine antibodies were then added and incubated at 4\u0026deg;C in the dark for 30 minutes. The antibodies included: GM-CSF (BD, 554507), GZMB (BD, 560213), IL-4 (BD, 560672), IFN-γ (BD, 564039), IL-17A (BD, 562933), and TNF-α (BD, 340512).\u003c/p\u003e \u003cp\u003eFor phospho-protein detection, the BD Phospho-Flow staining kit was used. After surface marker staining, cells were fixed with 1\u0026times; TFP Fix/Perm Buffer and incubated at 4\u0026deg;C for 50 minutes in the dark. Cells were then washed with TFP Perm/Wash Buffer, incubated with pre-chilled Perm Buffer III on ice for 20 minutes in the dark, and stained with phospho-specific antibodies including pSTAT3 (BioLegend, 698907) and pSTAT5 (BD, 612567) for 50 minutes at 4\u0026deg;C in the dark.\u003c/p\u003e \u003cp\u003eAfter all staining procedures, cells were washed once with 1\u0026times; PBS and resuspended in 200 \u0026micro;L of 1\u0026times; PBS. Flow cytometric analysis was performed using a BD LSRFortessa\u0026trade; X-20 or Cytek\u0026reg; Northern Lights\u0026trade; cytometer. Data were analyzed using FlowJo software (version 10.8.1).\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eRNA Sequencing (RNA-Seq)\u003c/h2\u003e \u003cp\u003eTo evaluate the changes in total B cells before and after stimulation with CpG in combination with IL-15, CD19⁺ B cells were isolated using CD19 magnetic microbeads (Miltenyi Biotec, Cat# 130-050-301). Total RNA was extracted from the sorted B cells using the TRIzol method, and RNA-Seq libraries were constructed. After PCR amplification, library quality was assessed using the Agilent 2100 Bioanalyzer. Libraries that passed quality control were subjected to paired-end sequencing (PE150) on the Illumina NovaSeq 6000 platform, with a target sequencing depth of 30\u0026ndash;50\u0026nbsp;million reads per sample. All library construction, sequencing, and bioinformatics analysis were performed by OBiO Technology (Shanghai, China). Bioinformatics analysis of raw sequencing data included quality control, alignment to the reference genome, and identification of differentially expressed genes (DEGs). DEGs were defined as those with \u003cem\u003ean adjusted P value (padj)\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e and |log₂ (fold change)| \u0026gt; 0.07. Subsequent analyses included Gene Ontology (GO) enrichment analysis and Gene Set Enrichment Analysis (GSEA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eStatistical analysis was conducted using GraphPad Prism version 10.1.1. Data distribution and variance homogeneity were assessed by Shapiro\u0026ndash;Wilk and Levene\u0026rsquo;s tests. Parametric or non-parametric tests were selected accordingly. Unpaired t tests or Mann\u0026ndash;Whitney tests were used for two-group comparisons, while one-way ANOVA with Dunnett\u0026rsquo;s or Kruskal\u0026ndash;Wallis with Dunn\u0026rsquo;s post-hoc tests were applied for multiple groups. Paired data were analyzed using paired t tests or Wilcoxon signed-rank tests, as appropriate. For multifactorial comparisons, two-way ANOVA followed by suitable corrections was used. Statistical significance was defined as p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eHepatic Expression of FcRL5 in AIH Patients and Its Clinical Correlation with AIH\u003c/h2\u003e \u003cp\u003eTo investigate the expression of FcRL5 in the liver, we performed immunohistochemical staining for FcRL5 in liver tissues from patients with AIH (n\u0026thinsp;=\u0026thinsp;51), CHB (n\u0026thinsp;=\u0026thinsp;16), NASH (n\u0026thinsp;=\u0026thinsp;7), and HC (n\u0026thinsp;=\u0026thinsp;6) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). We found that FcRL5\u003csup\u003e+\u003c/sup\u003e cells were significantly enriched in the portal areas and lobular inflammatory regions of AIH patients (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Notably, although FcRL5\u003csup\u003e+\u003c/sup\u003e cells were also detected in the NASH (\u003cem\u003ep\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.0011), CHB (\u003cem\u003ep\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.0005), and healthy control groups, their numbers were markedly lower compared to those in the AIH group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eClinical characteristics of patients with AIH, NASH, CHB, and healthy controls.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAutoimmune hepatitis\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;51)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNonalcoholic steatohepatitis\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;7)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eChronic hepatitis B\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;16)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eHealthy control\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;6)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003efemale/male\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e42/9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4/3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6/10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5/1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eage(year)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e52.24\u0026thinsp;\u0026plusmn;\u0026thinsp;1.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e40.29\u0026thinsp;\u0026plusmn;\u0026thinsp;5.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e41.38\u0026thinsp;\u0026plusmn;\u0026thinsp;2.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e39.17\u0026thinsp;\u0026plusmn;\u0026thinsp;3.32\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTB(\u0026micro;mol/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e17.69\u0026thinsp;\u0026plusmn;\u0026thinsp;2.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e11.47\u0026thinsp;\u0026plusmn;\u0026thinsp;1.63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e13.59\u0026thinsp;\u0026plusmn;\u0026thinsp;1.74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e12.87\u0026thinsp;\u0026plusmn;\u0026thinsp;1.14\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDB(\u0026micro;mol/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e9.09\u0026thinsp;\u0026plusmn;\u0026thinsp;1.80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.49\u0026thinsp;\u0026plusmn;\u0026thinsp;0.40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.56\u0026thinsp;\u0026plusmn;\u0026thinsp;0.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.27\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAKP(U/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e83.02\u0026thinsp;\u0026plusmn;\u0026thinsp;3.82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e65.57\u0026thinsp;\u0026plusmn;\u0026thinsp;5.80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e90.25\u0026thinsp;\u0026plusmn;\u0026thinsp;5.90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e77.67\u0026thinsp;\u0026plusmn;\u0026thinsp;11.15\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGGT(U/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e64.72\u0026thinsp;\u0026plusmn;\u0026thinsp;10.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e73.00\u0026thinsp;\u0026plusmn;\u0026thinsp;24.89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e59.64\u0026thinsp;\u0026plusmn;\u0026thinsp;22.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e89.00\u0026thinsp;\u0026plusmn;\u0026thinsp;25.59\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eALT(U/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e119.4\u0026thinsp;\u0026plusmn;\u0026thinsp;49.82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e98.43\u0026thinsp;\u0026plusmn;\u0026thinsp;22.20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e63.25\u0026thinsp;\u0026plusmn;\u0026thinsp;20.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e21.00\u0026thinsp;\u0026plusmn;\u0026thinsp;3.92\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAST(U/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e63.05\u0026thinsp;\u0026plusmn;\u0026thinsp;14.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e53.14\u0026thinsp;\u0026plusmn;\u0026thinsp;10.80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e41.54\u0026thinsp;\u0026plusmn;\u0026thinsp;2.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e18.17\u0026thinsp;\u0026plusmn;\u0026thinsp;2.95\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAlbumin(g/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e40.29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e44.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e41.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e45.10\u0026thinsp;\u0026plusmn;\u0026thinsp;1.69\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIgA(g/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.09\u0026thinsp;\u0026plusmn;\u0026thinsp;0.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.44\u0026thinsp;\u0026plusmn;\u0026thinsp;0.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIgM(g/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.97\u0026thinsp;\u0026plusmn;\u0026thinsp;0.40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIgG(g/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e13.96\u0026thinsp;\u0026plusmn;\u0026thinsp;0.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e14.08\u0026thinsp;\u0026plusmn;\u0026thinsp;1.82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e12.24\u0026thinsp;\u0026plusmn;\u0026thinsp;1.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e \u003cp\u003eData are shown as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further explore the potential relationship between FcRL5 expression and disease progression in AIH, we analyzed the correlation between the number of FcRL5\u003csup\u003e+\u003c/sup\u003e cells and the histological grades of hepatic inflammation and fibrosis stage, as well as various clinical biochemical parameters. The results demonstrated a significant positive correlation between the number of FcRL5\u003csup\u003e+\u003c/sup\u003e cells and both the degree of hepatic inflammation (r\u0026thinsp;=\u0026thinsp;0.4546, \u003cem\u003ep\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.0008) and fibrosis stage (r\u0026thinsp;=\u0026thinsp;0.4592, \u003cem\u003ep\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.0007) in AIH patients (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Additionally, analysis of clinical biochemical indicators showed that FcRL5 expression positively correlated with serum levels of albumin (ALB) (r = -0.3676, \u003cem\u003ep\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.008), total bilirubin (TB) (r\u0026thinsp;=\u0026thinsp;0.2814 \u003cem\u003ep\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.0455), direct bilirubin (DB) (r\u0026thinsp;=\u0026thinsp;0.3217, \u003cem\u003ep\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.0213), and immunoglobulin G (IgG) (r\u0026thinsp;=\u0026thinsp;0.3131, \u003cem\u003ep\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.0253) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003eGiven that FcRL5 is primarily expressed on B cells, we further characterized the phenotype of FcRL5\u003csup\u003e+\u003c/sup\u003e cells through immunofluorescence co-localization analysis. The results showed that the majority of FcRL5\u003csup\u003e+\u003c/sup\u003e cells co-localized with the B cell marker CD19 in AIH liver tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), and a subset of these cells also co-localized with the classical memory B cell marker CD27 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). However, co-localization with the plasma cell-specific marker CD138 was not observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Multiplex immunofluorescence further demonstrated that FcRL5 was predominantly expressed in CD19⁺CD27⁺ memory B cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003ePhenotypic and Functional Analysis of FcRL5⁺CD27⁺ Memory B Cells in the Liver of AIH Patients\u003c/h2\u003e \u003cp\u003eTo confirm that FcRL5 is primarily expressed in CD19⁺CD27⁺ memory B cells, we analyzed hepatic immune cells from AIH patients. Within the B cell gate, a distinct cluster of FcRL5⁺ cells were observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Cytometric t-distributed stochastic neighbour embedding (t-SNE) flow cytometry data analysis identified four B cell subsets based on CD27 and CD38 expression: memory B cells (CD27⁺CD38⁻), na\u0026iuml;ve B cells (CD27⁻CD38⁻), pre-germinal center B cells (CD27⁻CD38⁺), and plasma cells/plasmablasts (CD27⁺CD38⁺). Notably, the region with high FcRL5 expression was predominantly located within the memory B cell cluster (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Consistent with this finding, FcRL5⁺ cells exhibited high levels of CD21, CD27, and IgG, while expressing low levels of CD38, CD138, and IgD (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). When comparing AIH patients to HCs, the proportion of FcRL5⁺CD27⁺ cells among B cells was significantly increased in the AIH group (\u003cem\u003ep\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.0233) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Furthermore, a comparison between peripheral blood and liver immune cells from AIH patients revealed a marked enrichment of FcRL5⁺CD27⁺ B cells in the liver (\u003cem\u003ep\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.0167) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further investigate the phenotypic features of hepatic FcRL5⁺CD27⁺ B cells in AIH patients, we analyzed the expression of several functional markers. These cells showed significantly elevated expression of CXCR3 (\u003cem\u003ep\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.0110) and CD11c (\u003cem\u003ep\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.0037). Conversely, CD69 and CD62L expression was relatively lower but without statistical significance. Importantly, FcRL5⁺CD27⁺ B cells demonstrated robust secretion of GM-CSF (\u003cem\u003ep\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.0218), and also showed a trend toward increased production of pro-inflammatory cytokines IFN-γ and TNF-α (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). Taken together, these findings suggest that FcRL5⁺CD27⁺ B cells possess liver-homing potential and represent a pro-inflammatory subset characterized by active cytokine secretion.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eDynamic Changes in FcRL5⁺ Cells During Repeat Liver Biopsies in AIH Patients\u003c/h2\u003e \u003cp\u003eTo further investigate the dynamic changes of FcRL5⁺ cells during disease progression and their potential clinical relevance, we collected paired liver biopsy samples from AIH patients(n\u0026thinsp;=\u0026thinsp;15) who underwent a second liver biopsy. Compared to the first biopsy, both hepatic inflammation grade (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA) and fibrosis stage (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB) were generally reduced at the time of the second biopsy, indicating clinical remission following treatment with glucocorticoid. Immunohistochemical analysis revealed that both the staining intensity and number of FcRL5⁺ cells were higher in the first biopsy samples than in the second ones (\u003cem\u003ep\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.0012) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). Quantitative analysis confirmed a significant decrease in FcRL5⁺ cell numbers in the second biopsies (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo determine whether the change in FcRL5⁺ cell abundance is associated with treatment outcomes, we further compared the number of FcRL5⁺ cells in the initial biopsies between patients who achieved histological remission and those who did not. Although the non-remission group showed a higher average number of FcRL5⁺ cells than the remission group, the difference did not reach statistical significance (\u003cem\u003ep\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.9451) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eIn Vitro Induction and Phenotypic Characterization of FcRL5⁺CD27⁺ B Cells\u003c/h2\u003e \u003cp\u003eInterleukin-15 (IL-15), a crucial homeostatic cytokine, plays a pivotal role in sustaining the survival and functional integrity of tissue-resident immune cells. To further explore the immunophenotypic signature of FcRL5⁺CD27⁺ B cells, we cultured peripheral B cells in vitro using CpG combined with or without IL-15 stimulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). After four days of stimulation, combined stimulation with CpG and IL-15 doubled the proportion of FcRL5⁺CD27⁺ B cells (approximately 29%) compared to CpG stimulation alone (\u003cem\u003ep\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.0001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB), and the mean fluorescence intensity (MFI) of FcRL5 in total B cells was significantly increased (\u003cem\u003ep\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.0001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ePhenotypic analysis by flow cytometry showed that FcRL5⁺CD27⁺ B cells expressed significantly higher levels of CD11c (\u003cem\u003ep\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.0349) and CD62L (\u003cem\u003ep\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.0118) compared to other B cell subsets, with a downward trend in CXCR5. Cytokine profiling further demonstrated elevated levels of granzyme B (GZMB) (\u003cem\u003ep\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.0459), with increasing trends in IFN-γ and GM-CSF, and a significant decrease in IL-4 (\u003cem\u003ep\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.0217) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). These findings indicate that in vitro\u0026ndash;induced FcRL5⁺CD27⁺ B cells display a pronounced pro-inflammatory phenotype.\u003c/p\u003e \u003cp\u003eTo assess molecular changes pre- and post-induction, we performed RNA sequencing (RNA-Seq) on sorted CD19⁺ B cells. The volcano plot illustrated the distribution of differentially expressed genes following induction (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). Heatmap analysis revealed notable upregulation of the following gene categories: memory-related genes (CD27, CD38); B cell activation markers (FAS, CD80, CD86); chemokines and chemokine receptors (CXCR3, CCR5, ITGAE, CXCL9, CXCL10); cytokine secretion genes (CSF2 [GM-CSF], GZMB); and components of the JAK-STAT signaling pathway (JAK3, STAT3), along with the transcription factor TBX21 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eFunctional Characteristics of FcRL5⁺CD27⁺ B Cells and Regulation via the JAK-STAT Signaling Pathway\u003c/h2\u003e \u003cp\u003eTo elucidate the functional characteristics and regulatory mechanisms of FcRL5⁺CD27⁺ B cells, we conducted GO enrichment and GSEA. GO analysis indicated significant enrichment in biological processes such as \u0026ldquo;response to cytokine\u0026rdquo; and \u0026ldquo;immune system process\u0026rdquo; after induction (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Consistently, GSEA showed significant upregulation of gene sets related to B cell activation, cytokine regulation, lymphocyte migration, and the JAK-STAT signaling pathway in the induced group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). These findings are in line with our earlier gene expression analysis (heatmap), which showed marked upregulation of JAK3 and STAT3.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo confirm activation of the JAK-STAT pathway, we assessed phosphorylation levels of STAT3 and STAT5 via flow cytometry. Compared to the control group, phosphorylated STAT5 was significantly increased in total B cells following induction (\u003cem\u003ep\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.0005) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). Subset analysis revealed that FcRL5⁺CD27⁺ B cells exhibited significantly higher phosphorylation of both STAT3 (\u003cem\u003ep\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.0007) and STAT5 (\u003cem\u003ep\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.0084) compared to other B cell subsets (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003eTo further clarify the regulatory role of the JAK-STAT pathway in FcRL5⁺CD27⁺ B cells, we treated PBMCs with the STAT3-specific inhibitor C188-9 and the STAT5-specific inhibitor STAT5-IN-1. Under both culture systems the frequency of FcRL5⁺CD27⁺ B cells was significantly reduced in a dose-dependent manner (\u003cem\u003ep\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.0001) (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF).\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eMemory B cells serve as direct precursors to antibody-secreting plasma cells and play pivotal roles in various autoimmune diseases (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). For instance, in systemic lupus erythematosus (SLE), memory B cells enhance autoimmune responses via a positive feedback loop involving Toll-like receptor signaling and the CD40L pathway (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). Similarly, in immune thrombocytopenic purpura, the activation of CD95⁺ memory B cells exacerbates autoimmune responses (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). Notably, FcRL5⁺ T-bet⁺ memory B cells exhibit transcriptional profiles reminiscent of effector CD8⁺ T cells, and FcRL5⁺ memory B cells derived from tonsils are more prone to differentiate into antibody-secreting cells in vitro (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). In healthy donors, FcRL5⁺ cells are primarily enriched in the peripheral blood within atypical CD21⁻/\u003csup\u003elo\u003c/sup\u003eCD27⁻ tissue-like memory B cell populations (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOur study revealed a significant intrahepatic enrichment of FcRL5⁺ cells in patients with AIH, primarily localized to CD19⁺CD27⁺ memory B cells. The frequencies of intrahepatic FcRL5⁺ cells were substantially higher than those observed in peripheral blood, suggestive of a tissue-resident phenotype. Furthermore, FcRL5⁺ cell abundance was positively correlated with hepatic inflammation, fibrosis, and clinical biochemical markers such as IgG, DB, and ALB, indicating a close association with disease severity.\u003c/p\u003e \u003cp\u003eResearch on tissue-resident memory B (B\u003csub\u003eRM\u003c/sub\u003e) cells. provides valuable insights into the biological features of FcRL5⁺ cells. For example, lung-resident B\u003csub\u003eRM\u003c/sub\u003e cells established post-influenza infection highly express CD69, CCR6, and CXCR3 (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e), while studies on local respiratory immunity highlight the essential role of CXCR3 in the localization and functional maintenance of B\u003csub\u003eRM\u003c/sub\u003e cells in the lung mucosa (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). In the gut, the co-expression of CD45RB and CD69 has been identified as a hallmark of tissue-resident memory B cells (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). Similarly, our analysis revealed that intrahepatic FcRL5⁺CD27⁺ B cells in AIH patients display a distinct phenotype characterized by high expression of CXCR3 and CD11c, along with robust secretion of GM-CSF. Longitudinal analysis of paired liver biopsy samples from AIH patients before and after treatment revealed a significant reduction in FcRL5⁺ cells following disease remission. Although the mean abundance of FcRL5⁺ cells was higher in the non-remission group at the time of initial biopsy, the difference did not reach statistical significance, suggesting that FcRL5⁺ cells may reflect disease activity to some extent, but their value as a prognostic marker requires further validation.\u003c/p\u003e \u003cp\u003eAt the molecular level, Toll-like receptor 9 (TLR9) signaling activated by CpG is a potent stimulator of B cell proliferation and differentiation (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). Previous studies have shown that stimulation with FCRL3, FCRL4, or FCRL5 alone does not induce B cell responses; instead, their function relies on co-engagement with the B cell receptor or TLR9 (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e). IL-15 may play a dual role in AIH. Clinically, serum IL-15 levels and the number of IL-15⁺ B cells are positively correlated with ALT levels in patients (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e). Conversely, in a ConA-induced murine model of AIH, IL-15 protects against liver injury by reducing IL-4, IL-5, and TNF-α production by NKT cells (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). Our previous work demonstrated that an IL-15 plus TGF-β protocol could successfully induce CD8⁺ T\u003csub\u003eRM\u003c/sub\u003e cells. in vitro, resembling those found in AIH livers (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn the current study, we used in vitro stimulation with CpG and IL-15, combined with RNA-Seq analysis, to investigate the properties of FcRL5⁺CD27⁺ B cells. The induced cells exhibited pronounced pro-inflammatory characteristics, including high secretion of IL-4 and GZMB, as well as elevated CD11c expression. These features partially mirrored those of liver-derived FcRL5⁺CD27⁺ B cells, suggesting that the in vitro model may, to some extent, recapitulate their functional state in vivo. Nevertheless, discrepancies in the expression of CXCR5 and CD62L between in vitro-induced and liver-derived cells may be attributable to differences in culture conditions or the surrounding microenvironment. RNA-Seq further revealed that the induced B cells exhibited significant upregulation of genes involved in B cell activation, cytokine regulation, lymphocyte migration, and the JAK-STAT signaling pathway, including marked increases in JAK3 and STAT3 expression.\u003c/p\u003e \u003cp\u003eThe JAK-STAT pathway, a downstream effector of IL-15 signaling (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e), plays a crucial role in regulating B cell function. Specifically, STAT3 is essential for the activation of human memory B cells (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e), while STAT5 is implicated in the activation of germinal center B cells, governing proliferation and differentiation into memory B cells (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e). These findings align with our flow cytometry data showing high levels of phosphorylated STAT3 and STAT5. Importantly, the proliferation of FcRL5⁺CD27⁺ B cells was significantly inhibited by selective STAT3 and STAT5 inhibitors, suggesting that the JAK-STAT pathway may contribute to AIH pathogenesis by regulating the proliferation and function of this B cell subset.\u003c/p\u003e \u003cp\u003eThis study has several limitations. We did not evaluate whether FcRL5⁺CD27⁺ B cells directly contribute to liver injury through the secretion of pro-inflammatory mediators. Additionally, the effects of JAK-STAT inhibition on these cells were not validated in animal models. Differences between the in vitro induction system and the in vivo environment also limit the interpretation of some findings. Future studies employing AIH animal models or single-cell sequencing approaches are warranted to further elucidate the precise roles and mechanisms of FcRL5⁺CD27⁺ B cells in AIH pathogenesis.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis study identifies FcRL5⁺CD27⁺ memory B cells as key players in the pathogenesis of AIH. We demonstrated that FcRL5⁺ cells are significantly enriched in the livers of AIH patients and display a pro-inflammatory phenotype, characterized by high expression of CXCR3 and CD11c, as well as robust secretion of GM-CSF. Longitudinal analysis showed that the frequency of FcRL5⁺ cells declined notably following clinical remission, suggesting their potential as biomarkers of disease activity. Furthermore, in vitro experiments and RNA sequencing revealed that STAT3 and STAT5 signaling plays a pivotal role in regulating the proliferation of FcRL5⁺CD27⁺ B cells. These findings not only deepen our understanding of B cell\u0026ndash;mediated mechanisms in AIH but also provide a potential foundation for the development of FcRL5-targeted immunotherapies.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eAIH: Autoimmune Hepatitis\u003c/p\u003e\n\u003cp\u003eALB: Albumin\u003c/p\u003e\n\u003cp\u003eatMBCs: Atypical Memory B Cells\u003c/p\u003e\n\u003cp\u003eCHB: Chronic Hepatitis B\u003c/p\u003e\n\u003cp\u003eDAB: 3,3'-Diaminobenzidine\u003c/p\u003e\n\u003cp\u003eDB: Direct Bilirubin\u003c/p\u003e\n\u003cp\u003eDEGs: Differentially Expressed Genes\u003c/p\u003e\n\u003cp\u003eEDTA: Ethylenediaminetetraacetic Acid\u003c/p\u003e\n\u003cp\u003eFBS: Fetal Bovine Serum\u003c/p\u003e\n\u003cp\u003eFcRL5: Fc Receptor-Like 5\u003c/p\u003e\n\u003cp\u003eGO: Gene Ontology\u003c/p\u003e\n\u003cp\u003eGPA: Granulomatosis with Polyangiitis\u003c/p\u003e\n\u003cp\u003eGSEA: Gene Set Enrichment Analysis\u003c/p\u003e\n\u003cp\u003eHRP: Horseradish Peroxidase\u003c/p\u003e\n\u003cp\u003eIg: Immunoglobulin\u003c/p\u003e\n\u003cp\u003eIgG: Immunoglobulin G\u003c/p\u003e\n\u003cp\u003eIL-15: Interleukin-15\u003c/p\u003e\n\u003cp\u003eMBCs: Memory B Cells\u003c/p\u003e\n\u003cp\u003eMFI: Mean Fluorescence Intensity\u003c/p\u003e\n\u003cp\u003eMPA: Microscopic Polyangiitis\u003c/p\u003e\n\u003cp\u003eNASH: Non-Alcoholic Steatohepatitis\u003c/p\u003e\n\u003cp\u003ePBMCs: Peripheral Blood Mononuclear Cells\u003c/p\u003e\n\u003cp\u003ePBS: Phosphate-Buffered Saline\u003c/p\u003e\n\u003cp\u003ePE150: Paired-End 150\u003c/p\u003e\n\u003cp\u003eRNA-seq: RNA Sequencing\u003c/p\u003e\n\u003cp\u003eSLA: Soluble Liver Antigen\u003c/p\u003e\n\u003cp\u003eSLE: Systemic Lupus Erythematosus\u003c/p\u003e\n\u003cp\u003eTB: Total Bilirubin\u003c/p\u003e\n\u003cp\u003eTBST: Tris-buffered Saline with Tween\u003c/p\u003e\n\u003cp\u003eTRMs: Tissue-Resident Memory T Cells\u003c/p\u003e\n\u003cp\u003epadj: Adjusted P Value\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study strictly adheres to the principles outlined in the \u0026lt;Declaration of Helsinki\u0026gt; and the ethical guidelines of China. It has been approved by the relevant ethics committee. All patients and healthy donors participating in this study provided written informed consent, explicitly acknowledging that their peripheral blood and liver tissues would be used for scientific research. They were fully informed about the nature, purpose, potential risks, and benefits of the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003cbr\u003e\u003c/strong\u003eThe original data applied in this research are accessible from the corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests in this section.\u003cbr\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China grants (#82070581 and 81770564 to Qixia Wang) and Hospital-pharma Integration Project on Innovation Coordination (NO. SHDC2022CRT002 to Xiong Ma).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' contributions \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eQW, ZY, and XM were responsible for project design, research supervision, and manuscript review and revision; YL and XP were responsible for experimental design, execution, and data analysis; CG contributed to manuscript writing, RNA sequencing data analysis, and data verification; HW, YZ, and QX provided technical support, performed data analysis, and contributed to result validation, respectively. All authors made significant contributions to the conduct of the study and the completion of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank OBiO Technology (Shanghai, China) for providing technical support in RNA library construction, sequencing, and bioinformatics analysis.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eMuratori L, Lohse AW, Lenzi M. Diagnosis and management of autoimmune hepatitis. BMJ (clin Res Ed,). 2023 Feb 6;380:e070201. \u003c/li\u003e\n\u003cli\u003eTaylor SA, Assis DN, Mack CL. The Contribution of B Cells in Autoimmune Liver Diseases. Semin Liver Dis. 2019 Nov;39(4):422\u0026ndash;31. \u003c/li\u003e\n\u003cli\u003eYou Z, Li Y, Wang Q, Zhao Z, Li Y, Qian Q, et al. The Clinical Significance of Hepatic CD69+CD103+CD8+ Resident‐Memory T Cells in Autoimmune Hepatitis. 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IL-15-producing splenic B cells play pathogenic roles in the development of autoimmune hepatitis. JHEP Rep. 2023 Jul;5(7):100757. \u003c/li\u003e\n\u003cli\u003eLi B, Sun R, Wei H, Gao B, Tian Z. Interleukin-15 prevents concanavalin A-induced liver injury in mice via NKT cell-dependent mechanism. Hepatology. 2006 Jun;43(6):1211\u0026ndash;9. \u003c/li\u003e\n\u003cli\u003eHu X, Li J, Fu M, Zhao X, Wang W. The JAK/STAT signaling pathway: from bench to clinic. Signal Transduct Target Ther. 2021 Nov 26;6(1):402. \u003c/li\u003e\n\u003cli\u003eHuang W, de Vries C, Sharma RK, Wangriatisak K, Chatzidionysiou K, Malmstr\u0026ouml;m V, et al. JAK inhibitors and B cell function: a comparative study of their impact on plasma cell differentiation, cytokine production, and na\u0026iuml;ve B cell activation. Eur J Immunol. 2025 Mar;55(3):e202451437. \u003c/li\u003e\n\u003cli\u003eScheeren FA, Naspetti M, Diehl S, Schotte R, Nagasawa M, Wijnands E, et al. STAT5 regulates the self-renewal capacity and differentiation of human memory B cells and controls bcl-6 expression. Nat Immunol. 2005 Mar;6(3):303\u0026ndash;13. \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Autoimmune hepatitis, FcRL5, memory B cells, JAK-STAT pathway, inflammation, ","lastPublishedDoi":"10.21203/rs.3.rs-6509177/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6509177/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBackground: Dysregulation of B cells has been implicated in the development of autoimmune hepatitis (AIH), yet identification of potentially pathogenic subpopulations remains elusive. Fc receptor-like 5 (FcRL5), a B cell surface marker, represents a potential identifier of pathogenic subsets. This study aimed to investigate the function and immunophenotypic profile of FcRL5⁺B cells in AIH and evaluates their therapeutic targetability.\u003c/p\u003e\n\u003cp\u003eMethods: Immunohistochemistry was performed on liver biopsies from AIH, nonalcoholic steatohepatitis (NASH), chronic hepatitis B (CHB), and healthy controls. Flow cytometry was conducted on liver grafts from AIH patients, healthy donor livers, and peripheral blood samples. FcRL5⁺CD27⁺ B cells were induced in vitro using CpG and IL-15 stimulation. Phenotypic analysis was performed by flow cytometry, and RNA sequencing was conducted on total B cells after induction. STAT3 and STAT5 inhibitors were added at the start of induction to evaluate their effects on cell expansion.\u003c/p\u003e\n\u003cp\u003eResults: FcRL5⁺ cells were significantly enriched in the livers of AIH patients, predominantly within the CD19⁺CD27⁺ memory B cell compartment. These cells exhibited a pro-inflammatory phenotype, characterized by high expression of CXCR3 and CD11c, along with robust secretion of GM-CSF. Longitudinal analysis revealed a marked decrease in intrahepatic FcRL5⁺ cell frequency following clinical remission in AIH patients. RNA sequencing and functional assays indicated that the JAK-STAT pathway regulates the proliferation and function of FcRL5⁺CD27⁺ B cells, with STAT3 and STAT5 phosphorylation playing critical roles.\u003c/p\u003e\n\u003cp\u003eConclusion: This study highlights the importance of FcRL5⁺CD27⁺ memory B cells in the pathogenesis of autoimmune hepatitis and identifies them as promising targets for therapeutic intervention.\u003c/p\u003e","manuscriptTitle":"Enrichment and Clinical Relevance of FcRL5⁺CD27⁺ B Cells in Autoimmune Hepatitis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-13 01:34:00","doi":"10.21203/rs.3.rs-6509177/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b0e2ffe9-65a7-4be9-b418-840f21a2291f","owner":[],"postedDate":"May 13th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-06-13T19:10:42+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-13 01:34:00","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6509177","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6509177","identity":"rs-6509177","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}