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Naive- and Memory-like CD21low B Cell Subsets Share Core Phenotypic and Signaling Characteristics in Systemic Autoimmune Disorders This information is current as of September 29, 2021. Mirjam Freudenhammer, Reinhard E. Voll, Sebastian C. Binder, Baerbel Keller and Klaus Warnatz J Immunol published online 9 September 2020 http://www.jimmunol.org/content/early/2020/09/08/jimmun ol.2000343 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2020 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published September 9, 2020, doi:10.4049/jimmunol.2000343 The Journal of Immunology

Naive- and Memory-like CD21low B Cell Subsets Share Core Phenotypic and Signaling Characteristics in Systemic Autoimmune Disorders

Mirjam Freudenhammer,*,†,‡ Reinhard E. Voll,*,x Sebastian C. Binder,{ Baerbel Keller,*,x,1 and Klaus Warnatz*,x,1

An expansion of CD21low B cells has been described in a variety of diseases associated with persistent immune stimulation as in chronic infection, immunodeﬁciency, or autoimmunity. Different developmental stages of CD21low B cells have been highlighted in speciﬁc diseases; however, a systematic comparison of distribution, phenotype, and signaling capacity of these populations has not yet been performed to delineate the pivotal character of this unusual B cell population. Screening of more than 200 patients with low autoimmune disease demonstrated that the prevalence of patients with expanded CD21 B cells varies between diseases. The Downloaded from expansion was frequent in patients with systemic lupus erythematosus, in which it correlated to relative B cell lymphopenia and duration of disease. Different proportions of distinct developmental stages of CD21low B cells co-occur in nearly all patients with autoimmune disease. Although in most patients, naive-like and CD272 switched memory B cells were the most prominent CD21low subpopulations, there was no detectable association of the pattern with the underlying disease. Despite their distinct developmental stage, all CD21low B cells share a common core phenotype including the increased expression of inhibitory receptors,

associated with an elevated constitutive phosphorylation of proximal signaling molecules downstream of the BCR but impaired http://www.jimmunol.org/ Ca2+ mobilization and NF-kB activation after BCR stimulation. Further, this was accompanied by impaired upregulation of CD69, although CD86 upregulation was preserved. Beyond maturation-associated differences, the common core characteristics of all CD21low B cell populations suggests either a common ancestry or a shared sustained imprint by the environment they originated in. The Journal of Immunology, 2020, 205: 000–000.

he acute stimulation of the immune system induces a cryoglobulinemia (6, 7), or common variable immunodeficiency variety of adaptive changes in the composition, activation, (CVID) with autoimmune manifestations (3, 8) encompassing an and differentiation state of immune cells to properly de- increased percentage of B cells expressing autoreactive BCR entities. T by guest on September 29, 2021 fend the host against invading pathogens and to provide long- It was also found in chronic infections, including HIV (9), malaria lasting memory. Similarly, chronic stimulation as in the context (10), or CMV (11), associated with an increase in pathogen-specific of chronic infections or other inflammatory conditions drives al- B cells. Although the expansion of autoantigen-specific CD21low terations of the immune system. However, these changes often B cells in autoimmune disorders is implying a direct role of these differ in character and may also contribute to secondary devel- cells in autoimmune pathogenesis, the expansion of pathogen- opment of autoimmunity, inflammation, or malignant lympho- specific CD21low B cells in infectious diseases underlines their in- proliferation. In several diseases associated with chronic immune volvement in regular immune responses against certain pathogens. stimulation, the persistent expansion of a B cell subpopulation with The distinct expression profile of CD21low B cells is associated a low expression of CD21 has been identified. This expansion was with the expression of inflammatory-type homing receptors, which described in autoimmune disorders like systemic lupus eryth- favor cell migration to inflamed tissues (2, 7–9, 11). Indeed, ematosus (SLE) (1, 2), rheumatoid arthritis (RA) (3), primary CD21low B cells have been extracted from sites of inflammation, Sjo¨gren syndrome (pSS) (4), anti-neutrophil cytoplasm antibodies– like the synovial fluid of patients with RA or the bronchoalveolar associated vasculitis (AAV) (5), hepatitis C virus (HCV)–associated lavage of CVID patients with interstitial lung disease (8). CD21low

*Department of Rheumatology and Clinical Immunology, Medical Center – University M.F. performed the experiments and drafted the manuscript. B.K. and K.W. con- of Freiburg, Faculty of Medicine, University of Freiburg, 79106 Freiburg, Germany; ceived the original idea and supervised the project. M.F., B.K., and K.W. wrote the †Institute for Immunodeficiency, Center for Chronic Immunodeficiency, Medical manuscript. R.E.V. provided patient cohort and patient information. S.C.B. helped Center - University of Freiburg, Faculty of Medicine, University of Freiburg, with statistical analysis. All authors discussed the results and contributed to the final 79106 Freiburg, Germany; ‡Center for Pediatrics and Adolescent Medicine, Medical version of the manuscript. Center – University of Freiburg, 79106 Freiburg, Germany; xCenter for Chronic Address correspondence and reprint requests to Prof. Klaus Warnatz, Department of Immunodeficiency, Medical Center – University of Freiburg, Faculty of Medicine, { Rheumatology and Clinical Immunology, Medical Center – University of Freiburg, University of Freiburg, 79106 Freiburg, Germany; and Department of Systems Faculty of Medicine, University of Freiburg, Breisacherstrasse. 115, 79106 Freiburg, Immunology and Braunschweig Integrated Centre of Systems Biology, Helmholtz Germany. E-mail address: [email protected] Centre for Infection Research, 38106 Braunschweig, Germany The online version of this article contains supplemental material. 1B.K. and K.W. contributed equally. Abbreviations used in this article: AAV, anti-neutrophil cytoplasm antibodies– ORCIDs: 0000-0002-3208-2747 (M.F.); 0000-0003-1169-1786 (S.C.B.); 0000-0002- associated vasculitis; AF, Alexa Fluor; BV, Brilliant Violet; CTD, connective tissue 1172-865X (K.W.). disease; CVID, common variable immunodeficiency; HCV, hepatitis C virus; HD, Received for publication March 30, 2020. Accepted for publication August 11, 2020. healthy donor; MCTD, mixed CTD; MFI, mean fluorescence intensity; pSS, primary Sjo¨gren syndrome; RA, rheumatoid arthritis; SI, stimulation index; SLE, systemic This work was supported by the Deutsche Forschungsgemeinschaft (Grants TRR130 lupus erythematosus; SSc, systemic sclerosis; UCTD, undifferentiated CTD. P07 to K.W. and TRR130 P12 to R.E.V.) and the Bundesministerium fu¨r Bildung und Forschung (Grant BMBF 01E01303). Copyright Ó 2020 by The American Association of Immunologists, Inc. 0022-1767/20/$37.50

www.jimmunol.org/cgi/doi/10.4049/jimmunol.2000343 2 COMPARING CD21low B CELL SUBSETS IN AUTOIMMUNITY

B cells show a preactivated phenotype with an increased baseline using the FlowJo software package (version 7.6.5; Tree Star, Ashland, OR). low high low low expression of CD86 and a high basal phosphorylation of proteins of CD21 B cells were defined as CD19 CD21 CD38 lymphocytes. the proximal BCR signaling pathway (2, 4, 7, 12, 13). Inhibitory B cell phenotyping and defining subsets proteins like FcRL4 and CD32 (FcgRIIB) are upregulated and have been linked to the restricted differentiation and proliferative and Isolated PBMCs were incubated with unlabeled mouse mAbs against 2+ FcRL4 and Siglec10 and, in a second step, marked with fluorochrome- functional capacity with impaired Ca signaling upon BCR stimu- conjugated anti-mouse IgG. After washing fluorochrome-conjugated lation(3,4,6,8,9,14,15).CD21low B cells are furthermore char- mAbs specific for CD11c, CD19, CD21, CD22, CD27, CD32, CD38, acterized by a high expression of CD95 (Fas). Disease-related Ag CD62L, CD95, IgD, IgM, and IgG were added. Cells were analyzed by flow low hi low low specificities of BCR are enriched among the CD21low subpopulation cytometry. B cells were gated into CD21 (CD19 CD21 CD38 ) and CD21pos (CD19+CD21posCD38low/+) subsets, and for each subset, naive/ (2–4, 9), indicating an Ag-triggered selection and expansion into this naive-like (CD272IgM+), IgM memory (CD27+IgM+), switched memory pool. Lately, a high expression of the transcription factor T-bet has (CD27+IgM2), IgG memory (CD27+IgM2IgG+), CD272 switched mem- been demonstrated for CD21low B cells in different disorders (16–19). ory (CD272IgM2), and CD272 IgG memory (CD272IgM-IgG+) sub- Previous work has investigated CD21low B cells mainly in the populations were identified (for gating strategy, see Supplemental Fig. 1). , context of one disease and analyzed the prevalent specific B cell For analysis, subsets with 50 events were excluded. differentiation state associated with this disorder as determined Measurement of BCR-induced signaling according to the surface expression of the Ig isotype and CD27. 2 + Upon isolation, PBMCs were rested for 2 h at 37˚C to minimize nonspecific Thus, mostly CD27 IgG cells were found in HIV (9) and malaria B cell activation. Cells were stimulated with 15 mg/ml anti-k Ab at 37˚C (10), CD27+IgM+ in HCV-associated mixed cryoglobulinemia, for 7, 10, and 30 min as indicated and subsequently fixed and per- 2 and more naive-like CD27 IgM+IgD+ B cells in RA, CVID (3), meabilized using Cytofix and Perm Buffer III (BD Biosciences). Cells Downloaded from and pSS (4), although there are reports that different CD21low were labeled with mAbs against CD19, CD21, CD38, CD27, IgM, IgG, l, SYK(pY352), PLCg2(pY759), ERK1/2(pThr202/Y204), AKT(pS473), subpopulations may exist within the same patient (3, 5, 9). S6(pS235/236), and IkBa. To define the stimulated and the unstimulated Therefore, we asked the question whether certain autoimmune population, respectively, cells were differentiated in l2 and l+ cells. Upon disorders are associated with the preferential expansion of one gating on CD21low and CD21pos cells, naive, IgM memory, IgG memory, 2 differentiation stage of CD21low B cells and how much the altered and CD27 IgG memory subpopulations were identified by their expression of IgM, IgG, and CD27. For Ca2+ mobilization, loading of PBMCs phenotype, signaling, and activation overlap between the different http://www.jimmunol.org/ low with Indo-1 was performed as described before (15). Cells were stained CD21 populations to gain further insights into the core phe- with Abs against CD19, CD21, CD22, CD27, CD38, IgM, IgG, and l. notype of CD21low B cells. After baseline acquisition for 45 s, 8 mg anti-k was added for BCR stimulation. The following changes in intracellular Ca2+ concentrations were recorded for 7 min. Materials and Methods Study subjects Measurement of BCR-induced upregulation of activation markers EDTA blood was obtained from 218 patients with RA, connective tissue disease (CTD), or AAV seen at the outpatient clinics of the Department of Isolated PBMCs were incubated with 10 mg/ml anti-k for 36 h. Surface Rheumatology and Clinical Immunology, Medical Center, University of molecules were labeled with fluorochrome-conjugated mAbs specific for Freiburg (Freiburg, Germany). Exclusion criteria were a preceding therapy CD19, CD21, CD38, CD27, l, CD86, CD69, and ICAM. To account for by guest on September 29, 2021 with rituximab, a total dose of .5 g cyclophosphamide, or a current potential internalization of BCR, cells were permeabilized (Beckman therapy with cyclophosphamide or .10 mg/d prednisone equivalent. Coulter IntraPrep Permeabilization Reagent) before staining with anti-IgG Control blood was obtained from 24 healthy donors (HD). The study was and anti-IgM mAbs. Analysis and gating were performed as described approved by the ethics committee of the University of Freiburg (FR66/13), above. and all study subjects provided written informed consent before inclusion. Statistics Abs used in this study Data analysis was performed with GraphPad Prism version 7.0 for Windows The following Abs were used in this study: CD11c Alexa Fluor (AF) 700, (GraphPad, La Jolla, CA) and R (20) with libraries ggplot2 (21) and CD19 allophycocyanin, CD19 Brilliant Violet (BV) 421, CD19 BV605, ggiraphExtra (22). Normal distribution was analyzed using D’Agostino– CD21 PE/Cy7, CD27 PerCP/Cy5.5, CD38 PerCP/Cy5.5, IgM BV421, l Pearson normality test followed by paired or unpaired t test, respectively, FITC, and Siglec10 UNLB, all from BioLegend (San Diego, CA); CD19 Wilcoxon matched-pairs test or Mann–Whitney U test for not normally allophycocyanin-H7, CD21 PE/Cy7, CD27 BV605, CD32 PE, CD38 PE- distributed collectives, and Bonferroni–Holm post hoc test for multiple CF594, CD38 V450, CD69 FITC, CD86 PE, CD95 PE, IgG AF700, comparisons (Figs. 2–4). Mixed effect models followed by Holm–Sidak ICAM-1 PE, Akt (pS473) AF488, ERK1/2 (pThr202/Y204) AF647, IkBa PE, multiple comparisons test or Friedman test followed by Dunn multiple PLCg2 (pY759) AF647, SYK (pY352) PE, and S6 (pS235/236) allophyco- comparison test were used for datasets consisting of paired samples cyanin, all obtained from BD Biosciences (San Jose, CA); CD22 PE (Beckman (Supplemental Figs. 2–4) and repeated measures–ANOVA followed by Coulter, Krefeld, Germany); CD62L FITC (ImmunoTech, Marseille, France); Holm–Sidak multiple comparisons test or Kruskal–Wallis test followed by IgD FITC (Southern Biotech, Birmingham, AL); IgG FITC (Dako, Hamburg, Dunn multiple comparison test for unpaired samples (Fig. 1), respectively. Germany); IgM AMCA and IgM Cy-5 (Jackson ImmunoResearch Laborato- Correlations were measured with Spearman rank correlation coefficient ries,WestGrove,PA);l PE (Leinco Technologies, Fenton, MO); FcRL4 (Fig. 1), and a multiple linear regression model was used for analysis of UNLB, a kind gift from M. Cooper (Emory University School of Medicine); more than one variable (Fig. 1). The p values ,0.05 were considered secondary anti-mouse IgG AF647 (Life Technologies, Carlsbad, CA); and significant and depicted as *p , 0.05, **p , 0.01, and ***p , 0.001. anti-k F(ab9)2 for BCR stimulation was obtained from Southern Biotech. Isolation of PBMCs Results Presence of CD21low B cells in patients with rheumatic disease PBMCs were isolated by density-gradient centrifugation using Ficoll-Paque (Pancoll Human; PAN-Biotech, Aidenbach, Germany), according to We screened 218 patients with different autoimmune disorders for standard protocols. thepresenceofCD21low B cells in peripheral blood. In 69 pa- Screening of patients for expansion of CD21low B cells tients (31.7%), we found an increase in the proportion of CD21low Bcellsof.7.3% of all B cells (internal reference Whole blood staining was performed on 50 ml of EDTA blood using values: 1.0–7.3%, referring to the 5th and 95th percentile of OptiLyse C (Beckman Coulter), following the manufacturer’s instructions, 53 healthy controls). These patients are referred to as “CD21low after incubation with mAbs specific for CD19, CD21, CD38, and CD27. low Flow cytometric data were acquired on a LSRFortessa equipped with an patients.” Over 30% of CD21 patients were identified among UV laser (BD Biosciences, Heidelberg, Germany). FACS data were analyzed SLE patients (40.7%), seropositive (35.1%) and seronegative (33.3%) The Journal of Immunology 3

RA patients, patients with AAV (33.3%), and with pSS (31.3%) but Common phenotype of CD21low B cells independent of their less frequently in undifferentiated CTD (UCTD) or mixed CTD developmental stage (MCTD) (21.2%) or systemic sclerosis (SSc) (10.5%) (Table I). To compare characteristic phenotypic features of CD21low B cells, There were no significant differences regarding the percentage of the expression of CD19, CD11c, Siglec10, CD32, FcRL4, CD95, low low CD21 B cells in CD21 patients between the different disease and CD62L was assessed for each CD21low subpopulation and entities (Fig. 1A). their CD21pos counterparts in these 29 autoimmune CD21low pa- CD21low B cells correlate with B cell lymphopenia and tients and 24 HD. The increased expression of CD19, CD11c, duration of disease in SLE Siglec10, CD32, FcRL4, and CD95 as well as decreased expression of CD62L compared with the CD21pos counterparts was Correlations between the proportion of CD21low B cells and dis- common to all subpopulations in the analyzed disease conditions ease features were made for SLE and RA cohorts. We found an (Fig. 2). Interestingly, patient-derived CD21pos B cells often show inverse correlation (r = 20.413 and 20.294) with the frequency of an intermediate phenotype between CD21low B cells of patients total B cells in patients with SLE and RA, respectively (Fig. 1B). and CD21pos B cells of HD. The few CD21low B cells of HD In an additive multiple linear regression model including the displayed similar phenotypic changes like patient-derived CD21low variables age and duration of disease, we further identified an B cells when compared with the respective CD21pos population association between frequency of CD21low B cells and disease (Supplemental Fig. 2A and data not shown). b p duration (coefficient 1 = 0.666, = 0.016) in patients with SLE The mean fluorescence intensity (MFI) of the respective (Fig. 1C). No significant correlations were found between pro- markers, however, differed significantly between the four CD21low

low Downloaded from portion of CD21 B cells and age, sex, disease activity (SLEDAI subpopulations (Supplemental Fig. 3). To determine if these dif- and DAS28), inflammatory markers (C3d and CRP), type of au- ferences just reflect the underlying differentiation stage of the toantibodies (ANA, anti-dsDNA, anti-Ro/La, and rheumatoid B cell subpopulation, we further analyzed the expression patterns factor, anti-CCP) in SLE and RA, respectively, or organ manifes- of surface markers within the respective CD21low and CD21pos tations (arthritis, skin, neurologic manifestation, renal involvement, B cell subsets of patients and HD (Supplemental Figs. 2, 3, as hematologic/immunologic manifestation, serositis, secondary Sjo¨gren CD21pos B cells from patients and HD showed largely similar syndrome) in SLE (data not shown). patterns, we did not include patient’s CD21pos B cells in these http://www.jimmunol.org/ Diverse composition of CD21low subpopulations figures for reasons of clarity). The expression pattern of CD19 (high expression on IgM memory cells), CD11c (high expression Next, the composition of distinct subpopulations within the on switched memory cells), CD95 (high expression on memory CD21low population defined by the expression of CD27, IgM, and especially CD27+ IgG memory cells), and CD62L (high ex- and IgG was determined for 29 autoimmune CD21low patients pression on CD27+ IgG memory cells) was largely similar between (15 patients diagnosed with RA, 7 with SLE, 3 with other patients’ CD21low B cell subsets and CD21pos subpopulations. In CTDs, and 4 with AAV) as well as for 24 HD (for gating contrast, the inhibitory receptors FcRL4, Siglec10, and CD32 were Materials and Methods strategy, see Supplemental Fig. 1 and ). differentially expressed (Supplemental Fig. 3). The increased ex- low by guest on September 29, 2021 All patients had CD21 B cells belonging to all four sub- pression of these markers on CD21pos IgM memory cells when populations, however, with a quite heterogeneous distribution pos low compared with the other CD21 subpopulations is not reflected (Fig. 1D). In most patients, CD21 B cells consisted pre- low 2 among CD21 subpopulations, and the pattern varies for the dif- dominantly of naive-like or CD27 switched memory B cells. ferent receptors (Supplemental Fig. 3). CD32 and Siglec10 are Comparison of the mean percentages of the different subpop- expressed the highest on naive CD21low B cells, whereas FcRL4 pos ulations of the CD21 B cell population of patients with RA expression is higher on switched memory B cells. Very similar or SLE and HD demonstrated the previously reported reduction expression patterns were observed for CD21low subpopulations of of naive B cells in SLE (23) and RA (Fig. 1D). In contrast, in HD (Supplemental Fig. 2A and data not shown). the CD21low population of SLE and RA patients, the CD272 switched memory compartment was expanded, whereas the Increased constitutive signaling of low percentage of naive B cells was comparable between CD21low CD21 B cell subpopulations and CD21pos B cells (Fig. 1F, 1G). There was no detectable Several studies suggested elevated levels of basal phosphorylation of difference in the distribution of CD21low B cell subpopulations signaling molecules downstream of the BCR and impaired sig- between patients below and above 60 y of age (data not shown). naling following BCR engagement in CD21low B cells (2, 13–15).

Table I. Expansion of CD21low B cells in patients with autoimmune diseases

Autoimmune Disease Number of Patients Number of Patients with CD21low .7.3% Proportion of Patients with CD21low .7.3% RA 78 27 34.6 Seropositive RA 57 20 35.1 Seronegative RA 21 7 33.3 CTD 122 36 29.5 SLE 54 22 40.7 pSS 16 5 31.3 UCTD/MCTD 33 7 21.2 Systemic scleroderma (limited/diffuse) 19 2 10.5 AAV 18 6 33.3 GPA 8 2 25.0 Eosinophilic GPA 7 2 28.6 Other AAVs 3 2 66.7 Total autoimmune patients 218 69 31.7 GPA, granulomatosis with polyangiitis. 4 COMPARING CD21low B CELL SUBSETS IN AUTOIMMUNITY Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 1. CD21low B cells in patients with autoimmune disease. (A) Percentage (Median +95% conﬁdence interval) of CD21low B cells of whole B cell count in patients with seropositive (RA+) and seronegative (RA2) RA, SLE, pSS, UCTD/MCTD, or AAVand .7.3% CD21low B cells (CD21low patients) (Kruskal–Wallis test, followed by Dunn multiple comparison test). (B) Correlation of proportion of CD21low B cells with the absolute B cell count in patients with SLE and RA (Spearman rank correlation). (C) Multiple linear regression analysis of association between proportion of CD21low B cells, years since initial diagnosis (ID), and age in patients with SLE. (D) Distribution of naive-like, IgM memory, CD27+ switched memory, and CD272 switched memory B cells in the CD21low and CD21pos B cell compartment in patients with RA, SLE, AAV, or CTDs other than SLE. Each bar represents the composition of CD21pos or CD21low B cell subsets in one single patient. (E–G) Mean values of the proportions of naive-like, IgM memory, CD27+ switched memory, and CD272 switched memory B cell populations in CD21pos B cells of healthy donors (HD), RA, and SLE (D) and CD21pos and CD21low B cells of patients with RA (E) and SLE (F) (repeated measures ANOVA and Tukey multiple comparison tests). *p , 0.05, **p , 0.01, ***p , 0.001.

To address key signaling pathways in the different CD21low B cell compared with CD21pos B cells of patients and HD (Fig. 3B, subpopulations, we stimulated PBMCs of CD21low patients and 3C). This was also true for ERK phosphorylation with the ex- HD with anti-k and stained for l to distinguish between stimulated ception of the IgM memory B cells, whereas for AKT and S6, (l2) and unstimulated (l+) B cells (for gating strategy, see this was only seen in the naive populations (Fig. 3C). Protein Fig. 3A). levels of IkBa in CD21low B cells were relatively comparable Analysis of unstimulated (l+) B cell subsets revealed ele- to CD21pos B cells, with a slight tendency to lower expression vated basal phosphorylation levels of the early signaling mol- (Fig. 3C). These changes were consistent for all investigated ecules SYK and PLCg2inallCD21low B cell subpopulations patient groups, and the observed differences between CD21low The Journal of Immunology 5 Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 2. Phenotype of CD21low B cell subsets. (A) Representative FACS plot of CD21 and CD11c in different B cell subpopulations of a patient with AAV. (B) MFI of CD19, CD11c, Siglec10, CD32, FCRL4, CD95, and CD62L of naive-like, IgM memory (M mem), CD27+ IgG memory (CD27+ G mem), and CD272 IgG memory (CD272 G mem) B cells in patients’ (P) CD21low B cells in comparison with the respective subpopulations of CD21pos B cells in patients and HD. Red, seropositive RA; orange, seronegative RA; blue, SLE; yellow, AAV; purple, Sjo¨gren syndrome; gray, SSc; and green, MCTD/UCTD. Data were analyzed using paired or unpaired t test for normally distributed datasets and Wilcoxon matched pairs or Mann–Whitney test for not normally distributed data, followed by Bonferroni–Holm corrections for multiple testing between the three values within one subpopulation. Error bars indicate mean and SD. *p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001. and CD21pos B cells were also seen in HD-derived cells signaling molecules SYK, PLCg,ERK,andAKT(Fig.4A,4B), (Supplemental Fig. 2B). demonstrating their general BCR-induced signaling potential When comparing the phosphorylation pattern driven by B cell (Fig. 4A, 4B, Supplemental Fig. 4B). Because of the high basal differentiation, in CD21pos B cells, basal phosphorylation was phosphorylation levels in nonstimulated cells, the SI of SYK signiﬁcantly increased for all investigated molecules in IgM and PLCg was signiﬁcantly lower in CD21low B cells compared memory populations, whereas this pattern was not replicated in with the CD21pos compartment (Fig. 4B), indicating a pre- CD21low B cells (Supplemental Fig. 4A). served, however, reduced signaling capacity of CD21low B cells. This disturbed activation of the proximal signaling Reduced signaling capacity of CD21low B cell subpopulations cascade was commonly associated with an abrogated canonical after BCR stimulation NF-kB response (Fig. 4B) and poor increase of the intracellular To describe the activation capacity of the different B cell pop- Ca2+ concentration (Fig. 4C, 4D). Unlike the phosphorylation ulations, we calculated the stimulation index (SI) as the ratio of the response of proximal signaling components, ERK phosphory- 2 MFI of the respective phosphoproteins of l and l+ B cells after lation after BCR stimulation was preserved in naive-like and stimulation with anti-k. Similarly, the degradation of IkBa was IgM memory CD21low B cells but not the IgG memory com- 2 assessed as the ratio of the MFI of IkBa in l and l+ B cells. For partment. Although there was not a clearly altered pattern for Ca2+ signaling strength, the mean peak of the response curve was AKT phosphorylation, S6 phosphorylation was severely decreased compared. in all but the IgM memory CD21low populations when compared All CD21low subpopulations independent of the underlying with HD and NS to patient-derived CD21pos B cells. Interest- disease were capable of increasing the phosphorylation of ingly, for both signaling molecules of the AKT–PI3K–mTOR–S6 6 COMPARING CD21low B CELL SUBSETS IN AUTOIMMUNITY Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 3. Constitutive signaling in CD21low B cell subpopulations. (A) Gating strategy of unstimulated (l+) and stimulated (l2) CD21low and CD21pos B cells. For gating of subpopulations, see Supplemental Fig. 1. (B) Representative histogram overlays for PLCg2(pY759) in naive-like, IgM memory (M mem), CD27+ IgG memory (CD27+ G mem), and CD272 IgG memory (CD272 G mem) l+ CD21low B cells compared with the respective CD21pos B cell subsets of one representative patient with AAVand HD. Black lines indicate the peak values of the respective CD21pos B cell population of HD. (C) MFI of SYK(pY352), PLCg2(pY759), ERK(pThr202/Y204), AKT(pS473), S6(pS235/236), and IkBa in the different CD21low and CD21pos B cell subpopulations of autoimmune patients and HD, gated on unstimulated (l+) B cells. Red, seropositive RA; orange, seronegative RA; blue, SLE; yellow, AAV; purple, Sjo¨gren syndrome; gray, SSc; and green, MCTD/UCTD. Data were analyzed using paired or unpaired t test for normally distributed datasets and Wilcoxon matched pairs or Mann–Whitney test for not normally distributed data, followed by Bonferroni–Holm corrections for multiple testing between the three values within one subpopulation. Error bars indicate mean and SD. *p , 0.05, **p , 0.01, ***p , 0.001. pathway, the CD21pos compartments of patients showed a clearly regarding the phosphorylation capacity (Supplemental Fig. impaired phosphorylation compared with HD, with an exception 4B,4C).IntheCD21pos compartment, naive and IgM mem- for IgM memory B cells (Fig. 4B). The alterations seen in ory B cells displayed signiﬁcantly increased phosphorylation of CD21low B cells of HD resembled the ones described for patient- the proximal signaling molecules SYK and PLCg2 upon BCR derived CD21low B cells (Supplemental Fig. 2C and data not stimulation compared with the other subpopulations, whereas shown). phosphorylation of AKT and ERK was signiﬁcantly higher in IgG Comparison among the respective CD21pos and CD21low B cell memory B cells. IgM memory cells showed only a little increase subpopulations revealed differentiation-dependent differences of phosphorylation of S6 and degradation of IkBa. This signaling The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 4. BCR signaling in CD21low B cell subpopulations. (A) Representative histograms of pPLCg2 expression in the four different CD21low B cell subpopulations of one representative patient with AAV compared with the respective CD21pos subpopulations of the same patient and HD. (B) Statistics show the SI calculated from the ratio MFI l2/MFI l+ B cells for pSYK, pPLCg2, pAKT, pS6, pERK1/2, and IkBa after stimulation with anti-k, comparing CD21pos B cells from HD and from patients and CD21low B cells from patients in each subpopulation. (C) Kinetics of the ratio Indo bound/unbound of l+ (red) and l– (black) B cells after stimulation with anti-k shown for CD21low and CD21pos B cells of one representative patient with AAVand for CD21pos B cells of one representative HD. Arrows indicate the addition of anti- k.(D) Mean peak of the MFI Indo bound/unbound, comparing CD21pos B cells from HD and from patients and CD21low B cells from patients within each subpopulation. Red, seropositive RA; orange, seronegative RA; blue, SLE; yellow, AAV; purple, Sjo¨gren syndrome; gray, SSc; and green, MCTD/UCTD. Data were analyzed using paired or unpaired t test for normally distributed datasets and Wilcoxon matched pairs or Mann–Whitney test for not normally distributed data, followed by Bonferroni–Holm corrections for multiple testing between the three values within one subpopulation. Error bars indicate mean and SD. *p , 0.05, **p , 0.01, ***p , 0.001. 8 COMPARING CD21low B CELL SUBSETS IN AUTOIMMUNITY pattern between the different naive and memory B cell populations were described for the different diseases (e.g., naive-like in RA was similar in CD21low B cells for SYK and PLCg2 but different and CVID, nonswitched memory like in HCV-associated cry- for all other molecules (Supplemental Fig. 4B, 4C). In general, in oglobulinemia, or switched memory like in malaria) (3, 6, 8–10). CD21low B cells, the lowest response was detected in CD272 IgG The co-occurrence of different developmental stages of CD21low memory cells; the severe deficiency of S6 phosphorylation and B cells was first recognized in HIV patients, in which degradation of IkBa, however, was common for all CD21low CD27+CD21low B cells “activated memory B cells” have been B cell populations. This altered pattern was also seen in CD21low differentiated from CD272CD21low tissue-like memory B cells B cells of HD (Supplemental Fig. 2C and data not shown). (9, 29), and more recently, also naive like and nonswitched memory CD21low B cells were reported (30). In acute SLE, recent Altered expression of activation markers on reports distinguished populations referring to our naive like and CD21low B cell subpopulations CD272IgG/IgA CD21low B cells (16, 31). Our data demonstrate Before the initiation of differentiation and induction of effector for the first time, to our knowledge, that in healthy people and functions, an immediate consequence of the BCR-induced sig- autoimmune diseases, the CD21low compartment of every indi- naling cascade is the upregulation of activation markers, among vidual person comprises all four developmental stages and that these the costimulatory molecule CD86 and the adhesion mol- the predominant populations differ between patients. Although ecule CD69. naive and CD272 switched memory B cells typically make up + Expression of CD86 on unstimulated (l ) cells was increased in the largest portions of the CD21low compartment, CD21low low pos all CD21 subpopulations compared with the respective CD21 CD27+IgM+ and class-switched memory B cells co-occur in the subset of HD (Fig. 5). Upon stimulation, expression was increased same patient. Downloaded from pos low on all subpopulations, and only CD21 and CD21 IgM Although a few reports have already demonstrated some shared memory B cells failed to upregulate CD86 on their surface (Fig. 5, features between different CD21low populations in CMV and HIV Supplemental Fig. 4D). The highest levels of CD86 were con- (11, 30), we provide the first systematic report, to our knowledge, low sistently detected on stimulated CD21 B cells. In contrast, there that all four CD21low B cell subsets share a common core of was no difference in the expression of CD69 between unstimu- phenotypic markers in HD and across different autoimmune dis- pos low lated CD21 and CD21 B cells. CD69 was upregulated on eases. This includes the elevated expression of CD19, by defini- http://www.jimmunol.org/ pos pos nearly all CD21 cells after BCR stimulation, but CD21 IgM tion low expression of CD21 and CD38, the expression of CD11c, low memory B cells and all CD21 subpopulations failed to upreg- and increased expression of inhibitory receptors like Siglec10, ulate CD69 (Fig. 5B, Supplemental Fig. 4D). Similar patterns for FcRL4, and FcgRIIB (CD32). In SLE, a previous comparison of low CD86 and CD69 were observed among CD21 B cells of HD “activated naive B cells,” representing naive-like CD21low B cells, (Supplemental Fig. 2D and data not shown). and double-negative “DN2” cells, representing a subset strongly overlapping with CD21lowCD272IgM- B cells, demonstrated, Discussion besides similarities, some differences (16). This finding is in An increased proportion of CD21low B cells has been identified in agreement with our notion that some features of the different a variety of autoimmune disorders, including CTDs like SLE and CD21low subsets can be assigned to the underlying differentiation by guest on September 29, 2021 pSS, RA, and AAV (3–5, 24), but data comparing number, phe- stage, like the expression of transcription factors TRAF5, ZEB2, notype, and function between the different autoimmune disorders and BACH2, which matched the expression pattern in their are scarce. Culton et al. (5) described an expansion of CD19hi CD21pos counterparts. cells, which are strongly overlapping with CD21low B cells, in The similar expression patterns for CD19, CD11c, CD95, and 34% of patients with SLE and 25% of patients with AAV. Our data CD62L when comparing the respective CD21low B cell subsets of reflect Culton et al. (5) finding with the higher percentage of patients and CD21pos B cell populations of HD suggest a prev- CD21low patients among patients with SLE when compared with alent differentiation-specific regulation of the expression of these AAV, RA, or pSS. In SSc, however, we observed a much lower surface molecules. In contrast, the increased expression of the prevalence of CD21low patients, which is in contrast to data by inhibitory receptors FcRL4, CD32, and Siglec10 is mainly Rubtsov et al. (24) reporting an increase of CD11c+ B cells in controlled by activation rather than differentiation of the cell more than 30% of patients. Given the restriction of CD11c ex- because it is unique on IgM memory cells among CD21pos pression on B cells to the CD21low B cell compartment, this in- BcellsbutsharedbetweenallCD21low B cell populations. The consistent finding remains unexplained. activated status of CD21low Bcells,aswellasIgMmemory Expansion of CD21low B cells correlates with B cell lympho- B cells, is corroborated by the increased basal phosphorylation penia, which is a common finding in patients with SLE (1, 23) and of signaling molecules like SYK and PLCg2reportedbyusand also observed in patients with RA (25). This indicates that in- others (2, 13, 14). This shared feature indicates constitutively creased proportions of CD21low B cells may partly reflect a re- active BCR signaling of CD21low subpopulations independent duction of CD21pos B cells in some of the patients. A positive of the differentiation state, possibly by chronic/repetitive en- correlation of CD21low B cells with duration of disease in SLE gagement with (auto-)antigens or alternatively by constitutive independent of age corroborates former data of our group (1) and rewiring of signaling networks. is in line with the hypothesis of chronic immune stimulation This footprint is also associated with impaired signaling upon causing an expansion of CD21low B cells. The previously reported BCR restimulation in vitro as previously described by us and others association of CD21low B cells with disease activity (16, 26, 27) for single CD21low subpopulations in patients with CVID, SLE, was not observed in our study. This is probably due to our patient HIV, malaria, and HCV-associated cryoglobulinemia (2, 6, 7, 14, 15, selection excluding patients with therapies known to have a strong 32). In this study, we demonstrate that reduced BCR-dependent impact on the B cell compartment and thereby reducing the in- signaling concerning SYK, PLCg2, Ca2+, and especially ca- clusion of active patients. nonical NF-kBisalsocommontoallCD21low subpopulations Although the first descriptions of CD21low B cells did not dif- when compared with CD21pos cells of the same patient or HD. In ferentiate between B cell developmental stages (1, 9, 28), later, contrast, ERK phosphorylation upon BCR stimulation is pre- typically only the most prominent CD21low B cell subpopulations served and comparable between naive and CD27+ memory The Journal of Immunology 9 Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 5. Activation markers on CD21low B cell subpopulations. (A) FACS histograms showing the expression of CD86 in l+ (gray) and l2 (black) B cells after stimulation with anti-k for 36 h in the different subpopulations of CD21low and CD21pos B cells from a representative patient with AAV and CD21pos B cells from a representative HD. Black and gray lines indicate the peak values of the respective CD21pos B cell population of HD. (B) Statistics show the MFI of CD86 and CD69 in the different l+ and l2 populations as depicted in (A). Red, seropositive RA, orange, seronegative RA; blue, SLE; yellow, AAV; purple, Sjo¨gren syndrome; gray, SSc; and green, MCTD/UCTD. Data were analyzed using paired or unpaired t test for normally distributed datasets and Wilcoxon matched pairs or Mann–Whitney test for not normally distributed data, followed by Bonferroni–Holm corrections for multiple testing between the three values within one subpopulation. Error bars indicate mean and SD. *p , 0.05, **p , 0.01, ***p , 0.001.

CD21low B cells, although it is mainly seen in memory CD21pos receptors but respond to BCR stimulation. Unlike all other signaling B cells (33). This is remarkable because in contrast to other pathways, phosphorylation of AKT and S6 is reduced in both BCR-induced signaling pathways, which are commonly down- CD21pos and CD21low populations from patients when compared regulated by high Siglec expression, increased proapoptotic ERK with CD21pos B cells from HD, indicating a disease-associated al- signaling has been described after engagement of Siglec mole- teration of the PI3K signaling pathway. The general alteration of cules on naive B cells (34), indicating that preserved ERK sig- this pathway in CD21low patients requires further investigations. nalinginnaiveCD21low B cells may contribute to the increased In summary, we show that a substantial proportion of patients apoptosis of these cells in vitro (4, 8, 35). The increased ex- with different autoimmune diseases present with an expansion of pression of inhibitory receptors like Siglecs and FcRL4 is CD21low B cells comprising various proportions of naive-like, thought to act as a regulatory feedback mechanism on B cell IgM memory, CD27+, and CD272 class-switched memory sub- activationinCD21low B cells (36) because knockdown of cer- populations. The unique core phenotype of all CD21low B cell tain inhibitory receptors partially restored B cell function in subsets in healthy controls and patients suggests either a com- HIV-derived tissue-like memory B cells (37). This, however, mon environmental imprint during their differentiation from naive may not be the complete explanation of the impaired BCR sig- and memory CD21pos precursor cells as recently postulated for a naling of CD21low B cell populations because CD21pos IgM Th1-driven inflammatory environment (17) or a common ancestor memory B cells also exhibit increased constitutive phosphory- cell that subsequently differentiates into the four CD21low sub- lation of key signaling molecules and higher levels of inhibitory populations. This theory was supported by the discovery of related 10 COMPARING CD21low B CELL SUBSETS IN AUTOIMMUNITY expanded BCR clones between activated naive and DN2 cells and 15. Foerster, C., N. Voelxen, M. Rakhmanov, B. Keller, S. Gutenberger, S. Goldacker, J. Thiel, S. Feske, H. H. Peter, and K. Warnatz. 2010. B cell the possibility to differentiate both in vitro from resting naive cells receptor-mediated calcium signaling is impaired in B lymphocytes of type Ia and DN2 from activated naive B cells (16, 38). patients with common variable immunodeficiency. J. Immunol. 184: 7305–7313. Given the increasing knowledge about the role of BCR, TLR, 16. Jenks, S. A., K. S. Cashman, E. Zumaquero, U. M. Marigorta, A. V. Patel, low X. Wang, D. Tomar, M. C. Woodruff, Z. Simon, R. Bugrovsky, et al. 2018. IL-21, and IFN-g signaling in the development of CD21 Distinct effector B cells induced by unregulated toll-like receptor 7 contribute to B cells in vitro and in vivo (16, 17, 19, 26, 39), their capacity to pathogenic responses in systemic lupus erythematosus. [Published erratum ap- serve as Ag presentation cells because of preserved upregulation pears in 2020 Immunity 52: 203.] Immunity 49: 725–739.e6. 17. Unger, S., M. Seidl, P. van Schouwenburg, M. Rakhmanov, A. Bulashevska, of CD86 expression, and the enriched autoreactive BCR reper- N. Frede, B. Grimbacher, J. Pfeiffer, K. Schrenk, L. Munoz, et al. 2018. The TH1 toire among CD21low B cells, it is tempting to speculate that the phenotype of follicular helper T cells indicates an IFN-g-associated immune low dysregulation in patients with CD21low common variable immunodeficiency. expansion of CD21 B cells in patients with different auto- J. Allergy Clin. Immunol. 141: 730–740. immune diseases may point toward shared, pathogenetically 18. Knox, J. J., M. Buggert, L. Kardava, K. E. Seaton, M. A. Eller, D. H. Canaday, relevant mechanisms between these diseases, the clarification of M. L. Robb, M. A. Ostrowski, S. G. Deeks, M. K. Slifka, et al. 2017. T-bet+ B cells are induced by human viral infections and dominate the HIV gp140 which may allow for targeted treatment options. response. JCI Insight 2: e92943. 19. Obeng-Adjei, N., S. Portugal, P. Holla, S. Li, H. Sohn, A. Ambegaonkar, J. Skinner, G. Bowyer, O. K. Doumbo, B. Traore, et al. 2017. Malaria-induced Acknowledgments interferon-g drives the expansion of Tbethi atypical memory B cells. PLoS We thank our patients and HD for collaboration and the nurses and physi- Pathog. 13: e1006576. cians of the outpatient clinics of the Department of Rheumatology and Clin- 20. R Core Team. 2019. R: A language and environment for statistical computing. ical Immunology for patient care. We thank Dr. Erika Graf and Dominik R Foundation for Statistical Computing, Vienna, Austria. 21. Wickham, H. 2016. ggplot2: Elegant Graphics for Data Analysis. Springer- Stelzer from the Institute of Medical Biometry and Statistics, University Verlag, New York. Downloaded from of Freiburg for statistical advice. 22. Moon, K.-W. 2018. ggiraphExtra: Make Interactive ’ggplot2’. Extension to ’ggplot2’ and ’ggiraph’. R package version 0.2.9. Available at: https://CRAN.R- project.org/package=ggiraphExtra. Disclosures 23. Odendahl, M., A. Jacobi, A. Hansen, E. 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Rizzi, H. Eibel, R. Niesner, and K. Warnatz. 2017. Disturbed 39. Ambegaonkar, A. A., S. Nagata, S. K. Pierce, and H. Sohn. 2019. The differenti- canonical nuclear factor of k light chain signaling in B cells of patients with common ation in vitro of human tonsil B cells with the phenotypic and functional charac- variable immunodeficiency. J. Allergy Clin. Immunol. 139: 220–231.e8. teristics of T-bet+ atypical memory B cells in malaria. Front. Immunol. 10: 852. M mem Naive- like CD27 - G mem CD27 + G mem Transit. IgM IgM IgG IgG

CD27 - CD27 + sw mem CD27 sw mem CD38 CD38 CD38

CD21 pos SSC SSC CD38

CD21 low FSC CD19 CD21

M mem Naive- CD27 - G mem CD27 + G mem like IgM IgG IgG

CD27 - CD27 + sw mem CD27 sw mem CD38 CD38

Fig. S1: Gating strategy of subpopulations of CD21 low and CD21 pos B cells CD19 pos B cells were gated for CD21 low B cells defined by low expression of CD21 and CD38 and “CD21pos” including CD21 pos and CD21 low CD38 pos B cells. For both subsets naive-like, IgM memory (M mem), CD27 + switched memory (CD27 + sw mem) and CD27 - switched memory (CD27 - sw mem), the two latter populations were further gated on CD27 + IgG memory (CD27 + G mem) and CD27 - IgG memory (CD27 - G mem) B-cell subsets CD19 CD32 pPLC g pERK1/2 A 100000 **** **** *** **** **** **** * **** B **** **** ** **** 400 **** ** ** 80000 1000 300 g 60000 200 40000 500 MFI CD19 MFI MFI CD32 MFI MFI pERK MFI MFI pPLC MFI 100 20000

0 0 0 low low low pos low pos pos pos low low low low low low pos pos low pos low low pos pos low pos pos pos low pos pos pos pos low HD 21 HD 21 HD 21 HD HD 21 HD HD 21 HD HD 21 HD 21 HD 21 HD HD 21 HD HD 21 HD 21 HD 21 HD 21 HD HD 21 HD HD 21 HD 21 HD HD 21 HD 21 HD HD 21 HD 21 HD 21 HD 21 HD 21 HD 21 HD 21 HD 21 HD HD 21 HD HD 21 HD HD 21 HD HD 21 HD 21 HD HD 21 HD naive- M 27 +G 27 - G naive- M 27 +G 27 - G naive- M 27 +G 27 - G naive- M 27 +G 27 - G like mem mem mem like mem mem mem like mem mem mem like mem mem mem

pPLC g pERK1/2 Ca 2+ C 20 **** *** *** **** 15 ** ** *** *** ** ***

15 g 10

10 SIpERK SIpPLC 5 Mean Peak 5 Indo bound/unbound

0 0 low low pos low low low pos low pos low low low pos pos low pos pos pos pos pos low pos low pos HD 21 HD 21 HD HD 21 HD 21 HD 21 HD 21 HD 21 HD 21 HD 21 HD 21 HD 21 HD 21 HD HD 21 HD 21 HD 21 HD 21 HD 21 HD 21 HD 21 HD 21 HD HD 21 HD 21 HD 21 HD 21 HD naive- M 27 +G 27 - G naive- M 27 +G 27 - G naive- M 27 +G 27 - G like mem mem mem like mem mem mem like mem mem mem D naive-like IgM memory CD27 + IgG mem CD27 - IgG mem ** * * * *** MFI CD86 MFI low low pos pos low pos low pos pos low pos low low pos low pos HD 21 HD HD 21 HD 21 HD HD 21 HD 21 HD 21 HD 21 HD 21 HD HD 21 HD 21 HD 21 HD HD 21 HD HD 21 HD HD 21 HD HD 21 HD 21 HD l + l - l + l - l + l - l + l -

Fig. S2: CD21 low B cells in HD. A Statistics compare the MFI of CD19 and CD32 of CD21 pos and CD21 low B-cells in different B cells subpopulations from HD (paired t-test). B Statistics compare the MFI of pSYK and pERK1/2 of CD21 pos and CD21 low B-cells in different B cells subpopulations from HD, gated on unstimulated (l+) B cells (paired t-test). C Statistics compare the stimulation index (SI) calculated from the ratio MFI l-/MFI l+ B cells for pSYK and pERK1/2 after stimulation with anti-k of CD21 pos and CD21 low B-cells in different B cells subpopulations from HD (paired t- test). D Statistics show the MFI of CD86 and CD69 in the different l+ and l- B cell subpopulations from HD (paired t-test). P values were depicted as * p<0.05, ** p<0.01, *** p<0.001. MFI CD62L MFI CD32 MFI CD19 oprsn et auswr eitda <.5 *p00,* p<0.01, ** p<0.05, * as depicted were f values analysis P Mixed-effects test, comparisons patients. CD21 autoimmune the of within compartment subpopulations B-cell CD21different HD-derived of A pattern expression CD21patient-derived Surface S3: Fig. ttsiscmaeteMIo D9 D1,Sge1,C3,FR4 C FcRL4, CD32, Siglec10, CD11c, CD19, of MFI the compare Statistics ***

naive ***

naive *** naive *** *** * HD HD

M mem HD M mem M mem *** *** *** *** *** CD27 + G mem CD27 + G mem * CD27 + G mem *** **** * * CD27 - G mem CD27 - G mem CD27 - G mem P CD21P P CD21P P CD21P naive ***

naive naive *** *** ***

low M mem M mem M mem *** *** ** * + + + -el subsetsB-cells * low low CD27 G mem low CD27 G mem CD27 G mem * *** *** CD27 - G mem CD27 - G mem CD27 - G mem

MFI FcRL4 MFI CD11c ***

naive *** naive *** HD M mem HD M mem *** CD27 + G mem CD27 + G mem ** *** ***

pos CD27 - G mem CD27 - G mem oprmn fH n ihnteCD21 the within and HD of compartment P CD21P naive CD21P naive **

M mem M mem **** *** * + + low ***

CD27 G mem low CD27 G mem loe yHl-ia’ multiple Holm-Sidak’s by ollowed pos CD27 - G mem CD27 - G mem *p<0.001. **

-elsbouain and subpopulations B-cell MFI CD95 MFI Siglec10

naive naive *** *** *** ** 9 n D2 of CD62L and D95 HD M mem HD M mem * ** ** *** CD27 + G mem CD27 + G mem *** ** CD27 - G mem CD27 - G mem P CD21P P CD21P naive *** naive

M mem *** M mem *** *** * low +

+ * low

CD27 G mem low CD27 G mem *** CD27 - G mem CD27 - G mem HD P CD21 low HD P CD21 low HD P CD21 low A *** 250 *** * 200 * *** *** * * ** *** * ** *** *** *** 2

200 g *** *** *** 150 *** ***

150 100 100 MFI pAKT MFI MFI pSYK MFI pPLC MFI 50 50

0 0 * ** 300 * * *** * *** *** ** ** *** *** *** * *** ** * *** ** a

200 B k MFI pS6 MFI 100 I MFI MFI pERK MFI

0 *** B ** *** ** *** *** * *** *** *** *** *** ** 10 *** *** ** ***

2 *** g

5 SI pSYK SI pPLC SI pAKT

0 * *** * *** *** ** ** *** ** *** * ** *** ** a B k SI pERK SI pS6 SI I

C D ** *** ** ** ** ** *** ** *** *** 300

200 SI CD86 SI CD69

100

0 naive naive naive naive naive naive G G mem G G mem G G mem G mem G G mem M mem M G G mem G mem G G mem G G mem G G mem M mem M mem M M mem M G G mem G G mem M mem M M mem M - - - + - - + + + - + + Mean PeakIndo bound/unbound CD27 CD27 CD27 CD27 CD27 CD27 CD27 CD27 CD27 CD27 CD27 CD27

Fig. S4: Pattern of constitutive and BCR-mediated activation in HD-derived CD21 pos and patients‘ CD21 low B-cell subpopulations. A Statistics compare the MFI of pSYK, pPLCg2, pAKT, pS6, pERK1/2 and IkBa of different B-cell subpopulations within the CD21 pos compartment of HD and within the CD21 low compartment of autoimmune patients. B Statistics show the stimulation index (SI) calculated as the ratio MFI l-/MFI l+ B cells for pSYK, pPLCg2, pAKT, pS6, pERK1/2 and IkBa after stimulation with anti-k comparing the different subpopulations within CD21 pos and CD21 low B-cell compartments. C Mean peak of the MFI Indo bound/unbound, comparing the different subpopulations within the CD21pos and CD21 low B-cell compartments. D Statistics show the SI of CD86 and CD69 after stimulation with anti-k for 36 h, comparing the different subpopulations within the CD21 pos B-cell compartment of HD and the CD21 low B-cell compartment of autoimmune patients. Mixed-effects analysis followed by Holm-Sidak’s multiple comparisons test, P values were depicted as * p<0.05, ** p<0.01, *** p<0.001.