Circulating Human CD27−IgA+ Memory B Cells Recognize Bacteria with Polyreactive Igs

This information is current as Magdalena A. Berkowska, Jean-Nicolas Schickel, Christina of October 2, 2021. Grosserichter-Wagener, Dick de Ridder, Yen Shing Ng, Jacques J. M. van Dongen, Eric Meffre and Menno C. van Zelm J Immunol 2015; 195:1417-1426; Prepublished online 6 July 2015; doi: 10.4049/jimmunol.1402708 Downloaded from http://www.jimmunol.org/content/195/4/1417

Supplementary http://www.jimmunol.org/content/suppl/2015/07/03/jimmunol.140270 http://www.jimmunol.org/ Material 8.DCSupplemental References This article cites 57 articles, 31 of which you can access for free at: http://www.jimmunol.org/content/195/4/1417.full#ref-list-1

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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 © 2015 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Circulating Human CD272IgA+ Memory B Cells Recognize Bacteria with Polyreactive Igs

Magdalena A. Berkowska,*,1 Jean-Nicolas Schickel,† Christina Grosserichter-Wagener,* Dick de Ridder,‡,2 Yen Shing Ng,† Jacques J. M. van Dongen,* Eric Meffre,†,3 and Menno C. van Zelm*,3

The vast majority of IgA production occurs in mucosal tissue following T cell–dependent and T cell–independent Ag responses. To study the nature of each of these responses, we analyzed the gene-expression and Ig-reactivity profiles of T cell–dependent CD27+ IgA+ and T cell–independent CD272IgA+ circulating memory B cells. Gene-expression profiles of IgA+ subsets were highly similar to each other and to IgG+ memory B cell subsets, with typical upregulation of activation markers and downregulation of inhibitory receptors. However, we identified the mucosa-associated CCR9 and RUNX2 genes to be specifically upregulated in 2 + 2 +

CD27 IgA B cells. We also found that CD27 IgA B cells expressed Abs with distinct Ig repertoire and reactivity compared with Downloaded from those from CD27+IgA+ B cells. Indeed, Abs from CD272IgA+ B cells were weakly mutated, often used Igl chain, and were enriched in polyreactive clones recognizing various bacterial species. Hence, T cell–independent IgA responses are likely involved in the maintenance of gut homeostasis through the production of polyreactive mutated IgA Abs with cross-reactive anti- commensal reactivity. The Journal of Immunology, 2015, 195: 1417–1426.

he microbiome of the human gastrointestinal tract contains Ig variable domains and class-switch recombination (CSR) from the http://www.jimmunol.org/ large numbers of bacteria (up to 30,000 species) (1). The IgM, for example, to the IgA isotype (8). SHM and CSR are medi- T majority of these bacteria are coated with Igs (2) that are ated by activation-induced cytidine deaminase (AID) (9), which is generated in dynamic responses (3, 4). Indeed, the mucosal sur- upregulated through CD40 signaling following interaction with faces of the intestinal tract, the oral cavity, and lungs are major CD40L on activated CD4+ T cells. Such T cell–dependent (TD) sites of Ab production, mainly the secretory form of IgA (5). responses take place in germinal center reactions in lymphoid tissues. Each B cell carries surface Ig generated through V(D)J re- Alternatively, AID expression can be induced in T cell–independent combination of IgH and Igk and Igl L chain genes during stepwise (TI) B cell responses, which are associated with limited proliferation differentiation in the bone marrow (6, 7). Upon Ag recognition, and affinity maturation to lipid or carbohydrate structures (8, 10–13). by guest on October 2, 2021 these newly generated B cells undergo responses involving affinity TI class-switching toward IgA is well supported by the microenvi- maturation by induction of somatic hypermutations (SHMs) in the ronment of the gut, especially by dendritic cells (DCs) in the GALT. These DCs secrete retinoic acid (RA) that activates circulating B cells a b *Department of Immunology, Erasmus MC, University Medical Center, 3015 CN to induce expression of adhesion molecule 4 7 and chemokine Rotterdam, the Netherlands; †Department of Immunobiology, Yale University School receptor CCR9, which mediate gut homing (14). Upon activation via ‡ of Medicine, New Haven, CT 06511; and The Delft Bioinformatics Lab, Faculty of TLRs, DCs and monocytes secrete BAFF and APRIL, which bind Electrical Engineering, Mathematics, and Computer Science, Delft University of Technology, 2628 CD Delft, the Netherlands TACI on B cells and can induce CD40-independent class-switching b 1Current address: Department of Experimental Immunohematology, Sanquin Re- toward IgA (15–18). In addition, DC-derived TGF- and RA act in search and Landsteiner Laboratory, Amsterdam, the Netherlands. concert with IL-5, IL-6, and IL-10 to induce differentiation of B cells 2Current address: Bioinformatics Group, Wageningen University, Wageningen, the into Ab-secreting plasma cells (14, 18–20). Netherlands. Although ∼25% of intestinal IgA-producing plasmablasts are 3E.M. and M.C.v.Z. are joint senior authors. polyreactive, they show molecular signs of Ag-mediated selection Received for publication October 27, 2014. Accepted for publication June 8, 2015. (21), fitting with Ag-induced production rather than secretion of This work was supported by Grants AI071087, AI082713, AI095848, and AI061093 natural Abs independent of Ag stimulation. It is tempting to spec- from the National Institutes of Health-National Institute of Allergy and Infectious ulate that TI IgA is directed against cell wall components of com- Diseases (to E.M.), as well as by a fellowship from the Ter Meulen Fund–Royal Netherlands Academy of Sciences (to M.A.B.) and fellowships from the Erasmus mensal bacteria to support the formation of a biofilm and to disable University Rotterdam and Erasmus MC (to M.C.v.Z.). their translocation through the epithelial layer (22, 23). This would The microarray data presented in this article have been submitted to ArrayExpress prevent priming of systemic high-affinity TD responses to beneficial under accession numbers E-MEXP-3767 and E-MTAB-3637. gut microbiota. Indeed, MyD88/TRIF double-knockout mice defi- Address correspondence and reprint requests to Dr. Menno C. van Zelm or Dr. Eric Meffre, cient in TI IgA production spontaneously developed systemic Department of Immunology, Erasmus MC, University Medical Center, Wytemaweg 80, responses against gut microbiota (24). 3015 CN Rotterdam, the Netherlands (M.C.v.Z.) or Department of Immunobiology, Yale + University School of Medicine, 300 George Street, New Haven, CT 06511 (E.M.). We recently distinguished two circulating human IgA memory E-mail addresses: [email protected] (M.C.v.Z.) or [email protected] (E.M.). B cell subsets: conventional CD27+IgA+ cells were dependent on 2 The online version of this article contains supplemental material. T cell help, whereas unconventional CD27 IgA+ cells were present Abbreviations used in this article: AID, activation-induced cytidine deaminase; CSR, in CD40L-deficient individuals (25). Moreover, the limited repli- class-switch recombination; DC, dendritic cell; FR, framework region; RA, retinoic cation history of CD272IgA+ memory B cells, their low frequency acid; SHM, somatic hypermutation; TD, T cell dependent; TI, T cell independent. of SHM, and increased IgA2 usage were features reminiscent of + Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00 IgA B cells from the intestinal lamina propria (25, 26). We show in www.jimmunol.org/cgi/doi/10.4049/jimmunol.1402708 1418 GENE EXPRESSION AND Ig REACTIVITY OF IgA+ B CELLS

2 this study that both CD27+IgA+ and CD27 IgA+ B cell subsets are (InvivoGen) or sonicated lysates from cultured Enterobacter cloacae (ATCC typical memory B cells, as is evident from their gene-expression 13047), Enterococcus faecalis (ATCC 29212), Escherichia coli,orStrepto- profiles and detailed immunophenotypes. From single-cell–sorted coccus aureus or were obtained by multiple cycles of freezing and thawing + + 2 + lysates from Bacteroides fragilis (ATCC 2528) and Clostridium difficile CD27 IgA and CD27 IgA memory B cells we produced in vitro (ATCC 9689) at a concentration of 1 ng/ml. For indirect immunofluorescence rAbs to assess their reactivity to various Ags and bacterial strains. assays, HEp-2 cell–coated slides (Bion Enterprises) were incubated in We found that a large fraction of CD272IgA+ memory B cells a moist chamber at room temperature with purified rAbs at 50–100 mg/ml, express polyreactive Abs with a unique repertoire and reactivity according to the manufacturer’s instructions. FITC-conjugated goat anti- human IgG was used as detection reagent. toward commensal bacteria, suggesting that these B cells play an important role in maintaining mucosal immunity. Statistical analysis Statistical analyses were performed with the two-tailed Student t test, Materials and Methods Mann–Whitney U test, or x2 test, as indicated in the figure legends. The Cell sorting and gene-expression profiling p values , 0.05 were considered statistically significant. Three naive and six human memory B cell subsets were purified from post- Ficoll mononuclear cells on a FACSAria I cell sorter (BD Biosciences) (25, Results 27). Naive B cells were separated into CD38+CD272IgD+IgM+ transi- 2 Gene-expression profiling of naive and memory B cell subsets tional B cells, CD38dimCD27 IgD+IgM+CD5+ pre–naive B cells, and 2 2 CD38dimCD27 IgD+IgM+CD5 mature naive B cells. Memory B cells Circulating CD27+IgA+ and CD272IgA+ B cells both display dim + + + were separated into CD38 CD27 IgD IgM natural effector B cells, a memory B cell phenotype with distinct features, suggesting that CD38dimCD27+IgD2IgM+ IgM-only B cells, and CD38dimCD27+IgA+, dim + + dim 2 + dim 2 + they may originate from different types of immune responses (25). CD38 CD27 IgG , CD38 CD27 IgA , and CD38 CD27 IgG 2 + Downloaded from subsets. RNA was isolated from each sorted subset with the RNeasy Mini To study whether the TI origin of CD27 IgA B cells results in Kit (QIAGEN). Gene expression was quantified using Affymetrix HG- a typical memory B cell transcription program, we compared their U133 Plus 2.0 GeneChip arrays (containing 54,675 probe sets), as previ- gene-expression profile with three naive and five other memory ously described (7, 27, 28), and all data were deposited in the ArrayEx- B cell subsets. press database (http://www.ebi.ac.uk/arrayexpress) under accession numbers E-MEXP-3767 and E-MTAB-3637. Expression profiles of the Unsupervised clustering analysis based on 399 probe sets that three naive and six memory B cell subsets from three healthy donors were showed the greatest variation in expression between any two compared based on the perfect-match probe-intensity levels. RMA back- samples yielded three main clusters (Fig. 1A). Cluster 1 contains http://www.jimmunol.org/ ground removal and quantile normalization were performed, followed by the three naive B cells (transitional, prenaive, and mature naive), a per-probe set two-way ANOVA (with factors probe and cell type). This cluster 2 contains the natural effector and IgM-only memory resulted in average expression levels for each probe set in each cell type, as + + well as p values for the significance of the difference between cell types. B cells, and cluster 3 contains the Ig class-switched CD27 IgA , 2 2 The p values were adjusted for multiple testing using Sida ´k stepdown CD27+IgG+, CD27 IgA+, and CD27 IgG+ memory B cells adjustment (29), and all differences with adjusted p values , 0.05 were (Fig. 1A). Among class-switched memory B cells, CD27+IgA+ considered significant. cells mostly resembled CD27+IgG+ cells, and CD272IgA+ cells The maximum absolute difference in expression between any two cell 2 + types was calculated to select probe sets that showed a signal. Only probe were most similar to CD27 IgG cells (Fig. 1A). The expression 2 + sets that showed larger differences than a log threshold value of 0.7 were patterns of these 399 probe sets in CD27 IgA B cells correlated 2 by guest on October 2, 2021 selected for clustering. Correlation r between samples was calculated well with all Ig class–switched cells, showed medium correlation based on only these selected probe sets, and the data were hierarchically with the IgM+ memory B cell subsets, and differed the most from clustered (complete linkage) using 1 2 r as a distance measure (28). naive B cells (Fig. 1B, Supplemental Table I). Single-cell sorting All nine B cell subsets showed high gene-expression levels Post-Ficoll mononuclear cells from healthy donors were enriched for B cells of pan-B markers CD19, CD20, BAFF-R, CD79A, and CD79B by magnetic separation with CD19 or CD20 MicroBeads (Miltenyi Biotec) (Fig. 1C, Supplemental Table I), and the gene-expression levels and stained with CD20–PE–Cy7, CD27-allophycocyanin (both from Bio- for markers that were used to define the subsets (CD5, CD38, Legend), CD38-FITC (BD Pharmingen), CD27-allophycocyanin, and IgA- IgM, IgD, IgA, IgG, CD27) correlated well with their protein- PE (Southern Biotech) prior to purification. Single CD20+CD38dimCD27+ 2 expression levels. In addition, all memory B cell subsets showed IgA+ and CD20+CD38dimCD27 IgA+ memory B cells were sorted on a FACSAria flow cytometer (BD Biosciences) into 96-well PCR plates and the expected increase in activation markers and costimulatory immediately frozen on dry ice. molecules, such as CD80, CD86, TACI, FAS, and CD58, and downregulation of genes encoding inhibitory receptors CD22 and cDNA synthesis, Ig gene amplification, Ab production, and CD72 compared with naive B cell subsets (Fig. 1C) (25, 34). purification These expression patterns were confirmed at the protein level by RNA from single cells was reverse-transcribed in the original 96-well plate flow cytometry (Fig. 2A) (25). TLR gene-expression levels were in 12.5-ml reactions containing 100 U Superscript II RT (Life Technolo- similar for IgA+ and other memory B cells (Supplemental Table I), gies) for 45 min at 42˚C. RT-PCR reactions and primer sequences were as 2 + described previously (30–32), supplemented with an IGHA-specific primer although CD27 IgA cells contained fewer TLR1 and more (59-CTTTCGCTCCAGGTCACACTGAG-39) for the first PCR reaction. TLR10 transcripts than did mature naive B cells. However, Cloning strategy, expression vectors, Ab expression, and purification were membrane TLR-1 and TLR-10 expression levels were similarly performed as described previously (30, 31); Ig sequences were analyzed by low in naive and memory B cell subsets (Fig. 2A). In addition, IgBLAST comparison with GenBank. IgH-CDR3 was defined as the in- there were no signs of TLR signaling pathway deregulation in terval between the conserved arginine/lysine at position 94 in IGHV-FR3 + 2 + and the conserved tryptophan at position 103 in the IGHJ gene. SHM IgA cells. Furthermore, CD27 IgA memory B cells showed selection strengths were determined using the BASELINe program (http:// similar expression of signaling molecules involved in BCR and selection.med.yale.edu/baseline/) (33). CD40 signaling pathways to all other memory B cell subsets. ELISAs and immunofluorescence assays Comparison with mature naive B cells revealed upregulation of GAB2 and GRB2 (adaptor molecules in the BCR signaling Ab-reactivity analysis was performed as described previously with the highly pathway) and downregulation of LYN in CD272IgA+ B cells, polyreactive ED45 Ab as positive control for HEp-2 reactivity and poly- reactivity (30, 31). Abs were considered polyreactive when they recognized as well as downregulation of TANK in the CD40 signaling the three distinct Ags: dsDNA, insulin, and LPS. ELISA plates for bacteria- pathway. However, the majority of transcripts from both sig- reactivity testing were coated with purified flagellin from Bacillus subtilis naling pathways were not expressed significantly differently The Journal of Immunology 1419 Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 1. Gene-expression profiling of naive and memory B cell subsets. (A) Hierarchical clustering (complete linkage) using 1 2 correlation as a distance measure based on the 399 probe sets that showed the most variation between any two samples (threshold at log2 value 0.7). Clustering analyses was performed without bias for known genes or subsets. (B) Correlation of the gene-expression profiles of these 399 genes for CD272IgA+ memory B cells compared with the naive and memory B cell subsets. (C) Heat map with normalized expression levels of selected probe sets in naive and memory B cell subsets. Z-scores were maximized to 22 and 2. *Statistically significant difference between CD27+IgA+ and mature naive B cells, #statistically significant difference between CD272IgA+ and mature naive B cells, +statistically significant difference between CD27+IgA+ and CD272IgA+ B cells. between the subsets. Both IgA+ B cell subsets showed low IL4R Differential expression of mucosa-homing–related genes by and high IL6R and IL10RA expression, supporting the role of IL-6 B cell subsets + and IL-10 in IgA memory B cell differentiation (Supplemental Expression of chemokine receptors on lymphocytes determines Table I). their ability to migrate in response to stimuli. All analyzed B cell The Runx2 and Runx3 transcription factors act downstream of the subsets expressed lymph node–homing receptors CXCR4 and TGF-b andRAsignalingpathwaystoinduce TI class-switching to- CCR7, but their levels were significantly higher on naive than on ward IgA in the gut (35). Although RUNX3, TGFBR,andRARA class-switched memory B cells (Supplemental Table I). CCR7 transcript levels were similarly high in all analyzed B cell subsets, protein was present on nearly all mature naive B cells but only on RUNX2 was exclusively expressed by IgA+ B cells (Figs. 1, 2B, a fraction of cells within each memory B cell subset (Fig. 2A). Supplemental Table I), especially in CD272IgA+ B cells, supporting Stimulation of chemokine receptors induces surface expression of theirTIorigin.Thus,CD272IgA+ B cells appear to be true memory diverse adhesion molecules. All B cell subsets showed similarly high B cells; they display the highest expression of RUNX2,whichmay expression of genes encoding CD62L (SELL), a4b7 (ITGA4/ITGB7), play an important role in their development. and LFA-1 (ITGAL/ITGB2) involved in migration to lymph nodes 1420 GENE EXPRESSION AND Ig REACTIVITY OF IgA+ B CELLS Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 2. Expression levels of selected markers on memory B cell subsets. (A) Expression levels of selected costimulatory molecules (CD58, CD86), BCR signaling inhibitors (CD22, CD72), TLRs (TLR-6, TLR-10), and chemokine receptors (CCR7, CCR9) were analyzed by flow cytometry on IgM+,IgG+,andIgA+ memory B cell subsets. Each plot contains a filled graph representing the isotype control and a dark gray line graph representing mature naive B cells. (B) Expression levels of selected genes were analyzed by real time quantitative PCR. Each bar represents mean fold expression relative to control gene ABL; error bars are SEM. The number of analyzed samples is indicated below the name of the subset. Data were analyzed with Mann–Whitney U test. *p , 0.05, **p , 0.01.

(36, 37). In addition, ITGB1, which encodes the b1subunit (Fig. 3A, Supplemental Table II) (40), as well as to previously re- of the a4b1 integrin, was upregulated in all memory B cells ported naive B cells (30). More than half of the cells used a member (Supplemental Table I). Although none of the B cell subsets of the large IGHV3 subgroup, followed by IGHV4 (∼16%) and expressed the mucosal-homing marker CCR10 (38), CD272 IGHV1 (∼10% in CD27+IgA+ Bcells,∼20% in CD272IgA+ IgA+ B cells specifically expressed the small intestine–homing B cells). In both subsets, IGHV3-23, IGHV3-30,andIGHV3-33 receptor CCR9 (Supplemental Table I) (39). Membrane CCR9 were the most frequent IGHV3 genes, and IGHV4-59 was the most protein expression was only detectable on a fraction of cells frequent IGHV4 gene. IGHJ4 was the most often used IGHJ gene, within this B cell subset (7.9% of CD272IgA+ memory B cells; followed by IGHJ5 and IGHJ6. The IgH-CDR3 regions in rear- Fig. 2A). We conclude that CD272IgA+ B cells contain clones rangements from both IgA+ subsets showed similar length dis- with the capacity to home to the intestinal tract. tributions, with the majority of regions 10–14 aa in size, and they

+ + 2 + did not differ with regard to the content of positively charged amino Distinct Ig gene repertoires in CD27 IgA and CD27 IgA acids (arginine, lysine, and histidine) (Fig. 3B). CD272IgA+ Bcells B cells carried an average of 10 mutations, and ∼15% of sequences were The distinct maturation pathways of CD27+IgA+ and CD272IgA+ unmutated. In contrast, nearly all IGHV genes of CD27+IgA+ memory B cells were barely reflected in their transcription program. memory B cells were mutated and contained an average of 19 However, previous observations indicated that these two IgA+ mutations (Fig. 3C) (25). Notably, IgA1 transcripts showed a large memory B cell subsets harbored distinct Ig repertoires (25). To difference in SHM levels between the two subsets (data not shown) study the Ig repertoire and reactivity of CD27+IgA+ and CD272 (40). Despite difference in SHM levels, mutations were normally IgA+ memory B cells, we single-cell purified these from the blood targeted to hypermutable motifs and appeared to be properly se- of five healthy donors. lected during immune responses, as reflected by a high ratio of The IGHV subgroup and IGHJ gene distributions in CD27+IgA+ replacement to silent mutations $ 3.2 in their CDRs (Supplemental and CD272IgA+ memory B cells were similar to each other Fig. 1A, 1B, Supplemental Table III). The Journal of Immunology 1421

FIGURE 3. IgH gene repertoire and characteristics in CD27+IgA+ and CD272IgA+ memory B cells. (A) IGHV subgroup (upper panels) and IGHJ gene (lower panels) usage in IgA+ B cell subsets. The numbers of analyzed sequences are indicated in the center circles. (B) IgH-CDR3 length (upper panel) and charge (lower panels) distributions. (C) The number of SHMs in rearranged IGHV genes. Each gray dot represents an individual sequence; black horizontal lines represent median values. Data were analyzed with the x2 test (A and B) or the Mann–Whitney U test (C). ****p , 0.0001.

2 The Igk repertoire did not differ between the two IgA+ memory CD27 IgA+ B cells was primarily caused by abundant IGLV3-1 Downloaded from B cell subsets and showed predominant IGKV1 and IGKV3 sub- gene (20% versus 0% in CD27+IgA+ cells) (Fig. 4C). Further group and IGKJ1 and IGKJ4 gene usage. Still, CD272IgA+ analysis of ∼550 IGLV3 rearrangements bulk amplified from memory B cells showed increased Igl usage (Supplemental seven additional donors revealed that IGLV3-1 usage within Table II) (25, 41) and differed in the Igl repertoire from CD27+ IGLV3 genes was similar between CD27+IgA+ and CD272IgA+ IgA+ B cells (Fig. 4A) (30). Although almost 90% of IGL rear- B cells (Fig. 5). Thus, the observed increase in single-cell–sorted + + 2 + rangements in CD27 IgA cells involved the IGLV1 or IGLV2 CD27 IgA B cells may parallel the overall increase in IGLV3 http://www.jimmunol.org/ subgroups, the rearrangements in CD272IgA+ B cells reflected the usage by these B cells (Fig. 4B). In addition, CD272IgA+ B cells pattern of mature naive B cells, with 29% IGLV3 and 29% IGLV2 showed less usage of IGLJ1 than did CD27+IgA+ B cells (17% subgroup usage (Fig. 4B) (42, 43). The increased IGLV3 usage in versus 38%) (Fig. 4B). by guest on October 2, 2021

FIGURE 4. Igk and Igl L chain gene repertoire and characteristics in CD27+IgA+ and CD272IgA+ memory B cells. (A) IGKV subgroup (upper panels) and IGKJ gene (lower panels) use in IgA+ memory B cell subsets. The numbers of analyzed sequences are indicated in the center circles. (B) IGLV subgroup (upper panels) and IGLJ gene (lower panels) use. (C) IGLV gene use. (D) The number of SHMs in rearranged IGKV (top panel) and IGLV (middle panel) in memory B cell subsets, as well as in IGLV3-1 and non–IGLV3-1 genes of CD272IgA+ memory B cells (bottom panel). Each gray dot represents an individual sequence, and horizontal black lines represent median values. Data were analyzed with the x2 test (A and B) or the Mann–Whitney U test (D). ***p , 0.001, ****p , 0.0001. 1422 GENE EXPRESSION AND Ig REACTIVITY OF IgA+ B CELLS

FIGURE 5. Usage and characteristics of IGLV3-1 gene. (A) Frequency of IGLV3-1 gene in IGLV3 subgroup in mature naive, CD27+IgA+,and CD272IgA+ B cells from seven independent donors. IGL rearrangements were amplified with forward IGLV3-specific and two reverse IGLJ1- and IGLJ2/3-specific primers. In total, 557 unique productive rearrangements were analyzed. (B) Frequency of SHM in rearranged IGLV3-1 and non–IGLV3-1 genes in mature naive, CD27+IgA+, and CD27-IgA+ memory B cells from seven donors. Each black dot represents an individual sequence, and black horizontal lines represent median values. (C) Selection of SHMs in IGLV3-1 and other IGLV3 genes in CD27+IgA+ B cells (upper panel)andCD272IgA+ Bcells(lower panel), as measured with the BASELINe program (http://selection.med.yale.edu/baseline). *p , Downloaded from 0.05, ****p , 0.0001, Mann–Whitney U test. http://www.jimmunol.org/ Similar to IGHV,CD272IgA+ B cells carried significantly fewer regions (FRs), whereas selection in CDR was comparable between SHMs in the expressed IGKV and IGLV genes than did CD27+IgA+ IGLV3-1 and other IGLV3 genes (Fig. 5C). Hence, the decreased B cells (Fig. 4D), with slightly more mutations in IGLV than in IGKV mutation loads in IGH, IGK,andIGL, combined with the increased in CD27+IgA+ memory B cells. Analogous to IGHV,CD27+IgA+ and Igl usage, in CD272IgA+ B cells, suggest that these B cells were CD272IgA+ B cells showed similar selection of SHMs in their IGKV generated toward specific Ags through selection mechanisms that and IGLV genes, with high CDR replacement/silent ratios ranging appear distinct from those shaping CD27+IgA+ B cells. from 2.8 to 3.4 (data not shown) and positive selection for replace- 2 + ment mutations (Supplemental Fig. 1B, 1C). Interestingly, the IGLV3- CD27 IgA B cells express Abs with distinct self-reactive features 1 genes contained significantly fewer mutations than did other IGLV3 by guest on October 2, 2021 genes (Figs. 4D, 5B). These mutations in IGLV3-1 showed particu- To determine whether the Ig repertoire differences between CD27+ larly strong selection against replacement mutations in framework IgA+ and CD272IgA+ memory B cells reflect distinct Ab reactivity,

FIGURE 6. Autoreactivity in CD27+IgA+ and CD272IgA+ memory B cell subsets. (A) Reactivity to HEp-2 cell lysates of 88 Abs from CD27+IgA+ cells and 75 Abs from CD272IgA+ cells derived from five healthy donors. The dashed line represents the highly reactive ED45 positive control (31), and the red line represents the cut-off value of 0.5, above which Abs were considered HEp-2 reactive. (B) Frequencies of HEp-2–reactive CD27+IgA+ and CD272IgA+ cells in five donors. Data points representing values for the same donor are connected with a black line. **p , 0.01, two-tailed Student t test for paired samples. (C) Autoreactivity patterns of CD27+IgA+ and CD272IgA+ cells, as measured in an immunofluorescence assay with HEp-2 cell–coated slides. The numbers of analyzed Abs are indicated in the center. Data were analyzed with the x2 test. (D) Representative pictures from immunofluorescence assay staining (original magnification 340). The Journal of Immunology 1423 we cloned and expressed the amplified Ig H and L chain genes as CD272IgA+ B cells contained significantly more polyreactive rAbs. Although not all Ig genes could be expressed in vitro, the clones, which averaged 26% compared with 16% in CD27+IgA+ repertoire of successfully produced rAbs was representative of the memory B cells (Fig. 7). Polyreactive Abs often used members of total set with regard to their Ig gene usage, SHM frequency and the IGHV1 subgroup, mainly at the expense of IGHV4 (Fig. 7C). selection, and IgH-CDR3 characteristics. We tested Ab self- The presence of positively charged amino acids in IgH-CDR3 was reactivity by immunofluorescence and ELISA against the human not different between polyreactive and nonpolyreactive clones, larynx carcinoma cell line HEp-2. We found that CD27+IgA+ B cells whereas polyreactive Abs were significantly enriched in very long expressed significantly more HEp-2–reactive Abs than did their IgH-CDR3s ($20 aa) (9% in polyreactive versus 1% in non- CD272IgA+ counterparts, representing 35 and 26% of the clones, polyreactive cells; Fig. 7D). Interestingly, polyreactive Abs from respectively (p , 0.01; Fig. 6A, 6B). In CD272IgA+ B cells, the CD272IgA+ B cells harbored more IGHV SHMs than did non- autoreactive Abs used IGHV1 more frequently, but otherwise these polyreactive CD272IgA+ clones, at a level similar to that of did not differ from nonautoreactive Abs with regard to SHM num- CD27+IgA+ memory B cells (Fig. 7E). However, selection of bers or IgH-CDR3 length and amino acid composition (data not SHM was similar between polyreactive and nonpolyreactive shown). Autoreactive CD27+IgA+, but not CD272IgA+, B cells clones (Supplemental Fig. 1C, 1D, Supplemental Table III). showed limited selection for replacement mutations in CDRs We conclude that CD27+IgA+ and CD272IgA+ B cells express Abs (Supplemental Fig. 1C, 1D). Immunofluorescence analyses revealed with different self-reactive features; although CD27+IgA+ B cells are that 11.4% (10/88) of CD27+IgA+ memory B cells and only 5.3% (4/ enriched in autoreactive clones recognizing cellular components, 75) of CD272IgA+ memory B cells reacted with cytoplasmic and/or CD272IgA+ B cells often produce polyreactive Abs that are rarely nuclear Ags (Fig. 6C, 6D). In addition, CD272IgA+ B cells were cross-reactive with self and displaying a distinct Ig repertoire asso- virtually devoid of anti-nuclear clones, whereas these represent 4% ciated with increased SHMs compared with their nonpolyreactive Downloaded from of CD27+IgA+ B cells (Fig. 6C, 6D). Thus, CD272IgA+ memory counterparts.

B cells express Abs with decreased reactivity against cellular Ags + compared with those produced by CD27+IgA+ B cells. Polyreactive Abs from IgA memory B cells display To determine the multispecificity of rAbs, we assessed the anti-bacteria reactivity frequencies of polyreactive clones that recognized three structur- Because IgA+ B cells are assumed to produce Abs protecting mu- ally distinct Ags: dsDNA, insulin, and LPS (30). We found that cosa, we tested the reactivity of rAbs expressed by CD27+IgA+ and http://www.jimmunol.org/

FIGURE 7. Polyreactivity in CD27+ IgA+ and CD272IgA+ memory B cell subsets. (A) Abs showing triple reac- by guest on October 2, 2021 tivity against dsDNA (top panels), in- sulin (middle panels), and LPS (bottom panels) were defined as polyreactive. In total, 88 Abs from CD27+IgA+ memory B cells and 75 Abs from CD272IgA+ memory B cells were analyzed. Dashed lines represent the highly reactive ED45 positive control (31). (B) Fre- quencies of polyreactive CD27+IgA+ and CD272IgA+ Abs in five donors. Data points representing values for one donor are connected with a black line. (C) IGHV subgroup (upper panels) and IGHJ gene (lower panels) use in poly- reactive and nonpolyreactive IgA+ memory B cells. The numbers of ana- lyzed sequences are indicated in the center circles. (D) IgH-CDR3 length (upper panel) and charge (lower panel) distributions. (E) The numbers of SHMs in rearranged IGHV genes. Each gray dot represents an individual sequence, and the horizontal black lines represent median values. Data were analyzed with the two-tailed Student t test for paired samples (B), the x2 test (C), or the Mann– Whitney U test (E). *p , 0.05. 1424 GENE EXPRESSION AND Ig REACTIVITY OF IgA+ B CELLS

CD272IgA+ memory B cells with specific micro-organisms, in- Despite their postulated origin from TI immune responses, cluding commensal bacteria B. fragilis, E. cloacae,andE. faecalis, CD272IgA+ memory B cells displayed a gene-expression profile potentially pathogenic C. difficile, E. coli,andS. aureus,aswellas typical of other memory B cells. All memory subsets differed bacterial flagellin. Irrespective of the tested Ags, the frequencies of from naive B cells with regard to upregulation of costimulatory reactive Abs were higher in CD272IgA+ memory B cells than in molecules and downregulation of BCR signaling inhibitors and CD27+IgA+ memory B cells (Fig. 8A). CD272IgA+ cells showed naive B cell–specific transcription factors (34). These changes are high binding frequencies for commensal B. fragilis and E. faecalis thought to underlie the increased B cell responsiveness of memory strains, as well as potentially pathogenic E. coli and C. difficile,and B cells, which is crucial for adaptive immunity (34). In addition, a slightly lower frequency of Abs binding to S. aureus and the increased expression of IL6R and IL10R shared among E. cloacae. The frequencies of bacteria-reactive Abs were con- memory B cell subsets promotes the synergistic effects of IL-6 sistently higher in CD272IgA+ B cells than in CD27+IgA+ cells and IL-10 to induce terminal B cell differentiation into plasma for all six analyzed stains (Fig. 8B). Furthermore, nearly all of the cells (44–46). RUNX2 was expressed exclusively by IgA+ B cells, strongly bacteria-reactive Abs were polyreactive (Fig. 8C). Thus, and its expression level was significantly higher (2.4-fold) in polyreactive Abs enriched in CD272IgA+ B cells recognize CD272IgA+ B cells than in CD27+IgA+ B cells. RUNX2 acts mucosa-associated bacteria. downstream of the TGF-b and RA signaling pathways to induce TI class-switching toward IgA in the gut and promote the terminal Discussion differentiation of IgA+ B cells into plasma cells (19, 20, 35). We reported in this article the molecular characterization of un- Hence, the specific expression of RUNX2 in IgA+ memory B cells conventional CD272IgA+ B cells, which share a common gene- further supports this scenario regarding their generation. CCR9 expression profile with conventional CD27+IgA+ and IgG+ mem- mediates homing to the gut, and CCR9 transcripts were specifi- Downloaded from ory B cells, although they express a unique Ig repertoire favoring cally expressed in CD272IgA+ B cells, and CCR9 protein was anti-commensal reactivity. detected on the cell surface of some of these cells (14). Given the http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 8. Reactivity of CD27+IgA+ and CD272IgA+ memory B cells with commensal and potentially pathogenic bacteria. (A) Pie charts summarizing the frequencies of Abs from CD27+IgA+ and CD272IgA+ memory B cells with reactivities against flagellin, commensal bacteria (B. fragilis, E. cloacae, E. faecalis), and potentially pathogenic bacteria (E. coli, S. aureus, C. difficile). The numbers of analyzed Abs are indicated in the center circles. (B) Overall bacteria reactivities of CD27+IgA+ and CD272IgA+ memory B cells. (C) Reactivity levels against bacteria, as measured by ELISA for Abs from CD27+ IgA+ and CD272IgA+ memory B cells. Each gray dot represents a nonpolyreactive Ab, and each white dot represents a polyreactive Ab. Black horizontal lines represent the median values of all Abs together, and the dashed line represents a threshold value above which Abs were considered bacteria reactive. Data were analyzed with the x2 test (A) or the two-tailed Student t test for paired samples (B). **p , 0.01. The Journal of Immunology 1425 function of CCR9 to direct B cell migration toward mucous IgG+ plasmablasts (21). This high Ab polyreactivity may be bene- membranes (38, 39, 47), it is possible that protein expression is ficial for responses toward gastrointestinal bacteria because poly- downregulated in circulating CD272IgA+ B cells to allow their reactive Abs generated from a specific immune response could trafficking to other mucosal locations (48, 49). Altogether, our serve as natural Abs that recognize other bacterial strains, even data indicate that human memory B cell subsets share a common upon a first encounter. Interestingly, polyreactive anti-bacteria genetic program and that RUNX2 and CCR9 more clearly define clones in CD272IgA+ B cells display higher SHM frequencies CD272IgA+ memory B cells and likely play an important role in than do their nonpolyreactive CD272IgA+ counterparts, further mucosal immunity. supporting the importance of SHM in the generation of specific We further analyzed the Ig H and L chain gene repertoires and immunity. The role of SHM in the introduction of polyreactivity confirmed that CD272IgA+ B cells more frequently expressed Igl also was observed in several studies in mice and humans that than did CD27+IgA+ B cells and carried significantly fewer SHMs demonstrated that reversion of mutated Ig genes to their germline in their Ig (25). This could be a direct consequence of a TI origin counterparts results in a significant decrease in polyreactivity (42, associated with limited expression of AID (50). However, it is 56). Moreover, mice carrying a mutated form of AID that allows possible that not all IgA functions in the gut require extensive CSR, but not SHM, display severe intestinal lymphoid hyperplasia affinity maturation (51). It was postulated that long coevolution accompanied by overwhelming microbial expansion in the mucosa between host and microbiome may favor the selection of Ig var- (4). Thus, the production of mutated and Ag-selected, although iable genes that, in germline configuration, could recognize con- polyreactive, Abs by CD272IgA+ B cells may play an important served bacterial Ags (52). Multiple studies showed that unmutated role in controlling intestinal microbiota in humans. IgA isolated from mice colonized with a single bacterial strain can In summary, we showed that CD27+IgA+ and CD272IgA+ bind Ags with good affinity (3, 53). The distinct Igl repertoire memory B cell subsets contain highly similar transcription pro- Downloaded from expressed by CD272IgA+ B cells associated with their increased files, despite their postulated origin from TD and TI immune anti-commensal reactivity may also support this hypothesis. In- responses, respectively. However, the distinct Ab repertoire and deed, IGLV3-1 genes from CD272IgA+ memory B cells carried reactivity of these IgA+ memory B cell subsets reflect their unique very few SHMs and showed a very strong selection against re- physiological functions, with CD272IgA+ B cells likely being placement mutations in FRs. The abundance of IGLV3-1 was involved in the maintenance of gut homeostasis through the pro- described in diverse tissue-related conditions, such as amyloidosis duction of polyreactive mutated IgA Abs with cross-reactive an- http://www.jimmunol.org/ (54). Thus, differences in the IGL repertoire of CD272IgA+ tibacterial reactivity. B cells may reflect their involvement in local mucosal responses. The increased usage of Igl L chains in the intestinal tract was Acknowledgments proposed to result from ongoing receptor-revision processes (41, We thank Dr. L. Devine, C. Wang, and S.J.W. Bartol for cell sorting and 55). Our sequence analyses suggest otherwise for the following Dr. J.P. Hays for support with bacterial cultures. reasons: first, CD272IgA+ B cells are enriched in IGLV3-1 genes, which are located downstream of the Igl locus (41) and, in our Disclosures study, contained nontemplated nucleotides, reflecting activity of The authors have no financial conflicts of interest. by guest on October 2, 2021 the TdT enzyme. Because TdT is primarily expressed in B cell progenitors, we conclude that Igl gene rearrangements in CD272 IgA+ memory B cells were generated during early B cell devel- References opment in the bone marrow. Hence, the increased frequencies of 1. Arumugam, M., J. Raes, E. Pelletier, D. Le Paslier, T. Yamada, D. R. Mende, l 2 + G. R. Fernandes, J. Tap, T. Bruls, J.-M. Batto, et al. 2011. Enterotypes of the Ig L chains in CD27 IgA B cells are most likely the result of human gut microbiome. Nature 473: 174–180. selection processes rather than receptor revision. 2. van der Waaij, L. A., P. C. Limburg, G. Mesander, and D. van der Waaij. 1996. Differences in Ig repertoire between CD272IgA+ and CD27+ In vivo IgA coating of anaerobic bacteria in human faeces. Gut 38: 348–354. + 3. Hapfelmeier, S., M. A. Lawson, E. Slack, J. K. Kirundi, M. Stoel, IgA B cell subsets were associated with differences in Ab reac- M. Heikenwalder, J. Cahenzli, Y. Velykoredko, M. L. Balmer, K. Endt, et al. tivity. Because all of the studied Abs were expressed as IgG, we 2010. Reversible microbial colonization of germ-free mice reveals the dynamics of IgA immune responses. Science 328: 1705–1709. only took into account their Ag specificity and affinity, as medi- 4. Wei, M., R. Shinkura, Y. Doi, M. Maruya, S. Fagarasan, and T. Honjo. 2011. ated by the V region, and not avidity and effector functions as- Mice carrying a knock-in mutation of Aicda resulting in a defect in somatic sociated with the constant regions. Previous studies reported that hypermutation have impaired gut homeostasis and compromised mucosal de- + + fense. Nat. Immunol. 12: 264–270. human CD27 IgG B cells displayed an increased frequency of 5. Macpherson, A. J., K. D. McCoy, F. E. Johansen, and P. Brandtzaeg. 2008. The autoreactive clones (47%) compared with mature naive B cells immune geography of IgA induction and function. Mucosal Immunol. 1: 11–22. (20%) (56, 57). Similarly, we found that CD27+IgA+ B cells 6. Ghia, P., E. ten Boekel, E. Sanz, A. de la Hera, A. Rolink, and F. Melchers. 1996. Ordering of human bone marrow B lymphocyte precursors by single-cell poly- also expressed a higher frequency of HEp-2–reactive clones merase chain reaction analyses of the rearrangement status of the immuno- 2 (35%). In contrast, CD27 IgA+ B cells contained fewer autore- globulin H and L chain gene loci. J. Exp. Med. 184: 2217–2229. 7. van Zelm, M. C., M. van der Burg, D. de Ridder, B. H. Barendregt, E. F. de Haas, active clones (26%) and, therefore, were more similar to mature M. J. Reinders, A. C. Lankester, T. Re´ve´sz, F. J. Staal, and J. J. van Dongen. naive B cells (30). The lower frequency of autoreactive Abs in the 2005. Ig gene rearrangement steps are initiated in early human precursor B cell CD272IgA+ subset compared with CD27+IgA+ and CD27+IgG+ subsets and correlate with specific transcription factor expression. J. Immunol. 175: 5912–5922. memory B cells may be explained by their low SHM levels. 8. Cerutti, A. 2008. The regulation of IgA class switching. Nat. Rev. Immunol. 8: Autoreactivity in CD27+IgG+ memory B cells was shown to result 421–434. from the introduction of SHMs (56). Although autoreactive and 9. Muramatsu, M., K. Kinoshita, S. Fagarasan, S. Yamada, Y. Shinkai, and 2 + T. Honjo. 2000. Class switch recombination and hypermutation require nonautoreactive CD27 IgA B cells had similarly mutated Ig activation-induced cytidine deaminase (AID), a potential RNA editing enzyme. genes, reduced AID activity induced by TI responses in these Cell 102: 553–563. 10. Fagarasan, S., K. Kinoshita, M. Muramatsu, K. Ikuta, and T. Honjo. 2001. In situ B cells might provide a basis for the generation of fewer autore- class switching and differentiation to IgA-producing cells in the gut lamina active Abs than in conventional memory B cells. propria. Nature 413: 639–643. The frequency of polyreactive Abs expressed by CD272IgA+ 11. Fagarasan, S., and T. Honjo. 2000. T-Independent immune response: new aspects + + of B cell biology. Science 290: 89–92. B cells was significantly higher than in CD27 IgA B cells and 12. Chorny, A., I. Puga, and A. Cerutti. 2010. Innate signaling networks in mucosal + comparable with those previously observed for intestinal IgA and IgA class switching. Adv. Immunol. 107: 31–69. 1426 GENE EXPRESSION AND Ig REACTIVITY OF IgA+ B CELLS

13. Macpherson, A. J., D. Gatto, E. Sainsbury, G. R. Harriman, H. Hengartner, and class switching acting downstream of TGF-beta 1 and retinoic acid signaling. J. R. M. Zinkernagel. 2000. A primitive T cell-independent mechanism of intes- Immunol. 184: 2785–2792. tinal mucosal IgA responses to commensal bacteria. Science 288: 2222–2226. 36. Warnock, R. A., S. Askari, E. C. Butcher, and U. H. von Andrian. 1998. Mo- 14. Mora, J. R., M. Iwata, B. Eksteen, S. Y. Song, T. Junt, B. Senman, K. L. Otipoby, lecular mechanisms of lymphocyte homing to peripheral lymph nodes. J. Exp. A. Yokota, H. Takeuchi, P. Ricciardi-Castagnoli, et al. 2006. Generation of gut- Med. 187: 205–216. homing IgA-secreting B cells by intestinal dendritic cells. Science 314: 1157– 37. Bargatze, R. F., M. A. Jutila, and E. C. Butcher. 1995. Distinct roles of L-selectin 1160. and integrins alpha 4 beta 7 and LFA-1 in lymphocyte homing to Peyer’s patch- 15. He, B., R. Santamaria, W. Xu, M. Cols, K. Chen, I. Puga, M. Shan, H. Xiong, HEV in situ: the multistep model confirmed and refined. Immunity 3: 99–108. J. B. Bussel, A. Chiu, et al. 2010. The transmembrane activator TACI triggers 38. Kunkel, E. J., C. H. Kim, N. H. Lazarus, M. A. Vierra, D. Soler, E. P. Bowman, immunoglobulin class switching by activating B cells through the adaptor and E. C. Butcher. 2003. CCR10 expression is a common feature of circulating MyD88. Nat. Immunol. 11: 836–845. and mucosal epithelial tissue IgA Ab-secreting cells. J. Clin. Invest. 111: 1001– 16. Castigli, E., S. Scott, F. Dedeoglu, P. Bryce, H. Jabara, A. K. Bhan, 1010. E. Mizoguchi, and R. S. Geha. 2004. Impaired IgA class switching in APRIL- 39. Pabst, O., L. Ohl, M. Wendland, M. A. Wurbel, E. Kremmer, B. Malissen, and deficient mice. Proc. Natl. Acad. Sci. USA 101: 3903–3908. R. Fo¨rster. 2004. Chemokine receptor CCR9 contributes to the localization of 17. Castigli, E., S. A. Wilson, S. Scott, F. Dedeoglu, S. Xu, K. P. Lam, R. J. Bram, plasma cells to the small intestine. J. Exp. Med. 199: 411–416. H. Jabara, and R. S. Geha. 2005. TACI and BAFF-R mediate isotype switching 40. Wu, Y. C., D. Kipling, and D. K. Dunn-Walters. 2011. The relationship between in B cells. J. Exp. Med. 201: 35–39. CD27 negative and positive B cell populations in human peripheral blood. Front. 18. Litinskiy, M. B., B. Nardelli, D. M. Hilbert, B. He, A. Schaffer, P. Casali, and Immunol. 2: 81. A. Cerutti. 2002. DCs induce CD40-independent immunoglobulin class 41. Su, W., J. N. Gordon, F. Barone, L. Boursier, W. Turnbull, S. Mendis, switching through BLyS and APRIL. Nat. Immunol. 3: 822–829. D. K. Dunn-Walters, and J. Spencer. 2008. Lambda light chain revision in the 19. Borsutzky, S., B. B. Cazac, J. Roes, and C. A. Guzma´n. 2004. TGF-beta receptor human intestinal IgA response. J. Immunol. 181: 1264–1271. signaling is critical for mucosal IgA responses. J. Immunol. 173: 3305–3309. 42. Tsuiji, M., S. Yurasov, K. Velinzon, S. Thomas, M. C. Nussenzweig, and 20. Tokuyama, H., and Y. Tokuyama. 1995. Endogenous cytokine expression pro- H. Wardemann. 2006. A checkpoint for autoreactivity in human IgM+ memory files in retinoic acid-induced IgA production by LPS-stimulated murine B cell development. J. Exp. Med. 203: 393–400. splenocytes. Cell. Immunol. 166: 247–253. 43. Yurasov, S., H. Wardemann, J. Hammersen, M. Tsuiji, E. Meffre, V. Pascual, and M. C. Nussenzweig. 2005. Defective B cell tolerance checkpoints in systemic

21. Benckert, J., N. Schmolka, C. Kreschel, M. J. Zoller, A. Sturm, B. Wiedenmann, Downloaded from and H. Wardemann. 2011. The majority of intestinal IgA+ and IgG+ plasma- lupus erythematosus. J. Exp. Med. 201: 703–711. blasts in the human gut are antigen-specific. J. Clin. Invest. 121: 1946–1955. 44. Zhang, Y., J. X. Lin, and J. Vilcek. 1988. Synthesis of interleukin 6 (interferon- 22. Macpherson, A. J., L. Hunziker, K. McCoy, and A. Lamarre. 2001. IgA beta 2/B cell stimulatory factor 2) in human fibroblasts is triggered by an in- responses in the intestinal mucosa against pathogenic and non-pathogenic crease in intracellular cyclic AMP. J. Biol. Chem. 263: 6177–6182. microorganisms. Microbes Infect. 3: 1021–1035. 45. Burdin, N., F. Rousset, and J. Banchereau. 1997. B-cell-derived IL-10: pro- 23. Bollinger, R. R., M. L. Everett, S. D. Wahl, Y. H. Lee, P. E. Orndorff, and duction and function. Methods 11: 98–111. ` W. Parker. 2006. Secretory IgA and mucin-mediated biofilm formation by en- 46. Defrance, T., B. Vanbervliet, F. Briere, I. Durand, F. Rousset, and J. Banchereau. 1992. Interleukin 10 and transforming growth factor beta cooperate to induce vironmental strains of Escherichia coli: role of type 1 pili. Mol. Immunol. 43:

anti-CD40-activated naive human B cells to secrete immunoglobulin A. J. Exp. http://www.jimmunol.org/ 378–387. Med. 175: 671–682. 24. Slack, E., S. Hapfelmeier, B. Stecher, Y. Velykoredko, M. Stoel, M. A. Lawson, 47. Hieshima, K., Y. Kawasaki, H. Hanamoto, T. Nakayama, D. Nagakubo, M. B. Geuking, B. Beutler, T. F. Tedder, W. D. Hardt, et al. 2009. Innate and A. Kanamaru, and O. Yoshie. 2004. CC chemokine ligands 25 and 28 play es- adaptive immunity cooperate flexibly to maintain host-microbiota mutualism. sential roles in intestinal extravasation of IgA antibody-secreting cells. J. Science 325: 617–620. Immunol. 173: 3668–3675. 25. Berkowska, M. A., G. J. Driessen, V. Bikos, C. Grosserichter-Wagener, 48. Holtmeier, W., A. Hennemann, and W. F. Caspary. 2000. IgA and IgM V(H) K. Stamatopoulos, A. Cerutti, B. He, K. Biermann, J. F. Lange, M. van der Burg, repertoires in human colon: evidence for clonally expanded B cells that are et al. 2011. Human memory B cells originate from three distinct germinal center- widely disseminated. Gastroenterology 119: 1253–1266. dependent and -independent maturation pathways. Blood 118: 2150–2158. 49. Dunn-Walters, D. K., L. Boursier, and J. Spencer. 1997. Hypermutation, diver- 26. Kett, K., P. Brandtzaeg, J. Radl, and J. J. Haaijman. 1986. Different subclass sity and dissemination of human intestinal lamina propria plasma cells. Eur. J. distribution of IgA-producing cells in human lymphoid organs and various se- Immunol. 27: 2959–2964. cretory tissues. J. Immunol. 136: 3631–3635. 50. He, B., W. Xu, and A. Cerutti. 2010. Comment on “Gut-associated lymphoid by guest on October 2, 2021 27. Berkowska, M. A., C. Grosserichter-Wagener, H. J. Adriaansen, D. de Ridder, tissue contains the molecular machinery to support T-cell-dependent and T-cell- K. P. Mirani-Oostdijk, H. J. Agteresch, S. Bo¨ttcher, A. Orfao, J. J. van Dongen, independent class switch recombination”. Mucosal Immunol. 3: 92–94, author and M. C. van Zelm. 2014. Persistent polyclonal B-cell lymphocytosis: exten- reply 94–95. sively proliferated CD27+IgM+IgD+ memory B cells with a distinctive immu- 51. Stoel, M., H. Q. Jiang, C. C. van Diemen, J. C. Bun, P. M. Dammers, nophenotype. Leukemia 28: 1560–1564. M. C. Thurnheer, F. G. Kroese, J. J. Cebra, and N. A. Bos. 2005. Restricted IgA 28. Nodland, S. E., M. A. Berkowska, A. A. Bajer, N. Shah, D. de Ridder, J. J. van repertoire in both B-1 and B-2 cell-derived gut plasmablasts. J. Immunol. 174: Dongen, T. W. LeBien, and M. C. van Zelm. 2011. IL-7R expression and IL-7 1046–1054. signaling confer a distinct phenotype on developing human B-lineage cells. 52. Slack, E., M. L. Balmer, J. H. Fritz, and S. Hapfelmeier. 2012. Functional Blood 118: 2116–2127. flexibility of intestinal IgA - broadening the fine line. Front. Immunol. 3: 100. 29. Ludbrook, J. 1998. Multiple comparison procedures updated. Clin. Exp. Phar- 53. Bos, N. A., J. C. Bun, S. H. Popma, E. R. Cebra, G. J. Deenen, M. J. van der macol. Physiol. 25: 1032–1037. Cammen, F. G. Kroese, and J. J. Cebra. 1996. Monoclonal immunoglobulin A 30. Wardemann, H., S. Yurasov, A. Schaefer, J. W. Young, E. Meffre, and derived from peritoneal B cells is encoded by both germ line and somatically M. C. Nussenzweig. 2003. Predominant autoantibody production by early human mutated VH genes and is reactive with commensal bacteria. Infect. Immun. 64: B cell precursors. Science 301: 1374–1377. 616–623. 31. Meffre, E., A. Schaefer, H. Wardemann, P. Wilson, E. Davis, and 54. Perfetti, V., S. Casarini, G. Palladini, M. C. Vignarelli, C. Klersy, M. Diegoli, M. C. Nussenzweig. 2004. Surrogate light chain expressing human peripheral E. Ascari, and G. Merlini. 2002. Analysis of V(lambda)-J(lambda) expression in B cells produce self-reactive antibodies. J. Exp. Med. 199: 145–150. plasma cells from primary (AL) amyloidosis and normal bone marrow identifies 32. Tiller, T., E. Meffre, S. Yurasov, M. Tsuiji, M. C. Nussenzweig, and 3r (lambdaIII) as a new amyloid-associated germline gene segment. Blood 100: H. Wardemann. 2008. Efficient generation of monoclonal antibodies from single 948–953. human B cells by single cell RT-PCR and expression vector cloning. J. Immunol. 55. Su, W., L. Boursier, A. Padala, J. D. Sanderson, and J. Spencer. 2004. Biases in Methods 329: 112–124. Ig lambda light chain rearrangements in human intestinal plasma cells. J. 33. Hershberg, U., M. Uduman, M. J. Shlomchik, and S. H. Kleinstein. 2008. Im- Immunol. 172: 2360–2366. proved methods for detecting selection by mutation analysis of Ig V region 56. Tiller, T., M. Tsuiji, S. Yurasov, K. Velinzon, M. C. Nussenzweig, and sequences. Int. Immunol. 20: 683–694. H. Wardemann. 2007. Autoreactivity in human IgG+ memory B cells. Immunity 34. Good, K. L., D. T. Avery, and S. G. Tangye. 2009. Resting human memory 26: 205–213. B cells are intrinsically programmed for enhanced survival and responsiveness to 57. Scheid, J. F., H. Mouquet, J. Kofer, S. Yurasov, M. C. Nussenzweig, and diverse stimuli compared to naive B cells. J. Immunol. 182: 890–901. H. Wardemann. 2011. Differential regulation of self-reactivity discriminates 35. Watanabe, K., M. Sugai, Y. Nambu, M. Osato, T. Hayashi, M. Kawaguchi, between IgG+ human circulating memory B cells and bone marrow plasma cells. T. Komori, Y. Ito, and A. Shimizu. 2010. Requirement for Runx proteins in IgA Proc. Natl. Acad. Sci. USA 108: 18044–18048.

SUPPLEMENTAL FIGURE 1. Selection for replacement mutations. (A) Selection strengths in amplified rearrangements, and (B) selection strengths in cloned rearrangements as determined with the Bayesian estimation of Antigen-driven SELectIoN program (https://selection.med.yale.edu/baseline) (32). (C) Selection strengths in autoreactive (left panel) and polyreactive (right panel) antibodies derived from CD27+IgA+ B cells. (D) Selection strengths in autoreactive (left panel) and polyreactive (right panel) antibodies derived from CD27-IgA+ B cells. In all panels, red lines represent CDRs, blue lines represent FRs. 1

SUPPLEMENTAL TABLE I. Expression levels of selected genes in naive and memory-B-cell subsets.

fold change

Gene symbol Probe set trans. pre- mat. natural IgM- CD27+ CD27+ CD27- CD27- CD27-IgA+ CD27-IgA+ CD27-IgA+ naive naive effector only IgG+ IgA+ IgG+ IgA+ vs vs vs CD27+IgA+ CD27-IgG+ naive mat. pan B CD19 206398_s_at 1356.9 1521.8 1912.9 930.6 774.1 1226.8 1360.2 1272.3 1131.0 -1.2 -1.1 -1.7 MS4A1 (CD20) 228592_at 5809.8 4802.1 6516.7 4603.6 4550.8 10107.4 10008.6 9587.5 7915.4 -1.3 -1.2 1.2 CR2 (CD21) 205544_s_at 101.20 76.64 105.0 50.5 66.5 82.0 117.9 79.6 88.9 -1.3 1.1 -1.2 CD40 35150_at 203.0 212.3 265.1 206.5 184.2 213.5 207.4 220.1 176.6 -1.2 -1.2 -1.5 CD79A 205049_s_at 1889.1 2318.7 1878.6 1676.8 1577.1 897.7 929.1 1104.9 795.1 -1.2 -1.4 -2.4 CD79B 205297_s_at 2062.18 1520.96 1458.40 1161.46 862.22 722.05 745.00 1066.61 961.20 1.3 -1.1 -1.5 CD81 200675_at 1990.3 1391.2 1272.1 1513.4 1340.4 1381.0 1304.0 1560.8 1167.5 -1.1 -1.3 -1.1 TNFRSF13C (BAFFR) 1552892_at 239.8 276.8 336.4 298.8 327.7 255.4 235.6 331.0 288.6 1.2 -1.1 -1.2

B cell CD5 230489_at 133.0 100.2 35.7 51.0 51.2 37.6 32.8 37.5 36.9 1.1 1.0 1.0 subset CD38 205692_s_at 150.3 77.2 42.0 39.6 40.5 29.5 39.3 41.4 36.8 -1.1 -1.1 -1.1 IGHM 212827_at 9930.3 9629.5 10453.4 7616.8 6668.8 550.6 488.7 863.4 657.7 1.3 -1.3 -15.9 IGHD 213674_x_at 4212.8 4559.7 4419.2 2340.6 541.2 57.3 72.5 144.5 93.2 1.3 -1.5 -47.5 IGHA 217022_s_at 57.2 51.0 93.1 554.7 865.2 1922.5 6175.6 917.5 4632.9 -1.3 5.0 49.8 IGHG 211430_s_at 79.5 197.3 397.3 1008.3 902.0 3356.3 1011.4 3791.6 841.0 -1.2 -4.5 2.1 IGKC 205544_s_at 14485.2 13709.4 13117.2 14284.0 13377.4 12043.9 12019.7 11990.8 9414.2 -1.3 -1.3 -1.4 IGLC 209139_s_at 318.4 263.1 329.0 445.8 501.7 542.6 527.4 473.3 414.0 -1.3 -1.1 1.3 CD23 206760_s_at 383.2 738.4 721.6 164.1 100.1 90.5 94.7 139.8 81.3 -1.2 -1.7 -8.9 activation/ CD27 206150_at 49.0 53.8 47.6 425.9 368.8 363.8 344.9 107.1 128.6 -2.7 1.2 2.7 memory CD80 207176_s_at 32.8 42.9 31.7 64.6 66.3 133.3 97.8 104.8 81.9 -1.2 -1.3 2.6 CD86 210895_s_at 166.5 105.0 171.2 334.3 276.6 442.7 359.7 357.2 365.5 1.0 1.0 2.1 CD180 206206_at 224.6 221.8 199.7 291.6 236.1 176.5 184.4 203.3 177.1 1.0 -1.1 -1.1 FAS 204781_s_at 31.0 24.7 36.9 91.3 119.8 268.0 186.8 149.8 314.9 1.7 2.1 8.5 TNFRSF13B (TACI) 207641_at 72.3 58.9 70.4 194.9 177.1 214.6 187.2 138.1 207.9 1.1 1.5 3.0 CD58 205173_x_at 21.8 26.4 31.9 93.5 105.8 148.8 136.4 126.5 198.3 1.5 1.6 6.2

BCR- DAPP1 (BAM32) 222858_s_at 562.9 427.0 550.6 657.6 467.8 642.3 598.6 600.1 461.7 -1.3 -1.3 -1.2 signaling PIK3AP1 (BCAP) 226459_at 778.9 700.1 925.0 789.9 789.0 1053.7 1045.3 1039.3 988.2 -1.1 -1.1 1.1 BCL10 205263_at 1263.2 859.6 603.7 893.6 644.9 592.2 580.0 725.3 711.8 1.2 1.0 1.2 BLNK 207655_s_at 939.1 737.0 931.8 671.5 645.0 967.9 1141.1 926.8 819.2 -1.4 -1.1 -1.1 BTK 205504_at 582.1 484.4 496.4 379.0 360.1 453.9 435.1 406.5 414.9 1.0 1.0 -1.2 CARD11 223514_at 485.5 399.6 626.8 557.8 742.8 631.0 616.8 525.5 576.5 -1.1 1.1 -1.1 CDC42 206398_s_at 1400.4 1112.6 1105.6 1497.4 1410.4 1309.0 1223.8 1455.6 1658.2 1.4 1.1 1.5 GAB1 212587_s_at 96.8 46.7 28.0 24.6 26.3 17.5 22.4 14.3 18.6 -1.2 1.3 1.5 GAB2 205049_s_at 324.7 145.7 112.0 275.2 447.5 257.0 258.9 208.6 316.9 1.2 1.5 2.8 GRB2 205297_s_at 1719.1 1295.9 1387.7 2523.6 2707.2 1628.3 1802.4 1800.8 1915.1 1.1 1.1 1.4 LYN 208728_s_at 4985.1 3770.2 4259.0 3704.4 4436.6 2642.6 2763.1 3345.5 2872.1 1.0 -1.2 -1.5 MALT1 202723_s_at 339.4 282.0 367.4 445.5 399.6 326.9 300.7 361.4 334.7 1.1 -1.1 -1.1 PTEN 1557030_at 1127.4 972.0 916.9 1226.0 1035.6 930.4 1012.9 914.4 1044.0 1.0 1.1 1.1

2

RAC1 203853_s_at 1625.5 1499.3 1586.7 1665.3 1570.5 1122.7 1283.1 1424.1 1374.4 1.1 1.0 -1.2 PTPN6 (SHP1) 223049_at 598.3 911.9 667.6 464.7 479.2 735.3 626.3 675.0 581.9 -1.1 -1.2 -1.1 PTPN11 (SHP2) 202626_s_at 404.4 333.1 374.2 426.9 468.5 435.6 443.5 529.9 523.0 1.2 1.0 1.4 SYK 208309_s_at 1262.5 1005.2 1091.8 1836.2 1384.4 900.9 1062.1 877.5 746.8 -1.4 -1.2 -1.5 INPP5D (SHIP1) 225363_at 714.8 600.7 876.8 625.9 826.9 1001.4 1088.0 942.7 907.8 -1.2 1.0 1.0

BCR- CD22 38521_at 921.1 1123.1 1365.1 568.0 560.4 516.5 570.8 620.0 591.6 1.0 1.0 -2.3 signaling CD72 215925_s_at 602.7 463.9 469.0 238.6 140.9 71.5 159.3 177.8 172.7 1.1 1.0 -2.7 inhibition PTPRC (CD45) 212587_s_at 2878.7 2230.4 4002.0 3616.1 3147.2 3686.9 3588.3 2899.2 2431.0 -1.5 -1.2 -1.6 SIGLEC10 1552807_a_at 335.7 216.6 272.8 545.0 495.2 382.7 394.5 424.1 450.3 1.1 1.1 1.6 FCGRB 210889_s_at 377.2 384.1 490.8 544.2 493.6 384.1 449.2 431.7 513.3 1.1 1.2 1.0 PDCD1 207634_at 52.7 67.1 61.2 60.3 57.4 53.6 52.1 55.3 58.7 1.1 1.1 1.0 CEACAM1 (CD66A) 211881_x_at 316.4 343.5 382.1 248.7 179.0 131.8 129.8 172.6 129.5 1.0 -1.3 -3.0 LAIR1 210644_s_at 124.6 160.5 120.6 68.6 45.1 61.2 66.9 81.0 46.9 -1.4 -1.7 -2.6 LILRB3 (PIRB) 210225_x_at 61.7 65.5 68.4 76.8 62.5 56.8 64.9 69.6 77.3 1.2 1.1 1.1 LILRB1 (ILT2) 229937_x_at 140.4 133.1 237.9 253.0 225.7 311.3 390.0 419.0 380.4 1.0 -1.1 1.6

CD40 A20 202644_s_at 1467.8 1022.2 953.3 1750.1 1961.5 2522.5 2133.4 1061.3 1375.4 -1.6 1.3 1.4 signaling TRAF3IP2 (ACT1) 215411_s_at 227.0 237.5 310.2 266.1 329.2 229.3 259.0 285.5 242.4 -1.1 -1.2 -1.3 TANK 207616_s_at 529.4 422.6 715.7 584.4 616.1 668.7 579.2 656.9 519.8 -1.1 -1.3 -1.4 TRAF1 205599_at 55.7 64.4 49.0 52.1 56.2 83.6 69.3 71.8 67.4 1.0 -1.1 1.4 TRAF2 204413_at 54.2 49.0 52.6 52.6 51.3 52.1 58.8 48.4 55.8 -1.1 1.2 1.1 TRAF3 208315_x_at 145.2 89.9 124.6 175.2 198.5 175.4 160.2 193.8 215.6 1.3 1.1 1.7 TRAF5 204352_at 755.6 638.0 851.7 598.2 635.6 999.7 1109.2 1072.6 1118.1 1.0 1.0 1.3 TRAF6 1569861_at 14.0 19.8 15.3 14.1 12.1 12.7 13.3 15.5 13.8 1.0 -1.1 -1.1

TLRs TLR1 210176_at 190.0 253.8 308.0 132.2 119.1 124.1 111.5 124.5 79.2 -1.4 -1.6 -3.9 TLR2 204924_at 34.3 45.6 36.3 44.4 41.6 41.6 38.4 47.5 36.5 -1.1 -1.3 1.0 TLR3 206271_at 15.4 24.1 13.6 12.0 12.4 14.5 10.5 12.7 13.8 1.3 1.1 1.0 TLR4 1552798_s_at 27.2 28.7 25.1 27.7 25.9 23.0 22.0 22.2 24.1 1.1 1.1 1.0 TLR5 210166_at 26.2 46.6 23.8 27.6 25.8 23.1 20.7 24.3 25.1 1.2 1.0 1.1 TLR6 239021_at 50.6 61.5 71.4 75.9 72.7 84.3 87.2 68.2 63.6 -1.4 -1.1 -1.1 TLR7 220146_at 64.0 57.0 51.1 66.1 52.9 59.1 50.0 44.6 40.5 -1.2 -1.1 -1.3 TLR8 220832_at 19.3 19.3 19.2 17.6 19.2 13.5 16.1 18.1 18.2 1.1 1.0 1.1 TLR9 223903_at 45.6 51.4 44.9 48.1 48.5 39.5 43.0 40.8 49.5 -1.2 -1.2 -1.1 TLR10 223750_s_at 82.5 83.7 92.2 211.4 146.7 157.8 154.2 158.0 205.3 1.3 1.3 2.2

TLR MYD88 209124_at 691.9 544.0 552.4 620.6 684.3 569.8 616.8 715.0 769.3 1.2 1.1 1.4 signaling IRAK1 201587_s_at 1469.8 1158.1 1377.0 1661.2 1663.1 1464.4 1490.8 1345.8 1721.5 1.2 1.3 1.3 IRAK2 231779_at 46.5 71.6 104.5 163.9 233.4 170.3 139.1 276.7 265.8 1.9 1.0 2.5 IRAK4 219618_at 106.1 85.3 97.2 86.7 85.9 105.5 99.9 95.5 95.2 1.0 1.0 1.0 IKBKG (NEMO) 209929_s_at 149.5 131.1 155.6 124.8 123.1 147.2 160.2 184.4 165.2 1.0 -1.1 1.1 TIRAP 1554091_a_at 51.1 47.0 39.9 47.5 49.8 49.5 44.1 58.1 45.4 1.0 -1.1 1.1 TRAF6 205558_at 132.7 116.8 117.9 141.4 141.2 119.5 126.4 126.9 120.6 1.0 -1.0 1.0 TBK1 218520_at 146.2 93.8 151.2 181.8 229.4 326.9 310.0 309.7 341.4 1.1 1.1 2.3 3

RIPK1 209941_at 119.9 74.2 103.6 110.2 130.4 127.2 121.8 137.9 117.4 1.0 -1.1 1.1 interleukins IL1B 39402_at 37.4 36.4 40.0 37.0 38.1 35.3 28.3 33.8 32.4 1.1 1.0 -1.2 IL2 209849_s_at 39.3 36.0 41.1 34.9 27.9 42.2 40.0 38.9 39.4 1.0 1.0 1.0 IL3 207906_at 11.4 15.2 13.0 16.4 11.9 11.5 12.3 13.1 14.0 1.1 1.1 1.1 IL4 207539_s_at 10.8 14.7 14.2 12.7 13.3 11.7 10.8 12.7 12.5 1.2 1.0 -1.1 IL5 207952_at 16.9 20.2 17.9 20.3 17.2 17.0 15.9 13.9 19.0 1.2 1.3 1.1 IL6 205207_at 93.8 234.7 475.0 517.8 561.2 130.4 174.6 101.3 95.5 -1.8 -1.1 -5.0 IL7 206693_at 46.3 50.2 46.4 110.7 95.1 130.5 134.8 155.1 142.9 1.1 -1.1 3.1 IL8 211506_s_at 17.5 27.7 21.1 18.0 18.2 13.9 15.3 15.3 18.9 1.2 1.2 -1.1 IL9 208193_a 14.1 17.4 13.0 14.2 11.4 11.7 10.3 12.1 10.4 1.0 -1.2 -1.2 IL10 207433_at 20.7 16.8 12.7 23.9 23.7 14.6 13.0 13.5 14.9 1.1 1.1 1.2 IL11 206926_s_at 37.0 33.9 34.4 33.7 36.6 34.8 38.4 37.6 40.5 1.1 1.1 1.2 IL13 207844_at 39.0 51.8 41.2 41.2 41.2 44.9 43.6 39.6 46.6 1.1 1.2 1.1 IL15 205992_s_at 72.3 90.7 245.0 205.5 170.3 328.9 400.7 223.1 284.9 -1.4 1.3 1.2 IL16 209827_s_at 364.7 387.6 421.7 343.4 331.7 261.1 378.1 426.2 447.8 1.2 1.0 1.1 IL17A 208402_at 23.7 28.1 23.7 19.9 20.5 20.3 19.1 21.0 23.0 1.2 1.1 1.0 IL17B 220273_at 21.4 23.9 25.2 21.2 19.8 17.6 19.1 20.5 19.1 1.0 -1.1 -1.3 IL17D 227401_at 24.8 30.6 25.2 28.4 25.5 22.8 21.2 25.1 22.4 -1.1 -1.1 -1.1 IL17F 234408_at 17.9 29.6 20.4 24.4 20.6 16.2 16.1 16.7 16.6 1.0 1.0 -1.2 IL18 206295_at 20.0 16.7 20.1 23.9 17.8 20.5 16.6 15.6 17.6 -1.1 -1.1 -1.1 IL19 220745_at 37.9 45.9 40.9 40.9 36.0 33.1 35.9 39.7 38.8 1.1 1.0 -1.1 IL20 224071_at 20.2 22.8 18.5 19.2 19.8 18.6 20.0 17.3 19.3 1.0 1.1 1.0 IL21 221271_at 19.3 22.9 19.2 16.8 17.5 17.6 17.6 19.2 20.2 1.1 1.1 1.1 IL22 220056_at 37.8 44.2 40.9 34.3 37.7 33.7 32.9 34.3 39.4 1.2 1.1 1.0 IL23A 220054_at 52.4 48.4 76.5 79.9 78.1 107.0 92.3 91.8 108.5 1.2 1.2 1.4 IL24 206569_at 29.3 34.1 36.7 42.7 30.9 24.4 26.5 33.6 29.6 -1.1 1.1 -1.2 IL25 220971_at 37.5 39.8 26.7 33.1 34.6 31.4 33.4 29.9 36.9 1.1 1.2 1.4 IL26 221111_at 12.2 15.1 13.7 13.3 11.3 12.6 14.2 12.9 13.6 1.0 1.0 1.0 IL27 1552995_at 54.1 50.6 56.7 57.6 58.5 43.4 46.1 51.1 50.3 1.1 1.0 1.0 IL29 1552917_at 30.4 44.4 33.2 30.5 33.1 23.7 25.7 29.9 33.7 1.3 1.1 1.0 IL32 203828_s_at 27.8 29.2 30.3 29.0 27.6 23.3 22.4 28.0 24.4 1.1 -1.1 -1.2 IL33 209821_at 8.4 12.8 9.5 8.4 9.9 7.7 7.0 8.3 8.2 1.2 1.0 -1.1 IL34 237046_x_at 64.5 63.1 65.1 67.2 65.7 62.0 59.9 59.8 71.4 1.2 1.2 1.1 TNF 207113_s_at 28.7 36.9 33.4 46.0 55.0 38.3 48.8 35.5 36.0 -1.4 1.0 -1.1 interleukin IL1R1 215561_s_at 18.2 19.9 20.9 18.4 21.3 17.7 18.6 18.2 16.6 -1.1 -1.1 -1.3 receptors IL1R2 211372_s_at 23.4 31.4 24.4 22.7 24.3 22.7 19.9 23.5 24.7 1.2 1.0 1.0 IL1RB 39402_at 37.4 36.4 40.0 37.0 38.1 35.3 28.3 33.8 32.4 1.1 1.0 -1.2 IL2RA 206341_at 27.1 30.4 30.3 33.6 39.0 47.6 43.4 38.4 33.5 -1.3 -1.1 1.1 IL2RB 205291_at 34.0 45.0 33.9 49.4 46.4 59.2 56.6 55.1 77.9 1.4 1.4 2.3 IL2RG 204116_at 129.8 255.1 180.2 158.5 116.8 144.6 130.5 147.0 128.0 -1.0 -1.1 -1.4 IL3RA 206148_at 81.9 59.4 46.4 43.5 48.2 39.7 38.9 37.4 48.9 1.3 1.3 1.1 IL3R 207906_at 11.4 15.2 13.0 16.4 11.9 11.5 12.3 13.1 14.0 1.1 1.1 1.1 IL4R 203233_at 1800.4 2788.7 3255.0 316.8 261.6 219.3 161.5 529.4 282.3 1.7 -1.9 -11.5 IL5RA 211517_s_at 26.2 39.2 47.0 28.0 30.4 54.8 47.5 40.1 41.0 -1.2 1.0 -1.1 4

IL5R 207952_at 16.9 20.2 17.9 20.3 17.2 17.0 15.9 13.9 19.0 1.2 1.4 1.1 IL6R 226333_at 24.7 34.3 29.7 51.0 49.2 105.4 73.1 48.4 79.5 1.1 1.6 2.7 IL7R 205798_at 24.7 35.6 26.1 26.2 26.5 25.2 22.9 29.7 25.3 1.1 -1.2 1.0 IL9R 217212_s_at 38.2 32.1 34.3 33.8 31.8 34.7 33.9 29.2 33.8 1.0 1.2 1.0 IL10RA 204912_at 466.7 870.8 908.8 1392.7 1639.9 1321.2 1379.3 1544.4 1454.3 1.1 -1.1 1.6 IL10RB 209575_at 226.1 179.4 150.2 270.1 212.9 163.4 173.3 198.7 166.3 1.0 -1.2 1.1 IL11RA 204773_at 62.9 54.0 94.7 57.9 86.0 109.5 119.3 88.8 84.4 -1.4 1.0 -1.1 IL12RB1 206890_at 43.4 48.4 39.9 40.9 39.2 31.9 39.9 36.6 40.5 1.0 1.1 1.0 IL12RB2 206999_at 17.7 21.0 16.4 15.1 15.2 16.3 14.9 16.4 14.4 1.0 -1.1 -1.1 IL13RA1 201887_at 39.4 106.7 147.9 93.3 87.7 397.2 258.1 407.5 213.5 -1.2 -1.9 1.4 IL15RA 207375_s_at 49.2 55.6 47.1 53.3 59.8 82.4 77.1 72.9 70.5 -1.1 1.0 1.5 IL17RC 221926_s_at 64.5 63.3 46.2 48.4 48.2 42.2 43.6 43.1 39.8 -1.1 -1.1 -1.2 IL17RB 224361_s_at 34.3 47.2 35.0 35.7 44.2 30.8 33.2 32.9 31.3 -1.1 -1.1 -1.1 IL17RA 205707_at 180.9 142.1 149.2 144.1 146.8 145.2 148.9 145.6 143.5 1.0 1.0 1.0 IL21R 221658_s_at 245.6 154.1 98.0 40.9 28.3 22.0 19.4 33.3 42.4 2.2 1.3 -2.3 IL22RA1 220056_at 37.8 44.2 40.9 34.3 37.7 33.7 32.9 34.3 39.4 1.2 1.1 1.0 IL23R 1561853_a_at 20.0 27.1 22.2 20.8 19.4 16.8 15.9 18.9 18.0 1.1 1.0 -1.2

TGFBR1 224793_s_at 243.4 237.6 271.5 677.6 710.3 208.4 284.8 345.4 426.6 1.5 1.2 1.6 TGFBR2 208944_at 1296.5 1168.9 1535.6 1358.0 1467.1 675.7 843.2 914.0 881.7 1.0 1.0 -1.7 RARA 201605_x_at 756.4 720.7 558.6 640.1 612.5 488.1 490.4 532.2 551.6 1.1 1.0 1.0 trancription factors PRDM1 (BLIMP1) 217192_s_at 50.8 55.7 55.5 58.3 56.4 74.9 72.0 68.8 63.2 -1.1 -1.1 -1.1 FOXO1 202723_s_at 1119.5 1120.3 1458.5 558.7 536.1 371.7 393.7 446.2 324.8 -1.2 -1.4 -4.5 IRF4 204562_at 175.3 126.5 120.7 166.4 212.6 167.6 234.2 180.9 219.6 -1.1 1.2 1.8 KLF2 219371_s_at 4581.5 6246.3 6085.8 5168.3 4143.3 5815.8 4573.6 5177.5 3506.9 -1.3 -1.5 -1.7 KLF4 221841_s_at 1052.4 1579.9 1343.9 572.3 217.2 620.3 442.6 189.9 176.8 -2.5 -1.1 -7.6 KLF9 203542_s_at 969.8 1216.6 1640.5 1825.1 1318.6 869.2 928.8 1009.8 879.6 -1.1 -1.1 -1.9 NOTCH2 202443_x_at 791.3 743.8 745.5 887.4 821.0 827.4 945.0 943.5 873.0 -1.1 -1.1 1.2 PAK2 (p21) 208877_at 524.1 457.2 503.8 767.3 779.0 644.3 596.0 706.7 740.1 1.2 1.0 1.5 PAX5 221969_at 4639.4 3919.2 5064.9 3251.1 3161.1 3160.1 3450.2 3462.2 2899.5 -1.2 -1.2 -1.7 ZBTB16 (PZLF) 205883_at 29.6 56.9 177.4 41.1 30.9 25.2 31.2 43.8 39.4 1.3 -1.1 -4.5 RORA 210426_x_at 17.2 17.9 21.4 188.0 206.0 354.8 339.8 165.1 283.2 -1.2 1.7 13.2 RUNX1 209360_s_at 214.8 161.8 184.5 164.7 143.5 135.2 136.0 172.3 137.4 1.0 -1.3 -1.3 RUNX2 232231_at 12.9 15.4 15.2 22.3 78.1 44.2 204.2 60.1 483.2 2.4 8.0 31.9 RUNX3 204198_s_at 1188.5 1153.5 1444.0 1821.8 2343.7 2212.5 2005.9 1880.6 1948.0 1.0 1.0 1.3 SOX5 207336_at 64.7 33.9 33.6 150.1 76.6 47.3 48.2 74.6 68.2 1.4 -1.1 2.0 SPIB 205861_at 333.8 207.0 168.9 262.3 253.6 237.2 266.6 211.0 218.5 -1.2 -1.0 1.3 TBX21(Tbet) 220684_at 33.6 52.9 45.6 79.8 65.4 92.1 91.7 110.4 75.6 -1.2 -1.5 1.7 chemokine CXCR1 207094_at 59.5 74.5 49.1 57.5 62.3 44.5 46.0 50.5 46.1 1.0 -1.1 -1.1 receptors CXCR2 207008_at 18.7 22.7 21.5 18.2 19.3 21.4 19.9 18.1 22.4 1.1 1.2 1.0 CXCR3 217199_s_at 37.2 33.8 36.2 33.0 32.6 38.6 34.8 39.4 34.7 1.0 -1.1 1.0 CXCR4 209201_x_at 4233.9 5514.2 3675.7 3376.7 3564.5 1772.1 2327.8 2614.2 1614.5 -1.4 -1.6 -2.3 CXCR5 206126_at 429.7 367.1 459.7 788.3 952.2 370.0 510.4 429.8 436.7 -1.2 1.0 -1.1 5

CXCR6 211469_s_at 28.9 42.0 39.6 33.7 31.5 34.8 28.8 34.6 34.2 1.2 1.0 -1.2 CCR1 205099_s_at 19.4 18.5 21.0 22.9 22.2 20.8 21.6 19.8 23.4 1.1 1.2 1.1 CCR2 207794_at 19.0 25.8 22.8 19.1 15.6 19.2 23.6 22.2 21.1 -1.1 1.0 -1.1 CCR3 208304_at 18.5 28.1 18.2 17.6 19.8 17.5 17.4 20.6 21.7 1.2 1.1 1.2 CCR4 208376_at 40.7 49.3 35.2 41.0 36.6 37.1 37.4 42.4 43.4 1.2 1.0 1.2 CCR5 206991_s_at 27.8 36.5 28.2 30.2 32.4 27.4 26.2 30.0 27.9 1.1 -1.1 1.0 CCR6 206983_at 50.7 62.0 101.7 162.7 121.8 67.8 97.1 112.8 86.8 -1.1 -1.3 1.2 CCR7 206337_at 132.6 356.3 660.6 305.2 296.1 250.1 334.9 402.3 250.0 -1.3 -1.6 -2.6 CCR8 208059_at 19.4 32.8 18.0 17.9 19.7 19.5 21.5 19.5 20.8 1.0 1.1 1.2 CCR9 207445_s_at 45.7 29.7 20.1 29.8 44.8 17.4 35.6 20.5 87.3 2.5 4.3 4.4 CCR10 220565_at 27.4 34.0 30.6 28.6 31.2 34.8 34.9 26.9 26.1 -1.3 1.0 -1.2 CX3CR1 205898_at 27.2 31.5 27.1 18.3 20.8 24.7 29.0 27.6 42.4 1.5 1.5 1.6 XCR1 1561226_at 29.0 32.0 28.4 28.6 26.8 24.5 24.3 24.6 25.4 1.0 1.0 -1.1 adhesion ITGAL (CD11A) 213475_s_at 130.1 110.7 166.3 217.4 201.0 360.9 326.4 229.9 274.0 -1.2 1.2 1.6 molecules ITGA4 (CD49D) 213416_at 639.8 497.5 692.7 527.4 388.4 960.5 712.7 866.7 812.6 1.1 -1.1 1.2 ITGB1 (CD29) 211945_s_at 379.6 189.0 292.3 1075.1 1000.0 2920.9 2067.4 1560.3 1467.9 -1.4 -1.1 5.0 ITGB2 (CD18) 202803_s_at 546.6 557.5 598.7 604.5 408.5 534.3 493.6 739.1 591.8 1.2 -1.2 1.0 ITGB7 205718_at 261.7 171.0 170.0 174.7 193.2 157.3 147.5 152.5 149.6 1.0 1.0 -1.1 SELL (CD62L) 204563_at 778.4 1347.5 1240.4 823.7 780.0 918.5 892.2 964.3 666.0 -1.3 -1.4 -1.9 Statistically significant differences between CD27-IgA+, and mat. naive, CD27+IgA+ and CD27-IgG+ memory-B cells are depicted in bold. Fold change was calculated by determining the absolute value of the ratio of transcript expression between the indicated populations. Values were assigned a negative value if gene expression was lower in CD27-IgA+ as compared to the indicated control population.

6

SUPPLEMENTAL TABLE II. Repertoire and reactivity of antibodies from CD27+IgA+ and CD27-IgA+ B cells of 5donors.

Ig HEAVY LIGHT REACTIVITY VH DH RF JH (-) CDR3 (aa) (+) Vκ Jκ CDR3 (aa) Length No of SHM Length No of SHM Poly Hep2 IFA flagellin B.fragilis C.difficile E.cloacae E.faecalis S.aureus E.coli donor1-01 3-66 6-19 3 4 1 VLPFSPSGGWTPFDR 0 15 19 1-5 1 QQYDTYPWT 9 9 donor1-04 4-59 3-3 1 5 2 TVLLEWFFFEP 0 11 26 3-20 3 QQYGSSPFT 9 11 ------donor1-05 3-9 5-24 2 6 2 DGRYSYMDV 1 9 29 3-15 5 QQYNYWPLT 9 22 donor1-06 5-51 3-10 2 4 1 RSGYSASDF 1 9 41 1-5 4 QEYDSYSGS 9 24 donor1-07 3-13 5-12 2 4 2 VRYDHGYDL 2 9 22 1-39 2 QQSYSTLLYN 10 1 - + ------donor1-08 3-23 3-10 1 5 3 DRGVGEILNNWFDP 0 14 20 3-15 4 QQHSDWPLT 9 14 ------+ - - donor1-09 3-15 / / 4 1 TTGSVEGY 0 8 20 donor1-11 4-4 3-9 1 5 2 GDTYDWFFNS 0 10 31 3-20 4 QHYATSPLT 9 21 donor1-12 4-31 / / 4 1 RPGNRTFYLGVCDY 2 14 27 3-20 2 QYYGRSPYS 9 12 - - - - + + + + + + donor1-14 1-2 2-2 2 4 2 DHCSGTSCYAVD 0 12 28 1-33 4 QQYDDLRLT 9 12 ------donor1-17 3-7 5-24 2 4 0 GFGPGY 0 6 4 4-1 1 QQYYNTPPWT 10 9 - - - - + + + + - - donor1-19 3-30 3-16 1 4 4 DLGDDYPSLGVIDY 0 14 9 1-39 2 QQSYSTPPVFT 11 13 - + - - + + - + - + donor1-20 3-74 1-26 3 4 1 LRVGAAIPHGFDL 2 13 36 3-20 2 QQYDGSPYT 9 2 - + ------donor1-21 4-39 / / 1 1 HFYAETFQN 1 9 21 3-20 4 QHYATSPLT 9 24 CD27+IgA+ CD27+IgA+ donor1-26 3-30 / / 6 2 DRISPSGPKIVYVYYYMDV 2 19 39 3-11 3 QLGRTWPPA 9 18 ------+ + - + donor1-27 3-9 / / 6 2 GKDISRGGMDV 1 9 32 donor1-29 3-23 / / 4 3 AKDRTGGADTLDY 2 11 8 1-6 4 LQDYNYPRT 9 4 ------donor1-30 5-51 2-21 2 6 1 SYCASTSCQTRGYFGMDV 1 18 14 donor1-31 3-24 / / 4 1 SLRGAVTVRDY 2 11 12 3-20 3 QQFGSSRFT 9 6 - + - - - + - + + + donor1-34 3-15 3-10 3 5 0 QTRNTVVRGVPINGGLAP 2 18 16 4-1 4 QQYYSSPLT 9 10 + ------donor1-37 3-7 5-5 2 4 0 VRGYGNGHWVF 2 11 21 3-20 1 HQYVGSPWT 9 13 - + ------donor1-38 1-39 3 QQSYNFPRT 9 17 donor1-39 3-7 / / 4 1 IFSGLTFDY 0 9 28 1-8 3 QQYSSYPFT 9 14 ------donor1-41 3-15 3-10 3 3 2 VREGVIAAFDI 1 11 17 1-33 5 QQYDNLLRIT 10 4 ------+ - - - donor1-49 1-2 / / 5 2 RKGDLLGEG 2 9 4 1-12 4 QQANSFPLT 9 4 + - c ------donor1-52 4-59 3-16 3 4 1 GFGGVIADY 0 9 0 3-11 2 QQRSNWPPEYT 11 0 donor1-59 4-59 2-2 2 4 1 VREGYCSSTSCYTGN 1 15 1 3-15 3 QQYNNWPL 8 0 ------donor1-60 3-33 5-24 2 3 2 DGGGGHFDI 1 8 0 1-39 1 QQSYSTTWT 9 0 ------donor1-62 3-23 6-19 2 4 1 FSGWYRGSLDY 1 11 11 1-27 3 QKYNSAPFT 9 2 + - c + + + + + + + donor1-65 4-59 3-16 3 4 1 GFGGVIADY 0 9 0 3-20 3 QQHGSSPMT 9 12 donor1-71 1-2 6-19 3 5 2 DQVGRAVAGWFDP 1 13 9 3-15 2 QQHNNWPYS 9 4 - + - - - + + + - + DREQQLDHLVTGYYSYGMD donor1-72 3-21 6-13 1 6 4 V 1 20 18 1-5 1 QQYNGYSRT 9 5 + - N - + + + + - - donor1-73 3-48 1-7 2 4 2 DWWNWNSDN 0 9 10 4-1 2 QQYYSTPSYT 10 2 CD27-IgA+ CD27-IgA+ donor1-74 3-15 2-21 3 4 1 APAVTARPGLDY 1 12 22 1-5 2 QQYSDYPMYS 10 10 7

donor1-77 3-33 5-5 3 6 3 RDLDTATFYYYYGMDVW 1 15 6 3-20 2 QQYGSSQYS 9 2 ------donor1-79 4-39 6-19 3 4 1 HPIAVAGWGGFDY 1 13 19 1-33 4 QQYDNLPLS 9 6 ------donor1-80 4-39 3-3 2 5 2 PQGQRDYDFWSGHL 1 14 0 donor1-84 3-9 5-12 3 4 1 ARIVATITGVNYYFDS 1 16 16 donor1-85 3-9 / / 3 2 DMSRGLGMFAFDM 1 13 30 3-20 4 QQRGS 5 15 ------donor1-86 3-9 5-5 2 4 3 GSTSYGPDHELEN 0 13 20 3-15 4 QQYNKLPLT 9 8 donor1-87 1-18 2-21 2 3 1 GNGLSHCGGDCL 1 12 23 3-15 1 HQYTNWPT 8 10 donor1-91 3-74 4-4 2 6 2 MDYSNYVGHSGFDV 1 14 29 donor1-94 1-46 5-24 2 6 2 DSFANWGVMDV 0 11 20 3-20 2 QQYGSSMYS 9 9 ------donor2-03 4-59 4-17 2 3 3 GKGDLSGTDAFDI 1 13 13 1-5 1 QQYNTYWT 8 8 ------donor2-07 3-7 4-4 2 5 3 EWSNYADNWFDS 0 12 21 1-39 2 QQSYSTPT 8 23 - + ------donor2-13 3-7 3-16 2 4 2 PTYFDYIWGRPPFGSFDY 1 18 12 4-1 4 QQYYETPLT 9 10 - + - - - + - + - - donor2-17 4-31 6-13 2 5 2 DASSSPGWLDP 0 11 21 3-15 4 QQYNNWPPLT 10 7 - + ------donor2-19 3-21 1-26 2 6 2 SDYIGASYYFYMDV 0 14 31 1-5 2 QQYSTYPYT 9 14 - donor2-23 3-30 2-8 2 3 1 PCTGGVCYSSGLDV 0 14 9 1-39 4 QQSYSTPALS 8 7 - + - - - - - + + - donor2-24 3-30 6-13 2 4 2 ETTGYSSRSFDY 1 12 7 1-39 3 QQSFTSSTT 9 12 ------+ - donor2-29 4-59 3-3 3 4 1 ERIGAATDY 1 9 26 1-39 1 QQSYITPRT 9 15 ------donor2-30 1-69 3-10 3 6 3 DSNYHGSGSDYNSCCYMDV 1 19 9 1-5 1 QQYNRYST 8 5 ------

CD27+IgA+ donor2-37 1-24 3-3 2 4 2 HNRFWRDYSRFFDY 3 14 27 3-15 4 QQYNSWPPLT 10 18 + - - - + + + + + + donor2-38 1-46 4-4 2 5 1 GLSNYGWFDP 0 10 10 3-15 4 QHYNNWPLT 9 7 ------donor2-39 4-15 4-17 2 4 3 DWANGDSFSPFDY 0 13 23 1-33 4 QQYDTLPT 8 17 ------donor2-42 4-34 3-10 1 6 2 RAWFRESPYYYYYMDV 2 16 21 1-39 1 QQSYTTLWT 9 10 - + ------donor2-44 3-23 3-16 1 4 2 DVARSQLGKFDC 2 12 15 1-8 1 QQYYNYPWT 9 6 ------donor2-46 3-15 3-3 2 4 2 HGGYDFWSGYYDY 1 13 16 3-15 4 QQYNYWLT 8 9 - + c - + - - - - - donor2-60 3-23 6-6 3 5 1 SSIAARPTWFDP 1 12 12 1-39 2 QQFYTTLPERS 11 9 - - - - - + - - - - donor2-65 1-69 6-13 3 6 2 DRIAAATYLGYYYYYMDV 1 18 1 3-11 4 QQRSNWLT 8 0 4- donor2-68 30-4 / / 2 1 RVSRHVYWFFDV 3 12 25 3-7 1 QQDYNLPWT 9 16 donor2-90 3-30 6-19 3 4 2 DVAGIAFDY 0 9 6 1-6 1 LQDYNYPWT 7 4

CD27-IgA+ CD27-IgA+ donor2-92 3-11 4-17 2 3 5 LADYGDYGDDAFDI 0 14 7 donor2-95 3-48 3-3 2 4 1 RHDFWGGYTY 1 10 20 3-20 1 QHYSLQT 7 10 + - - + + + + + + + donor3-10 3-48 4-17 2 4 3 PDYGDYVGGLD 0 11 14 3-20 2 QQYGTSPPYT 10 6 donor3-12 3-23 2-15 2 4 1 GTQNYCSGATCYSLDY 0 16 20 donor3-18 3-23 3-22 2 4 1 RGDGASYY 1 8 21 1-27 1 QKYNGAPWT 9 11 ------donor3-23 4-34 4-17 2 4 3 RLGVETTTDYFDY 1 13 9 donor3-25 3-30 3-9 2 2 3 AWGLLTGDYWDDWYFHF 1 17 39 donor3-28 3-53 2-15 2 4 1 GSGGYCSGSTCYSAFDC 0 17 42 3-15 1 QQYNNWLWT 9 25 donor3-32 3-30 6-13 3 4 1 VPAGTRSYIDY 1 11 21 1-37 5 QRTDSVLIS 9 17 donor3-35 3-23 1-26 2 2 1 GHKYCPSGSCYPYWYLDV 1 18 36 3-15 1 QQYNTWPGT 9 16 + + N - + + - - - + CD27+IgA+ CD27+IgA+ donor3-37 4-61 5-12 1 5 3 GKIFMEWLEGFDP 0 13 8 4-1 4 QQYYGTPLT 9 5 - - - + - - - + - + donor3-39 3-23 2-15 2 4 1 GTQNYCSGATCYSLDY 0 16 21 3-15 5 QQYNYFIT 8 7 ------donor3-41 3-30 5-5 2 4 1 DRWGYRNGPTY 2 11 17 3-11 3 QLRTNWPPMLT 11 19 donor3-42 3-30 5-5 2 4 1 DRWGYRNGPTY 2 11 12 1-33 5 QHYDNFPIT 9 12

8

donor3-43 4-59 3-10 2 3 2 GSDVFDM 0 7 24 donor3-46 3-15 1-7 3 4 1 VQLITGTPTNYFDY 0 14 0 1-39 5 QQSYSTPLIT 10 3 ------donor3-48 3-23 4-17 3 4 2 VFYADFVFIFDH 1 12 37 3-20 1 HQYFESPWT 9 32 ------donor3-54 3-23 6-19 3 4 2 DRIAVAGIFDY 1 11 0 0 1 QQRSNWPPWT 10 0 ------+ + donor3-58 3-30 5-24 3 4 1 VVATVNSPFDY 0 11 10 10 3 QQYNIWPS 8 2 ------donor3-59 5-51 6-19 3 4 1 HKPAVGTCFDY 1 11 11 11 4 QQYYSSPQD 9 7 donor3-68 4-31 3-22 2 5 3 GLYDSSGYYYEGRYFDP 1 17 13 13 5 QQRSNWIT 8 9 donor3-73 3-15 3-10 1 5 1 HTTLNVGLWFDS 1 12 33 33 3 QQAHTTPFT 9 18 + - - + - + + + + + donor3-74 3-30 3-22 1 6 2 DRSGKSYYYYGMDV 2 14 18 18 4 QQYDYLPSFT 10 21 ------donor3-78 3-7 1-26 2 5 2 DLTSGRYFGWFDS 1 13 9 9 3 QQYYSSPRT 9 9 ------

CD27-IgA+ CD27-IgA+ donor3-83 3-23 3-10 1 4 3 DKGALLWFEGFDQ 1 13 15 15 2 QQYNSYLYT 9 18 ------+ - + donor3-84 1-58 2-21 2 5 1 AYCGGDCYFAS 0 11 11 donor3-92 4-59 3-10 2 5 2 GLYYYGDTGYPHWCFDP 1 17 21 21 1 HQYGNSPWT 9 16 + + - + - - + + + + donor3-95 3-7 6-13 2 4 3 DGGSRWYGDFDY 1 12 11 11 4 QQNYNTPLT 9 11 donor4-01 1-69 6-6 2 4 1 DSTRGGSSSSYILGY 1 15 14 1-39 3 QQSYTIPVT 9 8 - + ------donor4-02 3-30 2-15 3 5 4 DRAEDTILVIAVGWFDP 1 17 25 1-5 1 QQYNTYSKT 9 10 ------donor4-05 1-18 3-22 2 4 1 SLGSSGYHSVNFDY 1 16 16 1-37 1 QRTYNAPPWT 10 12 donor4-08 3-21 3-9 2 4 2 AVGFSGYDLLTGYYDY 0 16 30 3-20 5 QQYGRSVT 4 10 - - - - - + - - - - donor4-10 3-11 4-23 2 3 2 GGLWSREGWDI 1 11 20 4-1 4 QQYYETSLT 9 9 - - - - + - + - + + donor4-17 3-48 4-23 2 3 3 DARDYGGKPLAFDV 2 14 9 2-28 1 MQVLQDSSNV 10 6 ------1D- donor4-18 3-48 3-3 3 3 2 DFIISGVTRPLDI 1 13 17 17 1 LQHHSFPRT 9 13 + + c ------3- donor4-19 30-3 3-16 2 4 1 IWGTYLDY 0 8 8 3-15 3 QQYSNWRVT 9 13 - + ------donor4-20 1-46 1-26 3 3 2 RRGGSYYRDAFDI 3 13 27 3-20 3 QQYGSSPT 8 16 ------donor4-22 3-9 3-22 2 4 2 DMGRGGGSGSYYYNYFDS 1 18 24 3-11 4 QHRSNWPALS 10 14 - + ------donor4-23 3-49 2-2 3 6 1 VPAANPNFYSGMDV 0 14 15 1-39 3 QQSYSNLFT 9 10 donor4-24 3-53 4-17 3 4 1 DRGPTTVTPPGVFGF 1 15 22 1-5 4 QQYNNYLLS 9 23 donor4-28 3-30 6-13 2 5 2 ETIIAATGLYNWFDP 0 15 13 3-20 4 QQYASYPLT 9 8 ------

CD27+IgA+ CD27+IgA+ donor4-30 3-33 2-15 2 6 2 GGYCSGTSCQYYDGMDV 0 17 13 2-28 1 MQALQTPGT 9 2 ------donor4-31 4-34 3-3 1 5 4 VRRFLEWMALEENWFDP 2 17 21 3-11 2 LQRKYWPPYT 10 2 - - c ------c donor4-33 1-18 2-2 2 4 1 NRGFCSSTSCYSDY 1 14 23 1-9 4 QHLNTYPP 8 12 + + N + - + + + + + donor4-35 1-18 5-5 3 5 2 GVRFHSDMVDGRGNRLDP 4 18 18 3-11 4 QQRSSWPPTT 10 13 donor4-36 3-48 3-3 2 6 3 GMYYDFETGMDV 0 12 17 1-39 1 QQTYNVPRT 9 24 ------donor4-41 3-30 4-23 2 4 5 DLEVDGPESDYGY 0 13 31 2-30 2 MQGTHWPPLYT 11 17 4- donor4-46 30-4 6-19 2 5 1 TRGGGWFDP 1 9 18 4-1 2 QQYYSTPYT 9 9 - - - - - + - - - - 1D- donor4-47 5a 2-15 2 4 1 GYCGGGSCYLAFDS 0 14 11 12 4 QQANSFPLT 10 17 - + c ------+ donor4-48 3-23 / / 6 1 HILTVGPSLVVPFGMDV 1 17 21 1-12 4 QQAYEFPVT 9 21 donor4-49 1-69 2-21 2 6 2 APPSRDSFYYYYAMDV 1 16 3 donor4-51 4-34 6-6 3 4 2 DRPFVPYYFDY 1 13 1 IgA+ IgA+ donor4-54 3-23 1-26 3 4 1 QKSGSYDY 1 8 3 1-17 1 LQHNSYPRT 9 1 - + ------9

donor4-64 3-48 1-16 3 2 3 EYMITFGGVRDANWYFDL 1 18 0 1-5 1 QQYNSYPGT 9 1 ------GISVVVPAAIVLGYYYYGMD donor4-70 1-69 2-2 3 6 1 V 0 21 0 2-28 2 MQALQTPGT 9 0 + + - - + + + + - + donor4-71 3-72 3-16 3 3 2 EHRSQGGGGAFDI 1 13 2 donor4-76 3-21 5-18 1 3 4 DPGVDTGTGSTHDAFDI 1 17 18 1-39 4 QQSYSGGT 8 14 ------donor4-79 1-18 2-2 2 4 2 DGFCSSASCYMPFDY 0 15 9 3-11 4 QQRTNWPPVT 10 9 - + ------donor4-80 5-51 6-13 2 4 1 RLLAAVGRSLGPTFDY 2 16 2 1-5 1 QQYNSYST 8 1 ------donor4-81 1-3 3-3 3 4 1 TRITIYGVPPAAD 1 14 19 3-11 4 HHRSDWPLT 9 19 + + - + + + + + + + donor4-82 3-7 6-19 1 3 6 DGDSSGWEDYDAFDI 0 15 2 1-39 2 QQGFSTPPTT 10 6 ------donor4-83 3-20 2 QQYGSSPPYT 10 7 donor4-87 4-4 / / 4 1 RAPSGPFDF 1 9 29 1-39 2 QQSFSRPPT 9 21 - - - - + - + - - - donor4-89 3-21 6-13 1 4 1 GGTSWSIDS 0 9 20 4-1 1 QQYYNTPRT 9 7 donor4-91 3-30 2-15 2 5 2 GGGYCGGESCYESGP 0 15 23 1-39 1 QQTYSTPRT 9 15 ------donor4-95 1-18 4-17 2 4 4 DRTDYREPAWIDY 2 13 2 4-1 1 QQYYNTPRT 9 7 QHVTNWPPGT donor5-02 1-2 1-14 3 4 3 DLDNRVFDY 1 9 23 3-11 1 WT 12 13 donor5-03 3-33 1-26 1 4 1 GGRTVVGATTLTNFDY 1 16 23 3-20 4 QQFLISPPT 9 22 - + ------donor5-04 3-49 3-9 2 5 3 EGEGFYEILTGYPSLTK 0 17 12 3-11 1 QQRSNRPPWT 10 7 ------donor5-05 4-59 1-26 2 4 2 DRGGSFRGFDY 2 11 30 4-1 4 QQYLSAPLT 9 16 ------donor5-09 3-33 6-19 2 3 2 EASLAGRPRAFDI 2 13 11 1-9 2 QHLNSYFNT 9 2 - + ------donor5-10 3-33 3-22 3 4 1 RGITMMKNPFDY 1 12 15 1-39 1 QQSHSYPLA 9 12 - donor5-14 3-15 4-23 2 4 0 RGGKTWGPY 1 9 42 3-20 2 QQYGSAPYT 9 11 + + - - - + - - - - donor5-15 4-61 5-24 2 6 1 GGVAGPSTTSYNYYGLDV 0 18 11 1-9 1 QPLNSF 6 4 ------donor5-19 5-51 / / 4 2 QGDLGFQPFDY 0 11 7 1-39 1 QQSYYLPRT 9 4

CD27+IgA+ donor5-21 3-49 4-17 2 4 2 RWGSYGDHIFDY 2 12 9 1-27 1 QKYNTAPE 8 4 - + c ------donor5-22 3-21 5-24 3 4 3 EGDYGYNSPFDL 0 12 19 3-11 2 QQRTNWPPYT 10 5 ------donor5-33 3-53 3-16 2 3 3 ARDIYPGYDAFDI 1 13 6 3-11 2 LQRSTWPQYT 10 26 ------donor5-34 3-23 3-22 2 4 3 ERCYYDNGGCFGVDY 1 15 7 donor5-35 3-23 1-26 2 4 1 KIGGQYPFDY 1 10 31 3-15 2 QHYNWWPYT 9 17 ------donor5-44 1-46 3-16 3 4 2 EARGGAATLDF 1 11 11 1-39 2 QQSYSSPRT 9 10 + + c + + + + + + + donor5-48 1-46 3-16 3 4 2 EARGGAATLDF 1 11 17 1-39 2 QQSYSSPRT 9 10 + + N + + + + + + + donor5-49 3-21 3-22 2 4 1 HSSGYLFDY 1 9 2 3-11 2 QQRSNWPPMYT 11 2 - + c - + + - - - +

donor5-53 3-74 2-2 2 5 1 QKYISCYE 0 8 2 3-20 4 QQYGSSPPGLT 11 1 - + ------donor5-60 4-39 / / 4 2 RKHDGGGHHYFDN 4 15 37 3-20 4 HQYDNSPLT 9 33 - + ------GDISSGYLDYDWGNYRLNYY n n donor5-62 1-2 3-16 2 6 4 GMDV 1 24 3 2-28 4 MQALQTPST 10 1 - - - - d d - - - - donor5-63 3-23 5-5 2 4 4 GDEYSDYFDY 0 10 3 1-13 2 QQFNSYPYT 9 10 + + ------+ + donor5-66 1-69 5-24 3 4 1 DGVY 0 4 24 1-5 2 QQYSSFYT 10 14 + + - + + + + + + + donor5-67 3-7 3-3 2 4 1 DSFWSGLH 1 8 5 1-5 2 QQYNSYPYT 9 0 + - - + + + + + + + donor5-69 3-11 / / 4 1 VNSSDVLFHY 1 10 7 3-11 2 QQRSNWPLYT 10 2 ------

CD27-IgA+ CD27-IgA+ donor5-71 3-15 3-22 2 4 3 AWEYYDSSGYYYEPLPY 0 17 5 3-20 4 QQYGSSPLT 9 4 ------+ donor5-72 3-33 6-19 2 3 3 DHASGWYAEAFDI 1 13 12 donor5-75 3-48 6-13 1 3 1 GRSGYSSSFFDI 1 12 7 3-20 4 QQYGSSPGLS 10 2 ------10

donor5-76 1-2 1-14 3 4 3 DLDNRVFDY 1 9 21 donor5-77 4-4 2-15 2 6 1 CGGSGGSCLHYGMDV 1 15 0 1-39 1 QQSYSTPRT 9 1 ------donor5-81 3-23 5-24 3 4 3 GRDRYAFFDD 2 10 10 3-20 1 QQYGSSLWT 9 3 ------donor5-86 4-39 6-19 1 5 2 TPESSGWHPAGSGWFDP 1 17 18 1-16 4 QQYNSYPLT 9 12 ------VH DH RF JH (-) CDR3 (aa) (+) Vλ Jλ CDR3 (aa) E.faecalis S.aureus E.coli Length No of SHM Length No of SHM Poly Hep2 IFA flagellin B.fragilis C.difficile E.cloacae donor1-02 3-53 / / 6 1 KGRTSCPYGMDV 2 12 31 2-14 1 SSYTSRNTFV 10 21 + + N - - - - - + - donor1-03 4-4 3-3 1 5 1 SSTFLGVTGWSDP 0 13 24 1-40 1 QSYDSGLSGYV 11 14 ------donor1-07 3-13 5-12 2 4 2 VRYDHGYDL 2 9 22 3-25 2 QSGDSETTV 9 36 ------donor1-20 3-74 1-26 3 3 1 LRVGAAIPHGFDL 2 13 31 2-23 1 CSYTGDTNIYV 11 22 donor1-23 4-39 2-8 2 4 2 NIEGYCSNGVCYAADY 0 16 25 donor1-24 3-74 2-8 2 3 2 ARDFGAVGSTNAFDI 1 13 16 2-8 1 SSYAGSYIYV 10 13 + ------donor1-26 3-30 / / 6 2 DRISPSGPKIVYVYYYMDV 2 19 37 donor1-30 5-51 2-21 2 6 1 SYCASTSCQTRGYFGMDV 1 18 14 1-40 2 QSFDSSLNAVV 11 8 ------CD27+IgA+ donor1-32 3-30 2-15 3 4 2 EPIVVVGVPTFYFDY 0 15 23 2-23 1 CSYATGNRYV 10 14 ------donor1-36 1-51 7 ATWDSSLNIVV 11 27 donor1-40 3-23 4-17 3 4 1 HSSTMTTNFDS 1 11 14 3-21 3 QVWDTSSNHPL 9 15 donor1-44 4-39 3-22 3 5 0 QPITMMVVV 0 9 10 2-14 1 SSYTSSSTL 9 8 - + ------donor1-53 3-33 3-10 3 6 2 SGDVVRGGPPGYYYPMDV 1 18 16 donor1-56 3-48 4-17 2 3 4 DVQKYNDGDAFDV 1 13 34 1-40 1 QSYDGSLNIYV 11 18 ------donor1-66 3-33 / / 5 2 EALGFDP 0 7 0 3-1 2 QAWDSSFVV 9 0 - + ------donor1-68 1-2 4-4 3 4 3 DSRDMTTLDY 1 10 0 3-1 2 QAWDSSFVV 9 0 - + ------donor1-69 1-46 2-21 1 2 2 DRNILWWQGSFDL 1 13 17 2-14 2 SSYTSSSTVV 10 2 + + - + + + + + + + donor1-73 3-48 1-7 2 4 2 DWWNWNSDN 0 9 10 1-51 3 GTWDSSLSAGV 11 6 ------donor1-76 1-46 2-2 2 4 3 YCSSTSCYKGYSDSNDPFDQ 1 20 32 2-14 2 SSFTNRNPPLGE 12 33 n n

CD27-IgA+ CD27-IgA+ donor1-84 3-9 5-12 3 4 1 ARIVATITGVNYYFDS 1 16 16 1-51 2 GTWDDSLSVVV 11 17 - - - - d d - - - - donor1-88 3-23 2-21 2 4 2 DLLAYWTNPDY 0 11 24 1-44 1 GTRDDSGRYF 10 16 ------donor1-90 3-23 / / 4 2 SGVLGPETVATIFVDY 0 16 0 3-1 2 QAWDSSTVV 9 0 ------donor1-92 4-4 3-10 2 4 2 DHRGSGACDY 1 10 0 1-44 1 AWDDSLNGPY 10 0 - + - - + - - - - - donor1-96 3-23 / / 4 2 IPDRAGYFDS 1 10 0 3-1 3 QAWDSSTWV 9 0 donor2-08 3-74 6-6 2 4 1 SSYPYYFDH 1 9 23 2-8 2 SSNAGSDNFVV 11 10 donor2-10 3-64 3-3 3 4 1 FGVYYFDY 0 8 14 2-11 1 CSYAGSYTYV 10 6 + + - + + + + + + + donor2-11 4-34 4-17 2 3 5 ITWDGEMSDDFDV 0 13 31 7-46 3 LLSFGGARV 9 15 ------+ - - - donor2-16 3-21 5-24 3 6 1 PSTIHYFYHMDV 2 12 15 2-8 3 SSYAGSNNLV 10 7 - - - - + + + + - - donor2-20 3-33 5-24 2 4 2 DDGITVPAH 1 9 36 2-14 1 SSYTISSTSI 10 19 ------donor2-22 3-33 2-15 2 4 2 DCSGGSCYSADY 0 12 16 2-23 3 CSYAGSEAFWI 11 21 - + - - - + - + - + CD27+IgA+ CD27+IgA+ donor2-43 3-23 1-26 3 6 2 VGAVVLTYPPMEYMDV 0 16 19 1-47 2 ATWDDSLRGVV 11 16 donor2-45 3-33 3-3 2 5 2 ERSGNGGNWFDP 1 12 20 1-40 2 QSYDSRLRGVV 11 19 + ------

+ donor2-49 1-2 4-17 2 4 2 YYCARGGAGDFDF 1 13 32 1-44 2 ATWDDSLNGVV 11 21 - - ---+-- - - 11

donor2-58 3-30 3-10 3 4 1 AQGITIVQPFDC 0 12 12 2-14 2 SSYTSSSTMI 10 9 - + --- -++-+ 3- donor2-64 30-3 3-3 2 4 2 QGIVFDFWSRFFVDH 2 15 32 1-51 3 GTWDSSLSAGM 11 17 + + - + + + + + + + donor2-65 1-69 6-13 3 6 2 DRIAAATYLGYYYYYMDV 1 18 1 3-1 2 QAWDSSVVV 9 0 + + - - + + + + + + donor2-70 1-2 4-17 2 5 2 ADYGDYPNF 0 9 7 donor2-82 3-33 / / 4 1 RSRIGGTFYFDY 2 12 9 1-40 3 QSYDSSLSGWV 11 4 ------3- donor2-88 30-3 4-23 3 4 3 VGEPTVDPLFDY 0 12 1 3-1 2 QAWDSGTVV 9 5 ------donor2-91 2-11 1 CSYAGSYYV 9 5 donor2-92 3-11 4-17 2 3 5 LADYGDYGDDAFDI 0 14 7 1-40 1 QSYDSSLSGYV 11 9 ------donor3-14 3-23 3-9 1 4 2 VGPYFDILTAYDY 0 13 14 donor3-23 4-34 4-17 2 4 3 RLGVETTTDYFDY 1 13 9 1-51 3 GAWDSSLSAGV 11 12 ------+ - + + donor3-32 3-30 6-13 3 4 1 VPAGTRSYIDY 1 11 21 1-40 2 QSFDSSLSGHVL 12 8 ------donor3-33 5-51 6-19 2 6 2 QGNSYWYLGADFGLDV 0 16 17 2-8 1 SSYAGSYTYV 10 13 + + - + + + + + + +

CD27+IgA+ CD27+IgA+ donor3-44 1-46 3-10 2 4 2 ARETTMVTWDY 1 11 12 2-14 1 SSYTSSNSYV 10 10 ------WGGYPVAPMKNFFDL donor3-66 5-51 5-24 2 5 1 1 15 15 2-14 2 TSYSSSYTHVV 11 12 ------HSTYREVRFDP donor3-67 4-59 4-17 1 5 2 2 11 6 2-8 2 SSYAGSKNLV 10 9 ------

CD27-IgA+ CD27-IgA+ donor3-71 1-46 6-19 2 5 3 DAPRSSAWYTLPEHSPWLDT 2 20 23 2-11 2 ISYAGGYIFV 10 31 + - - + + + + + + +

donor4-13 3-48 4-23 2 4 1 SSPLAGNSYQVDY 0 13 23 1-51 2 GAWDSLLSAGA 11 25 AAWDNRPNAA donor4-14 3-23 1-26 2 4 1 GRYSGSYYLQTDS 1 13 31 1-44 2 V 11 10 ------donor4-34 3-74 2-21 2 4 3 DLEQCTGDCLLF 0 12 27 2-8 1 SSYVGSSKNYV 11 17 ------

CD27+IgA+ CD27+IgA+ donor4-44 3-30 3-22 2 4 2 ALYYYDSSGYPPDY 0 14 18 2-18 1 SSYTASGTNV 10 19 + + - - + - - + - - QVWDISNAHAT

donor4-50 3-7 5-18 3 4 2 VRGYSFGDVVDY 1 12 15 3-21 1 V 12 6 donor4-59 3-21 2-8 2 4 2 DQGWLWPKPDY 1 11 0 1-44 2 AAWDDSLNGLE 11 0 + + - + + - + + - - donor4-60 3-33 3-10 2 4 2 DQYFSGSGYNFDY 0 13 19 2-11 3 CSYAGSYTLL 10 13 ------donor4-67 3-21 3-10 2 3 1 INYNAFDI 0 8 6 2-23 3 CSYAGSSTLGV 11 7 ------donor4-71 3-72 3-16 3 3 2 EHRSQGGGGAFDI 2 13 2 3-1 1 QAWDSSTYYV 10 3 ------donor4-73 3-30 3-16 3 4 1 GMLTFGGVLTPQVYYFDF 0 18 15 1-44 2 ATWDGSLNGPV 11 7 + ------+ donor4-74 3-15 1-26 3 4 1 SGSYFFRLDS 1 10 17 2-14 2 SSYTSSSTLV 10 5 + - - - - + + + - + donor4-75 3-7 2-8 3 4 2 DMGLHGVSDY 1 10 15 3-21 3 HVWDGSSEHRV 11 16 ------donor4-76 3-21 5-18 1 3 4 DPGVDTGTGSTHDAFDI 1 17 18 3-27 3 YSAADNSLGV 10 2

CD27-IgA+ CD27-IgA+ donor4-77 4-4 4-17 2 6 3 RLDGDYYYGMDV 1 12 4 3-1 2 QAWDSSTVV 9 4 ------donor4-85 5-51 3-22 2 4 3 LGDYDSSGTLRD 1 12 3 2-14 2 SSYTSSSTLVV 11 6 donor4-86 4-59 4-17 3 4 1 ASTFTVTGDY 0 10 15 2-14 3 NSYTSTSSLV 10 8 - - - - + - - - - - donor4-89 3-21 6-13 1 4 1 GGTSWSIDS 0 9 20 6-57 3 QSYDSRVRV 9 24 + - - + + - - - - + donor4-92 3-49 3-10 3 4 3 DLMGEGFDY 0 9 10 1-47 3 AAWYDSLSGW 11 3 ------12

V AAWDDSLNGH donor4-93 4-61 1-7 1 6 2 DRVITGTTVAMDV 1 13 7 1-44 3 WV 12 4 - + ------SSYTGASTLHV donor5-12 4b 3-22 2 4 2 GATYDSSGFRYLPIEY 1 16 15 2-14 2 V 12 17 ------donor5-17 3-7 6-19 1 5 1 LGGSGWSLDH 1 10 13 2-14 2 NSFSSSSTLLV 11 11 CQVWDINSEAV donor5-24 3-53 6-13 2 6 1 HGIATAGTPFYYGMDV 1 16 7 3-21 2 F 10 18 ------donor5-27 3-33 3-22 2 3 1 DPPYSGYAFHI 1 11 16 1-44 2 AAWDESLNVVA 11 15 - + ------donor5-32 2-8 1 CSYAGTNSFV 10 19

CD27+IgA+ CD27+IgA+ donor5-34 3-23 3-22 2 4 3 ERCYYDNGGCFGVDY 1 15 7 7-46 3 LLSYSGVWV 10 11 ------donor5-40 5-51 1-26 3 5 2 QRVELRYSGTYVVLPQFDP 2 19 32 2-14 2 TSSTRDTVPVVV 12 19 donor5-42 3-15 7-27 2 6 0 KYLGV 1 5 0 2-14 2 SSYTSSSTLV 10 2 - + ------donor5-47 3-48 5-5 3 4 2 GGRTSVDNTDY 1 11 20 AAWDDSLSGLR donor5-50 3-11 6-19 1 4 2 GEGGPSSGWYPFPFDY 0 16 6 1-47 3 V 12 3 QSYDSSLSGYV donor5-61 3-23 / / 4 0 FPY 0 3 2 1-40 2 V 12 5 ------donor5-64 3-48 6-19 3 4 1 VQWLVLGPFDY 0 11 0 ------GTWDSRLTAG CD27-IgA+ CD27-IgA+ donor5-65 1-8 5-5 2 4 1 SRRGYSYVDF 0 10 5 1-51 3 WV 12 11 donor5-87 3-73 2-2 3 6 1 VVPATILSGMDV 0 12 7 3-25 3 QSADGSGPWV 10 8 RF, reading frame; -, non-reactive; +, reactive; c, cytoplasmic staining; nd, not determined

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SUPPLEMENTAL TABLE III. Targeting and selection of individual mutations in rearranged IGHV.

total sequences cloned antibodies

CD27+IgA+ CD27-IgA+ CD27+IgA+ CD27-IgA+ polyreactive non-polyreactive autoreactive non-autoreactive (126) (109) (88) (75) (33) (130) (52) (111) RGYW (%) 564.2/2647 (21.3) 339.5/1399 (24.3)* 363.8/1691 (21.5) 234.2/955 (24.5) 137.8/611 (22.6) 455.5/2039 (22.3) 186.4/851 (21.9) 406.9/1799 (22.6) WRCY (%) 379.1/2647 (14.3) 191.2/1399 (13.7) 235.4/1691 (13.9) 131.4/955 (13.8) 82.2/611 (13.4) 287.0/2039 (14.1) 125.6/851 (14.8) 243.6/1799 (13.5) WA (%) 355.3/2647 (13.4) 199.7/1399 (14.3) 232.5/1691 (13.7) 135.4/955 (14.2) 76.4/611 (12.5) 298.8/2039 (14.7) 109.6/851 (12.9) 265.7/1799 (14.8) TW (%) 216.3/2647 (8.2) 94.7/1399 (6.8) 142.3/1691 (8.4) 67.9/955 (7.1) 55.6/611 (9.1) 155.7/2039 (7.6) 64.4/851 (7.6) 145.8/1799 (8.1)

Transitions (%) 1339/2647 (50.6) 744/1399 (53.2) 856/1691 (50.6) 520/955 (54.5) 329/611 (53.8) 1050/2039 (51.5) 431/851 (50.6) 948/1799 (52.7) Transversions (%) 1308/2647 (49.4) 655/1399 (46.8) 835/1691 (49.4) 435/955 (45.5) 282/611 (46.2) 989/2039 (48.5) 420/851 (49.4) 851/1799 (47.3)

FR R/S (ratio) 1109/649 (1.7) 585/349 (1.7) 684/421 (1.6) 390/245 (1.6) 227/170 (1.3) 845/503 (1.7)* 357/215 (1.7) 715/458 (1.6) CDR R/S (ratio) 683/206 (3.3) 360/103 (3.5) 445/141 (3.2) 246/72 (3.4) 159/55 (2.9) 532/157 (3.4) 200/77 (2.6) 491/135 (3.6)* FR indicates framework region; CDR, complementarity determining region; R/S is the ratio between replacement (R) and silent mutations (S); The number of analyzed sequences is indicated in the brackets next to the population name. All analyses were performed with the JoinSolver program and the differences between each analyzed population as compared with centrocytes were statistically analyzed with the X2 test. Significant differences between CD27+IgA+ and CD27-IgA+ B cells, polyreactive and non-polyreactive B cells and autoreactive and non-autoreactive B cells are depicted in bold. *, p<0.05

14