Natural Anti-Intestinal Autoantibody Production from Marginal Zone B Cells

This information is current as Daiju Ichikawa, Masanao Asano, Susan A. Shinton, Joni of September 27, 2021. Brill-Dashoff, Anthony M. Formica, Anna Velcich, Richard R. Hardy and Kyoko Hayakawa J Immunol 2015; 194:606-614; Prepublished online 5 December 2014;

doi: 10.4049/jimmunol.1402383 Downloaded from http://www.jimmunol.org/content/194/2/606

Supplementary http://www.jimmunol.org/content/suppl/2014/12/05/jimmunol.140238 Material 3.DCSupplemental http://www.jimmunol.org/ References This article cites 41 articles, 18 of which you can access for free at: http://www.jimmunol.org/content/194/2/606.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

Natural Anti-Intestinal Goblet Cell Autoantibody Production from Marginal Zone B Cells

Daiju Ichikawa,*,†,1 Masanao Asano,*,‡,1 Susan A. Shinton,* Joni Brill-Dashoff,* Anthony M. Formica,* Anna Velcich,x Richard R. Hardy,* and Kyoko Hayakawa*

Expression of a germline VH3609/D/JH2 IgH in mice results in the generation of B1 B cells with anti-thymocyte/Thy-1 autoreactivity by coexpression of Vk21-5/Jk2 L chain leading to production of serum IgM natural autoantibody. In these same mice, the marginal zone (MZ) B cell subset in spleen shows biased usage of a set of Ig L chains different from B1 B cells, with 30% having an identical Vk19-17/Jk1 L chain rearrangement. This VH3609/Vk19-17 IgM is reactive with intestinal goblet cell granules, binding to the intact large polymatrix form of 2 glycoprotein secreted by goblet cells. Analysis of a mk B cell AgR (BCR) transgenic (Tg) mouse with this anti–goblet cell/mucin2 autoreactive (AGcA) specificity demonstrates that immature B cells expressing the Tg BCR become MZ B cells in spleen by T cell–independent BCR signaling. These Tg B cells produce AGcA as Downloaded from the predominant serum IgM, but without enteropathy. Without the transgene, AGcA autoreactivity is low but detectable in the serum of BALB/c and C.B17 mice, and this autoantibody is specifically produced by the MZ B cell subset. Thus, our findings reveal that AGcA is a natural autoantibody associated with MZ B cells. The Journal of Immunology, 2015, 194: 606–614.

ntibodies present in serum of normal animals in the natural autoantibodies to determinants present on bacteria or http://www.jimmunol.org/ absence of specific Ag immunization are called natural viruses enables a rapid protective response to infection (7). Thus, A Abs. Among these, Abs binding to self-antigens, pre- the presence of natural autoantibodies contributes both a house- dominantly IgM Igs encoded by germline , are termed “natural keeping function and also defensive immunity. Although it is autoantibodies” (1–3). Natural autoantibodies that bind to intracel- known that genetic background, such as MHC-linked genes, lular constituents, such as DNA, nuclear , and cytoskeletal affects the natural autoantibody repertoire (8), the details of how components, and to plasma proteins are common in vertebrates at such natural autoantibodies are generated and controlled remain all ages, from newborn to adult (2, 4). The presence of autoanti- a subject of continued debate. In mice, one clear source is B1 bodies to apoptotic or senescent cells, which expose such intra- B cells. These B cells are generated by self-ligand–mediated cellular constituents, and to oxidized low-density lipoprotein in signaling, thereafter serving as a source of natural autoantibodies by guest on September 27, 2021 serum, suggests that a fundamental role for natural autoantibody (9). We show in this study that marginal zone (MZ) B cells also may be rapid elimination of damaged cells and clearance of de- make a natural autoantibody, producing IgM with autoreactivity to graded self-molecules (1, 5, 6). Furthermore, cross-reactivity of (Muc2), a major component of intestinal goblet cell granules and secreted intestinal mucus. *Fox Chase Cancer Center, Philadelphia, PA 19111; †Keio University Faculty of The MZ is a region in spleen between the lymphoid-rich white Pharmacy, Tokyo 105-8512, Japan; ‡Juntendo University School of Medicine, Tokyo x pulp and the red pulp that consists of an open circulatory network 113, Japan; and Albert Einstein Cancer Center/Montefiore Medical Center, Bronx, that filters the blood (10). B cells residing in this MZ site encounter NY 10467 and trap pathogens circulating in blood, with or without the aid of 1D.I. and M.A. contributed equally to this work. Ag-presenting dendritic cells (DCs), and rapidly respond, serving Received for publication September 18, 2014. Accepted for publication November 5, 2014. as a defensive barrier (11). Large polymerized Muc2 that bears This work was supported by National Institutes of Health/National Cancer Institute abundant and variable glycans (12) is the secreted mucin in gut, Grants AI49335 (to K.H.), AI026782 (to R.R.H.), and CA129330 (to K.H.), and the a major component of intestinal mucus that functions to block Fox Chase Cancer Center Blood Cell Development and Cancer Keystone Program. microbacterial invasion (13). Such Muc2 in the gut lumen is Address correspondence and reprint request to Dr. Kyoko Hayakawa, Fox Chase constantly sampled by DCs in the intestine (14). Our data dem- Cancer Center, Reimann Building, 333 Cottman Avenue, Philadelphia, PA 19111. E-mail address: [email protected] onstrate that developing B cells with autoreactivity to this heavily glycosylated intestinal mucin become MZ B cells and accumulate The online version of this article contains supplemental material. at this site. This process is dependent on Btk, a kinase involved in Abbreviations used in this article: AGcA, anti–goblet cell/mucin2 autoreactive; ATA, anti- thymocyte/Thy-1 glycoprotein autoreactive; BCR, B cell AgR; BM, bone marrow; DBA, B cell AgR (BCR) signaling. Btk is essential for IgM and IgG3 Dolichos biflorus agglutinin; DC, dendritic cell; DSP, dithiobis succinimidyl propionate; natural Ab production in serum (15, 16). Thus, our data demon- DSS, dextran sulfate sodium; EP67 mkTg, VH3609/Vk19-17 mk Tg; FO, follicular; 13H8k, anti-k Ab recognizing limited set of k L chains including Vk21-5 and Vk19- strate BCR-ligand–mediated selection leads to autoreactive MZ 17; IB4 (BSI-B4), isolectin B4; MK19, IgM produced by SP2/0 cotransfected with B cell generation and natural autoantibody production. This bears m k VH3609 IgH and Vk19-17 IgL; MK21, IgM produced by SP2/0 cotransfected with a striking similarity to Btk-dependent positive selection of B1 VH3609m IgH and Vk21-5k IgL; Muc2, mucin 2; MZ, marginal zone; PAS, periodic acid– Schiff; PC, plasma cell; PNA, peanut agglutinin; RT, reverse transcription; Tg, trans- B cells by Ag, which contribute a different natural autoantibody in genic; UC, ulcerative colitis; VH3609id, anti-VH3609SM6C10 idiotype Ab; VH3609t, the same animal. In humans, the presence of anti-goblet cell Ab in VH3609/D/JH2 targeted insertion (knock-in) mouse line; WGA, wheat germ agglutinin. serum has been recognized for decades, originally discovered This article is distributed under The American Association of Immunologists, Inc., in colitis patients (17, 18). Our data in mice suggest that such anti- Reuse Terms and Conditions for Author Choice articles. goblet cell Abs in humans may also include a natural autoanti- Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00 body, as described in the Discussion. www.jimmunol.org/cgi/doi/10.4049/jimmunol.1402383 The Journal of Immunology 607

Materials and Methods Quantitative RT-PCR assay Mice expression was quantitated by real-time PCR, in duplicate, using The V 3609/D/J 2 targeted insertion (knock-in) mouse line (V 3609t) TaqMan assays from Applied Biosystems, an ABI 7500 real-time thermal H H H cycler, and ABI software (Life Technologies). Relative gene expression was made by homologous recombination in ES cells as previously reported b (19), with a slight modification to the targeting construct, by extending levels were normalized using -actin values as a standard. the short (right side) homology arm from 0.8 kB to 2.5 KB, transfecting the Measurement of serum transgene Ab R1 ES line (129/Sv 3 129/Sv-CP F1 origin). The floxed NeoR gene was eliminated by crossing the knock-in founder to CMV-CreTg.BALB mice. To measure transgene-derived Ab in EP67 mouse serum by ELISA, we m VH3609 transgenic (Tg) mice have been described previously (9). To coated the assay plate with P9-10C7(VH3609id), then detected it by either mk establish VH3609/Vk19-17 Tg mouse lines, we cloned a Vk19-17/Jk1 P9-13H8, in conjunction with biotin mouse anti-rat IgG1 (MCA194B; + a rearranged k L chain gene from a VH3609m MZ B cell–derived hy- Serotec, Raleigh, NC) or biotin mouse anti-IgM , followed by alkaline a bridoma, P16-8F5, by long-distance PCR. Vk19-17 is identical to Gen- phosphatase–conjugated . Quantitation of total IgM/k, IgM , and Bank MUSIGLAFE. Generation of mkTg mouse lines was carried out as IgMb levels by ELISA was described previously (20). previously described (20), by injection into (C3H 3 C57BL/6)F1 eggs. One mkTg founder line, EP67, was used in this study as representative of Intestinal tissue cryosection immunofluorescence analysis six anti–goblet cell/mucin2 autoreactive (AGcA) mk lines (all showed Dissected intestine was gently flushed of stool material by ice-cold PBS in increased MZ B cell generation and Tg IgM production in serum). The a 1-ml syringe, then frozen. Staining of ethanol-fixed frozen sections (8-mm V 3609/V 19-17 mk Tg (EP67 mkTg) mouse line and the V 3609t H k H thickness for intestine and 5–6 mm for other tissues) and microscope im- mouse line were backcrossed to C.B17 mice more than six generations aging was done as described previously (23). First, for comparing IgM before use in experiments. Btk mutant Xid and Rag-1 knockout mice produced by SP2/0 cotransfected with VH3609m IgH and Vk19-17k IgL were both on a BALB/C background. Muc2 and Muc1 knockout mice m

(MK19) versus IgM produced by SP2/0 cotransfected with V 3609 IgH Downloaded from were on a C57BL/6 background. C.B17, BALB/cAnN, C57BL/6 ICR, H and V 21-5k IgL (MK21) staining (both IgMa+), C.B17 (IgMb) mouse and C.B17 SCID were bred and maintained in our laboratory animal k colon cryosections were incubated with IgMa-containing samples (at 2–5 facility. Germ-free (Swiss Webster) mice and Sprague Dawley rats were mg/ml) for 1.5 h at 37˚C, followed by incubation with fluorochrome- purchased from Taconic. All animal experiments were conducted under conjugated anti-IgMa Ab, together with different fluorochrome-con- a protocol approved by the Fox Chase Cancer Center Institutional Ani- jugated lectins (30 min, at room temperature). Second, natural autoanti- mal Care and Use Committee. bodies were tested at 1/100 dilution of sera. RagKO mouse intestine was Flow cytometry analysis and reagents used to comparably test different IgM allotype serum samples from normal mouse strains, in combination with fluorochrome-conjugated anti- http://www.jimmunol.org/ Multicolor flow cytometry analysis, sorting, and mAb reagents have been IgM Ab. Third, IgM in 3- to 4-d culture supernatant of LPS-activated described previously (21). Rat anti-mouse Ig idiotype Abs used in this study B cells was adjusted to 2–10 mg/ml for staining. Fluorochrome (fluores- were made as previously described, by using SM6C10 IgM (VH3609/Vk21- cein or Alexa Fluor 350)-conjugated lectins for cryosection staining were SM6C10 5) as immunizing Ag (20). The anti-VH3609 idiotype Ab “VH3609id” isolectin B4 IB4 (BSI-B4) (Sigma-Aldrich), peanut agglutinin (PNA; is P9-10C7, a rat IgG2a. The P9-13H8 Ab 13H8k, a rat IgG1, reacts with Vector Laboratories), Dolichos biflorus agglutinin (DBA; Vector Labora- SM6C10 a restricted set of mouse k L chains, including Vk21-5 and Vk19- tories), and wheat germ agglutinin (WGA; Invitrogen). DAPI (Thermo 17P16-8F5. The rat P9-19A4 anti-thymocyte/Thy-1 glycoprotein auto- Scientific) was used to visualize nuclei. Stained slides were viewed with reactivity (ATA) idiotype Ab (20) does not react with VH3609/Vk19-17 a Nikon Optiphot epifluorescence microscope. Images were recorded using IgM, distinguishing AGcA from ATA among 13H8khi+ B cells. the QImaging Retiga-1300 cooled CCD camera and processed with Openlab software (Improvision). Images were obtained with either a 310 by guest on September 27, 2021 Single-cell Igk-chain sequencing or 320 objective lens (original camera magnification, 3264).

For VH3609m Tg mouse data in Supplemental Table I, cDNA was prepared Isolation of colonic crypts and amplified from individual sorted B cells, MZ B (CD21hiCD232AA42 + med + 2 + After opening the colon longitudinally and washing in Dulbecco’s PBS VH3609id ) and follicular (FO) B cells (CD21 CD23 AA4 VH3609id ) 2+ 2+ from spleen and immature B (AA4+CD212CD232) cells from bone without Ca and Mg , specimens were incubated with 0.04% sodium marrow (BM) and spleen, for k gene sequencing by nested PCR as hypochlorite for 15 min at room temperature, then incubated with 3 mM EDTA for 90 min at room temperature, with moderate shaking throughout described previously (9). The Vk-Ck PCR product was cloned using TA vector (Invitrogen, Carlsbad, CA); then individual plasmid clones the incubation. Then samples were vigorously shaken for 2 min, removing were sequenced. Sequencing efficiency from individual cells under this stromal materials, and colonic crypts/mucous granules were pelleted by 3 procedure was 80% for all B cell preparations. To assess mouse-to- centrifugation at 480 g for 3 min at 4˚C. k mouse variation of VH3609t mouse Ig usagebyMZBandFOBcell Electrophoresis and Western blotting subsets, we sorted individual Igk+cells directly onto AmpliGrid AG480F slides (Beckman Coulter) using a FACS-VantageSE flow cytometer or Crypts were transferred into a new tube, washed, incubated at room a FACSAria II (Becton Dickinson), followed by a reverse transcription temperature for 5 min, then centrifuged at 8000 rpm for 5 min to release (RT) reaction and two rounds of PCR amplification. RT and first-round mucus into the supernatant. Crypt supernatant was treated with 1% NP-40 PCR were performed using the Qiagen OneStep RT-PCR kit (Qiagen) in lysis buffer and applied to 3.3% SDS-PAGE with a 2.5% stacking gel, a1-ml reaction containing Vk forward primer, 59-GACATTGTGATGA- following a standard procedure, in the presence of 2-ME. Also, 10% and CCCAGTCTC-39 and Ck reverse primer, 59-CCATTTTGTCGTTCACTG- 15% SDS-PAGE were used to test for the presence of MK19-specific CCATC-39, which was also used as the RT primer for cDNA synthesis. bands. After blocking membranes with 5% nonfat dry milk/TBS/0.05% Second-round PCR amplification used the same Vk primer and a nested Tween 20 for 1 h at room temperature, Muc2 and MK19 immunoblotting Ck primer 59-GAAGCACACGACTGAGGCACC-39. The second-round was carried out with goat anti-Muc2 (N terminus peptide) in combination PCR fragments were purified and then sequenced using a nested Ck primer. with HRP-donkey anti-goat IgG Ab (both from Santa Cruz Biotechnol- The efficiency of obtaining sequence was between 70 and 90%. Kappa ogy), and by MK19 IgM in combination with HRP-goat anti-mouse IgM L chain genes were classified according to the ImMunoGeneTics In- (Santa Cruz Biotechnology), respectively. For lectin blotting, biotin con- formation system database (http://www.imgt.org) and a report from jugated-DBA lectin (Vector Laboratories) was used in combination with Thiebe et al. (22). HRP-streptavidin (Southern Biotech). BM cell transfer Cross-linking of Ab and immunoprecipitation Cell sorter purified B/T lineage–negative, stem/progenitor cell–enriched RagKO mouse colonic crypts were incubated with MK19 or MK21 IgM for fractions (CD192 IgM2 CD52 and low side scatter) from BM of EP67mk 1.5 h at 37˚C, washed with PBS, then incubated with PBS containing 1 mM Tg C.B17 mice and non-Tg C.B17 mice were cotransferred to C.B17 cross-linker, dithiobis succinimidyl propionate (DSP; Thermo Scientific), SCID mice (lightly irradiated, 3 Gy, 1 d before) by i.v. injection (1:10 for 30 min at 37˚C, and finally quenched by adding 20 mM Tris-HCl. For transfer ratio; 5 3 104 cells from EP67 and 5 3 105 cells from C.B17, immunoprecipitation, cells were lysed in NP-40 lysis buffer, and soluble per recipient). Transfer of EP67mk Tg mouse BM stem/progenitors alone lysate was incubated with rat anti-mouse IgM Ab (331.12) bound (5 3 105 cells/recipient) was used as a control. Analysis was done 5–8 wk G-Dynabeads (Invitrogen) for 18 h. Beads were washed four times in 0.5% after transfer. NP-40 lysis buffer. Bound material was eluted with 1% NP-40 lysis buffer 608 NATURAL ANTI-GOBLET CELL AUTOANTIBODY containing Laemmli buffer with 2-ME, boiled for 5 min; then supernatant cell sequence analysis (31/31 = 100%). In contrast, the majority was applied to SDS-PAGE, for IgM, Muc2, and DBA lectin blotting. of CD52 B220hi B cells (B2) in the peritoneal cavity show less Experimental colitis staining by 13H8k than ATA B cells (Fig. 1A) and do not express Vk21-5/Jk2 k. Mice were treated by oral administration of 4% dextran sulfate sodium In the spleen of the same mouse, an alteration of the 13H8k (DSS; 36–50 kDa molecular mass; MP Biomedicals, Solon, OH) in + drinking water for 5 d, which was thereafter replaced by normal distilled binding pattern diversity is seen in VH3609id B cells from the water. Examination of the intestine was carried out 3 (the declined body transitional immature stages (CD21lo CD24hiAA4+) that can weight), 8 (recovered body weight), and 14 d after cessation of DSS water. progress to become either mature FO B cells or MZ B cells. In + med/lo 2 contrast with VH3609id FO B (13H8k and 13H8k ), MZ Results B cells, including CD21hiCD23+ pre-MZ B cells, show predomi- BCR associated with the MZ B cell subset nant 13H8khi/med cells (arrow in Fig. 1A, Spl B/MZ B). Single-cell Expression in mice of a germline VH3609/D/JH2 m H chain as sequence data from four individual VH3609t mice showed fre- a transgene (VH3609mTg) promotes generation of B1 B cells with quent usage by MZ B cells of an identical k-chain, Vk19-17/Jk1, anti–Thy-1 autoreactivity (9). ATA recognizes highly glycosylated followed by Vkba9/Jk5 and Vk19-25/Jk1 (Fig. 1B). Further single- Thy-1(CD90) that is predominantly expressed on the membrane of cell Igk sequence analysis using VH3609m Tg mice, comparing + hi 2 immature thymocytes (24), in both native and also dying cells. VH3609id MZ B cells (CD21 CD23 ) with immature and FO ATA IgM is secreted into serum as a natural autoantibody by B1 B cells, is shown in Supplemental Table I; the selected Igk set B cells (9, 20). This ATA BCR uses a Vk21-5/Jk2 k L chain, associated with MZ B is shown in Fig. 1C. These results revealed generated by rearrangement of the Ig L chain locus (9). In targeted that ∼30% of MZ B cells have an identical Vk19-17/Jk1 rear- Downloaded from VH3609/D/JH2 insertion mice (knock-in, VH3609t), as shown in rangement (CDR3 region is shown in Fig. 1C), in contrast with Fig. 1A, among CD5+ B cells (B1a) in the peritoneal cavity, much lower and the lowest frequency in FO B and immature + VH3609 B cells expressing the engineered IgH are present B cells, respectively. This Vk19-17/Jk1Igk is another L chain + hi+ (VH3609id ) with a large proportion being 13H8k . This anti-k strongly recognized by13H8k. Ab recognizing limited set of k L chains including Vk21-5 and To assess whether the accumulation of B cells with this BCR in

Vk19-17 (“13H8k”), which we developed by immunization the MZ B subset occurs directly from immature transitional stage http://www.jimmunol.org/ with ATA IgM, reacts with a restricted set of mouse k L chains at cells or a secondary outcome of mature FO B cell activation, we various levels, including Vk21-5/Jk2 k.Vk21-5/Jk2 k expression in made the mk Tg mouse line, EP67, expressing this VH3609/Vk19- 13H8khi+ B1a B cells, as ATA B cells, was confirmed by single- 17 BCR. In EP67 mice, the majority of newly generated immature by guest on September 27, 2021

FIGURE 1. MZ B cell–associated BCR, VH3609/Vk19-17. (A) Analysis of B cell subsets (gated rectangles color coded on plots) in the peritoneal cavity + hi/med washout cells (PerC) and spleen from a VH3609t mouse. The VH3609id cell gate is marked by a vertical line. The majority of 13H8k Igk B cells are + also VH3609id in the MZ B subset (arrow). (B)Igk single-cell sequence data from MZ B and FO B subsets of four individual VH3609t mice. Percentages + 2 are from a total of 25–36 samples per mouse B cell subset gated as VH3609id(10C7) Igl . Three Igks enriched in the MZ B cell subset, in contrast with FO B cells (empty bars), are shown. Vk19-17/Jk1 Ig L chains used by individual MZ B cells were all identical. (C) Selected Igk-chain usage data comparing the immature, FO B, and MZ B cells in VH3609mTg mice (all data are provided in Supplemental Table I). Confirmation of highest usage of identical Vk19-17/ Jk1 Ig L chains by MZ B cells (in CDR3, the Vk-Jk junction encodes a proline [P] residue). The Journal of Immunology 609

B cells in BM express this Tg BCR. At 3 wk of age, early gen- rather than the areas labeled by WGA (Fig. 3E, middle panel); erated mk Tg+ CD212CD24hi immature B cells progressed to this apical region staining is similar to DBA binding areas that become CD21hi (and CD1d+ CD9+) in spleen without intermediate are enriched for a-N-acetylgalactosamine glycan (Fig. 3E, right generation of FO B cells (Fig. 2A, asterisk), comprising a signifi- panel). Most MK19 binding was Muc2 dependent, as shown cant fraction of the CD21hiCD232 MZ B cell pool (Fig. 2A). In by a large reduction of staining in Muc2 gene knockout mice, adult (2 mo) animals, mk Tg+ B cells became both MZ B cells and whereas transmembrane Muc1 deficiency did not alter binding FO B cells when competitor Tg2 B cells were absent as assessed (Fig. 3F). MK19 goblet cell reactivity was found with intestinal by surface phenotype by flow cytometry analysis, colocalization tissue from various mouse strains (data not shown), whereas no by immunohistology staining, and expression of MZ B cell–spe- reactivity was observed with rat (Fig. 3G), similar to ATA IgM cific genes (Fig. 2B). However, the addition of non-Tg B cells in (MK21) sharing IgH, which has reactivity to thymocytes of var- chimeric BM transferred mice restricted mkTg+ B cells to pre- ious mice, but not rat (27). Intestinal goblet cells from germ-free dominantly assume a CD21hi MZ B cell fate (Fig. 2C, asterisk). mice also contain granules stained by MK19 (Supplemental Fig. Furthermore, introducing Btk deficiency in the context of mkTg 2). MK19-reactive granules in germ-free colon are on the apical EP67 mice (EP67.Xid) resulted in nearly complete loss of 13H8khi region toward secretion into lumen as found in normal mice. mk Tg+ MZ B cells (Fig. 2D), whereas neither CD40 deficiency The importance of a fully glycosylated polymeric antigenic nor complete elimination of T cells by Rag-1 deficiency had this form for MK19 recognition, rather than either glycan or protein effect (Supplemental Fig. 1A). Taken together, these data indicate alone, was initially suggested by the absence of MK19 binding to that a common self-antigen or environmental Ag present from the paraffin-embedded intestine section samples, regardless of deglyco- neonatal through adult age promotes VH3609/Vk19-17 MZ B cell sylation treatment (data not shown). By Western blotting, a ∼600-kDa Downloaded from development directly from transitional stage cells in a BCR- band was detected corresponding to the major Muc2 glycoprotein dependent manner, without a requirement for T cell help. precursor, comprising the majority of Alcian blue/PAS binding colon mucin and the majority of DBA+ Muc2 (Fig. 4A). In con- Anti-goblet cell autoantibody to Muc2 trast, MK19 IgM failed to reveal a Muc2-dependent band using Previously we found that Thy-1 expressed at aberrantly low levels 3.3–15% SDS-PAGE, by this direct immunoblot procedure. Be-

compared with physiologic results in ATA MZ B cell generation cause most MK19 binding is Muc2 dependent, and MK19 binds to http://www.jimmunol.org/ rather than B1 B cells (21). Thus, the possibility of weak anti– the surface of granules in a goblet cell suspension, we hypothe- Thy-1 reactivity was the first explanation considered for MZ B cell sized that its binding requires a native Muc2 polymatrix glyco- hi generation in 13H8k VH3609/Vk19-17 BCR Tg mice. However, form normally expressed in mucus that is lost in the denatured the ATA idiotype Ab (20) did not bind this IgM, and Thy-1 de- form present in SDS-PAGE, which differs from reactivity of the ficiency did not alter MZ B cell generation (Supplemental Fig. Muc2 anti-peptide Ab. Therefore, we used a DSP cross-linking 1A), in contrast with production of VH3609/Vk21-5 ATA B cells approach with MK19 preincubated goblet cell granules/mucus to that are Thy-1 dependent (20, 21). Therefore, to investigate its examine MK19 linked material by anti-IgM immunoprecipitation. reactivity, we cotransfected the VH3609m and Vk19-17k genes Formation of Muc2 polymatrix renders a large fraction of mucin into the SP2/0 hybridoma line to produce secreted IgM, referred to insoluble to extraction (28) (Fig. 4B, right panel), and most MK19 by guest on September 27, 2021 as “MK19.” This Ab was compared with the IgM produced by IgM-bound material was also insoluble (Fig. 4B, left and middle SP2/0 cotransfected with ATA VH3609m and Vk21-5k, termed panels) and, therefore, intractable to immunoprecipitation analy- “MK21.” Both transfected IgMs are IgMa. The binding specificity sis. However, a small amount of MK19-containing material was of MK19, testing for possible autoreactivity associated with MZ soluble, as was a small amount of Muc2 (Fig. 4B). Immunopre- B generation, was first assessed by standard procedures, including cipitation with anti-IgM beads of this small amount of soluble cell/tissue/cell line staining and ELISA. Although MK19 binding MK19 bound lysate confirmed that the ∼600-kDa DBA+ Muc2 was below detection in most assays used, including assessment glycoprotein was directly bound by MK19, but not by MK21 of reactivity to the cytoplasm and nucleus of various tissues control (Fig. 4C). This MK19-Muc2 binding is not to glycan and cell lines (Supplemental Table IIA), it intensely stained in- alone, however, because it does not stain rat colon goblet cells testinal mucosal cell suspensions. This is in sharp contrast with (Fig. 3G) that contain DBA+ Muc2 that is similar to mouse (29). thymocyte-restricted staining by MK21, an Ab that shares the Therefore, we termed IgM with this “anti-goblet cell auto- same IgH (Fig. 3A). When we used ethanol-fixed cryosection reactivity,” predominantly to intact Muc2, as “AGcA.” staining, MK19 bound to intestinal goblet cells, together with contiguous luminal mucus, with staining distinctively highest in Natural AGcA production from MZ B colon, followed by small intestine, but absent in stomach tissue In the AGcA mkTg line EP67, Tg+ B cells (recognized by staining (Fig. 3B, 3C). for the Tg IgMa allotype) become plasma cells (PCs) in red pulp The major constituent of intestinal goblet cell granules is mucin, (Fig. 5A, PC). Secreted transgene IgM comprises most of the IgM and Muc2 is the predominant secretory mucin found in colon, in serum, and this secretion is T cell independent (Fig. 5B). Anti- small intestine, but not stomach (25). Therefore, we hypothesized goblet cell autoreactivity by IgMa in EP67 mouse serum was that VH3609/Vk19-17 Ab reacts with Muc2. Muc2 dimerizes in confirmed by colon staining, in comparison with ATA mkTg 3369 the endoplasmic reticulum and becomes heavily O-glycosylated mouse serum, where thymocyte-reactive IgMa is predominant, in the Golgi, followed by polymerization, generating a long-chain produced by B1 B cells (20) (Fig. 5C). Thus, AGcA B cells dif- glycoprotein structure (26). The Muc2 macromolecule rapidly ferentiate into cells secreting autoantibody into serum, as with expands during secretion, forming a highly polyionic network, ATA B cells. constituting the intestinal mucus. MK19 binds goblet granules In both mice and humans, the major fetal colonic mucin is Muc2, with a high content of O-linked glycans, recognized by labeling as in adults (30). Thus, AGcA-reactive granules, predominantly with periodic acid–Schiff stain (PAS), and separate from PNA- Muc2, are normally present in gut from the fetal/neonatal stage labeled areas that identify an early glycosylated form of mucin in throughout life as an abundant self-antigen. Because of this nat- the supranuclear Golgi region (Fig. 3D). In colon, there is pref- ural abundance, to assess whether AGcA B cell generation is also erential MK19 staining at the apical region of colonic crypts, occurring in non-Tg normal mice, LPS mitogen-stimulated culture 610 NATURAL ANTI-GOBLET CELL AUTOANTIBODY

supernatant from different B cell subsets was tested for AGcA activity. Colon staining analysis showed that IgM(s) with goblet cell reactivity, similar to VH3609/Vk19-17 AGcA, is produced by MZ B cells of BALB/c (and C.B17) mice, but not by B1 or FO B cells (Fig. 5D). Furthermore, AGcA autoantibody was de- tectable in serum, particularly in BALB/c, followed by C.B17 (Table I). In contrast with BALB/c or C.B17 strains, the AGcA reactivity was below detection limits in C57BL/6 mice, both in LPS-stimulated B cell subset supernatant (data not shown) and in serum (Table I). However, generation of AGcA MZ B cells and autoantibody production is not blocked in C57BL/6 mice. This was shown in an experiment backcrossing EP67 mk Tg mice, originally on a C.B17 background, for four generations to C57BL/6, whereupon similar AGcA MZ B cell generation and autoantibody production occurred (Supplemental Fig. 1B). Thus, generation of AGcA-autoreactive B cells can occur if the germ- line BCR repertoire is appropriate. In conclusion, AGcA belongs to the class of strain-biased natural autoantibodies that is pro-

duced by MZ B cells. Downloaded from Function of natural AGcA autoantibody The presence of natural IgM autoantibody has been shown to play a protective role (5–7). However, it also carries a risk to initi- ate injury in conjunction with complement (31). Because auto-

reactivity to goblet cell granules was originally found in children http://www.jimmunol.org/ with colitis (17), the question naturally arises whether the pres- ence of AGcA, normally produced as a natural autoantibody, carries a risk for initiating disease. Although AGcA B cells pref- erentially differentiate to become MZ B cells in the spleen en- vironment, some B cells with AGcA BCRs circulate and are found in intestinal tissue, where the Muc2 autoantigen is present. In adult EP67 mkTg mice, without competing non-Tg B cells, a significant fraction of AGcA B cells become FO B cells in ad-

dition to MZ B cells (Fig. 2B). These B cells circulate to form by guest on September 27, 2021 follicles in both peripheral and mesenteric lymph nodes, and AGcA B cells are also found in intestinal tissue, including the colon, forming isolated lymphoid follicles, located nearby epi- thelial goblet cells that contain the target Ag (Fig. 6A, upper panel). AGcA (IgMa) PCs also appear in the intestinal lamina propria with abundance of AGcA IgM in serum. However, the secreted multimeric IgM does not permeate intact epithelial walls to reach the goblet cells. This is clear from a lack of staining of goblet cell sections by anti-IgMa reagent, unless goblet cells ex- posed by sectioning are first incubated with AGcA-containing autoserum from an EP67 mouse, as shown in Fig. 6A (lower panel). Thus, even abnormal excessive production of natural

+ PCR analysis of selected genes was compared in sorted VH3609id MZ B and FO B cells from EP67 mice (n = 3 each, mean and SE) with wild type C.B17 mouse FO B cells. (C) Mixed cell transfer of adult mouse BM hematopoietic stem cell–enriched fractions using a 1:10 Tgmk EP67/non- Tg ratio. Six weeks after transfer, spleen B cell FACS analysis was per- + + a+ formed, gating for VH3609id (Tg , IgM ) cells (9% of total B cells) and 2 2 b+ VH3609id (Tg , IgM ) B cells. Representative data of four recipients FIGURE 2. Btk-dependent MZ B cell development by VH3609/Vk19-17 (n = 4) from two separate transfer experiments are shown. All mice showed + BCR B cells. (A) Spleen B cell FACS analysis of the VH3609/Vk19-17 predominant MZ B cell generation by the Tg B cells. Transfer of EP67 mkTg mouse line, EP67, at 3 wk of age. The CD21hi B cell area is marked BM alone generated both MZ B and FO B cells, as found in intact adult by red dotted line. Predominance of CD21hi pre-MZ B and MZ B cells in EP67 mice (data not shown). (D) Analysis of BM AA4+ immature B and 2 Tg+ mice (starred) relative to Tg littermate. (B) FACS analysis of 2 mo spleen B cells in 2-mo-old EP67 mkTg mice without or with Xid (Btk adult EP67 spleen B cells. Tgmk+ B cells, the predominant population, mutant) background. In contrast with BM, in spleen there is a reduction of comprise 40–50% in spleen, and half become either FO B or MZ B cells. total B cell numbers (2-fold lower in Xid), together with reduction of + MZ B cell phenotype cells are colocalized in the MZ with SIGNR1 mkTg B cell frequency, and an absence of VH3609/Vk19-17 MZ B cells macrophages (ER-TR9+), and FO B cell phenotype cells are present in (red ellipse region) in EP67.Xid mice. Representative data are shown from B220+ follicles with CD21hi FO DCs (203 objective lens). Quantitative analyses of four mice. The Journal of Immunology 611

AGcA IgM autoantibody in the EP67 Tg mice does not result in goblet cell loss, and mucosal pathology is not enhanced over a 15-mo period (data not shown). To further assess how AGcA plays in mucosal immunity/disease, we treated EP67 Tg mice, in comparison with 3369 Tg mice, with an oral administration of DSS. DSS is toxic to intestinal epithelial cells, causing an epithelial barrier integrity defect. Body-weight decreaseoccurred3daftera5-dDSStreatmentwasdis- continued, followed by recovery after 3 more days, in both mouse lines. Three to 8 d after ceasing DSS treatment, whereas 3369 mice normally produce natural ATA as the predominant IgM in serum (20), there was a decrease in IgM normally found in the intestine of DSS-treated 3369 mice, including ATA IgMa PCs (Fig. 6B, lower panel). In contrast, DSS-treated EP67.CB17 mice showed microvascular dilation with increased red blood infiltration throughout the intestine as occurred during ongoing DSS treat- ment, together with an increase in AGcA PCs in the lamina propria (Fig. 6B, upper panel). In the colon, there was no sign of

accelerated colitis; rather, we observed that most goblet and epi- Downloaded from thelial cells regenerated 8 d after ceasing DSS treatment. AGcA PCs surround the degenerated epithelial area, in contrast with the intact area in EP67 mouse colon (Fig. 6C, left side versus upper right side), suggesting clearance of damaged goblet cells. Sur- veying for an additional 2 wk showed that chronic ulcerative co-

litis (UC) was not induced in DSS-treated EP67.CB17 mice (data http://www.jimmunol.org/ not shown). In this DSS colitis model, strain differences are well- known, such that chronic colitis occurs more often in C57BL/6 than BALB/c backgrounds (32). The EP67 Tg BCR on BALB/c IgM allotype-congenic C.B17 mice had abundant serum AGcA and also showed similar resistance to chronic colitis development as BALB/c mice.

Discussion We show in this study that B cells with AGcA become MZ B cells in by guest on September 27, 2021 spleen and produce natural autoantibody in serum. This AGcA binds predominantly to intact large Muc2 glycoprotein. In the same mouse, expression of a BCR with anti-thymocyte autoreactivity results in B1 B cell generation. Both AGcA and ATA react with highly glycosylated proteins, specific to murine determinants, showing no binding to corresponding rat (27). Ac- cumulation of AGcA MZ B cells occurs from the transitional immature stage and is dependent on Btk. Therefore, a BCR-ligand signal is required for AGcA MZ B cell development, as with ATA B1 B cells where again functional Btk is critical (9). How im- mature AGcA B cells are exposed to Ag, thereby generating MZ B cells and not B1 B cells, remains to be determined. However, in AGcA MZ B cell generation, DCs and blood vessels in spleen seem likely to play roles in presenting self-antigen, followed by the Notch 2 signal that is key in MZ B cell development (33). Goblet cells continually secrete Muc2 into the intestinal mucosa, providing a mucosal barrier against bacterial invasion. As with bacteria in the mucosal lumen, DC dendrites also continually in- ternalize Muc2 from mucus (14, 34). If these DCs move into the lymphatic vessels and enter blood circulation, then transport of Muc2 to the spleen can occur. This may explain the early initiation FIGURE 3. AGcA by MZ B cell–associated VH3609/Vk19-17 IgM of AGcA MZ B cell development in spleen, filtering the blood, (MK19). (A) Colon mucosal cell suspension was stained by MK19, in contrast with thymus cell suspension that was stained by MK21. (B) Cryosection staining of colon. Goblet cells and contiguous luminal mucus were stained with MK19, but not MK21. IB4 (BSI-B4) using a 103 ob- objective lens. (E) Apical goblet cell staining by MK19 was similar to DBA jective lens. (C) MK19 staining of transverse sections of intestines and lectin binding in colon. MK19 staining (bottom middle) was merged with stomach using a 203 objective lens. (D) Colon goblet cells (horizontal PNA and WGA lectins (top middle). (F) MK19 staining of Muc1- and section) were shown to be PAS+ (paraffin section staining). MK19-stained Muc2-deficient mouse colon demonstrates predominantly Muc2-dependent granules in the goblet cells, which were distinct from PNA and IB4 bound binding of MK19. (G) Absence of MK19 staining of rat colon goblet cells. supranuclear areas and nuclei stained by DAPI, as assessed using a 203 (E)–(G) using a 103 objective lens. 612 NATURAL ANTI-GOBLET CELL AUTOANTIBODY

FIGURE 4. Predominant MK19 AGcA binding to intact Muc2 glycoprotein. (A) Western blot of colon NP-40 lysate. Mucus released from crypts by centrifugation was suspended in 1% NP-40 and applied to 3.3% SDS-PAGE with a 2.5% stacking gel for detection of glycosylated colon mucin(s). Comparison of wild-type B6 versus Muc2KO.B6. Anti-Muc2 (N terminus peptide). (B) Colonic crypts from Rag1 null mice were incubated with MK19 (or MK21), cross-linked (CL) with DSP, then suspended in NP-40 buffer for SDS-PAGE. Left set is Western blot of IgM H chain band using soluble and in- soluble lysate, showing the presence of MK19 IgM CL colon granules detected by anti-IgM. Right set of the same lysates preincubated with MK19 shows copresence of Muc2. MK19 IgM and Muc2 were both predominantly in the insoluble fraction. (C) Soluble material from MK19 IgM prebound (MK21 as a control) colon granules, followed by cross-linking, was incubated with anti-IgM–bound protein G Dynabeads for

18 h, then washed, eluted, and applied to SDS-PAGE analysis. Downloaded from Presence of MK19 bound to ∼600 kDa DBA+Muc2 is shown. IB, immunoblot; LB, lectin blot.

promoted by the presence of Muc2 in neonates as well as adults. natural autoantibody (T15id+ anti-PC) that is higher in BALB/c http://www.jimmunol.org/ Thus, differences in BCR signaling because of variation in affinity compared with C57BL/6 (35, 36). However, generation of T15id+ (21) and/or the site of Ag availability may generate MZ rather anti-PC B cells does occur in C57BL/6 mice, although at a much than B1 B cells. lower frequency (37). This IgM is one of the most studied natural Our data also demonstrate genetic influences on AGcA B cell autoantibodies in mice. Binding to apoptotic cells leads to the initial generation. Variation of MZ B–associated natural AGcA autoanti- generation of T15id+ anti-PC B cells, with anti-PC autoantibody body levels between mouse strains resembles B1 B cell–associated production, and cross-reactivity of this Ab to oxidized low-density natural autoantibody, T15 idiotype positive anti-phosphorylcholine lipoprotein helps to prevent atherosclerosis (38). Furthermore, re- by guest on September 27, 2021

FIGURE 5. Natural AGcA IgM production from MZ B cells. (A) The presence of AGcA PCs (high IgMa+ and CD192) in EP67 mouse spleen. CD19+ B cells are also stained with IgMa+ at a lower level than PCs. (B) Predominant AGcA IgM in serum of EP67 mice was independent of T cells (EP67.RagKO). mkTg IgM is IgMa on a C.B17 (IgMb) mouse background. n = 9 each. (C) Colon and thymus cryosection staining comparison between EP67 AGcA mkTg mouse and 3369 ATA mkTg mouse sera, both with Tg IgMa predominance in serum. (D) AGcA reactivity by IgM produced from normal mouse MZ B cells. Colon from Rag1 null mice stained by LPS-stimulated B cell culture supernatant IgM (adjusted to 2–10 mg/ml) from BALB/c mice. B1 (B1a) B cells in the peritoneal cavity and MZ B cells, and FO B cells in spleen were purified from the same mouse. Representative of three experiments (two with BALB/c, one with C.B17). (A), (C), and (D) using a 203 objective lens. RP, red pulp; WP, white pulp. The Journal of Immunology 613

Table I. Strain difference of natural AGcA IgM levels

AGcA Reactivity

Serum from +++ + +/22 EP67 Tg.CB17 100% (8/8) 0% 0% 0% BALB/cAnN 0% 71% (5/7) 29% (2/7) 0% C.B17 0% 20% (1/5) 80% (4/5) 0% C57BL/6 0% 0% 17% (1/6) 83% (5/6) Analysis of sera from individual 2- to 4-mo-old mice (both male and female, 5–8 mice/group) for capacity to stain RagKO colon goblet cells. All reactivity compared at 1/100 dilution with similar total IgM levels, and also in comparison with 1/100, 1/500, and 1/1000 diluted one EP67 mkTg serum as a standard sample. Reactivity levels by mouse sera marked as (+) were around 8- to 10-fold less than EP67 mkTg sera (+++), although still detectable. Undetected at 1/100 dilution was (2). All sera show no reactivity to rat colon goblet cells. activity to phosphorylcholine expressed by various microorganisms organisms, thereby playing a protective role as part of the MZ plays a protective role against pathogens (35). We show in this B cell subset. However, in turn, when aberrant Ag exposure or study that MZ B–associated AGcA is also higher in BALB/c than autoreactive T cell generation induces Ig class switching of high- C57BL/6, again demonstrating the importance of genetic back- affinity AGcA B cells, their presence has the potential to result in ground in generation of the natural autoantibody repertoire. One goblet cell loss by required ability to permeate epithelium, leading Downloaded from possibility is that the VH or VL segment required for the AGcA to pathogenicity. BCR is different enough in C57BL/6 mice to reduce its affinity In human pathology, the relevance of anti-goblet cell autoan- below that required for selection into the MZ B cell pool. Alter- tibody to UC has been an issue for decades, as a potential disease- natively, the frequency of B cells with an appropriate AGcA BCR is inducing autoantibody (17). Sera from children with UC contain lower in C57BL/6 mice, as found with T15id+ anti-PC B cells. Ab that reacts with fetal colon cells (17, 18), and anti-Muc2 re-

Understanding the presence of natural autoantibodies in verte- activity in UC patient serum has been demonstrated (39). The http://www.jimmunol.org/ brates, their roles, and genetic influences have been a subject of presence of anti-goblet cell autoantibody among first-degree rel- continuing study. As with B1 B cell–derived natural autoanti- atives of UC patients without obvious sign of disease has been bodies, the production of AGcA IgM may normally function as interpreted as indicating a genetic risk for development of UC a scavenger natural autoantibody, as suggested by experiments (40). However, a role for AGcA in enteropathy has not been with DSS-treated mice. Because cells of the intestinal tract are clearly established; rather, AGcA is often detected in the serum of constantly sloughing off and renewed by intestinal stem cells, healthy individuals (41). As demonstrated in this study with mice, the presence of AGcA IgM in animals with appropriate genetic anti-goblet cell autoantibodies in relatives of UC patients may in background may help to promote clearance of damaged cells. part be composed of natural Igs at genetically regulated levels, as When B cells producing natural AGcA switch to IgA in intestine, found for BALB/c versus C57BL/6 strains. The mouse model we by guest on September 27, 2021 the secreted Ig will reach the mucosal lumen and bind to Muc2. describe enables such further study of the significance of natural This may enhance the physical barrier provided by Muc2 and aid autoantibodies, testing whether it functions to suppress inflam- in preventing microbial invasion, an interesting possibility to matory disease, but carrying a risk for promoting disease. In ad- pursue. Considering the higher AGcA titer in BALB/c mice, the dition, in considering reactivity to the highly glycosylated Muc2 presence of AGcA autoreactive B cells may moderate or prevent molecule, potential cross-reactivity to pathogenic bacteria or development of chronic colitis, potentially acting as a regulatory viruses to enable defensive immunity by MZ B cells is another B cell. In addition, AGcA may have cross-reactivity to infectious area that merits further investigation.

FIGURE 6. Natural AGcA autoanti- body in intestine. (A) The presence of AGcA B cells (IgMa+) in isolated lym- phoid follicles in EP67 mouse colon, and absence of goblet cell prebound AGcA IgM (upper panel), unless the tissue section is first incubated with se- rum from EP67 mkTg mice containing AGcA autoantibody (lower panel). (B) Analysis of DSS-treated (8 d after ceasing DSS treatment) EP67 and 3369 mouse intestine. AGcA (but not ATA) plasma cells are increased in intestinal lamina propria (selected area, enlarged at right).(C) DSS-treated EP67 colon section staining (8 d after ceasing DSS treatment). AGcA B cell follicle (iLF, right side of the IgMa-stained figure) and AGcA PC infiltration into the damaged region, in comparison with the PNA+ undamaged goblet cell area. (A) and (C) using a 203 objective lens. (B) using a 103 and 203 objective lens. 614 NATURAL ANTI-GOBLET CELL AUTOANTIBODY

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Supplemental Information

Natural anti-intestinal goblet cell autoantibody production from marginal zone B cells

Daiju Ichikawa, Masanao Asano, Susan A. Shinton, Joni Brill-Dashoff, Anthony M. Formica, Anna Velcich, Richard R. Hardy & Kyoko Hayakawa

Tables. S1, S2 Figure S1. S2

Table S1 + VH3609µTg B cell Imm B FOB MZB Vk Family Vk Gene Total Jk1 Jk2 Jk4 Jk5 Total Jk1 Jk2 Jk4 Jk5 Total Jk1 Jk2 Jk4 Jk5 Vk1 bb1(1-110) 2 2 1 1 0 Vk2 bj2 (2-135) 0 1 1 0 bd2 (2-139) 2 1 1 5 2 1 1 1 0 Vk4/5 4-50 1 1 3 3 1 1 ap4 (4-56) 1 1 3 1 2 1 1 aa4 (4-61) 2 1 1 7 4 1 2 1 1 ac4 (4-63) 1 1 1 1 0 aq4 (4-68) 1 1 4 2 2 2 2 al4 (4-71) 1 1 0 0 am4 (4-72) 1 1 4 1 1 2 0 ai4 (4-74) 2 1 1 4 2 1 1 1 1 aj4 (4-77) 0 1 1 2 2 ae4 (4-79) 0 1 1 2 1 1 af4 (4-80) 4 2 1 1 2 2 0 at4 4 4 1 1 0 kj4 (4-58) 0 1 1 0 kk4 (4-60) 9 3 1 5 5 1 4 0 kn4 (4-70) 0 2 2 0 kb4 (4-86) 2 1 1 1 1 0 kf4 (4-91) 2 1 1 7 2 2 3 3 1 2 kh4 3 1 2 10 1 2 7 2 2 Vk8 8-19 0 1 1 2 1 1 8-24 0 0 2 2 8-28 1 1 5 1 1 3 1 1 8-30 3 1 2 2 2 1 4 1 2 1 8-34 1 1 0 0 Vk9 ba9 2 1 1 11 6 1 4 39 4 7 28 br9 1 1 1 1 1 1 cf9 (9-100) 2 2 0 0 bv9 (9-122) 0 2 1 1 0 cb9 (9-123) 0 0 1 1 cy9 (9-125) 0 1 1 0 cw9 (9-126) 0 1 1 0 Vk12/13 fl12 (12-89) 0 1 1 0 ci12 (12-98) 4 4 0 6 6 12-38 0 1 1 0 12-41 0 1 1 0 12-44 1 1 4 2 1 1 0 12-46 10 4 5 1 12 9 1 2 1 1 Vk19/28 19-15 2 1 1 1 1 2 2 19-17 0 3 3 56 54 2 19-20 0 1 1 0 0 19-23 3 1 2 2 1 1 3 2 1 19-25 1 1 6 4 1 1 14 11 3 19-29 1 1 0 1 1 19-32 6 2 2 2 19 3 7 2 7 5 3 0 2 Vk20 bt20 (20-123) 1 1 1 1 3 1 2 bw20(20-130) 0 1 1 2 2 Vk21 21-2 0 1 1 0 21-4 0 1 1 1 1 21-5 2 1 1 2 2 0 21-7 0 3 1 2 0 21-10 1 1 0 6 4 2 21-12 0 1 1 0 Vk23 23-37 0 2 1 1 0 23-39 1 1 0 0 23-43 1 1 7 1 3 1 2 0 23-45 4 1 3 3 2 1 0 23-48 5 1 2 2 6 1 3 2 0 Vk32 gr32 (32-103) 4 3 1 10 4 4 1 1 1 1 Vk38C gj38c (38-93) 5 4 1 10 4 2 3 1 4 2 1 1 VkRF RF (RF-104) 4 1 3 3 1 2 1 1 total 104 29 23 9 43 191 50 56 28 57 171 87 28 19 37

Table S1. Biased κ-chain usage by the MZ B cells in VH3609µTg mice. Summary of single-cell κ gene cDNA sequence data + of VH3609µ B cell populations from 2-3 mo old VH3609µTg mice. Immature B cell data were derived from bone marrow and + – – spleen AA4 CD21 CD23 B cells. Only genes detected in this analysis are listed. The Vk19-17, Vk19-25, and Vk9/ba9 genes observed to be overrepresented in MZ B cells, and the highest identical Vk19-17/Jk1 usage (31 %) as marked in square. Table S2 A. VH3609µ/ Sample Vk19 reactivity Organ : Colon +++ Small intestine ++ Ileum + Jejunum + Duodenum + Lung +/–

Stomach – Pancreas – Salivary gland – Kidney – Liver – Brain – Heart – Spleen – Thymus –

Cell : Erythrocyte – Bromelain treated erythrocyte – Myeloid cells – Lymphocytes – Natural killer cell – Platelet –

Cell line : Stroma line OP9, Human Hep2 all – Fibroblast line NIH3T3 (nucleus, cytoplasm, cytoskeleton) Miscellaneous : Mouse IgG, ssDNA all – Cytochrome C,

B. Colon Mouse : Germ free (Swiss mouse) ++ 4 and 12 day old C.B17 ++ RagKO ++ TFF3 (trefoil factor 3)KO +++ BALB/c & C.B17 +++ C57BL/6 +++ Rat : 2 mo old –

Table S2. VH3609/Vk19-17 IgM (MK19) autoreactivity, specific to intestine. A. Intestine specific staining, highest with colon. Cryosection staining analysis for the majority of organs and intracellular staining analysis using fixed cell lines. Flow cytometry analysis for various cell types. ELISA for miscellaneous antigens. All MK19 (and MK21 control) analysis were done at 2-5µg/ml. IgM autoantibodies to the nucleus or myosin were used as positive controls for fixed cell line analysis. B. Positive reactivity with germ free, neonatal, and RagKO mouse colons, and with colon from different mouse strains; rat colon is negative.

Figure S1

A EP67

WT CD40KO RagKO Thy-1KO

5 10

10 4 k

8 3

H 10

3 2 1 10 0

2 3 4 2 3 4 5 2 3 4 5 2 3 4 5 0 10 10 10 105 0 10 10 10 10 0 10 10 10 10 0 10 10 10 10 VH3609 id

5 10

10 4 4

2 10 3 D

C 2 10 0 2 3 4 2 3 4 5 2 3 4 2 3 4 5 0 10 10 10 105 0 10 10 10 10 0 10 10 10 105 0 10 10 10 10 CD21

B EP67.B6 mg/ml 5 5 5 10 10 10 IgMb 4 0.8

k 4 4 10 M 10 4 10 3 8 g 2

I a 2 IgM H 3 3 3 0.6 D 10 10 10 D 3 m C 1 2 C 2 2 10 10 u 0.4

10 r

0 0 0 e s 0.2 2 3 4 2 3 4 5 2 3 4 5 0 10 10 10 105 0 10 10 10 10 0 10 10 10 10 0 V 3609 id CD21 CD21 7 H 1 6 .B B C EP67

Fig. S1. VH3609/Vk19-17 µκ MZ B cell generation in absence of T cell help, Thy-1 antigen independence, and on both C.B17 and C57BL/6 background. A. Spleen B cells (B220+) in the EP67 µκTg mouse line on C.B17 (wild type, WT), CD40KO, RagKO, or Thy- 1KO background. All adult (2-4 mo.). WT, CD40KO, and Thy-1KO EP67 µκTg mice show similar B cell frequency/numbers in spleen (50%-60%), and 93% in RagKO (frequency of total spleen cells). All show predominant µκTg+ B cells without BCR editing, and CD21hi (CD24medCD23–) MZ B cell generation, comprising 30-35% of total B cells. B. Spleen B cells (B220+) in the EP67 line backcrossed to C57BL/6, fourth generation. Predominant µκTg+ B cells and CD21hi CD24medCD23– MZ B cell generation (30-40% of spleen B cells by three 2-4 mo old mice analysis), and natural autoantibody production in serum (right panel, IgMa+, 2-5 mo old mice, n=5 each), similar to EP67.CB17 mice.

GF colon GF small intestine ) ) l l A A a a t t ) G G l n n 9 a W W o o 1 c i z z i i t 9 9 K r r r 1 1 o o e M K K v h h ( ( ( M M

Fig. S2. MK19 reactivity with goblet cells in germ free intestine. Colon and small intestine (jejunum) staining of germ free Swiss Webster mouse with MK19 (and WGA). Vertical and horizontal sections. White light images of colon or intestinal tissue are superimposed in red to enhance contrast, visualizing non-fluorescent cellular structures. 20x objective lens.