Intrinsic Molecular Factors Cause Aberrant Expansion of the Splenic Marginal Zone B Cell Population in Nonobese Diabetic Mice

This information is current as Jessica Stolp, Eliana Mariño, Marcel Batten, Frederic Sierro, of September 26, 2021. Selwyn L. Cox, Shane T. Grey and Pablo A. Silveira J Immunol 2013; 191:97-109; Prepublished online 5 June 2013; doi: 10.4049/jimmunol.1203252

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Intrinsic Molecular Factors Cause Aberrant Expansion of the Splenic Marginal Zone B Cell Population in Nonobese Diabetic Mice

Jessica Stolp,*,†,1 Eliana Marin˜o,*,†,1 Marcel Batten,*,† Frederic Sierro,*,† Selwyn L. Cox,*,† Shane T. Grey,*,†,2 and Pablo A. Silveira*,†,2,3

Marginal zone (MZ) B cells are an innate-like population that oscillates between MZ and follicular areas of the splenic white pulp. Differentiation of B cells into the MZ subset is governed by BCR signal strength and specificity, NF-kB activation through the B cell–activating factor belonging to the TNF family (BAFF) receptor, Notch2 signaling, and migration signals mediated by , integrin, and sphingosine-1-phosphate receptors. An imbalance in splenic B cell development resulting in expansion

of the MZ subset has been associated with autoimmune pathogenesis in various murine models. One example is the NOD inbred Downloaded from mouse strain, in which MZ B cell expansion has been linked to development of type 1 diabetes and Sjo¨gren’s syndrome. However, the cause of MZ B cell expansion in this strain remains poorly understood. We have determined that increased MZ B cell development in NOD mice is independent of T cell autoimmunity, BCR specificity, BCR signal strength, and increased exposure to BAFF. Rather, mixed bone marrow chimeras showed that the factor(s) responsible for expansion of the NOD MZ subset is B cell intrinsic. Analysis of microarray expression data indicated that NOD MZ and precursor transitional 2-MZ subsets were partic- ularly dysregulated for controlling cellular trafficking, including Apoe, Ccbp2, Cxcr7, Lgals1, Pla2g7, Rgs13, S1pr3, Spn, Bid, http://www.jimmunol.org/ Cd55, Prf1, and Tlr3. Furthermore, these B cell subsets exhibited an increased steady state dwell time within splenic MZ areas. Our data therefore reveal that precursors of mature B cells in NOD mice exhibit an altered migration set point, allowing increased occupation of the MZ, a niche favoring MZ B cell differentiation. The Journal of Immunology, 2013, 191: 97–109.

pon completing their differentiation program in bone subsets (1). Similar to humans, the FO subset in mice forms the marrow (BM), human and mouse B cells migrate to the major fraction of mature B cells, which are a circulating pop- U spleen in a transitional (TR) state, where they undergo ulation that characteristically resides within FO areas of the spleen further maturation to become follicular (FO) or marginal zone (MZ) and other secondary lymphoid organs. Their positioning adjacent to T cell–enriched periarteriolar lymphoid sheaths means that they by guest on September 26, 2021 are ideally situated for generating T-dependent Ab responses. In *Garvan Institute of Medical Research, Immunology Program, Darlinghurst, New contrast, the MZ subset is a longer-lived spleen-restricted pop- South Wales 2010, Australia; and †St. Vincent’s Clinical School, University of New South Wales, Sydney, New South Wales 2052, Australia ulation of B cells that inhabit the interface of the red and white 1J.S. and E.M. contributed equally to this paper and share primary authorship. pulp adjacent to the marginal sinus, known as the MZ (2). B cells 2S.T.G. and P.A.S. contributed equally to this paper and share senior authorship. comprising this innate-like population have a number of distinc- tive properties. First, they are enriched for low-affinity polyre- 3Current address: Dendritic Cell Biology and Therapeutics Group, ANZAC Research Institute, Concord, NSW, Australia. active BCR and can mount rapid and robust T-independent (mostly Received for publication November 26, 2012. Accepted for publication May 5, 2013. IgM) Ab responses to blood-borne pathogens (2). Second, they This work was supported by grants from the National Health and Medical Research migrate to FO areas upon activation, where they can participate in Council of Australia and the Juvenile Diabetes Research Foundation. S.T.G. is an T-dependent Ag responses due to their capacity to function as Australian Research Council Future Fellow. J.S. and S.L.C. were recipients of post- + graduate research awards from the University of New South Wales. E.M. was sup- potent APC for naive CD4 T cells (3). Third, they constitutively ported by a fellowship from the Ross Trust. shuttle blood-borne complement-coated Ags encountered in the The microarray data presented in this article have been submitted to Expression MZ into FO areas for recognition by other B cells (4). Finally, the Omnibus (http://www.ncbi.nlm.nih.gov/geo/) under accession number GSE43396. surface phenotype of MZ B cells differs from FO B cells and is Address correspondence and reprint requests to Dr. Pablo A. Silveira or Prof. Shane characterized in mice by lower levels of CD23 and IgD and higher T. Grey, ANZAC Research Institute, Gate 3 Hospital Road, Concord NSW 2139, Australia (P.A.S.) or Garvan Institute of Medical Research, 384 Victoria Street, levels of CD24, CD21/35, CD1d, and IgM (5). Darlinghurst, NSW 2010, Australia (S.T.G.). E-mail addresses: pablo.silveira@ A common feature of several murine models of autoimmunity sydney.edu.au (P.A.S.) or [email protected] (S.T.G.) is a dysregulated balance of B cells within the FO and MZ The online version of this article contains supplemental material. compartments. In particular, significant expansion of MZ B cells Abbreviations used in this article: BAFF, B cell–activating factor belonging to the is observed in New Zealand Black (NZB), (NZB 3 New Zea- TNF family; BAFF-R, BAFF receptor; BAFF-Tg, BAFF transgenic; BM, bone mar- row; FO, follicular; HEL, hen egg lysozyme; IgHEL-Tg, IgHEL transgenic; LN, land White [NZW])F1, MRL-lpr,andBcell–activating factor be- lymph node; MZ, marginal zone; NOR, nonobese resistant; NZB, New Zealand longing to the TNF family (BAFF; TNFSF13B) transgenic (BAFF- Black; NZW, New Zealand White; qRT-PCR, quantitative RT-PCR; S1P, sphingo- Tg) mouse strains (6–8). In these models, MZ B cells contribute sine-1-phosphate; S1PR3, sphingosine-1-phosphate receptor-3; T1, transitional 1; T2, transitional 2; T3, transitional 3; TACI, transmembrane activator and calcium to autoimmune pathology by harboring increased proportions of modulator and cyclophilin ligand interactor; T1D, type 1 diabetes; TR, transitional; clones displaying self-reactive BCR specificities that produce WT, wild-type. significant quantities of autoantibodies and infiltrate target organs Copyright Ó 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00 (9). B cells exhibiting a MZ phenotype also form a significant part www.jimmunol.org/cgi/doi/10.4049/jimmunol.1203252 98 GENETIC CONTROL OF MZ B CELLS IN NOD MICE of the target organ infiltrate in patients developing Sjo¨gren’s syn- (Minneapolis, MN). A human BAFF/mouse IgG2a-Fc fusion (AB Bio- drome and Grave’s disease (10, 11). Imbalanced production of sciences, Fresno, CA) capable of binding to mouse BAFF-R, TACI, B cell subsets may therefore be an important factor that underlies and B cell maturation Ag (TNFRSF17) was used to examine total BAFF- binding capacity of B cell subsets following secondary staining with an anti- several autoimmune phenotypes. mouse IgG2a-biotin and streptavidin-allophycocyanin (BD Biosciences). NOD mice are another inbred strain that displays enhanced MZ For in vivo labeling of B cells, mice were injected i.v. with 1 mg anti-CD19 B cell differentiation from an early age (12, 13). Interestingly, FITC (1D3; BD Biosciences) in 200 ml PBS. After 2 min, mice were culled several lines of evidence implicate MZ B cells in this strain as and spleen cells were obtained for staining with other B cell Abs described above. Stained cells were run on FACSCanto I or II flow cytometers (BD important contributors to the T cell–mediated b cell destruction Biosciences). Flow cytometric data were analyzed using FlowJo software associated with the development of type 1 diabetes (T1D). This (Tree Star, Ashland, OR). includes studies that show the following: 1) the MZ B cell subset undergoes a large secondary expansion prior to the onset of dis- BM chimeras ease, which is associated with their aberrant migration into the (NOD 3 B6)F1 mice were subjected to a 12 Gy lethal dose of irradiation pancreas and its draining lymph nodes (LN) (12); 2) transgenic from a 137Cs source. The following day, recipients were injected i.v. with 3 7 B cell clones specific for the primary T1D autoantigen insulin are a total of 1 10 BM cells extracted from femurs and tibias of NOD and B6 strains, mixed at a 1:1 ratio, and stained with anti-CD4 (GK1.5) and preferentially selected into the MZ subset (14); 3) MZ B cells in anti-CD8 Abs (53-6.7; eBioscience). Blood and tissues were harvested NOD mice have the capacity to present insulin to autoreactive from hosts for flow cytometric analysis at 8 wk postreconstitution. CD4+ T cells as effectively as the FO subset (12); and 4) prefer- ential depletion of this subset using an anti-CD21/35 mAb sig- Calcium flux nificantly decreased the incidence of cyclophosphamide-induced To measure calcium flux in B cells, cell suspensions from pooled LN Downloaded from T1D in NOD mice (15). In addition, accumulation of MZ B cells (inguinal, axillary, brachial, cervical, and mesenteric) or spleens were has also been associated with pathology causing Sjo¨gren’s syn- stained with anti-B220 and washed with HBSS containing 1% FCS (Life Technologies, Carlsbad, CA). These were resuspended at 1 3 107 cells/ml drome in nonautoimmune-prone C57BL/6 (B6) mice containing the in HBSS containing 4 mM Fluo-4 AM and 3 mg/ml Pluronic F-127 (Life Aec1 and Aec2 disease susceptibility loci from NOD mice (16). Technologies) and incubated for 20 min at room temperature. Cells were Despite several studies showing an association of MZ B cells then diluted to 2 3 106 cells/ml in HBSS/1% FCS and incubated for an additional 40 min at 37˚C. After three washes with HBSS/1% FCS, cells with autoimmune pathogenesis in NOD mice, little is known http://www.jimmunol.org/ about the actual factors responsible for their dysregulated accu- were finally suspended in HEPES-buffered salt solution containing 137 mM NaCl, 5 mM KCl, 1 mM Na2HPO4, 5 mM glucose, 1 mM CaCl, 0.5 mulation in this strain. To develop a better understanding of the mM MgCl, 1 g/L BSA, and 10 mM HEPES (pH 7.4). After 15-min in- underlying causes for augmented MZ B cell production in NOD cubation at 37˚C, cells were run for 30 s on a FACSCalibur flow cytometer mice, we analyzed various factors known to influence differenti- (BD Biosciences) to establish basal calcium levels. Cell suspensions were ation into this subset. Using BM chimeric mice, it was determined then mixed with the indicated concentrations of polyclonal anti-mouse IgM F(ab9)2 fragments (Jackson ImmunoResearch Laboratories) or hen that this phenotype is caused by B cell intrinsic factors. As a re- egg lysozyme (HEL; Sigma-Aldrich) before being run on the cytometer for sult, we obtained the transcriptional profiles of FO and MZ subsets 4 min to measure calcium flux over time via changes in Fluo-4 AM and TR precursors from NOD and B6 mice, which revealed a po- fluorescence. To control for Fluo-4 AM loading, the maximum flux for tential role for molecules involved in cell migration in the ex- B cells from each mouse strain was determined by stimulating cells with by guest on September 26, 2021 pansion of the NOD MZ B cell subset. 1 mg/ml ionomycin. Immunoblotting Materials and Methods Red cell–depleted splenocyte suspensions from wild-type (WT) or IgHEL- Mice Tg strains were stained with biotinylated anti-CD3ε (145-2C11) and CD11b (M1/70, BD Biosciences) Abs, followed by anti-biotin Microbeads Mice were housed under specific pathogen-free conditions at Garvan In- (Miltenyi Biotec, Bergisch Gladbach, Germany). Separation of unlabeled stitute or Australian Bio-Resources (Moss Vale, Australia). NOD/Lt, B cells from samples was achieved to a .93% purity (as determined by nonobese resistant (NOR)/Lt mice, and B6 mice were purchased from flow cytometry) by negative depletion with an autoMACS cell sorter Australian Bio-Resources or Walter and Eliza Hall Institute (Melbourne, (Miltenyi Biotec). Twenty million B cells from each strain were then re- Australia). DBA/2, BALB/c, and CBA mice were purchased from Animal suspended in PBS with or without 10 mg/ml Affinipure anti-IgM F(ab9)2 Resources Centre (Perth, Australia). (NOD 3 B6)F1 were generated by fragments (Jackson Immunoresearch; WT B cells) or varying concen- crossing NOD and B6 mice. NOD and B6 IgHEL transgenic (IgHEL-Tg) trations of HEL (Sigma-Aldrich; IgHEL-Tg B cells) for 5 min at 37˚C. mice have been described (17). NOD.H2b congenic mice (18) were pur- Cells were then pelleted, resuspended in cell lysis buffer containing pro- chased from The Jackson Laboratory. B6.BAFF2/2 (19) and B6.BAFF-Tg tease and phosphatase inhibitors (1% Nonidet P-40, 10 mM Tris-HCl, 150 (20) mice were provided by S. Kalled (Biogen Idec, Weston, MA) and mM NaCl, 0.1% NaN3,2mg/ml aprotinin, 1 mM sodium pervanadate, 10 backcrossed to NOD/Lt mice for 10 generations to generate NOD.BAFF2/2 mM sodium fluoride, 1 mM PMSF, 0.5 mm EDTA), and incubated at 4˚C and NOD.BAFF-Tg strains, respectively. Experiments were approved by for 30 min. Concentration of in lysates was determined by stan- the Garvan Institute/St. Vincent’s Hospital Animal Ethics Committee. dard Bradford assay (Bio-Rad). Equivalent levels of protein from stimu- lated and nonstimulated samples were subjected to electrophoresis on 4– Flow cytometry 12% gradient NuPAGE Bis-Tris gels (Life Technologies) and transferred Single-cell suspensions from the indicated tissues were washed in PBS onto BioTrace polyvinylidene difluoride membranes (Pall, Ann Arbor, MI). After blocking with 5% BSA/0.2% TBST, membranes were probed containing 1% BSA and 0.1% NaN3 (Sigma-Aldrich, St. Louis, MO), blocked with an anti-CD16/32 Ab (2.4G2; BD Biosciences, San Jose, CA), with biotinylated anti-phosphotyrosine Ab (4G10; Upstate Biotechnology, and stained with the following fluorochrome-conjugated streptavidin or Charlottesville, VA) in TBST, followed by secondary staining with strep- Abs at predetermined optimal concentrations: anti-IgM allophycocyanin tavidin conjugated to HRP (Santa Cruz Biotechnology, Santa Cruz, CA) (II/41), anti-IgMa FITC (DS-1), anti-CD24 biotin (30-F1), anti-H2Kb bi- and Supersignal West Pico chemiluminescent substrate (Thermo Fisher otin (AF6-88.5), anti-H2Kd FITC (SF1-1.1), anti-B220 allophycocyanin- Scientific, Rockford, IL). In some cases, membranes were stripped with 0.2 Cy7 (RA36B2), and streptavidin-PerCP from BD Biosciences; anti-CD21/ M NaOH and restained with biotinylated anti–phospho-ERK (polyclonal 35 PE or Pacific Blue (ebio8D9), anti-CD23 PE-Cy7 (B3B4), and CD93- 9101) and anti–phospho-JNK (polyclonal 4671; Cell Signaling Technol- allophycocyanin (AA4.1) from eBioscience (San Diego, CA); anti-CD1d ogy, Danvers, MA). Bands were visualized following exposure of mem- PerCP-Cy5.5 (1B1) from BioLegend (San Diego, CA); polyclonal anti- branes to x-ray film (Kodak, Rochester, NY). IgM FITC Fab fragments from Jackson ImmunoResearch Laboratories BAFF ELISA (West Grove, PA); and anti–transmembrane activator and calcium modu- lator and cyclophilin ligand interactor (TACI; TNFRSF13B)-PE (166010) ELISA plates (Nalge Nunc International, Rochester, NY) were coated with and anti–BAFF-R (TNFRSF13C)-FITC (204406) from R&D Systems 5 mg/ml purified rat anti-mouse BAFF overnight (clone 5A8) and blocked The Journal of Immunology 99 with 1% BSA in PBS at 37˚C for 2 h. A total of 20 ml/well mouse sera or largely been confined to comparisons with B6 and BALB/c strains standard mouse recombinant BAFF serial dilutions was added to the plates (12, 13, 21, 22). To determine the extent of this abnormality, we and incubated for 1 h at 37˚C. Wells were subsequently incubated for 1 h performed a more comprehensive comparison of splenic B cell with 10 mg/ml biotinylated anti-mouse BAFF (clone 1C9) and then HRP- labeled streptavidin (Santa Cruz Biotechnology). Concentrations of BAFF development in NOD mice to that of a variety of different mouse were determined using the tetramethylbenzidine substrate (BD Bio- strains, which, in addition to BALB/c and B6, included DBA/2, sciences). The reaction was stopped by adding 2 M H2SO4, and the ab- CBA as well as the closely related (88% genetically identical, sorbance at 450 nm was read using a Tecan Spectra Image microtiter plate including H2), but T1D-resistant, NOR strain. This analysis was reader (Bio-Tek Instruments, Winooski, VT). performed in 5-wk-old mice, an age that precedes the onset of Microarray autoimmune pathology associated with NOD mice. Compared Total B cells were enriched from eight pooled NOD or B6 mouse spleens with most other strains, NOD mice contained lower proportions of (7 wk old) using the negative depletion magnetic bead strategy described TR and FO B cell subsets, but higher proportions of MZ and MZ above. These cells were then stained with a mixture of polyclonal anti- precursor (T2-MZ) B cells in spleens (Fig. 1A). As a result of mouse IgM-FITC Fab fragments from Jackson ImmunoResearch Labora- tories; anti-CD24 biotin (30-F1; with streptavidin-PerCP secondary) and a general decrease in the cellularity of NOD spleens compared B220-allophycocyanin-Cy7 (RA36B2) from BD Biosciences; and anti- with other strains at this age (Fig. 1B), total numbers of FO and CD21/35 PE (eBio8D9) and anti-CD23 PE-Cy7 (B3B4) from eBio- TR B cells were found to be significantly lower than all the other sciences. Transitional 1 (T1), transitional 2 (T2)-FO, transitional 3 (T3), FO, mouse strains tested (Fig. 1C). In contrast, due to their increased T2-MZ, and MZ subsets were then separated with a FACSAria cell sorter proportion, total numbers of T2-MZ and MZ B cells in NOD (BD Biosciences). This process was repeated in an independent experiment to generate a duplicate sample of each B cell subset from NOD and B6 mice. spleens were equivalent or slightly higher than other strains, ex- RNA was extracted from each FACS-purified B cell sample using TRIzol cept CBA. NOD mice displayed the lowest FO:MZ ratio of all the Downloaded from (Life Technologies) as per manufacturer’s instructions. RNA quality and strains tested, highlighting the perturbed nature of splenic B cell quantity were determined using a RNA 6000 Nano Chip with 2100 Bio- analyzer (Agilent Technologies, Santa Clara, CA). RNA was then amplified, biotin labeled, and fragmented with the GeneChip two-cycle target la- beling and control kit (Affymetrix, Santa Clara, CA) as per manufacturer’s instructions. Resulting cRNA was hybridized to Mouse Genome 430 2.0 Arrays (Affymetrix), washed, stained on GeneChip Fluidics Station 400 http://www.jimmunol.org/ (Affymetrix), and scanned with a GeneChip Scanner (Affymetrix). Anal- ysis of data was performed using GeneSpring software (Agilent Tech- nologies) and submitted to the Omnibus public database (http://www.ncbi.nlm.nih.gov/geo/) under accession number GSE43396. All samples were normalized by Robust Multiarray Analysis on a per-gene basis to the median of the total samples. Genes were considered .2-fold upregulated/downregulated only if duplicate samples agreed and raw sig- nals of at least one of the compared samples was .100. Gene labels used are those prescribed by the Mouse Genomic Nomenclature Committee. Functional analysis of datasets was performed using Ingenuity Systems

Pathway Analysis software (Ingenuity Systems, Redwood City, CA). by guest on September 26, 2021 Quantitative real-time PCR FACS-purified B cell subsets from WT and IgHEL-Tg NOD and B6 mice were prepared as above, except that TR subsets (T1 and T2-FO) were pooled. RNA from B cell subsets was extracted using TRIzol (Life Tech- nologies) and then transcribed into cDNA using Moloney murine leukemia virus reverse transcriptase (New England BioLabs, Ipswich, MA) via the first-strand synthesis protocol provided by the manufacturer. Levels of gene transcripts were determined by performing quantitative RT-PCR (qRT-PCR) in a PRISM7900 HT machine (Life Technologies) using trip- licate reactions containing 50–100 ng cDNA and 0.9 mM each primer in LightCycler-RNA SYBR Green I Master Mix (Roche, Mannheim, Ger- many). Primers for each gene were designed such that one would bind across an exon–exon boundary (sequences in Supplemental Table I). All reactions were standardized to expression of the Hprt housekeeping gene. CXCL12 chemotaxis assay Single-cell suspensions of splenocytes from six NOD and B6 mice were prepared separately and resuspended in tissue culture media for 1 h at 37˚C in tissue culture plates (BD Biosciences) to remove adherent cells. Cells were then washed and placed in 0.5% FCS/RPMI 1640. Five hundred thousand cells were added to the upper chamber of Transwell plates (Corning Costar, Lowell, MA). Chemotaxis agent CXCL12 (PeproTech, Rocky Hill, NJ) was diluted in 0.5% FCS/RPMI 1640 and added to the bottom chamber at 0, 100, or 500 ng/ml. Splenocytes were allowed to FIGURE 1. transmigrate across 3-mm filters in Transwell plates for 4 h. Postmigration, Splenic MZ B cell generation is augmented in NOD mice. A cells were collected from the bottom chamber and stained for B220, IgM, ( ) Representative flow cytometry contour plots showing CD21 and CD23 + CD21, CD23, and CD24 to distinguish B cell subsets by flow cytometry. staining on B220 gated splenocytes of 5-wk-old NOD mice (five male and Transwell assays were performed in duplicate for each sample, and the six female) compared those of six B6, five BALB/c, six DBA/2, five CBA, average of the two results was used. and five NOR female mice. Numbers adjacent to gates delineate mean percentage of TR, FO, and MZ B cell subsets of all mice in each group. (B) Results Total numbers of splenic B cell subsets in individual mice from the indi- C Aberrant ratio of FO to MZ B cells produced in NOD mice cated strains. ( ) FO to MZ B cell ratio. Lines and error bars on graphs mark the mean and SEM, respectively. ****p , 0.0001, ***p , 0.001, Studies on the aberrant development of splenic B cells in NOD **p , 0.01, *p , 0.05 compared with the female NOD group (one-way mice leading to augmented production of MZ over FO B cells have ANOVA, Dunnet posthoc test). 100 GENETIC CONTROL OF MZ B CELLS IN NOD MICE development in these animals (Fig. 1C). Male NOD mice showed B cells due to the following: 1) defective expression of the latter no significant differences in splenic B cell subset numbers (Fig. by NOD mice (26), and 2) the fact that CD24 and CD93 are ex- 1B), nor FO:MZ ratio (Fig. 1C), compared with female NOD mice. pressed similarly on B6 TR B cells (Supplemental Fig. 1A). Con- Altered B cell differentiation is therefore not under the control of sistent with previous papers from our group (12) and others (13), sex hormones, which underlie the contrasting T1D susceptibilities this analysis showed the persistence of aberrant splenic B cell exhibited by male and female NOD mice (23, 24). differentiation in NOD mice as they get older, leading to the sig- nificant expansion of T2-MZ and MZ B cell proportions (Fig. 2A) Aberrant splenic B cell development in NOD mice is and numbers (Fig. 2B) in NOD compared with B6 spleens, independent of BCR specificity and underlying autoimmune whereas FO B cells eventually approached levels comparable to inflammation B6. B1 B cells were present at higher proportions (Fig. 2A), but To gain a better understanding of the events leading to altered B cell similar numbers (Fig. 2B) in NOD compared with B6 spleens. All development on the NOD background, we conducted a more thor- other TR precursor stages in the spleen, including T1, T2-FO, and ough flow cytometric evaluation of TR precursors (including T1, CD23highCD24low, remained greatly diminished in proportion T2-FO, [IgMhighCD21low]CD23highCD24low, T3, and T2-MZ) as (Fig. 2A) and numbers (Fig. 2B) in NOD compared with B6 mice. well as mature B cell subsets (including FO, MZ, and B1) in Moreover, in contrast to B6 mice, which contained higher pro- spleens of 7-wk-old female NOD compared with nonautoimmune- portions and numbers of T1 compared with the T2-FO subset, prone B6 mice. Subsets were distinguished in both strains utilizing NOD mice possessed equivalent levels of both subsets, in keeping the gating strategy described by Allman and colleagues (25), with with previously reported tolerance defects at the T1 to T2-FO the exception that CD24 was used instead of CD93 to gate TR transition in this strain (13, 22). NOD mice also exhibited sig- Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 2. Increased generation of MZ B cells in NOD mice occurs independently of BCR specificity or H2g7 MHC haplotype. (A, C, and E) Rep- resentative flow cytometry contour plots and (B, D, F) total numbers of T1, T2-FO, CD23highCD24low, B1, T3, FO, T2-MZ, and MZ B cell subsets in spleens of the following: (A, B) five 7-wk-old NOD and B6 female mice; (C, D) four 7- to 8-wk-old NOD and B6 IgHEL-Tg female mice; and (E, F) nine 7- wk-old NOD (H2g7) and NOD.H2b female mice. Percentages in contour plots mark the average for all mice in each group. Lines and error bars in graphs mark the mean and SEM, respectively. ***p , 0.001, **p , 0.01, *p , 0.05 (t test). The Journal of Immunology 101 nificantly reduced proportions (Fig. 2A) and numbers (Fig. 2B) of fore of interest to determine whether the strength of BCR signaling the T3 B cell subset (also known as An1) compared with B6 mice in NOD B cells was diminished compared with B6 B cells, leading [a population that displays several features of anergy (27)]. This to increased selection of MZ B cells. This was initially examined corroborates our previous studies reporting defects in B cell anergy by comparing the extent of tyrosine phosphorylation in B cell within the NOD strain (22, 28). protein extracts via immunoblot. Equivalent levels of total pro- Aberrant mechanisms of B cell self-tolerance in NOD mice tein phosphorylation were observed following stimulation of NOD during the early developmental stages in the BM (22, 29) and TR and B6 WT B cells with cross-linking anti-IgM F(ab9)2 fragments stages in the spleen (Fig. 2A, 2B) (13, 22, 28) give rise to in- (Fig. 3A). This was also the case when membranes were reblotted creased frequencies of self-reactive clones in their mature B cell with Abs that specifically detect phosphorylated versions of the compartment (30–33). Given that self-reactive B cells have a integral BCR signaling molecules JNK and ERK. Moreover, similar greater propensity to develop into the MZ subset (14, 34–36), we levels of protein phosphorylation were observed when NOD and asked whether skewed production of this subset in NOD mice B6 IgHEL-Tg B cells were stimulated with various concentrations might be caused by increased production of self-reactive B cells. of soluble HEL Ag (Fig. 3B), which provides a weaker BCR signal To investigate this question, we analyzed splenic B cell subsets in than anti-IgM F(ab9)2 fragments. Equivalent levels of BCR signals 7- to 8-wk-old female NOD and B6 mice expressing the same in splenic and LN NOD and B6 B cells were also confirmed by prearranged Ig transgene, IgHEL (Fig. 2C, 2D). This transgene measuring calcium flux by flow cytometry following stimulation forces the majority of B cells in both strains (.94%) to express of WT B cells with anti-IgM F(ab9)2 fragments (Fig. 3C). Although a BCR (of the IgMa IgDa allotype) that is highly specific for the we did observe a slightly higher persistence of calcium flux in nominal exogenous Ag, HEL (17, 37). T1 and T2-FO B cell stimulated NOD compared with B6 WT splenic B cells, this was Downloaded from numbers were comparable in NOD and B6 IgHEL-Tg spleens, not the case for stimulated LN WT B cells. Differences in B cell flux whereas T3 and CD23highCD24low subset numbers were low to between these tissues may be due to the fact that spleens contain negligible, most likely due to the forced expression of nonself- various B cell subsets that are present at different proportions in reactive BCR specific for HEL. Proportions and numbers of FO NOD and B6 mice, whereas LN (pooled from noninflammatory sites) B cells were also similar between these strains. However, despite contain only FO B cells in both strains. Alternatively, differences

expressing identical BCR, transgenic HEL-specific B cells devel- http://www.jimmunol.org/ oping in NOD.IgHEL mice showed significantly greater pro- portions (9.1 6 0.2 versus 4.7 6 0.4% of total splenic lympho- cytes; p , 0.001, t test; Fig. 2C) and numbers (Fig. 2D) of the MZ subset than those in B6.IgHEL mice, resulting in a significantly lower FO:MZ ratio (7.2 6 0.8 versus 4.1 6 0.02, respectively; p , 0.05, t test). The proportions of the T2-MZ subset were also significantly higher in NOD.IgHEL compared with B6.IgHEL mice (4.5 6 0.2 versus 2.6 6 0.03% of total splenic lymphocytes; p , 0.01, t test; Fig. 2C), whereas thedifferenceinnumbers by guest on September 26, 2021 showed a strong trend toward significance (p = 0.06, t test; Fig. 2D). Thus, the bias of NOD splenic B cell development toward the MZ subset occurs independently of BCR specificity and B cell autoimmunity. Self-tolerance is also defective in the T cell compartment of NOD mice, leading to T cell–mediated destruction of insulin- producing b cells and onset of T1D. Exchanging MHC suscepti- bility alleles within the H2g7 haplotype of NOD mice for their corresponding resistance alleles in the B6 H2b haplotype com- pletely abrogates T cell autoimmunity against b cells in this strain (18). To determine whether the skewed ratio of FO:MZ B cells in NOD mice is a secondary consequence of underlying T cell au- toimmunity, we compared splenic B cell development in 7-wk-old congenic NOD female mice that expressed the H2b MHC haplo- type (NOD.H2b) to WT NOD mice expressing H2g7. Whereas the levels of T1 B cells seemed to differ between NOD and NOD.H2b mice, proportions (Fig. 2E) and numbers (Fig. 2F) of all other splenic B cell subsets, including T2-FO, CD23highCD24low, T3, FO, T2-MZ, MZ, and B1, were not significantly altered by ex- pression of the H2b resistance haplotype on the NOD background (Fig. 2E, 2F). This indicated that expansion of the MZ B cell pop- FIGURE 3. Enlarged MZ B cell population in NOD mice is not asso- ulation in NOD mice was not secondary to the breakdown in T cell ciated with altered strength of BCR signaling. Purified B cells from spleens tolerance nor the ensuing b cell autoimmunity that occurs in this strain. of (A)WTor(B) IgHEL-Tg B6 and NOD mice were stimulated for 5 min at 37˚C with either anti-IgM F(ab9)2 fragments (10 mg/ml) or HEL at the Disparity of splenic B cell subset distribution in NOD versus indicated concentrations, respectively. Total protein extracts from cells B6 mice does not correlate with altered BCR signal strength were subjected to immunoblot analysis with Abs specific for phospho- tyrosine, or phosphorylated JNK or ERK. (C) Intracellular calcium levels BCR signal strength and specificity play an important role in were measured for 4 min in B220+ gated cells from spleens or pooled LN determining selection of B cells into FO or MZ subsets (38, 39). of WT or IgHEL-Tg mice of the indicated strains following stimulation

Weak BCR signaling favors selection of MZ B cells, whereas with anti-IgM F(ab9)2 fragments (5 mg/ml) or HEL (50 ng/ml), respec- stronger signaling promotes FO B cell development. It was there- tively, at the time marked by the arrow. 102 GENETIC CONTROL OF MZ B CELLS IN NOD MICE could be due to a greater proportion of activated/memory autoreac- differentiation bound more BAFF-Fc than their B6 counterparts tive B cells in NOD spleens. Consistent with the latter explanation, (Fig. 4B, 4C), raising the possibility that enhanced sensitivity of similar levels of calcium flux were generated by naive NOD and B6 NOD B cells to BAFF may skew selection into the MZ subset. To IgHEL-Tg B cells from spleen and LN following BCR stimulation explore this possibility, we examined splenic B cell subsets in with their cognate Ag (Fig. 3C). Alterations in BCR signal strength NOD mice producing elevated levels of BAFF due to a BAFF are therefore unlikely to contribute significantly to aberrant devel- transgene (NOD.BAFF-Tg), or reduced levels caused by knocking opment of splenic B cell subsets in NOD versus B6 mice. out one copy of the BAFF gene (NOD.BAFF+/2). Surprisingly, variation of BAFF expression in this manner did not significantly Altered splenic B cell development in NOD mice occurs alter selection of splenic B cells into FO and MZ compartments in independently of BAFF signals NOD mice, because these subsets were found in similar propor- The capacity of BAFF to activate the noncanonical NF-kB pathway tions in NOD.BAFF-Tg and NOD.BAFF+/2 mice compared with by binding to BAFF-R is critical for mediating B cell survival past WT NOD mice (Fig. 5A). This was in stark contrast to BAFF-Tg the TR stages (40). However, this cytokine also plays an inde- and BAFF+/2 mice on the B6 background, which displayed a 4- pendent role in regulating development of MZ B cells by virtue of fold increase and 2-fold decrease, respectively, in proportions of its ability to activate both the canonical and noncanonical NF-kB MZ B cells in spleens compared with WT mice (Fig. 5B, 5C), pathways through engagement of the BAFF-R and TACI (41, 42). consistent with past studies (8, 20). The insensitivity of B cells in To determine whether skewed MZ B cell production in NOD mice NOD mice to changes in BAFF levels led us to investigate the may be due to elevated production of BAFF, we compared serum overall importance of BAFF for splenic B cell differentiation in levels of BAFF in 6-wk-old prediabetic female NOD mice with this strain. For this, we generated NOD mice containing two mu- Downloaded from those in aged-matched B6 and BALB/c strains. Interestingly, de- tated copies of the BAFF gene (NOD.BAFF2/2) and thus rendered spite the greater expansion of MZ B cells in NOD compared with incapable of producing this cytokine. Similar to BAFF2/2 mice on B6 or BALB/c mice, the former were found to contain lower or the B6 background, the absence of BAFF in NOD mice resulted in equivalent levels of BAFF in their sera compared with these strains a significant reduction in proportion and total numbers of B cells (Fig. 4A). Moreover, serum BAFF levels did not increase signif- and their expression of CD21 and CD23 (Fig. 5A, 5D). Using the

icantly in NOD mice until they became diabetic after 16 wk of age alternate markers CD1 and CD24 to distinguish proportions (Fig. http://www.jimmunol.org/ (Fig. 4A). Nevertheless, BAFF serum concentrations in diabetic 5E) and numbers (Fig. 5F) of splenic B cell subsets, we found that NOD mice were still lower or equivalent to B6 or BALB/c levels B cells remaining in NOD.BAFF2/2 mice retained significantly at 6 wk of age. Therefore, elevated BAFF levels are not the cause greater capacity to undergo maturation into FO and MZ subsets of skewed MZ B cell production in the NOD strain. When, how- than those in B6.BAFF2/2 mice. Moreover, the ratios of mature ever, flow cytometry was used to measure surface levels of receptors FO and MZ B cell subsets generated in NOD.BAFF2/2 mice did for BAFF, we observed that both T2-MZ and mature MZ B cells in not differ significantly from WT NOD mice (Fig. 5G). This was in NOD mice expressed higher levels of BAFF-R and TACI com- stark contrast to B6.BAFF2/2 mice, in which a minute proportion pared with those of the B6 strain (Fig. 4B, 4C). This was con- of B cells matured into the FO, but not the MZ subset (Fig. 5E, sistent with our finding that NOD B cells at both these stages of 5F). Thus, although BAFF is important for mediating B cell sur- by guest on September 26, 2021

FIGURE 4. Altered splenic B cell development occurs independently of BAFF signals in NOD mice. (A) Mean serum concentrations of BAFF in 6-wk- old, 16-wk-old, and diabetic NOD female mice compared with 6-wk-old C57BL/6 and BALB/c female mice (n = 5/strain). *p , 0.05, ***p , 0.001 compared with 6-wk-old NOD group (one-way ANOVA, Dunnet posthoc test). (B) Representative histograms and (C) average mean fluorescence intensity (MFI) of anti–BAFF receptor and TACI Abs as well as BAFF-Fc binding to gated TR, FO, T2-MZ, and MZ B cell subsets in five 6-wk-old B6 and NOD female mice as determined by flow cytometry. Error bars mark SEM. **p , 0.01, ***p , 0.001 (t test). The Journal of Immunology 103 Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 5. Mature FO and MZ B cells still develop in BAFF-deficient NOD mice. (A) Representative flow cytometry contour plots showing mean percentages of total IgM+ splenic B cells and their subsequent breakdown into TR, FO, and MZ subsets in 7- to 8-wk-old NOD.BAFF+/+, NOD.BAFF+/2, and NOD.BAFF2/2 littermates as well as NOD.BAFF-Tg female mice (n = 5/group). Representative flow cytometry contour plots showing percentages of TR, FO, and MZ B cell subsets in B220+ splenocytes of (B) 10-wk-old B6 versus B6.BAFF+/2 mice (n =3)or(C) 6-wk-old B6 versus B6.BAFF-Tg mice (n = 2/group). Percentage of B220+ B cells in spleens of mice is shown on top of plots. (D) Total numbers of splenic (B220+) B cells in BAFF+/+ versus BAFF2/2 mice on NOD and B6 backgrounds. (E) Contour plots showing alternative strategy for distinguishing B cell subsets using CD1d- and CD24- specific Abs in B220+ gated splenocytes. Mean percentages of TR, FO, and MZ subsets in five NOD, seven NOD.BAFF2/2, six B6, and four B6.BAFF2/2 female mice shown. (F) Total numbers of TR, FO, and MZ B cell subset in individual mice using the CD1d and CD24 gating strategy, as well as (G) the FO: MZ ratio. Lines and error bars represent mean and SEM, respectively. *p , 0.05, ***p , 0.001 (one-way ANOVA, Tukey posthoc test). vival in NOD mice, as in other strains, B cell maturation past the generated chimeras by using a 1:1 mixture of T cell–depleted NOD TR stage and differentiation into the MZ subset in this strain are and B6 BM to reconstitute lethally irradiated (NOD 3 B6)F1 not as dependent on this cytokine as they are in the B6 strain. hosts. This strategy ensured that hosts would be tolerant to both types of donor cells as well as allowing hematopoietic cells gen- Evidence of a role for B cell–intrinsic factors in mediating erated from NOD, B6, and (NOD 3 B6)F1 sources to be distin- altered splenic B cell development in NOD mice guished using Abs that differentiate between H2-Kb,Kd, and Kb/d The negative results to date indicated that preferential generation of MHC class I allotypes, respectively (Fig. 6A). Although equal MZ B cells in NOD mice could not be attributed to factors in- numbers of NOD and B6 BM were used to reconstitute each host, cluding BCR specificity or signal strength, underlying T cell au- after 8 wk, the percentage of splenic cells derived from each donor toimmunity, nor elevated BAFF signals. To determine whether this differed markedly between individual chimeras (Fig. 6A, 6B). The phenotype was caused by factors intrinsic or extrinsic to B cells, we variance in levels of reconstitution could be due to two opposing 104 GENETIC CONTROL OF MZ B CELLS IN NOD MICE mechanisms previously described to influence mixed bone marrow B220 and the gating strategy shown in Fig. 2A and Supplemental chimeras involving the NOD background, as follows: 1) the rel- Fig. 1B. Activation of B cells through BCR was avoided by stain- atively strong resistance of the NOD background to engraftment ing B cells with anti-IgM Fab9 fragments, which have been dem- with allogeneic BM compared with T1D-resistant strains; 2) a onstrated to be incapable of inducing phosphorylation of sig- NOD hematopoietic stem cell defect that provides allogeneic stem naling proteins or calcium flux (44). cells with a competitive advantage if they are able to successfully Data from microarrays were first validated by assessing ex- engraft recipients (43). Nevertheless, irrespective of the levels of pression of genes that encode markers commonly used to differ- splenocytes derived from each BM donor, differentiating NOD- entiate splenic B cell subsets. Gene expression levels for each derived B cells generally yielded greater proportions of MZ B marker correlated well with protein levels normally found for these cells and lower proportions of TR subset compared with B6 subsets in NOD and B6 mice (Fig. 7A). To identify genes that are B cells in hosts (Fig. 6B). Indeed, in most chimeras, NOD-derived important for MZ B cell development, we compared gene ex- B cells outcompeted B6-derived B cells to comprise the majority pression fold changes between T2-MZ versus T2-FO precursor as of the MZ population (Fig. 6A). The altered differentiation of well as the mature FO versus MZ populations in NOD and B6 splenic B cells from NOD and B6 mice in the same chimeric hosts, mice (Fig. 7B). These particular subsets were selected for com- regardless of reconstitution levels, confirmed that skewed develop- parison as they represent the early and late stages of the FO or MZ ment of splenic B cells into the MZ subset in NOD mice was caused developmental pathways, respectively (5). The majority of genes by factors intrinsic to B cells. that were upregulated or downregulated in T2-MZ or MZ samples compared with T2-FO and FO samples, respectively, exhibited

Identifying cell-intrinsic molecular factors responsible for similar changes in expression (i.e., ,2-fold difference in fold Downloaded from skewed development of the MZ B cell subset in NOD mice change) between NOD and B6 samples (Fig. 7B). This group of To identify potential B cell–intrinsic molecular factors responsible genes encoded various protein markers used to distinguish MZ for increased selection of B cells into the MZ program of differ- populations, including Cr2 (Cd35), Cd1d, Cd9, and Cd36, which entiation in NOD versus B6 mice, we used Affymetrix mouse 430 were upregulated in T2-MZ and MZ subsets, and Fcer2a (Cd23), 2.0 microarrays to compare transcriptional profiles of FO and MZ which was correspondingly downregulated in MZ B cells (see

B cells in both strains as well as their TR precursors (T1, T2-FO, green labeled genes in Fig. 7B). Nevertheless, there was a group http://www.jimmunol.org/ T3, and T2-MZ). cRNA from two highly purified samples of each of these genes that displayed altered expression between T2-MZ B cell subset was obtained by FACS, as outlined in Materials and versus T2-FO and/or MZ versus FO subsets between NOD and B6 Methods, using Abs specific for IgM, CD21/35, CD23, CD24, and groups (i.e., $2-fold difference in fold change). Most of this by guest on September 26, 2021

FIGURE 6. Enlarged MZ B cell subset in NOD mice is B cell intrinsic. (B6 3 NOD)F1 recipients were lethally irradiated and reconstituted with a 1:1 mixture of NOD and B6 BM cells. Eight weeks postreconstitution, splenocytes of B6 or NOD origin were distinguished in chimeras by flow cytometry b d using Abs specific for H2-K or K MHC class I allotypes, respectively (residual host F1 B cells were positive for both markers). The distribution of TR, FO, and MZ subsets in B220+ gated B cells of NOD or B6 origins was analyzed utilizing CD21 and CD23 markers. (A) Contour plots of NOD or B6 BM- derived B cell subsets from representative chimeric recipients exhibiting different levels of BM reconstitution from each strain. (B) Graphs showing the percentage of TR, FO, or MZ subsets relative to total B cells of NOD or B6 origin plotted against the percentage of total lymphocyte reconstitution from that strain for each chimeric recipient. Lines of best fit are plotted to show the overall trend for NOD (solid line)- and B6 (dashed line)-derived subsets. ****p , 0.0001, comparison of goodness of fit of NOD versus B6 samples with NOD line of best fit using Mann-Whitney U test. The Journal of Immunology 105 Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

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FIGURE 7. Identifying molecular factors that are dysregulated during splenic B cell development in NOD mice. MACS-enriched B cells from two inde- pendent pools of eight NOD or B6 spleens were purified into T1, T2-FO, T3, FO, T2-MZ, and MZ B cell subsets using the FACS gating strategy shown in Fig. 2A. An example of the purity achieved for each subset is shown in Supplemental Fig. 1B. RNA from each sample was subjected to two-cycle amplification and hybridized to mouse genome 430 2.0 Affymetrix microarray chips. (A) Heatmap representing normalized expression of genes that encode markers used to differentiate splenic B cell subsets. Mean expression of two samples from each subset shown. (B) Dot plots comparing fold change in expression of genes between T2-MZ versus T2-FO (top)orMZversusFO(bottom) subsets in NOD and B6 mice. Dots outside dashed lines represent genes that exhibited differences in fold changes of .2 between these strains. Mean changes of two independent samples of B cell subsets shown. A selection of genes that also exhibited $2-fold differences in expression between NOD versus B6 T2-MZ and/or MZ samples is labeled in red (upregulated in NOD) or blue (downregulated in NOD). Genes encoding protein markers commonly used to differentiate T2-MZ and MZ from other subsets that are equivalently expressed in NOD and B6 B cells are labeled green. (C) Heatmaps representing normalized expression of all genes that were shown to have fold change differences of $2 in NOD and B6 T2-MZ versus T2- FO or MZ versus FO comparisons or both, in addition to showing $2-fold differences in expression between NOD versus B6 T2-MZ and/or MZ samples. These were separated into those genes that were upregulated (left)ordownregulated(right) in T2-MZ or MZ compared with T2-FO or FO subsets, respectively. Probe names are shown for transcripts not associated with a known or predicted gene. Mean expression level of two independent samples is shown for each subset. group of genes also showed $2-fold difference in expression in highly expressed in both T2-MZ and MZ subsets of NOD com- direct comparisons of NOD versus B6 T2-MZ or MZ subsets pared with B6 mice comprised Asb2, Ccbp2, Cxcr7, Dusp16, (selection of these genes is shown with red [upregulated in NOD] Marcks, Pla2g7, S1pr3, and 1700106N22Rik, whereas those with or blue [downregulated in NOD] labels in Fig. 7B, and all are lower expression in NOD versus B6 included Myadm and Gm8369 listed on heatmaps on Fig. 7C). Genes identified as being more (Fig. 7B, 7C). Twenty additional genes were identified as being dif- 106 GENETIC CONTROL OF MZ B CELLS IN NOD MICE ferentially upregulated or downregulated in NOD versus B6 T2-MZ proportion of CD1dhigh T2-MZ or MZ B cell subsets trafficking comparisons only, whereas 51 genes were differentially expressed through this niche or simply due to the larger numbers of these in NOD versus B6 MZ comparisons only (Fig. 7C). Differential subsets present in NOD compared with B6 mice. To distinguish expression in a selection of these genes (including Asb2, Apoe, Ccbp2, between these possibilities, we took a snapshot of the proportions Cxcr7, Dusp16, Lgals1, Pla2g7, Sema7a, Tlr3,and1700106N22Rik) of T2-MZ and MZ subsets residing within the MZ and FO niches was confirmed using qRT-PCR in independent NOD and B6 B cell in spleens of NOD versus B6 mice by performing a short in vivo subset cDNA samples (Supplemental Fig. 2). Moreover, our pre- Ab pulse-labeling strategy similar to that described by Cinamon vious study had already confirmed differential expression of S1pr3 et al. (4). This involved injecting mice from both strains i.v. with in NOD versus B6 MZ and T2-MZ subsets by qRT-PCR (12). anti-CD19 FITC Abs and culling them 2 min later, resulting in the Assessment of the data using Ingenuity Systems Pathway Anal- selective labeling of B cells residing within the splenic MZ or red ysis led us to conclude that the over- and underexpressed genes in pulp in that period, but not those shielded from the circulation in NOD T2-MZ and MZ subsets contained a predominance of genes FO areas. B cells were subsequently washed and labeled with other reported to affect cellular movement of leukocytes (p =1.043 1026, Abs ex vivo to reveal their subset lineage by flow cytometry. As Fisher’s exact test), which included the 12 following genes: Apoe, was expected, the primary population of B cells labeled with Bid, Ccbp2, Cd55, Cxcr7, Lgals1, Rgs13, Pla2g7, Prf1, S1pr3, CD19 FITC in B6 mice was of the MZ subset (∼29%; Fig. 8B, Spn, and Tlr3 (Supplemental Table II). Eight of these genes were 8C), followed by those within the T2-MZ subset (∼21%), indi- differentially expressed at the T2-MZ stage, indicating that gene cating that about one-third to one-fifth of these cells, respectively, changes affecting migration occur early in MZ B cell develop- reside within the MZ or red pulp regions during the 2-min time- 2 ment. Genes reported to affect cell death (n =24,p =1.523 10 6), frame. The majority of FO B cells in B6 mice were not stained Downloaded from and particularly apoptosis (n = 22, p = 1.41 3 1026), were also with CD19 FITC, except for a small percentage (∼7%) that bound enriched in this dataset (including Apoe, Bid, Bmf, CCNB1Ip1, low levels of the Ab most likely encountered while in the circu- Cd55, Dusp10, E2F2, Eaf2, Lgals1, Myb, Pla2g7, Pmepa1, Prf1, lation or in extrafollicular foci. NOD mice contained a signifi- Psap, S1pr3, Satb1, Sdc1, Sema7a, Sp110, Spn, Tlr3, and Zbtb10). cantly increased proportion of CD19 FITC-labeled B cells of the We did not find differences in expression of the Notch2 gene, nor MZ and T2-MZ subsets (∼37 and 32%, respectively; Fig. 8B, 8C)

genes expressing Manic or Lunatic Fringe [which enhance Notch2 compared with the B6 strain. These findings confirmed that an http://www.jimmunol.org/ ligand binding (45)], between NOD and B6 B cell populations. increased proportion of MZ and T2-MZ B cell subsets in NOD mice traffics through MZ or red pulp niches at any one time. In- NOD B cells have enhanced capacity to migrate into MZ niche terestingly, NOD mice also contained a greater proportion of FO The enrichment of differentially expressed genes involved in the B cells that were dimly labeled with CD19 FITC (∼18%; Fig. 8B, movement of leukocytes by T2-MZ and MZ B cell subsets in NOD 8C), which may reflect the increase in numbers of activated auto- mice raised the possibility that these cells may display enhanced reactive B cells in the circulation or extrafollicular foci in this strain. migration into MZ or red pulp niches. At these sites, B cells gain access to various molecular factors that drive both the differenti- Discussion ation and survival of the MZ B cell subset (38). Proteins encoded The expansion of MZ B cells in NOD mice has been associated by guest on September 26, 2021 by S1pr3 and Cxcr7 (named sphingosine-1-phosphate receptor-3 with various autoimmune pathologies (12–16). However, the cause [S1PR3] and CXCR7, respectively) have been shown to play of their expansion in this strain was not well understood. Past studies specific roles in the migration and/or retention of B cells within using knockout or transgenic mouse strains bred on nonautoimmune- the MZ niche (4, 46, 47). Our previous study showed that NOD prone backgrounds have demonstrated that the decision of TR MZ B cells had an increased capacity for migration toward sphin- B cells to differentiate into MZ B cells is governed through the gosine-1-phosphate (S1P) compared with those from B6 mice integration of various signals delivered through the BCR, BAFF in chemotaxis assays, which was dependent on their elevated ex- receptors, and Notch 2, in addition to chemokine, S1P, and integrin pression of S1PR3 (12). CXCR7 acts as a scavenging receptor that receptors that attract cells to the MZ niche (38). We have under- binds and degrades CXCL12 and CXCL11, preventing them from taken a systematic analysis of these factors to gain insight into the interacting with CXCR4 and CXCR3,respectively(48,49).A potential causes of MZ B cell expansion in the NOD background. recent report by Wang et al. (47) demonstrated that CXCR7 plays Mice harboring genetic deletions in molecules that negatively a role in guiding B cells to the MZ by restricting CXCL12 from regulate BCR signaling (including Aiolos, CD22, and Src homol- binding to CXCR4 and that blocking CXCR7 with small molecule ogy 2 domain-containing protein tyrosine phosphatase 1) generally inhibitors decreased localization of MZ B cells within this niche. favor differentiation of FO B cells, whereas genetic ablation of Consistent with their higher gene expression of Cxcr7 (Fig. 7B, positive regulators of BCR signaling (including Bruton’s tyrosine 7C, Supplemental Fig. 2), NOD T2-MZ and MZ B cells were kinase, Syk, Vav, CD45, CD21, and phospholipase Cg2) generally found to exhibit a significantly decreased capacity to migrate to- favors MZ B cell differentiation (38, 39). Although there are some ward CXCL12 compared with B6 counterparts in chemotaxis caveats to this trend (50, 51), these data have led to the hypothesis assays (Fig. 8A). In contrast, lower expression of Cxcr7 by NOD that weaker BCR signaling favors differentiation of MZ B cells T2-FO and FO B cells increased their capacity to migrate toward and stronger signaling favors FO B cell differentiation (38). De- CXCL12 compared with those from B6. From these studies, we spite significantly increased differentiation of MZ B cells in NOD predict that increased expression of S1PR3 and CXCR7, causing mice, our experiments examining protein phosphorylation and altered chemotaxis toward S1P and CXCL12, respectively, could calcium flux induced by BCR stimulation (Fig. 3) revealed that be two important intrinsic factors that confer NOD T2-MZ and B cells from this strain did not display diminished BCR signal MZ B cells with an increased capacity for migration and/or re- strength compared with those of the B6 strain, which generate tention within the MZ and red pulp niches. significantly lower levels of this subset. Past immunohistological studies of spleens in NOD mice have Preferential expansion of MZ B cells has been observed in BCR revealed abnormally large accumulations of CD1dhigh B cells transgenic mouse strains that primarily produce B cells specific for within MZ areas (12, 21). Nevertheless, it remained unclear from certain autoantigens such as insulin (14), Smith Ag (52), or dsDNA these studies whether the accumulation was due to an increased (53). These studies indicated that B cells with particular autore- The Journal of Immunology 107 Downloaded from http://www.jimmunol.org/

FIGURE 8. Enlarged MZ B cell numbers in NOD mice may be due to enhanced B cell migration into MZ niches. (A) Nonadherent splenocytes from six individual B6 or NOD mice were added in duplicate to the top chambers of Transwell chemotaxis plates. Cells were allowed to migrate toward CXCL12, placed in the bottom chamber at the indicated concentrations, for 4 h. Following chemotaxis, cells that migrated into the bottom chamber were stained for B220, CD21, CD23, CD24, and IgM to enumerate the distinct B cell subsets by flow cytometry, as shown in Fig. 2A. Graphs represent mean percentage of each B cell subset that migrated compared with total cells within the starting population. Migration for T2-MZ and MZ B cell subsets is shown in a different graph to that of T2-FO and FO B cell subsets to better illustrate the differences between NOD and B6 groups. *p , 0.05, **p , 0.01, ****p , 0.0001 comparison of NOD versus B6 subset at the same concentration (t test). (B and C) B6 and NOD mice were injected i.v. with 1 mg anti-CD19 FITC Ab and

sacrificed 2 min later for spleens. Splenocytes were stained with Abs to B220, IgM, CD21, anti-CD23, and CD24 conjugated to other fluorochromes to by guest on September 26, 2021 differentiate B cell subsets exposed to anti-CD19 FITC in vivo using flow cytometry. (B) Representative histograms showing proportion of CD19 FITC+ cells in FO, T2-MZ, and MZ B cell subsets in three injected B6 and NOD mice. (C) Graph showing average proportion of CD19 FITC+ cells (+SEM) in each B cell subset of three B6 and NOD mice. One of three experiments with similar results shown. **p , 0.01, *p , 0.05 (two-way ANOVA, followed by Bonferroni posttest). active specificities could be positively selected into the MZ subset. enlarged MZ B cell compartments, including MRL-lpr, NZB, and NOD mice possess defects in various mechanisms of B cell self- (NZB 3 NZW)F1 (6, 7), display high circulating levels of BAFF tolerance, leading to increased development, survival, and activa- (55, 56). In contrast to these strains, the expanded MZ B cell tion of autoreactive clones (13, 22, 28, 29). Indeed, in this study, our compartment in NOD mice was not associated with elevated se- detailed flow cytometric analysis of TR precursors in NOD mice rum levels of BAFF (Fig. 4A). However, we and others have de- provided further evidence of tolerance defects at the T1 to T2-FO scribed higher surface levels of the receptors BAFF-R and TACI in transition as well as highlighting their diminished capacity to recruit NOD B cell subsets compared with the B6 strain, which increased cells into the anergic T3/An1 population (Fig. 2A, 2B). However, their capacity to bind BAFF (Fig. 4B, 4C) (57). Although this the increased propensity of NOD mice to produce autoreactive B characteristic has the potential to increase BAFF signaling, we do cells is unlikely to account for MZ B cell expansion, because BCR not believe this is the sole cause for MZ B cell expansion in NOD transgenic B cells rendered specific for the disease-irrelevant Ag mice for two reasons. First, in contrast to the B6 background, HEL on this background still showed enhanced MZ B cell differ- increasing or decreasing systemic BAFF levels in NOD mice by entiation (Fig. 2C, 2D). In addition, the fact that MZ B cell expansion introduction of a BAFF transgene or a single gene knockout, re- wasalsoobservedinNOD.H2b congenic mice (Fig. 2E, 2F), which spectively, produced minimal effects on MZ B cell expansion (Fig. develop neither insulitis nor clinical T1D (18), indicates that this 5A–C). Second, deleting both copies of the BAFF gene in NOD phenotype is not a secondary consequence of T cell autoimmunity. mice, which decreased, but did not completely abrogate B cell BAFF promotes B cell survival by activating the noncanonical maturation, resulted in a similar output ratio of FO:MZ B cells NF-kB pathway through the BAFF-R (40). More recently, this to that of WT NOD mice (Fig. 5D–G). This was in marked con- interaction has also been shown to facilitate MZ B cell differen- trast to B6.BAFF2/2 mice, in which the great majority of B cells tiation through activation of the canonical and noncanonical NF- were prevented from maturation and the few that did were of the kB pathways (42). The latter function is particularly evident in B6 FO phenotype. Thus, enhanced MZ B cell differentiation of NOD mice that transgenically overexpress BAFF, which, in addition to mice can occur independently of BAFF. containing greater overall numbers of B cells, contained a dispro- A study by Quinn et al. (13) suggested that expansion of MZ portionately expanded MZ B cell compartment that contributes B cells in NOD mice may be caused by impaired egress of TR to autoimmune phenotypes (8, 10, 54). Similarly, various mouse B cells from the BM in this strain. Hence, diminished numbers of strains that are predisposed to systemic autoimmunity and contain NOD TR B cells in the spleen would face less competition for 108 GENETIC CONTROL OF MZ B CELLS IN NOD MICE factors promoting MZ B cell differentiation such as BAFF. How- cells whose encoded protein (regulator of G-protein signaling 13) ever, upon analyzing splenic B cell differentiation in mixed NOD has the capacity to regulate Gi signaling through CXCR4 and and B6 BM chimeras, we showed that, despite having to compete CXCR5 (60). It will be of interest to elucidate how differential for external factors in the same environment as B6-derived TR expression of these gene networks affecting leukocyte movement B cells, NOD-derived TR B cells still possessed a greater capacity combines to increase trafficking of NOD B cells through the to differentiate into the MZ subset (Fig. 6A, 6B). This result im- marginal sinus and potentially cause expansion of the MZ subset. plied that abnormal MZ B cell expansion in NOD mice is not due to In summary, our data indicate that MZ B cell expansion in the increased exposure of TR B cells to external factors, but due to NOD mouse strain derives from B cell–intrinsic factors. In this molecules that are intrinsically expressed by NOD B cells. strain, MZ B cells and their precursors (T2-MZ) exhibit an altered Previous reports (12, 13, 21, 22), as well as the current study migration set point allowing increased movement and retention (Figs. 1, 2), have shown that enhanced differentiation of the MZ within the marginal sinus, a niche favoring MZ B cell differenti- B cells in NOD mice is preceded by a significant increase in the T2- ation. This is distinct from other autoimmune-prone mouse models, MZ precursor subset. It was therefore essential to conduct micro- including NZB, (NZB 3 NZW)F1,MRL-lpr,andBAFF-Tgmouse array analyses on B cells at TR as well as mature stages to identify strains, in which MZ B cell expansion is governed by extrinsic intrinsic molecular factors that contribute to enhanced MZ B cell factors such as BAFF overproduction (6–9). In humans, aberrant development in NOD mice. A comparison of gene expression in T2- pathogenic activity by MZ B cells in Sjo¨gren’s syndrome and MZ versus T2-FO as well as MZ versus FO subsets in NOD and B6 Grave’s disease has also been associated with elevated levels of miceledtotheidentificationof20genesdifferentiallyexpressedat systemic BAFF (10, 11, 60, 61). However, given that not all pa- the T2-MZ stage, 51 genes differentially expressed at the mature tients with these diseases exhibit elevated levels of BAFF, it is Downloaded from MZ stage, and 10 genes that were differentially expressed at both possible that irregularities in pathways regulating movement of stages (Fig. 7C). Ingenuity pathway analysis of differentially ex- B cells could constitute another mechanism that leads to expansion pressed genes NOD versus B6 MZ and T2-MZ B cells revealed an and pathogenic activity of the MZ subset. In humans developing enrichment in molecules involved in cell migration of leukocytes, T1D, the specific contribution of the MZ subset to B cell patho- including Apoe, Ccbp2, Cxcr7, Lgals1, Pla2g7, Rgs13, S1pr3, Spn, genesis has not been studied. Nevertheless, it is fascinating that

Bid, Cd55, Prf1,andTlr3. The first 8 of these 12 genes were dif- CXCR7, identified by our study as one of the major dysregulated http://www.jimmunol.org/ ferentially expressed in T2-MZ precursors, indicating that migration/ genes associated with aberrant MZ B cell movement and generation chemotaxis may be altered at this early stage of MZ development. in NOD mice, contains single-nucleotide polymorphisms that are Consistent with altered migration, previous immunohistological associated with increased T1D susceptibility in humans (62). Fur- studies have shown that NOD mice harbor greater numbers of ther characterization of the molecular pathways underlying aberrant CD1high MZ B cells situated adjacent to the marginal sinus com- production of MZ B cells in NOD mice has the potential to elu- pared with most other mouse strains (12, 21). Using an in vivo Ab cidate new therapeutic avenues for specifically targeting this subset pulse-labeling strategy, we showed that an increased proportion of in humans in the context of autoimmune disease and cancer. T2-MZ and MZ B cells migrated through the MZ or red pulp by guest on September 26, 2021 niches within a 2-min timeframe in NOD compared with B6 mice Acknowledgments (Fig. 8A, 8B). This is important because it is in these niches that We acknowledge Christopher Brownlee for technical support with flow cytom- B cells gain access to limiting factors necessary for the promotion etry and the staff of the Garvan Institute Biological Testing Facility and Aus- of MZ differentiation, such as the Notch2 ligand deltex-like-1 tralian Bio-Resources for help with animal husbandry. We also thank Marcus expressed by endothelial cells (45). The increased capacity of Hayward for technical assistance with immunoblots, Prof. Alan Baxter for sta- NOD B cells to migrate into niches that favor MZ development tistical advice, Dr. Warren Kaplan for guidance with analyzing microarray may also explain why, in NOD and B6 mixed BM chimeras, the experiments, and Prof. Antony Basten for critical assessment of the paper. presence of NOD MZ B cells suppressed development of B6 MZ B cells (Fig. 6A, 6B). Two particular molecules that have been Disclosures shown to contribute to the migration and retention of B cells The authors have no financial conflicts of interest. within the MZ niche are S1PR3 and CXCR7 (4, 46, 47), which were both highly expressed in NOD T2-MZ and MZ subsets com- pared with B6 counterparts in our microarray and qRT-PCR studies References (Fig. 7B, 7C, Supplemental Fig. 2) (12). In a previous study, we 1. Monroe, J. G., and K. Dorshkind. 2007. 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