The Adaptor Sh2d3c Is Critical for Marginal Zone B Cell Development and Function

This information is current as Amin Al-Shami, Carrie Wilkins, Jeannette Crisostomo, of September 29, 2021. Dhaya Seshasayee, Flavius Martin, Nianhua Xu, Adisak Suwanichkul, Stephen J. Anderson and Tamas Oravecz J Immunol 2010; 185:327-334; Prepublished online 26 May 2010;

doi: 10.4049/jimmunol.1000096 Downloaded from http://www.jimmunol.org/content/185/1/327

Supplementary http://www.jimmunol.org/content/suppl/2010/05/26/jimmunol.100009 Material 6.DC1 http://www.jimmunol.org/ References This article cites 42 articles, 16 of which you can access for free at: http://www.jimmunol.org/content/185/1/327.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 © 2010 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

The Adaptor Protein Sh2d3c Is Critical for Marginal Zone B Cell Development and Function

Amin Al-Shami,* Carrie Wilkins,* Jeannette Crisostomo,* Dhaya Seshasayee,† Flavius Martin,† Nianhua Xu,* Adisak Suwanichkul,* Stephen J. Anderson,* and Tamas Oravecz*

Sh2d3c is an adaptor protein that has been implicated in T cell activation and shown to associate with different components of the integrin signaling pathway ex vivo. However, the in vivo significance of Sh2d3c expression in the regulation of the immune re- sponse and/or hematopoietic cell lineage development is not known. In this study, we show that expression of Sh2d3c is more critical for development and function of marginal zone B (MZB) cells than for T cell maturation. Mice deficient in Sh2d3c ex- pression (Sh2d3c2/2) had a reduced number of MZB cells, and the residual MZB cells failed to properly capture polysaccharide Ags. Activation-induced proliferation, cytokine production, and migration of Sh2d3c2/2 splenic B cells were also significantly Downloaded from reduced in vitro compared with wild-type (Sh2d3c+/+) cells. In contrast, T cell development and function were largely normal in Sh2d3c2/2 mice. The thymi of Sh2d3c2/2 mice showed no maturational abnormalities, the number of splenic T cells was only modestly reduced, and the T cells responded normally to in vitro polyclonal activation. The observed B cell deficiency in the Sh2d3c2/2 mice led to diminished humoral immune response against thymus-independent type 2, but not thymus-dependent Ags, which highlights the primary in vivo role of Sh2d3c in regulating B cell development and function. The Journal of Immunology, 2010, 185: 327–334. http://www.jimmunol.org/

h2d3c, also known as Chat, Shep1, and novel Src homology NSP , including Sh2d3c, participate in similar signaling 2-containing protein (NSP) 3 (1, 2), is a member of the events elicited by various stimuli in different cell types (2, 10, 11). S NSP family of adaptor proteins along with NSP1 (3) and In particular, Sh2d3c has been implicated in cell migration, adhe- NSP2 (4). These proteins are structurally related by having an N- sion, and integrin signaling pathways (5–7). terminal Src homology 2 domain, a central serine/proline-rich re- T lymphocyte precursors originate in the bone marrow (BM) and gion containing ERK phosphorylation sites, and a C-terminal re- migrate to the thymus, where they develop into mature CD4+ and +

gion bearing homology with the guanine-like exchange factor CD8 cells before moving to the periphery (12). In rodents, B cells by guest on September 29, 2021 domain of Cdc25 (1). Two distinct isoforms of Sh2d3c generated also originate in the BM and arrive in the spleen as immature by alternative splicing were reported to date, a 78-kDa isoform transitional or newly formed B cells before differentiating into ei- expressed in various tissues and a 115-kDa isoform detected ex- ther follicular B (FOB) cells or marginal zone B (MZB) cells (13). clusively in hematopoietic cells (2). FOB cells are located in the white pulp area of the spleen, whereas Functional studies of Sh2d3c to date focused on its role in signal MZB cells are noncirculating cells that are positioned around the transduction pathways (2, 5). Sh2d3c was shown to interact with marginal sinus through which most of the blood passes before the Pyk2 (6) and enhance its phosphorylation in continuing to the red pulp area (14). In this way, MZB cells in the CD3- and CD28-activated Jurkat T cells. Signaling via CD3 also red pulp can be exposed to blood-borne pathogens (15). Upon activates Sh2d3c in Jurkat cells, which, in turn, regulates the JNK encountering bacterial capsular polysaccharide Ags (16, 17), pathway, leading to IL-2 production (6). In addition, the C- MZB cells can differentiate rapidly to Ab-producing plasma cells terminal domain of Sh2d3c constitutively associates with Crk- independent of T cell help (15, 18) and start secreting IgM (19, 20). associated substrate (Cas)-L (5–7), which is a member of the We were interested to examine whether the in vivo immune Cas family of scaffold proteins along with p130-Cas and Efs/ phenotype of mice deficient in Sh2d3c expression was consistent Sin (8, 9). In fact, all NSPs interact with one or more Cas proteins with the role of this protein in T cell function, as predicted by in a cell type-specific manner. This commonality suggests that previous in vitro studies. Surprisingly, Sh2d3c2/2 mice did not exhibit significant alterations in T cell content or function. How- ever, Sh2d3c2/2 mice displayed a significant impairment in MZB *Lexicon Pharmaceuticals, The Woodlands, TX 77381; and †Department of Immu- nology, Genentech, South San Francisco, CA 94080 cell formation and thymus-independent type 2 (TI-2) immune 2/2 Received for publication January 14, 2010. Accepted for publication April 27, 2010. responses. Furthermore, Sh2d3c MZB cells exhibited weaker ability to retain polysaccharide Ags and to respond to chemotactic Address correspondence and reprint requests to Dr. Amin Al-Shami, Lexicon Phar- maceuticals, 8800 Technology Forest Place, The Woodlands, TX 77381. E-mail signals in vitro. Our data show that Sh2d3c plays a previously un- address: [email protected] known role in generating and regulating the function of MZB cells. The online version of this article contains supplemental material. Abbreviations used in this paper: BM, bone marrow; Cas, Crk-associated substrate; ES cell, embryonic stem cell; FOB, follicular B; KO, knockout; MFI, mean fluores- Materials and Methods 2/2 cence intensity; MZB, marginal zone B; NSP, novel Src homology 2-containing Generation of Sh2d3c mice protein; SDF, stromal cell-derived factor; TI-2, thymus-independent type 2; TNP, 2/2 trinitrophenyl; WT, wild type. The Sh2d3c line was generated by homologous recombination as part of Lexicon and Genentech’s collaboration to knock out and analyze the Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 function of 500 secreted and transmembrane proteins. The targeting vector www.jimmunol.org/cgi/doi/10.4049/jimmunol.1000096 328 A CRITICAL ROLE FOR Sh2d3c IN MZB CELL DEVELOPMENT Downloaded from http://www.jimmunol.org/

FIGURE 1. Schematic drawing of site-directed mutagenesis, location of primers for genotyping strategy, and expression analysis. A, A schematic representation of the Sh2d3c locus is shown, including relevant restriction sites used for confirmation of Southern hybridization analysis (SphI). The 59 internal and 39 external probes used for Southern hybridization analysis were designed inside and outside the target vector arms of homology, respectively, and were generated by genomic PCR. B, Southern hybridization demonstrating proper targeting of the ES cell clones. Four correctly targeted clones were identified using the 59 internal and 39 external probes. Clones 2A1, 2A2, and 2F9 were injected into blastocysts, and clone 2A1 ultimately achieved germline transmission. Lex-2 represents control ES cell DNA. C, RT-PCR performed on RNA extracted from thymus, spleen, and lungs from Sh2d3c+/+ and Sh2d3c2/2 mice using specific primers. Primers for actin were used as a control. D, Immunoblot analysis of lysates from spleens extracted from Sh2d3c+/+ and Sh2d3c2/2 mice using anti-Sh2d3c Abs, followed by anti-GAPDH Abs. by guest on September 29, 2021 was derived using the l KOS system (21). The l KOS phage library was mice of mixed genetic background (129/SvEvBrd and C57BL/6J) repre- screened by a PCR primer pair overlapping exon 4 of the murine Sh2d3c senting both sexes of littermate Sh2d3c2/2 and Sh2d3c+/+ animals. Pro- (GenBank accession number NM-013781 [www.ncbi.nlm.nih.gov/ cedures involving animals were conducted in conformity with Lexicon’s Genbank/]; Sh2d3c-1 [59-CTT ACT TTG CCT TGT AGG AAC CC] and Institutional Animal Care and Use Committee guidelines that are in com- Sh2d3c-2 [59-GGC ACG TGA GCA CAT AGT CCC]). One genomic pliance with the state and federal laws and the standards outlined in the clone, pKOS-82, was isolated from the library and confirmed by sequence Guide for the Care and Use of Laboratory Animals. analysis. To generate the target vector, first gene-specific arms (59-GCC AAT CTC GCT CTC CCC GTG CAG TTC TCC AAG GA-39 and 59-CCC Expression analysis of Sh2d3c by RT-PCR ACC AAC GAC AAC AGC ATT ACA GAC TAC CCC CT-39) were appended by PCR to a yeast selection cassette containing the URA3 To confirm disruption of the Sh2d3c gene in the knockout (KO) mice, total marker. The yeast selection cassette and pKOS-82 were cotransformed RNA were collected from lung, spleen, and thymus from three WT and into yeast, and clones that had undergone homologous recombination to three homozygous mice using a bead homogenizer (BioSpec Products, replace the 2395-bp genomic region containing exons 4–6 with the yeast Bartlesville, OK) and TRIzol reagent (Invitrogen, Carlsbad, CA), ac- selectable marker were isolated. After replacing the yeast cassette with an cording to the manufacturer’s instructions. Reverse transcription was per- embryonic stem (ES) cell selection cassette containing lacZ, the targeting formed to produce cDNA using 5 mg total RNA with SuperScript II (Invi- vector was linearized by NotI digest and electroporated into 129/SvEvBrd trogen) and random hexamer primers, according to the manufacturer’s (Lex-1) ES cells. G418/FIAU-resistant ES cell clones were isolated, and instructions. PCR amplification was performed using 1 ml of the RT prod- correctly targeted clones were identified and confirmed by Southern blot uct at initial denaturing step of 95˚C for 2.5 min, followed by 35 cycles of analysis. A 330-bp 39 external probe was generated outside the pKOS-82 95˚C (30 s), 59˚C, (30 s), and 70˚C (1 min) with oligonucleotide primers arm of homology using PCR primers Sh2d3c-27 (59-CCC CTA GTG GAC complementary to exons 4 and 5 of Sh2d3c (59-AGG AAC TGA AGC AAC AAG C) and Sh2d3c-28 (59-CCC ATA GGT GTG TGC C AT GTT TCA GCA GCA CGG-39 and 59-TAG GCT GGT GAG TGA GTC TCG G- AAA). A 357-bp 59 internal probe was generated using PCR primers 39). Primers to the mouse b actin gene (GenBank accession number Sh2d3c-22 (59-AGA TTT CTG GGC ATT GAC TCT AGT) and Sh2d3c-23 M12481 [www.ncbi.nlm.nih.gov/Genbank/], 59-GGC TGG CCG GGA (59-TAG GCC AGG GCA TTA AGT CT). Southern blot analysis using the CCT GAC GGA CTA CCT CAT-39 and 59-GCC TAG AAG CAC TTG 27 + 28 39 probe detected a 14-kb wild-type (WT) band and a 5.3-kb CGG TGC ACG ATG GAG-39) were also used as internal control for targeted band in SphI-digested genomic DNA, whereas the 22 + 23 59 sample handling. probe detected a 14-kb WT band and an 11.7-kb targeted band in SphI- digested genomic DNA. Three targeted ES cell clones were microinjected Immunizations, Ig, and trinitrophenyl measurements into C57BL/6 (albino) blastocysts. Resulting chimeras were mated to C57BL/6 (albino) females to generate mice that were heterozygous Basal, anti-trinitrophenyl (TNP), and anti-OVA Ig concentrations in serum (Sh2d3c+/2) for the exon 4-6 deletion mutation. Sh2d3c+/2 mice were were measured by ELISA using Abs from Southern Biotechnology Asso- intercrossed, yielding Sh2d3c2/2 mice. ciates (Birmingham, AL). Mice were immunized by i.p. injection of 100 mg The animals were maintained at the American Association for the TNP-Ficoll, TNP-LPS, or OVA in CFA, and Ag-specific Abs were cap- Accreditation of Laboratory Animal Care-accredited animal facility at Lex- tured on plates coated with TNP or OVA (BD Biosciences, San Jose, CA). icon Pharmaceuticals. All experiments were carried out on 6- to 16-wk-old TNP retention on MZB cells was assessed, as described previously (22), The Journal of Immunology 329

FIGURE 2. KO of Sh2d3c disrupts MZB cell development in the spleen. A, Representative FACS plots show the CD23/CD21 on B220- positive splenocytes and CD21/aIgM mAb staining patterns of the indicated genotype. Fraction of cells in different maturation stages is defined by the elliptical gates and indicated close to the respective gates. B, Cell counts in the bar graph were obtained from 35 mice of each genotype pooled from nine independent experiments, giving similar results. Data are Downloaded from presented as mean 6 SEM; numbers above bars indicate p values. C, Representative pictures of frozen spleen sections from indicated mice stained with combination of Ab to IgM (green) and IgD (red), Moma-1 (green) and B220 (red), or MARCO (green) and B220 (red), shown http://www.jimmunol.org/ at 310 magnification. Similar results were ob- tained in three additional mice of each geno- type. Arrow points to IgMhighIgDlow cell staining readily apparent in the marginal zone area of WT spleen. by guest on September 29, 2021

with the exception of using PE-labeled streptavidin (BD Biosciences) for (PeproTech, Rocky Hill, NJ) in the bottom chamber. Cell proliferation detection. was assessed, as described before (24), using mAbs to CD3 and CD28 at equal concentrations (as indicated), LPS (1 mg/ml), aIgM mAb (5 mg/ml), Immunofluorescent histochemistry or a mixture of CD40 mAb and IL-4 (2.5 mg/ml and 5 ng/ml, respectively). B cell subsets were purified with an enrichment kit (StemCell Technologies, Spleens were rapidly frozen in Tissue-Tek OCT compound (Sakura Fine- Vancouver, BC, Canada). The concentration of cytokines in cell culture technical, Torrance, CA) and sectioned. Sections were stained with Abs to supernatants was measured 48 h after activation using CBA mouse inflam- Moma-1 (biotinylated; Abcam, Cambridge, MA), MARCO (unconjugated; mation kits (BD Biosciences). AbD Serotec, Raleigh, NC), B220 (PE), IgM (FITC), or IgD (PE), and with secondary anti-rat Abs or streptavidin (FITC; all from BD Biosciences), as Ca2+ mobilization assay required. Slides were examined using Axiovert inverted fluorescent micro- scope (Zeiss, Thornwood, NY). Measurements were made by using a Fluo-4 NW Ca2+ assay kit (Invitro- gen). B cell-enriched splenocytes (1 3 107) were activated with 10 mg/ml BM transplantation aIgM mAb, and maximal fluorescence was assessed by addition of 100 ng/ml ionomycin. Basal fluorescence measured at the first time point was Recipient mice were irradiated, as described before (23), with the following subtracted from each subsequent measurement. exception: 2 3 106 donor BM cells were injected 8 h after the second dose of irradiation. The average percentage of Neo+ cells derived from donor Western blot analysis Sh2d3c2/2 BM was 93% in the reconstituted animals. Enriched B cells from spleen (4 3 107 cells/ml) were stimulated with FACS analysis 10 mg/ml LPS, and Western blotting of lysed samples was performed using polyclonal Abs to Pyk2 and phosphorylated Pyk2 (Cell Signaling Tech- These procedures were performed, as described previously (23); all Abs nology, Beverly, MA), HRP-conjugated goat anti-rabbit Ab (Bethyl Lab- were from BD Biosciences. Indicated B cell subsets were sorted to 99% oratories, Montgomery, TX), and ECL detection system (GE Life purity by using a FACSAria (BD Immunocytometry Systems, San Jose, Sciences, Pittsburgh, PA). Spleen lysates from Sh2d3c+/+ and Sh2d3c2/2 CA). mice were tested by anti-Sh2d3c (Santa Cruz Biotechnology, Santa Cruz, CA) and anti-GAPDH (Bethyl Laboratories) Abs. Cell migration, proliferation, and cytokine production assays Statistical analyses Migration of splenocyte subsets was assessed after a 4-h incubation in 5-mm pore-size 96-well plates (NeuroProbe, Gaithersburg, MD), with 1 ng/ml Statistical significance of group differences was evaluated by unpaired, two- murine stromal cell-derived factor (SDF)-1a or 100 ng/ml CXCL13 tailed, Student t test. A p value of ,0.05 was considered significant. 330 A CRITICAL ROLE FOR Sh2d3c IN MZB CELL DEVELOPMENT Downloaded from http://www.jimmunol.org/

FIGURE 3. Sh2d3c regulates the TI-2 response. A, The basal concentration of the different Ig isotypes in the serum of the indicated mice was determined by ELISA. Data are presented as mean 6 SEM. B, Anti-TNP Ab titers were determined by ELISA at the indicated time points after immunization of WT and KO mice with TNP-Ficoll. Serum dilutions were 1/1000 for IgG1 and 1/100 for the rest of the Ig subclasses. Data were obtained from 14–20 mice of by guest on September 29, 2021 each genotype pooled from at least three independent experiments giving similar results. Data are presented as mean 6 SEM; pp , 0.05; ppp , 0.005, Sh2d3c2/2 versus WT. C, Retention of TNP by MZB cells is deficient in Sh2d3c2/2 mice. Representative FACS histograms of cell surface anti-TNP and isotype-matching control mAb reactivities are shown as detected 30 min after TNP-Ficoll injection. D, Frozen sections from spleens of indicated mice injected with TNP-Ficoll and analyzed 30 min later. Red fluorescence identifies TNP accumulation in the marginal zone area (310 magnification). MFI, mean fluorescence intensity.

Results B220+CD21highCD23med/low MZB cells in the spleen of Sh2d3c2/2 Sh2d3c2/2 mice do not show gross abnormalities mice compared with WT littermates (Fig. 2). A decrease in the high high 2 2 proportion of the IgM CD21 T2/MZB cell population, We generated Sh2d3c / mice by targeted disruption of exons 4–6 which includes MZ precursor cells in addition to MZB cells in the of the murine homolog of the Sh2d3c gene (GenBank accession 2 2 spleen, was also readily apparent in the Sh2d3c / mice. Al- number NM_013781 [www.ncbi.nlm.nih.gov/Genbank/]) by ho- though the absolute number of FOB cells also appeared to be mologous recombination in ES cells (21), followed by germline 2 reduced, the difference did not reach statistical significance. Fur- transmission of the mutant gene into chimeric, Sh2d3c+/ ,and 2 2 thermore, no differences were observed in the immature T1 or Sh2d3c / mice (Fig. 1A). Successful targeting of the Sh2d3c gene mature B cell populations (Fig. 2B). Immunohistological exami- in the KO mice was confirmed by Southern hybridization analysis of nation of the spleen showed that the region of IgMhighIgDlow MZB ES cell (Fig. 1B), expression analysis of the gene transcript (Fig. 1C), cells that form a rim around the follicles was greatly diminished in and Western blot analysis of the Sh2d3c protein (Fig. 1D). Mating of 2 the absence of Sh2d3c (Fig. 2C), which correlates with the flow Sh2d3c+/ mice generated pups of the three possible genotypes with 2 2 cytometric analysis data. In contrast, the resident metallophilic ratios that fit well with normal Mendelian frequencies. Sh2d3c / macrophages that are important components of the marginal zone mice exhibited no substantial difference in growth rate and size. (26), identified by the marker Moma-1, were correctly located at Likewise, comprehensive clinical diagnostic and pathologic analysis the border between the marginal and follicular zones in both WT (described in detail in Ref. 25), including tests of inflammation, and mutant mice (Fig. 2C). Similarly, the distinct MARCO+ mar- behavior, obesity, diabetes, bone, cell proliferation, and cardiovas- ginal zone macrophages that are required for retaining MZB cells cular function, did not demonstrate significant differences in 2 2 (27) were also detected at comparable levels in WT and Sh2d3c- Sh2d3c / mice relative to their WT littermates (data not shown). deficient spleens (Fig. 2C). Of note, the blood, BM, and lymph Absence of Sh2d3c leads to impaired MZB cell development nodes of Sh2d3c2/2 mice did not demonstrate obvious B cell Hematologic and FACS analysis of lymphoid organs revealed deficiencies, and the peritoneal cavity also contained normal num- decreased cellularity and markedly reduced representation of ber of cells of the B1 B cell phenotype (data not shown). These The Journal of Immunology 331 Downloaded from

FIGURE 5. Sh2d3c is essential for chemokine-induced B cell migration. Splenocytes migrating to the indicated stimuli were collected, counted, and http://www.jimmunol.org/ analyzed by FACS. Total splenocyte numbers (upper left panel) were normalized to the number of migrating cells in the WT vehicle control FIGURE 4. Sh2d3c is indispensable for LPS-induced MZB cell activa- samples in each experiment. The rest of the bar graphs show the fraction of tion. A, Enriched B cells from spleen were cultured in the presence of indicated cell populations migrating to the different stimuli. Data were the indicated stimulators for 48 h, and cell proliferation was assessed by obtained from 9–11 mice of each genotype pooled from three independent [3H]TdR uptake for the last 10 h. Data were obtained from 20 mice of each experiments giving similar results, using three replicates of each sample. 6 genotype pooled from four independent experiments giving similar results. Data are presented as mean SEM; numbers above bars indicate p values. B, Enriched spleen B cells were stimulated with LPS, as above, and con- centration of the indicated cytokines was measured in the cell culture by guest on September 29, 2021 mice compared with their Sh2d3c+/+ littermates. The Ag-specific supernatants at the end of the assay (n = 11 mice). C, Sorted FOB (B220+CD21medCD23high) and MZB (B220+CD21highCD23med/low) cells IgG2a response was also reduced in the KO mice, but the decrease were cultured with LPS, and cell proliferation was assessed, as above did not achieve statistical significance. Importantly, the humoral re- (n = 6 mice). Background signals (FOB and MZB cells without LPS) were sponse to OVA, a thymus-dependent Ag, and to TNP-LPS, a thymus- between 30 and 100 cpm. Data are presented as mean 6 SEM; numbers independent type 1 Ag, showed no significant differences at either above bars indicate p values.

findings indicate that Sh2d3c is critical for MZB cell develop- ment, but less so for the homeostasis of other B cell subsets. Transplantation of BM cells from Sh2d3c2/2 mice to irradiated WT recipients recapitulated the MZB cell phenotype of the complete KOs, with the additional observation that the KO chi- meras showed a modest reduction in the fraction of B220+ cells in the BM (Supplemental Fig. 1). These findings demonstrate that the defect in MZB cell formation in Sh2d3c-deficient mice is intrinsic to hematopoietic cells.

Sh2d3c2/2 mice exhibit deficient humoral immune response to TI-2 Ags MZB cells have been associated with the TI-2 Ab response (18); therefore, we immunized Sh2d3c2/2 and Sh2d3c+/+ mice with TI-2 Ag TNP-Ficoll and measured the levels of TNP-specific Abs in the serum 7, 14, and 28 d after immunization. The basal serum concentrations of Ig subclasses were similar in unchal- 2/2 +/+ lenged Sh2d3c and Sh2d3c mice (Fig. 3A). On days 7 and 2 2 FIGURE 6. Pyk2 activation is diminished in Sh2d3c / splenic B cells. 14 after immunization with TNP-Ficoll, there was a clear trend of 2/2 A and B, Enriched B cells from the spleens of indicated mice were acti- reduced TNP-specific Ig in Sh2d3c mice that was statistically vated with LPS. Lysates of these cells were analyzed for Pyk2 and significant for IgG1, IgG2b, and IgG3 on day 7 (Fig. 3B). By day phospho-Pyk2 expression at the indicated time points by Western blotting. 28 after immunization, the levels of TNP-specific IgM, IgG1, A, Western blot of a representative experiment. B, Densitometry graph of 2 2 IgG2b, and IgG3 Abs all were significantly lower in Sh2d3c / data pooled from four independent experiments giving similar results. 332 A CRITICAL ROLE FOR Sh2d3c IN MZB CELL DEVELOPMENT

the LPS activation study with purified MZB and FOB cells. Consistent with previous reports (28), WT MZB cells proliferated more to LPS than WT FOB cells. However, Sh2d3c-deficient MZB cells proliferated poorly after LPS activation compared with WT MZB cells. The cell proliferation defect was restricted to MZB cells because purified FOB cells from Sh2d3c+/+ and Sh2d3c2/2 mice proliferated similarly when activated with LPS (Fig. 4C). Of note, the deficient LPS response of Sh2d3c2/2 MZB cells was not due to differences in TLR4 expression, as confirmed by FACS analysis (data not shown). Sh2d3c2/2 B cells are refractory to CXCL13-induced chemotactic response in vitro Proper migratory response of B cell precursors is a prerequisite for normal MZB cell development (14, 18). Furthermore, Sh2d3c has been implicated in cell migration in COS cells (5). Therefore, we 2/2 2/2 examined the ability of Sh2d3c splenocytes to migrate in re- FIGURE 7. T cell development appears to be normal in Sh2d3c a a mice. Indicated number and distribution of cell subsets were determined by sponse to the chemokines SDF-1 and CXCL13 in vitro. SDF-1 FACS analyses from at least four experiments (four to five mice per group is produced in the red pulp of the spleen and is involved in Downloaded from each). Cell proliferation of splenocytes to the indicated concentrations of migration of B cells (29, 30), whereas CXCL13 is required for CD3 and CD28 mAbs was assessed by measuring [3H]TdR uptake (n =14 the localization of B cells in the spleen (31, 32). The motility of mice from three experiments). Data are presented as mean 6 SEM. The Sh2d3c-deficient splenocytes to both SDF-1a and CXCL13 was representative FACS plots show normal CD4/CD8 mAb-staining patterns significantly reduced when compared with WT cells (Fig. 5). of thymocytes from mice of both Sh2d3c genotypes. The fractions of FACS analysis revealed that the deficiency in the migratory thymocytes of different maturational stages are listed in the appropriate response was not limited to MZB cells. Both FOB and MZB http://www.jimmunol.org/ quadrant of the two-color histograms. cells were largely refractory to the CXCL13-induced migratory signal in the absence of Sh2d3c. In contrast, only Sh2d3c2/2 FOB time points (Supplemental Fig. 2), which demonstrates the TI-2- cells appeared to show deficient migratory response to SDF-1a, 2 2 specific nature of the immunodeficiency in the Sh2d3c / mice. although the response of MZB cells was generally poor to this The Ag-retention capacity of MZB cells is decreased in the chemokine irrespective of Sh2d3c expression (Fig. 5). The sur- a absence of Sh2d3c face density of CXCR4 and CXCR5, the receptors for SDF-1 and CXCL13, respectively, was comparable between WT and Sh2d3c- Shortly after injection, TI-2 polysaccharide Ags are taken up by deficient cells (data not shown), ruling out a role for Sh2d3c in MZ macrophages and B cells, and retained on the cell surface regulating the expression of these receptors. by guest on September 29, 2021 to facilitate the generation of a robust Ab response (22). To com- pare the capacity of Sh2d3c2/2 and WT MZB cells to retain Ag Absence of Sh2d3c alters the kinetics of Pyk2 phosphorylation in vivo, mice were injected with TNP-Ficoll i.v., and their sple- Pyk2 is a signaling component of the integrin pathway that is nocytes were examined for the presence of TNP on the cell sur- involved in regulating cell morphology and cell migration to face. Staining with anti-TNP Ab indicated that MZB cells in 2/2 chemotactic signals (33). Deficiency in Pyk2 has been shown to Sh2d3c mice were less efficient at retaining the Ag than their result in a diminished number of MZB cells (22). Because Sh2d3c WT counterparts (Fig. 3C). Furthermore, immunofluorescent his- reportedly interacts with Pyk2 (6), it seemed plausible that the tochemistry revealed that in WT spleens TNP-Ficoll was localized 2/2 2/2 defect in MZB cell development in Sh2d3c mice might be exclusively in the marginal zone area (Fig. 3D), whereas Sh2d3c associated with an alteration in Pyk2 signaling. To test this hy- spleens showed weak and thin rim of positive staining that some- pothesis, and to provide a validation in primary B cells for the times appeared to be in a dotted pattern, most likely reflecting the reports linking Sh2d3c and Pyk2 (6), we examined the phos- TNP-Ficoll bound to MZ macrophages. phorylation of Pyk2 in B cell-enriched splenocytes of Sh2d3c2/2 and Sh2d3c+/+ mice (Fig. 6). In WT B cells, LPS induced a time- Sh2d3c expression is essential for the robust LPS response of dependent phosphorylation of Pyk2 that was observed as early as MZB cells 1 min after activation and was maintained throughout the test 2 2 We explored the response of B cells from Sh2d3c / and Sh2d3c+/+ period (60 min). In contrast, a weaker phospho-Pyk2 signal was mice to in vitro stimulation by using a variety of polyclonal stimuli, detected in Sh2d3c2/2 B cells, which was apparent only 5 min including LPS. When treated with LPS, cell proliferation (Fig. 4A)as after LPS stimulation and continued to strengthen over the entire well as the production of the inflammatory cytokine IL-6 (Fig. 4B) 60-min time course. were greatly reduced in splenic B cell cultures of Sh2d3c-deficient mice compared with WT littermates. In contrast, no differences Sh2d3c is dispensable for normal T cell development were observed in the proliferation rate when the cells were stimulated Previous reports have shown that overexpression of Sh2d3c with a mixture of CD40 mAb and IL-4 or with anti-IgM mAb (Fig. increases TCR-induced IL-2 production in Jurkat T cells (6), thus 4A). We also examined whether the absence of Sh2d3c would affect implicating this adaptor protein in T cell function. However, the early signaling events, such as calcium mobilization via the BCR. distribution of the major thymocyte subpopulations was normal in Enriched B cells from Sh2d3c+/+ and Sh2d3c2/2 mice did not differ the Sh2d3c2/2 mice (Fig. 7), and there were no substantial differ- in their kinetic response to anti-IgM–elicited calcium mobilization ences in the naive and memory T cell numbers in the blood or (Supplemental Fig. 3). distribution of the same cell subsets in the spleen of Sh2d3c2/2 To test whether MZB cells were responsible for the observed versus Sh2d3c+/+ mice (data not shown). Furthermore, despite deficiency in the LPS response of Sh2d3c2/2 B cells, we repeated a modest reduction in numbers (Fig. 7), splenic T cells from The Journal of Immunology 333

Sh2d3c2/2 mice proliferated normally to activation via the CD3/ the thymus and blood of Sh2d3c2/2 mice did not show significant CD28 pathway (Fig. 7) and produced similar levels of IL-2 (data changes and splenic T cells responded normally to TCR-mediated not shown). Together, our data suggest that Sh2d3c plays a non- activation in vitro. Together our data suggest that Sh2d3c does not critical role in T cell development or function. play a critical role in T cell development or function. It is possible that the role of Sh2d3c in T-lineage cells is redundant and alter- native pathways are in place to compensate for its absence, or that Discussion the signaling mechanism has been affected in cell lines, such as Comprehensive physiological analysis of Sh2d3c-deficient mice Jurkat. demonstrated that Sh2d3c has an essential and relatively specific In conclusion, our results reveal a nonredundant role for Sh2d3c role in MZB cell development. The residual MZB cells in the KO in MZB cell development and function. We propose that Sh2d3c is mice also had impaired function, as demonstrated by decreased required for proper localization of cells, particularly MZB cells ability to retain and get stimulated by polysaccharide Ags, and to in vivo, and support the concept that Sh2d3c is a component of respond to CXCL13-mediated chemotactic signals. a signaling pathway that also includes Pyk2 and Cas-L. Although The pathway leading to the generation of MZB cells proceeds our data show a clear and relatively selective role for Sh2d3c in through a series of checkpoints and is regulated by multiple factors, MZB cell formation, the exact role of Sh2d3c in downstream sig- including BCR signal strength, Notch signaling, and localization of naling pathways of integrins or chemokine receptors is yet to be precursors (reviewed in Refs. 14, 18, 34). For example, weak BCR determined. The decrease in polysaccharide binding on MZB cells signaling favors MZB cell formation (35, 36), whereas strong in Sh2d3c-deficient mice suggests a reduced function for comple-

BCR signals promote the generation of FOB cells while curtailing ment receptors on these cells, but the underlying causes are not yet Downloaded from MZB cell development (36–38). Our data indicate that neither clear. Finally, in light of the apparent absence of a nonimmuno- the early steps (Ca2+ mobilization) nor the later stages (e.g., logical phenotype in Sh2d3c null mice, the significance of alter- proliferation) of BCR signaling via anti-IgM are dependent on native splicing of Sh2d3c RNA and the relatively ubiquitous Sh2d3c expression, arguing against defective BCR signaling as expression of one of the described Sh2d3c isoforms remains to the primary contributor to the MZB cell deficiency in the KO be defined.

mice. Furthermore, the macrophage populations that are known http://www.jimmunol.org/ to play a role in generating MZB cells (26, 27) are unlikely to be Acknowledgments 2/2 responsible for the defect observed in Sh2d3c mice because We thank the phenotypic analysis groups at Lexicon Pharmaceuticals for they appeared to have normal distribution in the marginal zone carrying out the comprehensive clinical diagnostic tests on the mutant area of the spleen. mouse lines. We thank the Pathology Department for help in preparing The deficiency of MZB cells in Sh2d3c2/2 mice is more likely slides from tissue sections. related to the role of Sh2d3c in regulating cell migration (10). The importance of motility in the generation of MZB cells was dem- Disclosures onstrated by the observation that treatment of WT mice with The authors of this article are or have been employees of, and received pertussis toxin results in selective loss of MZB cells, suggesting stock options from, Lexicon Pharmaceuticals or Genentech. by guest on September 29, 2021 a strong dependence of MZB cell formation on chemotactic sig- nals and G protein-linked receptors (22, 39). B cells in Sh2d3c KO References mice had decreased ability to migrate in response to chemotactic 1. Dodelet, V. C., C. Pazzagli, A. H. Zisch, C. A. Hauser, and E. B. Pasquale. 1999. signals, which suggests a role for this protein in the localization A novel signaling intermediate, SHEP1, directly couples Eph receptors to R-Ras of MZB cell precursors to the appropriate area of the spleen. It and Rap1A. J. Biol. Chem. 274: 31941–31946. is possible that Sh2d3c regulates cell migration via its interacting 2. 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