By CD22 Signaling Βα Receptor and MHC Class II/Ig

Cognate B Cell Signaling via MHC Class II: Differential Regulation of B Cell Antigen Receptor and MHC Class II/Ig- βα Signaling by CD22 This information is current as of October 2, 2021. David M. Mills, John C. Stolpa and John C. Cambier J Immunol 2004; 172:195-201; ; doi: 10.4049/jimmunol.172.1.195 http://www.jimmunol.org/content/172/1/195 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2004 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Cognate B Cell Signaling via MHC Class II: Differential Regulation of B Cell Antigen Receptor and MHC Class II/ Ig-␣␤ Signaling by CD221

David M. Mills, John C. Stolpa, and John C. Cambier2

Recent studies demonstrate that MHC class II molecules can signal via associated Ig-␣␤ dimers, signal transducers previously thought to function only in B cell Ag receptor (BCR) signaling. Surprisingly, the biologic outputs of MHC class II and BCR ligation (by thymus-dependent Ags) differ, e.g., MHC class II signaling leads to robust proliferation and extension of pseudopods. It seemed possible that these differences might be due, at least in part, to differential use of inhibitory coreceptors thought to modulate membrane Ig signals. In this study, we demonstrate that CD22, an inhibitory BCR coreceptor, neither associates with nor functions in MHC class II/Ig-␣␤ signaling. Interestingly, CD22 is actively excluded from cell surface MHC class II aggregates. The Journal of Immunology, 2004, 172: 195–201. Downloaded from

uring cognate T cell-B cell interactions, multiple recep- mains of MHC class II (3). Recent studies demonstrate that sig- tor-ligand pairs become localized at the cell contact in- naling by MHC class II in primed cells occurs by association of the D terface. Many of these proteins become further ordered B cell Ag receptor (BCR) signal-transducing substructure, Ig-␣/ into subregions, termed central and peripheral supramolecular ac- Ig-␤ dimers, with MHC class II, and that transmembrane signaling 3 tivation clusters (SMACs) (1, 2). This organization increases the by MHC class II is induced upon cognate TCR interactions (9). http://www.jimmunol.org/ local concentration of signaling molecules and ensures that certain Extensive mutational analyses have determined that the ability of effectors are sequestered away from some proteins and colocalized MHC class II to associate with Ig-␣␤ is required for I-A-mediated with others. Although T cell SMAC formation can occur in the calcium mobilization and induction of whole-cell protein tyrosine absence of APCs (2), it is currently unknown whether transmem- phosphorylation in primed B cells.4 Although aggregation of both 2ϩ brane signaling in one or both cells influences the formation, sta- BCR and MHC class II on primed cells induces [Ca ]i mobili- bilization, and movement of molecules into and out of SMACs in zation and tyrosine kinase activation, the responses differ at the vivo. However, although T-B conjugate formation does not depend biologic level. Although both MHC class II aggregation and BCR on the T cell recognizing its specific Ag, the formation of SMACs ligation (by thymus-dependent Ags) leads to increased expression at cell contact points is seen only upon Ag recognition (1, 2). Thus, of activation markers, only MHC class II signals cause extension by guest on October 2, 2021 Ag-driven aggregation of both MHC class II and TCR may be of dendrite-like pseudopodia (6). important for productive T-B collaboration. We hypothesized that differences in MHC class II and BCR Aggregation of MHC class II on resting B cells leads to pro- signaling may be due to differential use of coreceptors. Signal transduction of cAMP and activation of protein kinase C (3Ð5). How- duction by the BCR can involve several coreceptors, including CD19/ ever, in B cells activated by Ag and/or IL-4, aggregation of MHC 21, CD22, Fc␥RIIB, and CD72. The coreceptors involved in MHC 2ϩ 2ϩ class II induces intracellular Ca concentration ([Ca ]i) mobi- class II signal transduction are not well characterized. lization, tyrosine kinase activation, dendritic extensions, and pro- CD22 is a 140-kDa sialic acid-binding Ig-like lectin superfamily liferation (6Ð8). Because B cells actively engulf TCR-coated member that binds ␣2,6-linked sialic acid residues (in particular beads (9), it is possible that the formation of pseudopods by B cells amino acid contexts) and is tyrosyl phosphorylated upon BCR ag- following MHC class II ligation in vitro reflects molecular events gregation (reviewed in Ref. 10). CD22 contains three immunore- important for in vivo dynamics of T-B conjugates. Triggering of ceptor tyrosine-based inhibitory motifs in its cytoplasmic domain. these primed cell responses is independent of the cytoplasmic do- Mice deficient in CD22 display increased sensitivity to BCR ag- 2ϩ gregation (measured by [Ca ]i mobilization and proliferation), but no noticeable differences in some thymus-dependent immune Integrated Department of Immunology, University of Colorado Health Sciences Cen- responses (11, 12). Although some studies suggest that CD22 may ter and National Jewish Medical and Research Center, Denver, CO 80206 interact with sialic acid associated with membrane (m)Ig, and Received for publication June 17, 2003. Accepted for publication October 3, 2003. thereby modulate BCR signaling selectively, these findings are The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance controversial. The potential interaction of MHC class II with CD22 with 18 U.S.C. Section 1734 solely to indicate this fact. has not been explored. CD22 is phosphorylated by the Src family 1 These studies were supported by National Institutes of Health Grant AI20519. J.C.C. kinase Lyn and exerts its negative effects in part by recruiting the is an Ida and Cecil Green Professor of Immunology. tyrosine phosphatase Src homology 2 domain-containing phospha- 2 Address correspondence and reprint requests to Dr. John C. Cambier, Integrated tase-1 (SHP-1) to signaling complexes. This inhibitory signaling Department of Immunology, University of Colorado Health Sciences Center and Na- tional Jewish Medical and Research Center, 1400 Jackson Street, Denver, CO 80206. E-mail address: [email protected] 3 2ϩ Abbreviations used in this paper: SMAC, supramolecular activation cluster; [Ca ]i, 2ϩ 2ϩ 2ϩ 4 ␣ intracellular Ca concentration; [Ca ]o, extracellular Ca concentration; BCR, B J. Stolpa, D. Mills, and J. Cambier. The connecting peptide region of the chain of cell Ag receptor; m, membrane; SHP-1, Src homology 2 domain-containing phos- MHC class II molecules is required for association with the signal-transducing het- phatase-1. erodimer Ig-␣/␤. Submitted for publication.

loop sets thresholds for autoimmunity in mice (13). SHP-1 sub- anti-MHC class I and biotinylated anti-CD22 were selected, which yielded strates are not well defined, but are proposed to include CD22, comparable staining by flow cytometry. Cells were washed with IMDM ϫ 6 CD72, Vav, B cell linker protein, Syk, Ig-␣, and Ig-␤ (14, 15). and resuspended at 1.5 10 /ml for analysis on an LSR flow cytometer (BD Biosciences, Mountain View, CA). Signaling was triggered by addi- In this report, we addressed whether the inhibitory BCR core- tion of avidin and anti-␮ as indicated in the figures. Data analysis was ceptor CD22 modulates MHC class II signal transduction, and performed using FlowJo software (Tree Star, San Carlos, CA). whether CD22 usage can account for any of the signaling differ- For analysis of Ca2ϩ release from intracellular stores, 0.5 M EGTA was ences observed following BCR and MHC class II aggregation. We added 1 min before stimulation to buffer the free [Ca2ϩ] in the medium 2ϩ 2ϩ report that CD22 becomes tyrosyl phosphorylated upon BCR but (extracellular Ca concentration ([Ca ]o)) to 60 nM (isotonic with re- spect to the cell cytoplasm). Four minutes poststimulation, 1 M CaCl2 was not MHC class II aggregation. Consistent with these findings, we ϩ ϩ added to adjust [Ca2 ] to 1.3 mM, allowing analysis of Ca2 influx. show that the CD22 effector SHP-1 becomes tyrosyl phosphory- o lated in response to BCR but not MHC class II aggregation. Stud- ies using knockout mice indicate that, although CD22 negatively Immunoprecipitation and immunoblotting regulates BCR-mediated [Ca2ϩ] mobilization and proliferation, it i IL-4-primed splenic B cells were resuspended at 20 ϫ 106/ml in IMDM does not affect MHC class II-induced responses. We were able to (10% FCS). Cells were incubated with biotinylated anti-I-Ab/d at 20 ␮g/ml detect CD22 enrichment in IgM but not MHC class II immuno- for 20 min at room temperature. Samples were washed once and resus- precipitates. Interestingly, although CD22 was recruited to mIgM pended at 20 ϫ 106 cells/ml in IMDM. Stimulations were performed for 5 aggregates, it was actively excluded from MHC class II aggregates min at 37¡C using 5 ␮g/ml anti-␮ or 20 ␮g/ml avidin. Samples were lysed on stimulated cells. Finally, forced coaggregation of CD22 and for 10 min on ice in 1 ml 0.5% 3-[(3-cholamidopropyl)dimethylammonio]- MHC class II induced Ca2ϩ mobilization responses similar to 1-propanesulfonate lysis buffer (10 mM NaF, 0.4 mM EDTA, 2 mM so- dium orthovanadate, 10 mM tetrasodium pyrophosphate, 1 mM PMSF, 1 Downloaded from those induced by BCR aggregation, and inhibited formation of mM aprotinin, 1 mM ␣-1-antitrypsin, and 1 mM leupeptin) followed by pseudopods. centrifugation at 14,000 rpm for 10 min in an Eppendorf centrifuge. CD22 was immunoprecipitated using directly coupled anti-CD22 Ab-conjugated Materials and Methods Sepharose beads. CD19 and SHP-1 were immunoprecipitated with 10 ␮lof ␮ Cell preparation and culture rabbit serum or purified IgG prebound to 20 l of protein A-Sepharose beads. Immunoprecipitations were performed for 1 h (or overnight for Fig. Ϫ/Ϫ 5B)at4¡C with constant mixing. Beads were pelleted by brief centrifuga- Splenic B cells from Ig-transgenic 3-83, C57BL6, and CD22 mice were http://www.jimmunol.org/ used in these experiments. Spleens were excised from 2- to 4-mo-old mice; tion and washed three times with 1 ml of lysis buffer. Samples were boiled single-cell suspensions were prepared, depleted of erythrocytes using in reducing sample buffer and fractionated by 10% SDS-PAGE. Proteins Gey’s solution, and washed once in IMDM. T cells were lysed using anti- were transferred to polyvinylidene difluoride membranes and visualized Thy1 mAbs and guinea pig complement. B cells were isolated by Percoll using specific Abs followed by ECL (NEN, Boston, MA). Relative induc- gradient centrifugation, washed with IMDM, and cultured at 2Ð3 ϫ 106 tion of CD22 phosphorylation was calculated by obtaining a ratio of the cells/ml (7% CO2;37¡C) in IMDM containing 100 U/ml penicillin, 100 intensity of the anti-phosphotyrosine signal to the anti-CD22 immunoblot ␮g/ml streptomycin, 50 ␮g/ml gentamicin, 2 mM L-glutamine, 1 mM so- signal. These ratios were then divided by the ratio obtained for unstimu- dium pyruvate, 50 ␮M 2-ME, and 10% FCS (HyClone Laboratories, Lo- lated samples to obtain relative induction. Band intensities were calculated gan, UT). Priming of MHC class II signal transduction was accomplished using NIH Image. by culturing cells for 12Ð18 h in 50 U/ml recombinant murine IL-4. For

pseudopod formation assays, both IL-4 and 10 ␮g/ml low-affinity anti-IgM by guest on October 2, 2021 were used to prime B cells. All samples were incubated for 15 min with Fluorescence microscopy anti-Fc␥ Abs before stimulation to block IgG binding to these receptors. For plate-bound stimulations, tissue culture plates were coated with anti- Primed B cells were incubated with biotinylated anti-MHC class II for 20 MHC class II, anti-CD22, and anti-H-2K Abs (diluted in PBS) for 18 h at min at room temperature and washed once with IMDM. Samples were 37¡C. Wells were washed three times with PBS to remove nonbound Ab stimulated with either polyclonal rabbit Cy5-anti-IgM, FITC-streptavidin, molecules. or Cy3-streptividin for 30 min at 37¡C. Cells were placed on poly-L-lysine- coated coverslips for 2 min at 37¡C and fixed with 4% paraformaldehyde, Abs and reagents followed by permeabilization for 5 min with 0.2% Triton X-100. Cover- The following mAbs were purified from hybridoma supernatant and used slips were blocked with PBS (2.5% FBS) for 5 min and probed with Cy5- for stimulation, immunoprecipitation, and staining experiments: anti-I-Ad/b anti-IgM, biotinylated anti-MHC class II, anti-CD22, anti-CD19, or anti- (D3.137), anti-CD22 (Cy34), low-affinity anti-␮ H chain (Bet-2), high af- MHC class I Abs for1hatroom temperature. Samples were washed finity anti-␮ (b-7-6), anti-Fc␥RII/III (2.4G2), and anti-H-2Kd/b/k several times in PBS and probed with appropriate secondary reagents for (M1.24.3.9.8). Purified polyclonal rabbit anti-SHP-1 used for immunopre- 1 h at room temperature. After washing several times, coverslips were cipitation and immunoblotting was from Upstate Biotechnology (Lake mounted on microscope slides with mounting solution (2 mg/ml o-phen- Placid, NY). Anti-CD19 and anti-CD22 cytoplasmic tail sera used for im- ylenediamine in 90% glycerol). Images were captured and analyzed using munoblotting were raised in New Zealand White rabbits. Anti-MHC class a Leica (Deerfield, IL) DMRXA microscope, a SensicamQE camera II (I-A␣) for immunoblotting was kindly provided by Dr. I. Mellman (Yale (Cooke, Auburn Hills, MI), and Slidebook imaging software (Intelligent University, New Haven, CT). Additional reagents used for immunoblotting Imaging Innovations, Denver, CO). and staining were as follows: donkey anti-mouse IgG1-HRP (Zymed, San Francisco, CA), anti-mouse IgM-HRP (Zymed), protein A-HRP (Zymed), Cy3-streptavidin (Caltag, Burlingame, CA), Cy3-goat anti-mouse IgG1 Proliferation (Caltag), Cy5-goat anti-rabbit IgG (Jackson ImmunoResearch Laborato- ries, West Grove, PA), and Cy5-donkey anti-rat IgG (Jackson ImmunoRe- Splenic B cells from wild-type and CD22Ϫ/Ϫ mice were negatively se- search Laboratories). rIL-4 was purified from J558L-IL-4 plasmacytoma lected using anti-CD43-coated microspheres (Miltenyi Biotec, Auburn, supernatant. Protein A- and streptavidin-Sepharose used for immunopre- CA) and magnetic separation according to the manufacturer’s recommen- cipitation were from Amersham Pharmacia Biotech (Uppsala, Sweden). dations. Cells were determined by flow cytometry to be Ͼ95% B220ϩ ϫ 6 ␮ 2ϩ (data not shown). Cells were incubated at 5 10 /ml in HBSS with 2 M Analysis of [Ca ]i CFSE (Molecular Probes) for 5 min at 22¡C. Cells were then washed with 6 Cells were resuspended at 5 ϫ 106/ml in IMDM (2.5% FCS) and incubated HBSS and cultured for 18 h at 2 ϫ 10 /ml with low-affinity anti-␮ (Bet-2; for 45 min at 37¡Cin5␮M Indo 1-AM (Molecular Probes, Eugene, OR). 2.5 ␮g/ml) and IL-4 (50 U/ml) in IMDM (10% FCS, 2% nonessential For aggregation of MHC class II, cells were incubated with biotinylated amino acids, and 50 ␮M 2-ME). Samples were then transferred to non- anti-MHC class II (1 ␮g/ml) for 15 min at room temperature. For coag- coated or anti-I-A-coated 24-well plates and cultured for 72 h for 3 days. gregation of MHC class II and CD22 or MHC class I, cells were incubated Proliferation was analyzed using a FACScan cytometer (BD Biosciences) with biotinylated anti-MHC class II Abs, as described above, and biotin- and FlowJo analysis software (Tree Star). Data are presented as percentage ylated anti-MHC class I or biotinylated anti-CD22. Doses of biotinylated of maximum population. The Journal of Immunology 197

may selectively limit the BCR response or enhance the MHC class II response. Alternatively, this mechanism may not be influx tar- geted, but merely late acting. The different intensities of MHC class II- and mIgM-mediated calcium mobilization observed in Fig. 1A may reflect different de- grees of cell surface receptor aggregation (resulting from the use of different ligands). However, the response magnitude differences are not entirely explained by Ab and/or aggregate dissimilarity, because under stimulatory conditions used in these experiments, the entire cell surface pool of each receptor is recruited into a single membrane aggregate (data not shown). Additionally, this difference in magnitude was seen when multiple anti-Ig and anti- I-A Abs were compared (data not shown) and may indicate that MHC class II has a greater intrinsic ability to transduce signals ϩ FIGURE 1. MHC class II aggregation leads to greater Ca2ϩ influx than leading to Ca2 mobilization. BCR ligation. A, IL-4-primed splenic B lymphocytes from 3-83 ␮␦ Ig transgenic mice were loaded with Indo 1-AM and stimulated with 8, 5, or MHC class II-induced Ca2ϩ mobilization is normal in CD22Ϫ/Ϫ 2 ␮g/ml anti-␮ (b-7-6) or preincubated with 1 ␮g/ml biotinylated anti- B cells MHC class II (D3.1.3.7) and stimulated with 4, 2, or 1 ␮g/ml avidin. B, 2ϩ Cells were primed and stimulated as described above using 5 ␮g/ml anti-␮, It has been shown previously that CD22 limits extracellular Ca Downloaded from 1 ␮g/ml biotinylated anti-MHC class II, and 2 ␮g/ml avidin. Before anal- influx mediated by BCR ligation (16). Based on these observa- 2ϩ ␮ ysis, [Ca ]o was buffered to 60 nM by adding 6.5 l of 0.5 M EGTA. Four tions, and those shown in Fig. 1, we hypothesized that, in contrast 2ϩ minutes following initiation of analyses, [Ca ]o was increased to 1.3 mM to the BCR, MHC class II signal transduction is not regulated by by adding 1.5 ␮l of 1 M CaCl . These data are representative of four 2ϩ 2 CD22. To address this hypothesis, we compared [Ca ]i following separate experiments. MHC class II and BCR ligation in CD22Ϫ/Ϫ and wild-type B cells. We observed modestly elevated basal [Ca2ϩ] in resting CD22Ϫ/Ϫ i http://www.jimmunol.org/ cells, perhaps implying that these cells experience more robust Results and Discussion tonic signals. As reported by others, CD22Ϫ/Ϫ B cells displayed Aggregation of MHC class II and IgM induces dissimilar Ca2ϩ enhanced Ca2ϩ influx upon BCR aggregation (Fig. 2A). The 2ϩ Ϫ/Ϫ influx in primed splenic B cells [Ca ]i mobilization response to BCR aggregation in CD22 B To explore molecular mechanisms underlying differences between cells was similar to the response of wild-type cells to MHC class BCR and MHC class II signaling, we compared Ca2ϩ mobilization II aggregation. In contrast to BCR stimulation, MHC class II-in- 2ϩ Ϫ/Ϫ responses to BCR and MHC class II aggregation in purified splenic duced Ca mobilization was similar in CD22 and wild-type B B cells. We found that aggregation of MHC class II induces more cells (Fig. 2A). These findings were not a result of maximal stim- robust Ca2ϩ mobilization than aggregation of BCR in primed cells ulation, because we obtained similar results at suboptimal stimu- by guest on October 2, 2021 at multiple stimulatory doses (Fig. 1A). At its maximal stimulatory latory doses (Fig. 2B). These results suggested that CD22 is func- dose, anti-␮-induced calcium mobilization did not approach that tionally linked to BCR, but not MHC class II signal transduction induced by anti-MHC class II. pathways. To investigate the basis of the dissimilar Ca2ϩ mobilization seen in response to MHC class II and IgM aggregation, we analyzed the CD22 is tyrosyl phosphorylated in response to BCR but not intracellular release and influx components of the response sepa- MHC class II aggregation rately following maximal stimulation with anti-␮ or anti-I-A Abs. To more directly address the hypothesis that CD22 does not neg- We observed greater Ca2ϩ influx in response to MHC class II atively regulate MHC class II signal transduction, we analyzed aggregation compared with BCR aggregation, but comparable re- tyrosyl phosphorylation of CD22 and CD19 (a positive regulator lease from intracellular stores (Fig. 1B). These results suggest two of BCR signaling) in response to receptor aggregation in primed possibilities. First, a post-intracellular Ca2ϩ release mechanism splenic B cells. Although CD19 was tyrosyl phosphorylated in

FIGURE 2. CD22 negatively regulates BCR but not MHC class II-induced mobilization of intracellular free Ca2ϩ. A, Splenic B lymphocytes from wild-type or CD22Ϫ/Ϫ mice were primed with IL-4 and cultured as described in Fig. 1. Cells were stimulated with 5 ␮g/ml anti-␮ (b-7-6) or preincubated with 1 ␮g/ml biotinylated anti-MHC class II (D3.1.3.7) and stimulated with 2 ␮g/ml avidin. B, Cells from wild-type and CD22Ϫ/Ϫ mice were loaded with INDO 1-AM and preincubated with 1 ␮g/ml anti-MHC class II. Sam- ples were stimulated with either 4, 2, or 1 ␮g/ml avidin. Data are representative of four experiments. 198 CD22 USAGE DETERMINES MHC CLASS II/BCR SIGNALING DIFFERENCES Downloaded from

FIGURE 3. Differential tyrosyl phosphorylation of the CD19 and CD22 coreceptors following MHC class II and BCR ligation. IL-4-primed 3-83 splenic B cells stimulated with anti-␮ (b-7-6) (5 ␮g/106 cells/ml) or pre- ␮ 6 incubated with biotinylated anti-MHC class II (1 g/10 cells/ml) and http://www.jimmunol.org/ stimulated with avidin (1 ␮g/106 cells/ml) for 5 min before lysis in 1% Nonidet P-40. CD22 and CD19 (A) or SHP-1 (B) were immunoprecipitated from lysates. Following fractionation by SDS-PAGE and transfer to polyvinylidene difluoride, membranes were immunoblotted with anti- phosphotyrosine Abs, stripped, and reprobed with anti-CD22, anti-CD19, or anti-SHP-1 polyclonal rabbit sera. Data are representative of five separate experiments. by guest on October 2, 2021 response to both BCR and MHC class II aggregation, CD22 was only phosphorylated upon BCR aggregation (Fig. 3A). This finding indicates that the MHC class II and BCR signaling complexes share some accessory molecules, including CD19, and use others uniquely, such as CD22. These results appear inconsistent with recently published findings of Bobbitt et al. (17), who report that both CD19 and CD22 are tyrosyl phosphorylated following MHC class II aggregation. One explanation is that Bobbitt et al. (17) used a B lymphoma to FIGURE 4. Association of CD22 with the BCR but not MHC class II biochemically study coreceptor cooperation with MHC class II, signaling complex. A, IL-4-primed 3-83 B cells were stimulated as de- whereas the studies presented here used primary B cells. Thus, it scribed in Fig. 3 before lysis in 0.25% 3-[(3-cholamidopropyl)dimethyl- is possible that the degree to which CD22 modulates MHC class II ammonio]-1-propanesulfonate. I-A or IgM molecules were precipitated us- signaling differs between ex vivo B cells and this lymphoma. ing biotinylated Abs coupled to streptavidin-Sepharose beads. Proteins The findings presented above predict that downstream signaling were detected using anti-IgM (Jackson ImmunoResearch Laboratories), anti- 6 events influenced by CD22 cooperation would be different upon I-A (Rivoli), and anti-CD22 rabbit sera. B, IL-4-primed 3-83 B cells (10 / ␮ MHC class II and BCR aggregation. To address this possibility, we ml) were preincubated with 1 g/ml biotinylated anti-MHC class II (D3.1.3.7) and stimulated for 30 min with FITC-avidin or polyclonal Cy5- analyzed SHP-1 tyrosyl phosphorylation upon aggregation of ei- anti-IgM (Jackson ImmunoResearch Laboratories). Cells were fixed onto ther MHC class II or the BCR. As reported by others, we observed microscope coverslips and stained for CD22. C, Samples were stimulated increased tyrosyl phosphorylation of SHP-1 upon BCR ligation as described above and stained for MHC class I (H-2K) or CD19. Data are (Fig. 3B). Although tyrosyl phosphorylation of SHP-1 was induced representative of four separate experiments, and numbers indicate percent- upon MHC class II aggregation, it was considerably less robust, ages of cells with representative phenotype. consistent with the finding that CD22 is not involved in MHC class II signal transduction.

CD22 coimmunoprecipitates with IgM, but not MHC class II, ted IgM and MHC class II immunoprecipitates using anti-CD22 and is excluded from MHC class II aggregates Abs. Additionally, we used ﬂuorescence microscopy to assess the As discussed previously, CD22 associates directly with the BCR spatial localization of CD22, plasma membrane IgM (mIgM), and signaling complex (18). The data described above suggested that MHC class II in nonstimulated and stimulated B cells. CD22 may not associate physically with the MHC class II signal We found that CD22 was present in anti-␮ immunoprecipitates transduction complex. To address this possibility, we immunoblot- from B cells before and following aggregation of mIgM (Fig. 4A). The Journal of Immunology 199

Conversely, we were unable to detect CD22 in MHC class II im- a critical MHC class II-peptide density threshold. Finally, such munoprecipitates either before or after aggregation of I-A. How- spatial segregation of CD22 from MHC class II may occur only if ever, in agreement with previously reported findings, we found the MHC ligand is laterally mobile. It will be important to deter- that nearly all of the cell surface CD22 colocalized with mIgM mine the molecular basis for the spatial segregation of MHC class following BCR aggregation (19). In contrast, CD22 was actively II and CD22. excluded from I-A aggregates formed following MHC class II ligation (Fig. 4B). In control experiments, the localization of MHC 2ϩ class I was unaffected by MHC class II aggregation (Fig. 4C). Forced coaggregation of CD22 and MHC class II reduces Ca Additionally, a portion of the plasma membrane CD19 was en- influx, leads to CD22 tyrosyl phosphorylation, and inhibits riched in MHC class II aggregates (Fig. 4C). These results dem- pseudopod formation onstrate that CD22 is a constitutive member of the BCR, but not As an additional challenge to the hypothesis that CD22 differen- the MHC class II signal transduction complex. Conversely, CD19 tially regulates Ig-␣␤ signals mediated by BCR and MHC class II, likely exists on the cell surface in distinct pools, associated with we compared Ca2ϩ mobilization responses of primed B cells to receptors such as MHC class II, mIgM, and CD21 (17, 20Ð22). MHC class II aggregation and forced MHC class II/CD22 coag- In previous studies (9), we found that the cell surface localiza- gregation. We found that coaggregation of MHC class II and CD22 tion of CD22 on the primed lymphoma line K46 was not altered diminishes the Ca2ϩ influx response compared with aggregation of following stimulation with TCR-coated microspheres. These data MHC class II alone (Fig. 5A). Analogous inhibition was not ob- corroborate biochemical evidence presented here in Figs. 2, 3, and served upon independent aggregation of I-A and CD22 (data not 6 but do not reveal the spatial segregation of CD22 and I-A ob- shown). In specificity controls, coaggregation of MHC class I with served in Fig. 4B. Although we cannot exclude the possibility that MHC class II also did not significantly alter the response. The Downloaded from 2ϩ this is due to differences in cell systems used (ex vivo B cells vs [Ca ]i mobilization response to coaggregation of MHC class II 2ϩ K46), it is possible that only stimulation with relatively high-af- and CD22 was similar to the [Ca ]i mobilization response to finity ligands induces the spatial segregation of CD22 and MHC BCR aggregation. To address whether CD22-mediated inhibition class II. Alternatively, because D3.137 Abs bind to all cell surface of MHC class II signals in this experiment was due to inhibitory I-Ab/d molecules but DO.11.10 TCR monomers ligate only those signaling, we analyzed CD22 phosphorylation following coaggre- I-Ad molecules presenting the OVA peptide, the exclusion of gation with MHC class II. We observed induced phosphorylation http://www.jimmunol.org/ CD22 from MHC class II aggregates may be detectable only above of CD22 upon forced coaggregation with MHC class II (Fig. 5B), by guest on October 2, 2021

FIGURE 5. Forced coaggregation of CD22 and MHC class II leads to inhibition of Ca2ϩ influx and pseudopod formation. IL-4-primed 3-83 splenic B cells were preincubated with biotinylated anti-MHC class II (D3.1.3.7) (1 ␮g/106 cells/ml) and either biotinylated anti-MHC class I or biotinylated anti-CD22 Abs (Cy34). Receptors were coaggregated by the addition of avidin (4 ␮g/106 cells/ml). mIgM was aggregated as described in Fig. 3. A, Analysis of intracellular free Ca2ϩ was performed as described in Fig. 1B. B, Immunoprecipitations were performed using anti-CD22-coupled Sepharose beads. Immunoblots were performed as described in Fig. 3. Relative inductions of phosphorylation were as follows: nostim, 1.0; anti-I-A, 1.4; anti-IgM, 6.01; anti-I-A/CD22, 5.01. C, 3-83 splenic B cells were primed with IL-4 and low-affinity anti-␮ (Bet-2) and transferred in triplicate to Ab-coated plates. Samples were incubated for 30 min at 37¡C. Data are means of triplicate assays (ϩ1 SD) of the percentage of cells containing dendritic extensions. D, Examples of primary data summarized in C. Results are representative of three separate experiments. 200 CD22 USAGE DETERMINES MHC CLASS II/BCR SIGNALING DIFFERENCES suggesting that MHC class II is capable of recruiting the appro- proliferation of wild-type and CD22-deficient B cells is compara- priate kinases for initiation of CD22-mediated inhibitory signaling. ble following MHC class II ligation (Fig. 6). The modest increases To address the biologic significance of differential CD22 usage in MHC class II-mediated proliferation of B cells from CD22Ϫ/Ϫ by the BCR and MHC class II, we assessed the effect of forced mice may reflect enhanced priming of MHC class II signaling by CD22 coaggregation on the ability of B cells to form pseudopods low affinity anti-␮. Most importantly, proliferative responses of upon ligation of MHC class II. We analyzed pseudopod formation CD22-deficient B cells following mIgM aggregation approach the by primed splenic B cells cultured on plates coated with anti-MHC levels seen following I-A aggregation on wild-type B cells. These class II Abs alone, or anti-MHC class II and anti-CD22 Abs. As data extend our previous findings (Figs. 2Ð4) by demonstrating shown in Fig. 5, C and D, we observed dose-dependent inhibition that differential use of CD22 by I-A and mIgM has both biochem- of pseudopod formation by forced CD22/MHC class II coaggre- ical and biologic consequences for B cell responses. Additionally, gation. Specificity controls illustrated that pseudopod formation is the data demonstrate that negative regulation by CD22 can fully not inhibited by coaggregation of MHC class I and MHC class II. account for some differences in the consequences of MHC class II As expected, BCR aggregation did not induce pseudopod forma- and mIgM signal transduction (Ca2ϩ mobilization and proliferation. These data demonstrate that CD22 does not regulate MHC tion) but not others (pseudopod formation). class II signal transduction under normal circumstances, but can be Finally, although IL-4 is sufficient to prime B cells for MHC forced to do so. class II-induced calcium mobilization, optimal priming of MHC ␣␤ If BCR and I-A signaling via Ig- differs only in usage of class II-mediated pseudopod formation and proliferation occurs CD22, one would predict that CD22-deficient B cells would form following combined stimulation with IL-4 and anti-IgM (data not pseudopods on anti-IgM-coated plates. Interestingly, CD22Ϫ/Ϫ B

shown and Ref. 6). The reasons underlying these differential re- Downloaded from cells did not change their morphology following IgM aggregation quirements are currently unclear. (data not shown). In agreement with data presented in Figs. 2 and The studies described in this report were undertaken to deter- 6, CD22-deﬁcient B cells formed pseudopodic extensions nor- mine how BCR and MHC class II transduce signals with distinct mally following MHC class II aggregation, even at suboptimal biologic consequences despite their use of the same transmem- stimulatory doses (data not shown). Because forced CD22 coag- brane transducers, Ig-␣ and Ig-␤. Our experiments suggest that gregation can inhibit MHC class II-mediated pseudopod forma- 2ϩ

BCR- mediated Ca influx is constrained relative to that induced http://www.jimmunol.org/ tion, we conclude that lack of CD22 inhibitory signals is important through MHC class II. Studies using knockout mice indicated that for these morphologic transitions. However, because CD22 defi- CD22 does not modulate MHC class II-mediated responses. We ciency is not sufficient to convey this phenotype following BCR further show that CD22 becomes tyrosyl phosphorylated upon aggregation, other qualitative differences must exist between MHC BCR but not MHC class II aggregation, and that this correlates class II and BCR signal transduction that mediate changes in cell with tyrosyl phosphorylation of the CD22 effector SHP-1. In ad- morphology. dition, we provide evidence that CD22 is associated with BCR B cells proliferate comparably following MHC class II and BCR aggregates, but is actively excluded from MHC class II aggregates. aggregation in the absence of CD22 function Importantly, forced coaggregation of MHC class II and CD22 re- ϩ sulted in a Ca2 mobilization response similar to that induced by by guest on October 2, 2021 To further explore the role of CD22 in biologic responses of B BCR ligation, and inhibited pseudopod formation upon MHC class cells, we analyzed proliferation of wild-type or CD22Ϫ/Ϫ B cells II aggregation. Finally, CD22 deficiency had little effect on the following mIgM and MHC class II ligation. Purified splenic B ability of B cells to proliferate following MHC class II ligation. cells were labeled with CFSE, primed with IL-4 and low-affinity Most significantly, differential CD22 usage, at least in part, does anti-IgM (to mimic a thymus-dependent Ag), and cultured on non- underlie differences in proliferation to BCR and MHC class II coated or anti-I-A-coated tissue culture plates. As reported by oth- aggregation. ers (11, 12), we found that CD22-deficient B cells are more sen- The observation that CD22 associates with mIgM but not MHC sitive to anti-␮-induced proliferation (Fig. 6). However, the class II may be explained by recent studies which demonstrate that the lectin-binding domain of CD22 is required for participation in BCR signaling (23, 24). Thus, it is possible that CD22 associates with mIgM via interactions with sialic acid moieties that are not present in MHC class II extracellular domains, or are present in an unfavorable amino acid context. The finding that CD22 is actively excluded from MHC class II aggregates indicates that CD22 function is subject to additional levels of regulation beyond the pres- ence or absence of appropriate sialic acid ligands. This is corrob- orated by recent evidence that the longer cytoplasmic domains of mIgG disallow association of mIg with CD22 in B cell lymphoma models (25). It is perhaps not surprising that mechanisms have evolved to enhance signal output through MHC class II. In physiologic responses, TCR-MHC class II interactions are typically of low af- FIGURE 6. CD22-deficient B cells proliferate normally following finity. Furthermore, only a very small proportion of MHC class II Ϫ/Ϫ MHC class II aggregation. Splenic B cells from wild-type or CD22 on an APC is associated with peptide recognized by a single T cell. mice were loaded with CFSE and primed for 18 h with IL-4 (50 U/ml) and Thus, a limited number of MHC class II molecules are recognized low-affinity anti-IgM (Bet-2; 2.5 ␮g/ml) at 106 cells/ml. Samples were transferred to noncoated (shaded histograms) or anti-I-A (D3.1.3.7)-coated by TCR. It is noteworthy that recent evidence indicates many (black histograms) tissue culture plates. Cells were harvested after 72 h, MHC class II molecules bearing irrelevant peptides are recruited to and proliferation was analyzed by flow cytometry. Data are representative T-B contact regions (26). However, it is not known whether these of three independent experiments. noncognate interactions lead to MHC class II signaling. The Journal of Immunology 201

We report here the novel observation that unique qualities of 9. Lang, P., J. C. Stolpa, B. A. Freiberg, F. Crawford, J. Kappler, A. Kupfer, and ligand-binding substructures (e.g., mIgM or MHC class II) deter- J. C. Cambier. 2001. TCR-induced transmembrane signaling by peptide/MHC class II via associated Ig-␣/␤ dimers. Science 291:1537. mine, by secondary associations with accessory proteins, the qual- 10. Nitschke, L., H. Floyd, and P. R. Crocker. 2001. New functions for the sialic ity of signals transduced by primary signal transducing substruc- acid-binding adhesion molecule CD22, a member of the growing family of Si- ␣␤ glecs. Scand. J. Immunol. 53:227. tures (e.g., Ig- ). Our studies underscore the importance of 11. Nitschke, L., R. Carsetti, B. Ocker, G. Kohler, and M. C. Lamers. 1997. CD22 is receptor-associated modulators in diversification of biochemical a negative regulator of B-cell receptor signalling. Curr. Biol. 7:133. and biologic responses that follow receptor ligation. 12. Otipoby, K. L., K. B. Andersson, K. E. Draves, S. J. Klaus, A. G. Farr, ␣␤ J. D. Kerner, R. M. Perlmutter, C. L. Law, and E. A. Clark. 1996. CD22 regulates Current evidence indicates that Ig- dimers play dual roles thymus-independent responses and the lifespan of B cells. Nature 384:634. during B cell activation by transducing signals following Ag en- 13. Cornall, R. J., J. G. Cyster, M. L. Hibbs, A. R. Dunn, K. L. Otipoby, E. A. Clark, counter and during cognate T-B collaboration. The findings re- and C. C. Goodnow. 1998. Polygenic autoimmune traits: Lyn, CD22, and SHP-1 are limiting elements of a biochemical pathway regulating BCR signaling and ported here suggest that CD22 limits Ig-␣␤ signals and subsequent selection. Immunity 8:497. B cell activation following Ag encounter, but once vetted, maxi- 14. Wu, Y., M. J. Nadler, L. A. Brennan, G. D. Gish, J. F. Timms, N. Fusaki, ␣␤ ␣␤ J. Jongstra-Bilen, N. Tada, T. Pawson, J. Wither, et al. 1998. The B-cell trans- mal Ig- signaling occurs upon TCR binding. Thus, Ig- signal membrane protein CD72 binds to and is an in vivo substrate of the protein ty- strength/quality may be limited during the early phases of thymus- rosine phosphatase SHP-1. Curr. Biol. 8:1009. dependent B cell responses to circumvent activation of potentially 15. Tamir, I., J. M. Dal Porto, and J. C. Cambier. 2000. Cytoplasmic protein tyrosine phosphatases SHP-1 and SHP-2: regulators of B cell signal transduction. Curr. harmful autoreactive cells. Opin. Immunol. 12:307. 16. Nadler, M. J., P. A. McLean, B. G. Neel, and H. H. Wortis. 1997. B cell antigen receptor-evoked calcium influx is enhanced in CD22-deficient B cell lines. J. Im- Acknowledgments munol. 159:4233. 17. Bobbitt, K. R., and L. B. Justement. 2000. Regulation of MHC class II signal

We thank Drs. Joseph Dal Porto and Stephen Gauld for critical reading of Downloaded from this manuscript. transduction by the B cell coreceptors CD19 and CD22. J. Immunol. 165:5588. 18. Leprince, C., K. E. Draves, R. L. Geahlen, J. A. Ledbetter, and E. A. Clark. 1993. CD22 associates with the human surface IgM-B-cell antigen receptor complex. Proc. Natl. Acad. Sci. USA 90:3236. References 19. Nitschke, L., R. Brossmer, C.-P. Danzer, S. Kelm, and J. Gerlach. 2003. Sialic- 1. Monks, C. R., B. A. Freiberg, H. Kupfer, N. Sciaky, and A. Kupfer. 1998. Three- acid binding of C22 is required for inhibition of the BCR signal. In Keystone dimensional segregation of supramolecular activation clusters in T cells. Nature Symposium: B Cells and Antibodies: Laboratory to Clinic, January 17, 2003. 395:82. Keystone, CO.

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