CD19 Can Regulate B Independent of Complement Activation

This information is current as Minoru Hasegawa, Manabu Fujimoto, Jonathan C. Poe, of September 25, 2021. Douglas A. Steeber and Thomas F. Tedder J Immunol 2001; 167:3190-3200; ; doi: 10.4049/jimmunol.167.6.3190 http://www.jimmunol.org/content/167/6/3190 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 © 2001 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. CD19 Can Regulate B Lymphocyte Signal Transduction Independent of Complement Activation1

Minoru Hasegawa, Manabu Fujimoto, Jonathan C. Poe, Douglas A. Steeber, and Thomas F. Tedder2

B are critically regulated by signals transduced through the CD19-CD21 cell surface complex, where complement C3d binding to CD21 supplies an already characterized ligand. To determine the extent that CD19 function is controlled by complement activation, CD19-deficient mice (that are hyporesponsive to transmembrane signals) and mice over- expressing CD19 (that are hyperresponsive) were crossed with CD21- and C3-deficient mice. Cell surface CD19 and CD21 expression were significantly affected by the loss of CD21 and C3 expression, respectively. Mature B cells from CD21-deficient littermates had ϳ36% higher cell surface CD19 expression, whereas CD21/35 expression was increased by ϳ45% on B cells from C3-deficient mice. Negative regulation of CD19 and CD21 expression by CD21 and C3, respectively, may be functionally significant Downloaded from because small increases in cell surface CD19 overexpression can predispose to autoimmunity. Otherwise, development and function in CD19-deficient and -overexpressing mice were not significantly affected by a simultaneous loss of CD21 expression. Although CD21-deficient mice were found to express a hypomorphic cell surface CD21 at low levels that associated with mouse CD19, C3 deficiency did not significantly affect B cell development and function in CD19-deficient or -overexpressing mice. These results, and the severe phenotype exhibited by CD19-deficient mice compared with CD21- or C3-deficient mice, collectively demonstrate that CD19 can regulate B thresholds independent of CD21 engagement and complement http://www.jimmunol.org/ activation. The Journal of Immunology, 2001, 167: 3190Ð3200.

lymphocyte development and function are critically reg- CD21 is expressed on FDCs and mature B cells, with expression ulated by signals transduced through the CD19-CD21 first by IgMhighIgDlow transitional B cells (3, 22). CD21 is com- B complex (1–3). The CD19-CD21 posed of an extracellular domain containing 15 or 16 repeating complex is composed of at least four : CD19, CD21 (com- structural elements termed short consensus repeats (SCRs), a trans- plement receptor 2), CD81, and CD225 (Leu13) (4–7). CD19 is a membrane region, and a 34-aa cytoplasmic domain (23, 24). CD21

member of the Ig superfamily expressed exclusively on B cells and and CD35 ( 1) are alternative splice products by guest on September 25, 2021 follicular dendritic cells (FDC)3 (8). In B cells, CD19 is expressed of the same Cr2 in mice, but are encoded by different by early pre-B cells from the time of heavy chain gene rearrange- in humans (25, 26). In mice, CD35 is generated by the addition of ment until differentiation (6, 9, 10). CD19 has an six SCRs to the amino-terminal end of the CD21 protein. Well- ϳ240-aa cytoplasmic domain that is critical for CD19-CD21 com- characterized ligands for CD21 are the iC3b/C3d,g cleavage frag- plex signaling (8, 11, 12). Specifically, CD19 functions as a spe- ments of complement. These C3 cleavage products form covalent cialized adapter protein for the amplification of Src family kinases bonds with foreign Ags or immune complexes to generate C3d-Ag and as an interaction molecule for multiple signaling pathways complexes that are proposed to bind CD21 and regulate B cell crucial for modulating intrinsic and Ag receptor-induced signals function by signaling through the CD19 complex (1, 2, 7). CD35 (13–18). The cytoplasmic domains of human CD19 (hCD19) and binds and C4b, and serves as a cofactor for the hydrolysis of mouse CD19 (mCD19) are highly homologous (19). In fact, C3b-Ag complexes into C3d-Ag complexes (10, 27). This process hCD19 can replace mCD19 function when expressed at the ap- Ϫ/Ϫ is important for the processing of Ag-Ab complexes and the final propriate site density in CD19 mice (20). Cell surface CD19 is deposition of C3d-Ag complexes on the surface of B cells and present in molar excess of CD21 at all stages of B cell develop- FDCs through CD21. A recent study indicates that human CD21 ment, although CD19-CD21 complexes are presumed to represent can physically associate with mCD19, and can restore humoral 1:1 complexes (4, 21). Ϫ Ϫ immune response in CD21/35 / mice (28). Studies using mice that lack or overexpress CD19 indicate that Department of Immunology, Duke University Medical Center, Durham, NC 27710 CD19 and/or CD19-CD21 complexes regulate signal transduction Ϫ/Ϫ Received for publication May 3, 2001. Accepted for publication July 19, 2001. thresholds governing . B cells from CD19 The costs of publication of this article were defrayed in part by the payment of page mice are hyporesponsive to a variety of transmembrane signals, charges. This article must therefore be hereby marked advertisement in accordance which leads to significant defects during the later stages of B cell with 18 U.S.C. Section 1734 solely to indicate this fact. maturation, clonal expansion, and differentiation (29–31). By con- 1 This work was supported by National Institutes of Health Grants CA81776 and ϩ/ϩ CA54464. trast, transgenic mice that overexpress hCD19 (hCD19TG ) are hyperresponsive to transmembrane signals and display severe al- 2 Address correspondence and reprint requests to Dr. Thomas F. Tedder, Department of Immunology, Duke University Medical Center, Box 3010, Durham, NC 27710. terations during early stages of B cell development, which leads to E-mail address: [email protected] diminished numbers of B cells in the peripheral pool (9, 29, 32). 3 Abbreviations used in this paper: FDC, follicular dendritic cell; KLH, keyhole lim- The development of B1 cells is severely decreased in CD19Ϫ/Ϫ pet hemocyanin; hCD19, human CD19; hCD19TG, transgenic mice that overexpress mice, whereas there is an increased frequency of B1 cells within hCD19; mCD19, mouse CD19; SCR, short consensus repeat unit; TD, -depen- ϩ ϩ dent; TNP-LPS, 2,4,6-trinitrophenol-conjugated LPS. the peritoneum and spleen of hCD19TG / mice (9, 29, 30). In

Copyright © 2001 by The American Association of Immunologists 0022-1767/01/$02.00 The Journal of Immunology 3191 two independent lines of CD21/35Ϫ/Ϫ mice, lymphocyte develop- ϳ ment, phenotypes, and numbers are normal (33, 34). A 40% Abs used in this study included: mouse IgA anti-mCD19 (MB19–1; Ref. reduction in the frequency of peritoneal B1 cells has been observed 9), mouse anti-hCD19 (HB12b; Ref. 48), rat anti-mouse CD21/35 (7E9: Ϫ Ϫ Ϫ Ϫ in one line of CD21/35 / mice (33). Both lines of CD21/35 / IgG2a, 7G6: IgG2b; provided by Dr. T. Kinoshita, Osaka University, mice exhibit markedly impaired primary and secondary humoral Osaka, Japan: Ref. 49), FITC-conjugated anti-mouse CD21/35 (7G6; BD immune responses and formation, especially IgG PharMingen, San Diego, CA), PE -conjugated anti-mCD19 (1D3; BD PharMingen), PE-conjugated anti-hCD19 (B4; Coulter, Miami, FL), PE- responses to T cell-dependent (TD) Ags (33–35). This is not due conjugated anti-CD5 (53-7.3; BD PharMingen), biotinylated or FITC-con- Ϫ/Ϫ to a defect in B cell Ag receptor signaling in CD21/35 mice jugated anti-mouse IgM (Southern Biotechnology Associates, Birming- because their B cells respond normally to IgM and/or CD40 cross- ham, AL), biotinylated or FITC-conjugated anti-B220 (CD45RA, RA3- linking (33). Pretreatment of mice with either a CD21/35-specific 6B2; provided by Dr. R. Coffman, DNAX Research Institute, Palo Alto, CA), and biotinylated anti-mouse IgD (Southern Biotechnology mAb or a CD21-IgG fusion protein also blocks secondary humoral Ϫ/Ϫ Associates) Abs. Anti-CD21/35 Ab binding (7E9) was visualized using responses (36–38). C3 mice have a normal phenotype and se- FITC- or PE-conjugated goat anti-rat IgG (H ϩ L) Abs (Caltag Laborato- rum Ig levels, whereas mice that lack C4, which is required for C3 ries, Burlingame, CA) diluted to the appropriate concentration for optimal activation, have decreased IgG1, IgG2a, and IgG3 levels (39, 40). immunostaining. PE-conjugated streptavidin (Southern Biotechnology As- Both C3Ϫ/Ϫ and C4Ϫ/Ϫ mice have modest TD immune responses sociates) was used to reveal biotin-coupled Ab staining. with defects in germinal center formation. In addition, complement Immunofluorescence analysis and CD21/35 regulate the elimination of self-reactive B cells be- lpr/lpr Single-cell suspensions of lymphocytes from spleen, bone marrow, peri- cause systemic lupus erythematosus-prone C57BL/6 mice toneal lavage, and peripheral lymph nodes were isolated before two-color with CD21/35 or C4 deficiencies have exacerbated disease and immunofluorescence analysis. Leukocytes (0.5–1 ϫ 106) were stained at Downloaded from increased autoantibody production (41, 42). Spontaneous autoim- 4°C using predetermined optimal concentrations of Abs for 20 min. Blood munity has been observed in C4Ϫ/Ϫ mice, but not in CD21/35Ϫ/Ϫ erythrocytes were lysed after staining using FACS lysing solution (BD Biosciences, San Jose, CA). Cells with the forward and side scatter mice, presumably due to an impaired clearance of immune com- properties of lymphocytes were analyzed on a FACScan flow cytometer plexes (43). (BD Biosciences) with fluorescence intensity shown on a 4-decade log The exact mechanisms by which complement and complement scale. Fluorescence contours for 5000 cells/sample are shown as 50% log receptors affect humoral immune responses remain uncertain. C3d density plots. Positive and negative populations of cells were determined http://www.jimmunol.org/ using unreactive isotype-matched mAbs (Caltag Laboratories) as controls binding to CD21 supplies an already characterized ligand for the for background staining. Background levels of staining were delineated CD19 complex, thereby linking complement activation and B cell using gates positioned to include Ͼ98% of the control cells. function. However, CD19 may also have signaling roles and li- Immunization of mice gand-binding activities independent of CD21 (1, 8). Alternatively, FDC expression of CD21/35 may be required for the generation of Two-month-old littermates were immunized i.p. with 50 ␮g of T cell- humoral immunity. In one study, normal humoral immune re- independent type 1 Ag, 2,4,6-trinitrophenol-conjugated LPS (TNP-LPS; Sigma, St. Louis, MO) in saline. Other littermates were immunized i.p. sponses require CD21/35 expression on FDCs (44). Another study with 100 ␮g of the TD Ag, DNP-keyhole limpet hemocyanin (DNP-KLH; has reported that B cell but not FDC expression of complement Calbiochem-Novabiochem, La Jolla, CA) in CFA and were boosted 21 receptors is required for humoral immune responses (45). In a third days later with DNP-KLH in adjuvant. Serum was collected before and by guest on September 25, 2021 study, optimal humoral immune responses required a combination after immunization. of complement-derived CD21 ligands on FDCs and CD21 on B Mouse Ig isotype-specific ELISAs cells (46). Furthermore, CD21 may provide an Ag-independent IgM and IgG1 concentrations in sera were determined by ELISA using signal required for the survival of B cells in germinal centers (47). affinity-purified mouse IgM and IgG1 (Southern Biotechnology Associ- To determine the extent that CD19 function is controlled by com- ates) to generate standard curves as described (29). Relative Ig concentra- Ϫ Ϫ Ϫ Ϫ plement activation, CD21/35 / and C3 / mice were generated tions in individual samples were determined by comparing the mean OD that either lack or overexpress CD19. The phenotypes of these values obtained for duplicate wells to a semilog standard curve of titrated mice demonstrate that CD19, CD21, and C3 expression are inter- standard Ab using linear regression analysis. TNP- and DNP-specificAb titers of sera were measured as described (31), using 96-well microtiter related and may thus form a regulatory loop that influences B cell ELISA plates (Costar, Cambridge, MA) coated with 5 ␮g/ml TNP-BSA function. In addition, the functional properties of these mice dem- (Biosearch Technologies, San Rafael, CA) and DNP-BSA (Calbiochem- onstrate that CD19 can regulate B cell signaling thresholds inde- Novabiochem). ELISA color development was allowed to progress until pendent of complement activation. the wells containing the highest Ab levels reached OD values of ϳ2.0. These OD values were determined to be within the linear range of the ELISA using sera over multiple dilutions. Serum IgM and IgG anti-dsDNA Materials and Methods levels were determined by ELISA using 96-well microtiter plates coated with 5 ␮g/ml calf dsDNA (Sigma), as described (9). Mice Immunoprecipitation and Western blot analysis CD21/35Ϫ/Ϫ (129 ϫ C57BL/6), C3Ϫ/Ϫ (129 ϫ C57BL/6), CD19Ϫ/Ϫ (C57BL/6, Ͼ6 generations), and hCD19TGϩ/ϩ (C57BL/6, Ͼ12 genera- Splenic B cells were purified by removing T cells with anti-Thy 1.2 Ab- tions) mice were generated as described (29, 32, 33, 40). Specifically, the coated magnetic beads (Dynal, Lake Success, NY). Purified (wild-type, hCD19TGϩ/ϩ mice used were from the TG-1 line, which expresses 2.6- Ͼ95%; CD21/35Ϫ/Ϫ, Ͼ93%; hCD19TGϩ/ϩ, Ͼ89% B220ϩ) splenic B fold higher levels of CD19 (20). CD19Ϫ/ϪCD21/35Ϫ/Ϫ, CD19Ϫ/ϪC3Ϫ/Ϫ, cells were lysed in buffer containing 1% digitonin, 10 mM triethanolamine, hCD19TGϩ/ϩCD21/35Ϫ/Ϫ, and hCD19TGϩ/ϩC3Ϫ/Ϫ littermates were gen- 150 mM NaCl, 5 mM EDTA, and protease inhibitors (pH 7.8). For im- erated through breedings of homozygous single-mutant mice to generate munoprecipitations, the cell lysates were precleared twice with mouse IgG heterozygous offspring at each locus. Heterozygous offspring were crossed plus protein G-Sepharose beads (Amersham Pharmacia Biotech, Uppsala, to generate littermates homozygous at each locus and wild-type control Sweden), followed by incubation with protein G beads plus 7G6 Ab for 3 h offspring. In all cases, results with wild-type littermates from each breeding at 4°C. CD19 was immunoprecipitated using anti-mCD19 (MB19-1) or group (CD19Ϫ/Ϫ, hCD19TGϩ/ϩ, CD21/35Ϫ/Ϫ, and C3Ϫ/Ϫ) were similar anti-hCD19 (HB12b) Abs covalently attached to Affigel 10 beads (Bio- and were therefore pooled. Thereby, any potential background genetic ef- Rad, Hercules, CA). After washing the beads with lysis buffer four times, fects were distributed throughout the test population without regard to the immunoprecipitates were subjected to SDS-PAGE, with subsequent elec- disrupted gene loci. All mice used were 2–3 mo of age unless indicated trophoretic transfer to nitrocellulose membranes. The membranes were in- otherwise, and were housed in a specific pathogen-free barrier facility. All cubated with goat anti-mouse CD21 polyclonal antisera (D-19 or M-19; studies and procedures were approved by the Animal Care and Use Com- Santa Cruz Biotechnology, Santa Cruz, CA), followed by incubation with mittee of Duke University. HRP-conjugated donkey anti-rabbit IgG Abs (Jackson ImmunoResearch 3192 CD19 REGULATES SIGNAL TRANSDUCTION INDEPENDENT OF COMPLEMENT

Laboratories, West Grove, PA). Blots were developed using an ECL CD19 expression on B220high mature bone marrow B cells from (Pierce, Rockford, IL). CD21/35Ϫ/Ϫ mice was significantly increased (19% higher) rela- PCR amplification and sequencing of CD21 transcripts tive to wild-type littermates (Fig. 1, A and B). CD19 expression was similar on immature B220low B cells from CD21/35Ϫ/Ϫ and Cytoplasmic RNA free of DNA contamination was isolated from spleno- Ϫ Ϫ wild-type littermates (Fig. 1A), consistent with a lack of or low- cytes of wild-type and CD21/35 / littermates using a RNeasy Mini kit (Qiagen, Chatsworth, CA) according to the manufacturer’s instructions. level CD21/35 expression by immature bone marrow B cells (50). Ϫ Ϫ Equal amounts of RNA were used for cDNA synthesis and PCR amplifi- CD19 expression was also increased on CD21/35 / B cells from cation. The region spanning 9/10 in the cDNA (25) was amplified blood, spleen, and lymph nodes by 16, 36, and 31%, respectively using a sense primer identical with sequence in 8 (5Ј-GGA CAG CTG Ј (Fig. 1). By contrast, mCD19 expression by mature B cells from TTA ATT CTT CTT GTG-3 ) and an antisense primer identical with se- Ϫ/Ϫ quence in exon 11 (5Ј-TCA TAA GTA TAT CCA GTC AAC TGG-3Ј)to bone marrow, blood, spleen, and lymph nodes of C3 mice was generate a 622-bp fragment in wild-type cDNA. The conditions used for not significantly different from wild-type levels (Fig. 1B). Thus, PCR amplification were: 94°C for 3 min, then 30 cycles at 94°C for 1 min, CD21/35 expression regulates cell surface CD19 expression levels, 58°C for 1 min, followed by 72°C for 1 min. The PCR products were whereas C3 deficiency does not affect CD19 expression by the electrophoresed and visualized by ethidium bromide staining. Amplified PCR products were purified from agarose gels using the QIAquick gel majority of B cells. purification kit (Qiagen) and were sequenced directly in both directions Whether CD21/35 or C3 expression affected hCD19 expression using an ABI 377 PRISM DNA sequencer after amplification using the in transgenic mice that overexpress CD19 was also assessed. PerkinElmer Dye Terminator Sequencing system with AmpliTaq DNA hCD19 expression levels on B cells from bone marrow or periph- polymerase and the same primers that were used for the initial PCR eral tissues of hCD19TGϩ/ϩ mice were not affected by the absence amplification. Downloaded from of CD21/35 or C3 expression (data not shown). However, B cells Statistical analysis from blood, spleen, and lymph nodes of hCD19TGϩ/ϩ mice ex- ANOVA was used to analyze the data, with Student’s t test used to deter- pressed mCD19 at 29, 20, and 20% lower levels, respectively, than mine the level of significance for differences between sample means. B cells from wild-type littermates (Fig. 1), as previously reported (20). In the absence of CD21/35 expression, mCD19 was ex- Results pressed at wild-type levels in hCD19TGϩ/ϩ mice in these tissues http://www.jimmunol.org/ CD21/35 expression negatively regulates mCD19 expression (Fig. 1). C3 deficiency did not significantly alter mCD19 expres- Because CD19 and CD21/35 physically associate on the cell sur- sion levels in hCD19TGϩ/ϩ mice. Thus, CD21/35 expression neg- face, it was assessed whether loss of CD21/35 expression affected atively influenced endogenous mCD19 expression levels, but did CD19 expression. As determined by immunofluorescence staining, not influence cell surface hCD19 expression. by guest on September 25, 2021

FIGURE 1. Cell surface CD21/35 expression influences mCD19 expression by B220ϩ lym- phocytes. A, Single-cell lymphocyte suspensions were isolated from littermates of each mouse ge- notype and examined simultaneously by two- color immunofluorescence staining with flow cy- tometric analysis. Mouse CD19 expression was visualized using PE-conjugated 1D3 mAb. Ver- tical dashed lines in each histogram are provided for reference. These results are representative of those obtained with cells from at least five lit- termates of each genotype. B, Relative cell sur- face mCD19 densities were determined by com- paring mean linear fluorescence intensity channel numbers for 1D3-PE staining between B cells from wild-type and transgenic littermates of each genotype. Values represent the mean (ϮSEM) percentage of wild-type expression lev- els obtained from at least five sets of littermates of each genotype. Mean values significantly dif- p Ͻ ,ء) ferent from wild-type levels are indicated .(p Ͻ 0.01 ,ءء ;0.05 The Journal of Immunology 3193

CD19 expression influences CD21/35 expression CD19 regulates B cell development independent of CD21/35 or Because CD21/35 expression negatively influences cell surface C3 expression mCD19 levels, the effect of CD19 deficiency on CD21/35 expres- In the current studies using CD19Ϫ/Ϫ, hCD19TGϩ/ϩ, CD21/ sion was assessed. CD21/35 expression levels on blood B cells 35Ϫ/Ϫ, and C3Ϫ/Ϫ littermates, CD19 deficiency and overexpres- from CD19Ϫ/Ϫ mice were 25% lower than on B cells from wild- sion had significant effects on the development and expansion of type littermates (Fig. 2). Nonetheless, CD19 deficiency did not peripheral B cells, whereas CD21/35 and C3 deficiencies had mod- significantly affect CD21/35 expression by B cells in spleen or est effects (Fig. 3). In all cases, combined CD19 deficiency or lymph nodes (Fig. 2). Similar results were obtained when using overexpression with CD21/35 or C3 deficiencies did not have ad- either the 7E9 or 7G6 mAbs (data not shown) that bind distinct ditive influences on B cell development in the bone marrow, blood, regions of the CD21/35 molecule (49). Thus, basal mCD19 ex- spleen, or peritoneal cavity (Fig. 3 and data not shown). Although pression levels do not significantly modulate CD21/35 expression the frequency and number of B220lowCD5ϩ B1 cells is signifi- levels. By contrast, mature B cells from hCD19TGϩ/ϩ mice ex- cantly inhibited or augmented by CD19 deficiency or overexpres- pressed ϳ57% less CD21/35 than wild-type B cells (Fig. 2). That sion, respectively, the frequency and number of B220lowCD5ϩ B CD19 overexpression results in a significant decrease in CD21/35 cells was not significantly affected by CD21/35 or C3 deficiencies expression may relate to the hyperresponsive status of these B (Fig. 3). Thus, the effects of CD19 deficiency or overexpression on cells. B cell development were not significantly influenced by CD21/35 or C3 expression. C3 expression negatively influences CD21/35 expression Downloaded from Because C3 cleavage fragments are ligands for CD21/35, the effect of C3 deficiency on CD21/35 expression was assessed. Blood, CD19 regulates IgM and IgD expression independent of CD21 spleen, and lymph node B cells from C3Ϫ/Ϫ mice had significantly or C3 expression increased CD21/35 expression (46, 33, and 31%, respectively) Consistent with CD19 regulating intrinsic signaling thresholds, when compared with wild-type B cells (Fig. 2). Similar results mature B cells from CD19-deficient or -overexpressing mice have were obtained when using either the 7E9 or 7G6 mAbs (data not significantly altered surface IgM and IgD levels (Fig. 4). By con- http://www.jimmunol.org/ shown). Similarly, CD19Ϫ/Ϫ littermates that were C3-deficient had trast, blood, spleen, and lymph node B cells from CD21/35Ϫ/Ϫ or increased CD21/35 expression (Fig. 2B). CD21/35 expression was C3Ϫ/Ϫ littermates had wild-type levels of IgM and IgD expression higher on B cells from hCD19TGϩ/ϩC3Ϫ/Ϫ mice than (Fig. 4). In all cases, combined CD19 deficiency or overexpression hCD19TGϩ/ϩ littermates, although the levels of CD21/35 expres- with CD21/35 or C3 deficiencies did not have significant additive sion remained below those of wild-type littermates (Fig. 2B). Thus, influences on IgM or IgD expression by B cells from blood, spleen, C3 deficiency resulted in significantly increased CD21/35 expres- or lymph nodes (Fig. 4). Therefore, the effects of CD19 deficiency sion by mature B cells. or CD19 overexpression on B cell Ag receptor expression were not by guest on September 25, 2021

FIGURE 2. Cell surface mCD19 expression influ- ences CD21/35 expression by B220ϩ lymphocytes. A, CD21/35 expression was assessed as in Fig. 1 using FITC-conjugated 7G6 anti-CD21/35 mAb. Vertical dashed lines in each histogram are provided for ref- erence. These results are representative of those ob- tained with cells from at least five littermates of each genotype. B, Relative cell surface CD21/35 densities were determined by comparing mean linear fluores- cence intensity channel numbers for 7G6-FITC stain- ing between B cells from wild-type and transgenic littermates of each genotype. Values represent the mean (ϮSEM) percentage of wild-type expression levels obtained from at least five sets of littermates of each genotype. Mean values significantly different ,ءء ;p Ͻ 0.05 ,ء) from wild-type levels are indicated p Ͻ 0.01). 3194 CD19 REGULATES SIGNAL TRANSDUCTION INDEPENDENT OF COMPLEMENT Downloaded from

FIGURE 3. B cell numbers in littermates with altered CD19, CD21/35, and C3 expression. Single-cell lymphocyte suspensions were isolated from littermates of each mouse genotype and examined simultaneously by two-color immunofluorescence staining with flow cytometric analysis and gating as described (9). The histograms indicate mean (ϮSEM) percentages or numbers of lymphocytes expressing the indicated cell surface markers from each tissue. B cell numbers were calculated based on the total number of cells harvested from each tissue, except for blood values that indicate numbers of cells http://www.jimmunol.org/ per milliliter. Values represent results from 36 wild-type, 12 CD19Ϫ/Ϫ, 11 CD19Ϫ/ϪCD21/35Ϫ/Ϫ, 4 CD19Ϫ/ϪC3Ϫ/Ϫ, 18 hCD19TGϩ/ϩ, 9 hCD19TGϩ/ Ϫ Ϫ ϩ ϩ Ϫ Ϫ Ϫ Ϫ Ϫ Ϫ ϩCD21/35 / , 11 hCD19TG / C3 / , 18 CD21/35 / , and 17 C3 / littermates. Mean values significantly different from wild-type levels are indicated .(p Ͻ 0.01 ,ءء ;p Ͻ 0.05 ,ء) significantly influenced by CD21/35 or C3 expression. Thus, al- hCD19TGϩ/ϩC3Ϫ/Ϫ littermates. Thus, CD21/35 or C3 loss did though CD19 expression levels regulate signaling thresholds that not significantly affect autoantibody production in influence cell surface IgM and IgD density, the loss of CD21 or C3 hCD19TGϩ/ϩ littermates. does not alter thresholds to an extent that affects Ag receptor by guest on September 25, 2021 expression. CD19 function dominates during humoral immune responses The influence of CD19, CD21/35, and C3 expression on humoral CD19 function dominates during B cell differentiation immune responses was assessed by immunizing littermates with a The effects of CD21/35 or C3 deficiency on B cell differentiation T cell-independent type 1 Ag, TNP-LPS, and a TD Ag, DNP- Ϫ Ϫ ϩ ϩ in CD19 / and hCD19TG / littermates were assessed (Fig. KLH. Following immunization with TNP-LPS, wild-type, CD21/ 5A). Although serum IgM levels were decreased by 70% in 35Ϫ/Ϫ, and C3Ϫ/Ϫ littermates generated comparable primary IgM Ϫ Ϫ ϩ ϩ CD19 / mice and increased by 96% in hCD19TG / littermates, responses (Fig. 6A). CD19Ϫ/Ϫ littermates demonstrated modest Ϫ Ϫ Ϫ Ϫ CD21/35 / and C3 / littermates had wild-type IgM levels IgM responses to TNP-LPS that were not significantly affected by Ϫ Ϫ Ϫ Ϫ (Fig. 5A). Serum IgM levels in CD19 / CD21/35 / and the additional loss of CD21 or C3 expression. Given the signifi- Ϫ Ϫ Ϫ Ϫ Ϫ Ϫ CD19 / C3 / littermates were similar to those of CD19 / lit- cantly reduced numbers of peripheral B cells in hCD19TGϩ/ϩ ϩ ϩ Ϫ Ϫ termates. Likewise, IgM levels of hCD19TG / CD21/35 / and mice relative to wild-type littermates (Fig. 3), hCD19TGϩ/ϩ mice ϩ ϩ Ϫ Ϫ hCD19TG / C3 / littermates were comparable to those of generate significant humoral immune responses (31). IgM re- ϩ ϩ Ϫ Ϫ ϩ ϩ hCD19TG / littermates. CD19 / and hCD19TG / littermates sponses by CD19TGϩ/ϩ, hCD19TGϩ/ϩCD21/35Ϫ/Ϫ, and had 89% decreased and 132% increased IgG1 levels, respectively. hCD19TGϩ/ϩC3Ϫ/Ϫ littermates were comparable, but elevated Ϫ Ϫ Unexpectedly, C3 / mice had 98% higher IgG1 levels than compared with responses of wild-type littermates. Ϫ Ϫ CD21/35 / or wild-type littermates (Fig. 5A). However, combin- Following DNP-KLH immunizations, primary and secondary ing CD21/35 and C3 deficiencies with alterations in CD19 expres- IgM responses were similar in wild-type, hCD19TGϩ/ϩ, CD21/ sion did not significantly alter IgG1 levels beyond the CD19-in- 35Ϫ/Ϫ,C3Ϫ/Ϫ, hCD19TGϩ/ϩCD21/35Ϫ/Ϫ, and hCD19TGϩ/ϩ duced changes. C3Ϫ/Ϫ littermates (Fig. 6B). By contrast, primary and secondary IgM responses to DNP-KLH were modest in CD19Ϫ/Ϫ mice and CD19 function dominates during autoimmunity their CD19Ϫ/ϪCD21/35Ϫ/Ϫ and CD19Ϫ/ϪC3Ϫ/Ϫ littermates. Because hCD19TGϩ/ϩ mice produce autoantibodies including CD19Ϫ/Ϫ, CD19Ϫ/ϪCD21/35Ϫ/Ϫ, and CD19Ϫ/ϪC3Ϫ/Ϫ littermates anti-dsDNA Abs (9, 51), the effects of CD21/35 or C3 loss on also showed similar profound defects in their primary and second- anti-dsDNA Ab production were assessed in 5-mo-old littermates ary IgG1 responses to DNP-KLH. Primary IgG1 responses to (Fig. 5B). IgM and IgG anti-dsDNA Abs were increased by 510 DNP-KLH were similarly delayed in hCD19TGϩ/ϩ, CD21/35Ϫ/Ϫ, and 340% in hCD19TGϩ/ϩ mice compared with wild-type lit- and C3Ϫ/Ϫ littermates (Fig. 6B). These effects were not additive termates, respectively. By contrast, anti-dsDNA Ab levels were because primary IgG1 responses were similar in hCD19TGϩ/ϩ, not significantly different in wild-type, CD19Ϫ/Ϫ, CD21/35Ϫ/Ϫ, hCD19TGϩ/ϩCD21/35Ϫ/Ϫ, and hCD19TGϩ/ϩC3Ϫ/Ϫ litter- or C3Ϫ/Ϫ littermates. Anti-dsDNA Ab levels were also compa- mates. Nonetheless, secondary IgG1 responses were compara- rable in hCD19TGϩ/ϩ, hCD19TGϩ/ϩCD21/35Ϫ/Ϫ, and ble in hCD19TGϩ/ϩ, hCD19TGϩ/ϩCD21/35Ϫ/Ϫ, and The Journal of Immunology 3195

hCD19TGϩ/ϩC3Ϫ/Ϫ littermates. Thus, humoral immune re- sponses were largely regulated by expression of CD19 with min- imal contributions from CD21/35 or C3.

CD21 preferentially associates with mCD19 in hCD19TGϩ/ϩ B cells Expression of CD21/35 on hCD19TG B cells selectively affected mCD19 but not hCD19 expression (Fig. 1). Because we have pre- viously stated that hCD19 can associate with mouse CD21/35 at detectable levels when expressed in mouse B cells that are mCD19Ϫ/Ϫ (20), it was assessed whether CD21/35 preferentially associates with mCD19 when both hCD19 and mCD19 are ex- pressed. CD19-associated proteins were immunoprecipitated from digitonin-lysed hCD19TGϩ/ϩ and wild-type B cells, transferred to nitrocellulose, and probed using two different antisera reactive with either the amino- or carboxyl-terminal regions of CD21. Pre- cipitated mCD19 coimmunoprecipitated CD21/35 proteins of ϳ 190, 165 (Fig. 7A, upper arrow), 157, 147, 141, and 120 kDa Downloaded from from wild-type B cells (Fig. 7A). These proteins were also immu- noprecipitated using the anti-CD21/35 mAb 7G6 (Fig. 7A). The 190-kDa band most likely represents CD35, whereas the other forms of CD21/35 protein are likely to result from the differential use of exons by CD21 and CD21/35 protein isoforms as described (25, 26). Although coprecipitated at lower levels, a similar spec- http://www.jimmunol.org/ trum of CD21/35 proteins was immunoprecipitated from hCD19TG B cells using anti-mCD19 mAb when the autoradio- graphs were visualized after further exposure. Reduced levels of FIGURE 4. Cell surface IgM and IgD expression levels in littermates coprecipitated CD21/35 protein are most likely to reflect reduced with altered CD19, CD21/35, or C3 expression. Relative cell surface IgM CD21/35 expression by hCD19TG B cells (Fig. 2). By contrast, or IgD densities were determined by comparing mean linear fluorescence coprecipitation of CD21/35 was never detected with hCD19 im- intensity channel numbers for immunofluorescence staining between B munoprecipitated from wild-type or hCD19TGϩ/ϩ B cells, al- cells from wild-type and transgenic littermates of each genotype. Values though the anti-hCD19 Ab efficiently precipitated hCD19 (data not Ϯ represent the mean ( SEM) percentage of wild-type expression levels ob- shown). Coprecipitation of CD21/35 was never detected with by guest on September 25, 2021 tained from at least five sets of littermates of each genotype. Mean values hCD19 immunoprecipitated from wild-type or hCD19TGϩ/ϩ B ,ءء ;p Ͻ 0.05 ,ء) significantly different from wild-type levels are indicated p Ͻ 0.01).

FIGURE 5. Serum Ig and anti-dsDNA Ab levels in littermates with altered CD19, CD21/35, or C3 expres- sion. A, Values represent mean (ϮSEM) serum Ig lev- els for at least nine littermates of each genotype as determined by Ab isotype-specific ELISAs. B, Serum anti-dsDNA Ab levels were determined by ELISAs at 5 mo of age. Each histogram represents the mean (ϮSEM) OD value obtained for sera from at least five littermates of each genotype. Mean values significantly p Ͻ ,ء) different from wild-type levels are indicated .(p Ͻ 0.01 ,ءء ;0.05 3196 CD19 REGULATES SIGNAL TRANSDUCTION INDEPENDENT OF COMPLEMENT

FIGURE 6. Humoral immune responses in lit- termates with altered CD19, CD21/35, or C3 ex- pression. Values represent mean (ϮSEM) serum Ab levels in at least five littermates of each ge- notype. A, Mice of each genotype were injected i.p. with 50 ␮g of TNP-LPS in saline on day 0 and bled on days 0, 7, 14, and 21. Relative anti- TNP IgM levels were determined by ELISA. B, Mice of each genotype were injected i.p. with 100 ␮g of DNP-KLH in CFA on days 0 and 21 and bled on days 0, 7, 14, and 28. Relative anti-

DNP IgM and IgG1 levels were determined by Downloaded from ELISA. Mean values significantly different from ,ءء ;p Ͻ 0.05 ,ء) wild-type levels are indicated p Ͻ 0.01). http://www.jimmunol.org/

cells even when the autoradiographs for mCD19 immunoprecipi- 35Ϫ/Ϫ mice were generated by targeting exons encoding SCRs tations were significantly overexposed. Therefore, CD21/35 may 9–10 of CD21, cDNA generated from CD21/35Ϫ/Ϫ B cells was associate with hCD19 at detectable levels in the absence of RT-PCR amplified using a forward primer specific for the exon by guest on September 25, 2021 mCD19 expression, but CD21/35 preferentially associates with encoding SCR 8 and a reverse primer specific for SCR 11. mCD19 when both hCD19 and mCD19 are expressed. This sug- CD21/35 transcripts were readily detected in wild-type and CD21/ ϩ/ϩ gests that hCD19 regulates signaling thresholds in hCD19TG 35Ϫ/Ϫ B cells, although exon 9/10 appeared to be selectively B cells independent of an association with CD21/35. spliced out in transcripts from CD21/35Ϫ/Ϫ mice (Fig. 7D). Se- Ϫ/Ϫ Ϫ Ϫ quence analysis of the PCR products verified that CD21/35 CD21/35 / mice express hypomorphic CD21/35 mice generated CD21/35 transcripts splicing out the exon encod- A surprising observation was that CD19 coprecipitated a number ing SCRs 9–10, but with in-frame coding sequence (Fig. 7E). Ϫ/Ϫ of proteins from CD21/35 B cells, although these proteins were Thus, CD21/35Ϫ/Ϫ mice produce hypomorphic CD21/35Ϫ/Ϫ pro- consistently 14 kDa smaller than those coprecipitated from wild- teins lacking the SCRs encoded by Cr2 exons 9/10 (25). type B cells (Fig. 7A, lower arrow). These proteins were CD21 because they reacted with two different antisera specific for either the amino-or carboxyl-terminal regions of CD21 and were precip- Discussion itated by the 7G6 Ab (Fig. 7A and data not shown). In four dif- This study demonstrates that CD19 predominantly regulates B cell ferent immunoprecipitations, the CD21-like proteins were abun- signal transduction independent of complement activation. The dant and were readily coprecipitated with mCD19 from CD21/ Ϫ/Ϫ Ϫ Ϫ Ϫ Ϫ hallmark characteristics of CD19 B cells were not significantly 35 / B cells. Thus, CD21/35 / B cells appear to express a hypomorphic CD21/35 protein product. altered when combined with CD21/35 or C3 deficiencies (Figs. The genotype of the CD21/35Ϫ/Ϫ mice was verified using 3–6). More important is that hCD19 expression by mouse B cells Southern blot analysis (Fig. 7B) as described in the original paper results in CD19 overexpression and a dramatic hyperresponsive characterizing these mice (33). Immunofluorescence staining of B phenotype (29). However, the hallmark characteristics of hyperre- cells from CD21/35Ϫ/Ϫ mice using two independent anti-CD21/35 sponsive B cells in hCD19TG mice were not significantly altered mAbs verified surface expression of the hypomorphic CD21/35 when combined with CD21/35 or C3 deficiencies (Figs. 3–6). protein (Fig. 7C). When assessed using the 7E9 mAb in two-color Therefore, the hyperresponsive phenotype of B cells that overex- immunofluorescence staining experiments, overall CD21/35 ex- press CD19 is unlikely to result from increased sensitivity to com- pression by B220ϩ blood and spleen B cells from CD21/35Ϫ/Ϫ plement activation. In addition, biochemical studies revealed that mice was 37 Ϯ 6% of wild-type levels (n ϭ 3, data not shown). little if any hCD19 associates with CD21/35 in hCD19TG B cells When assessed using the 7G6 mAb, blood and spleen B220ϩ B when mCD19 is expressed (Fig. 7A). Thus, hCD19 is not equiv- cells from CD21/35Ϫ/Ϫ mice expressed CD21/35 at 24 Ϯ 2% of alent to mCD19 in generating CD19-CD21 complexes in mouse B wild-type levels (n ϭ 5, data not shown), which was consistent cells, although hCD19 expression at wild-type levels can restore with the immunoprecipitation results (Fig. 7A). Because CD21/ normal function in CD19Ϫ/Ϫ mice (20). These findings do not rule The Journal of Immunology 3197 Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 7. CD21/35Ϫ/Ϫ mice express hypomorphic CD21/35. A, Coimmunoprecipitation of CD19 and CD21 from detergent lysates of purified splenic B cells (5 ϫ 107/lane) from wild-type, hCD19TGϩ/ϩ, and CD21/35Ϫ/Ϫ littermates. Cell lysates were incubated with the indicated Abs covalently bound to beads. The immunoprecipitated proteins or total cell lysate (2 ϫ 106 cells/lane) were subjected to SDS-PAGE and transferred onto nitrocellulose. The membrane was incubated with goat anti-mouse CD21 polyclonal antisera (M-19), followed by incubation with HRP-conjugated secondary Ab. Similar results were obtained in four experiments. B, Southern blot analysis of tail DNA from wild-type, CD21/35ϩ/Ϫ, and CD21/35Ϫ/Ϫ littermates digested with BamHI and hybridized with probe as previously described (33). The upper band (10 kb) corresponds to the targeted allele, and the lower band (8 kb) represents the wild-type Cr2 allele. C, Flow cytometric analysis of CD21/35 expression on spleen cells from wild-type (thin unbroken lines) and CD21/ 35Ϫ/Ϫ (thick lines) littermates. Cell surface expression of CD21/35 was visualized using 7E9 (visualized using FITC-labeled secondary Ab) and 7G6 (FITC-conjugated) Abs. Cell staining with unreactive isotype-matched control Abs is shown as a dashed histogram. Similar results were obtained when the splenocytes were stained with the primary Ab in the presence of 5% mouse serum or Fc blockade. These results represent those obtained with at least 10 littermates examined in separate experiments. D, Cytoplasmic RNA from splenocytes was reverse transcribed and amplified by PCR with primers specific for CD21/35 exons 8 and 11. The PCR products were electrophoresed and visualized by ethidium bromide staining, with the expected sizes indicated. E, The amplified PCR products described in D were purified from agarose gels and sequenced on both strands. out the possibility that complement activation and CD21/35 en- receptor internalization. During inflammatory responses or in pa- gagement generate transmembrane signals through CD19, but sug- tients with systemic , serum immune com- gest that CD19 predominantly regulates signal transduction plexes can be loaded with C3d. In fact, B cells from patients with through complement-independent mechanisms. systemic lupus erythematosus have decreased CD21 (51–53) and Cell surface CD21/35 expression levels were significantly af- CD19 expression levels (51), presumably due to the continuous fected by C3 and CD19 expression. Specifically, CD21/35 expres- presence of CD21/35 ligands that induce CD21 internalization sion was increased by 31–46% on peripheral B cells from C3- (54). In lupus-prone MRLlpr/lpr mice, CD21/35 expression levels deficient littermates (Fig. 2). This suggests that ongoing C3d,g are also specifically decreased on B cells, even before the devel- fragment generation may chronically engage CD21/35, resulting in opment of clinical or serological manifestations of disease (50). 3198 CD19 REGULATES SIGNAL TRANSDUCTION INDEPENDENT OF COMPLEMENT

The 44–58% decrease in CD21/35 expression (Fig. 2) and 20– down-regulated surface IgM expression is a consequence of im- 29% decrease in mCD19 expression (Fig. 1) by B cells from paired or augmented transmembrane signaling through the B cell hCD19TGϩ/ϩ mice may also result from increased CD21/35 re- Ag receptor complex, respectively (1, 63). Each of these results is ceptor internalization because these mice produce autoantibodies consistent with the conclusion that CD19 can function indepen- that may activate complement. Furthermore, CD21/35 expression dently of CD19/CD21 complex expression, although CD21/35 in autoimmune LynϪ/Ϫ mice (55–57) is reduced by 60% (our un- uses the CD19 complex to generate transmembrane signals published data). Thus, C3, CD21/35, and CD19 expression may be through the CD19 cytoplasmic domain (11). components of a common regulatory loop that influences B cell Primary and secondary humoral immune responses to TD and signaling thresholds. CD21/35 engagement may result in increased T-independent Ags were severely compromised in CD19Ϫ/Ϫ mice, CD19 turnover, which may limit signal transduction or partially particularly IgG1 responses to TD Ags (Fig. 6), as previously de- desensitize B cells chronically stimulated through the CD19-CD21 scribed (30, 31). By contrast, serum IgG1 responses to a TD Ag complex. were only delayed in CD21/35Ϫ/Ϫ and C3Ϫ/Ϫ littermates, and CD19 expression levels were significantly influenced by these mice generated normal primary IgM responses and second- CD21/35 and C3 expression (Fig. 1). CD19 expression levels were ary responses (Fig. 6). Delayed IgG1 responses to DNP-KLH in 16–36% higher on peripheral B cells of CD21/35Ϫ/Ϫ mice (Fig. CD21/35Ϫ/Ϫ and C3Ϫ/Ϫ littermates did not hinder the IgG1 re- 1), whereas CD21/35 deficiency did not influence cell surface IgM sponses of hCD19TGϩ/ϩ littermates when these genetic alter- or IgD expression levels (Fig. 4). Human CD19 expression by ations were combined. Therefore, there were dramatic quantitative hCD19TG B cells was not affected by CD21/35 deficiency, con- and qualitative differences between the immune responses of sistent with a lack of detectable physical interaction between these CD19-deficient mice and CD21/35Ϫ/Ϫ or C3Ϫ/Ϫ littermates. Al- Downloaded from xenogeneic molecules (Fig. 7A). Therefore, CD21/35 may influ- though CD19 deficiency always has profound effects on humoral ence mCD19 expression by regulating cell surface complex turn- immune responses, TD-immune responses and affinity maturation over. This contrasts with CD81-deficient mice where cell surface in CD21/35Ϫ/Ϫ mice vary depending on the Ag dose and use of CD19 expression is reduced by half (58–60). Increasing CD19 adjuvants (39, 64–66). Thus, alterations in CD19 expression can expression by 16–36% on CD21/35Ϫ/Ϫ B cells may be function- influence Ab production irregardless of CD21/35 or C3 expression. Ϫ/Ϫ ally significant because ϳ20% increases in CD19 expression pre- Although the CD21/35 mice used in the current studies have http://www.jimmunol.org/ dispose mice to autoimmunity, and similar increases in CD19 ex- been well characterized, these mice expressed low levels of a hy- pression correlate with autoantibody production in humans (51). In pomorphic CD21/35Ϫ/Ϫ molecule (Fig. 7). An appropriate geno- general, CD19 overexpression increases endogenous levels of ac- type was verified in CD21/35Ϫ/Ϫ mice by Southern blot analysis tivated Lyn, dysregulates tolerance, and results in autoantibody (Fig. 7B), as originally described (33). Cell surface CD21/35 ex- production (9, 51, 61). This may explain in part why CD21/35 pression was verified using two mAbs that react with different deficiency contributes to autoimmunity (41, 42). Normally, CD19 extracellular regions of the protein, with one Ab binding to the expression levels are developmentally regulated and tightly con- ligand-binding site in SCR domains 1–2 (Fig. 7C). PCR and nu- trolled (10, 20). In mice, the majority of early B lineage cells cleotide sequence analysis indicate that CD21/35 transcripts in the express mCD19 at relatively high levels in the bone marrow, with CD21/35Ϫ/Ϫ mice had spliced out exons 9/10 of CD21 (Fig. 7, D by guest on September 25, 2021 a 2.5-fold increase in expression during the transition from an im- and E), which is the Neor insertion site that generates the targeted mature B220low to a mature B220high CD21ϩ B cell (9, 25). There- (33). Thereby, CD21/35Ϫ/Ϫ mice produce a 14-kDa fore, it is interesting that peritoneal B1 cells express lower levels smaller cell surface CD21 protein that retains its ability to asso- of CD21 (50), yet express incrementally higher levels of CD19 ciate with mCD19 (Fig. 7A) and retains SCR domains 1 and 2 that than conventional B cells (9). Thus, regulated CD19 and CD21/35 normally mediate CD21 interactions with ligands (67, 68). In expression levels may balance intrinsic signal transduction thresh- many cases, exon length can affect RNA splicing in transcripts olds and B cell responsiveness to transmembrane signals. initiated from the endogenous promoter (69). An artificially large B cell development, surface IgM expression, and serum Ig lev- exon with an inserted selective marker may not be recognized by els were almost normal in CD21/35Ϫ/Ϫ and C3Ϫ/Ϫ littermates the splicing mechanism and may be skipped, thereby deleting the (Figs. 3–5), as previously described (33, 34, 39, 40). Although a mutated exon from the mRNA transcript. With CD21/35, this al- decrease in peritoneal B1 cell numbers has been reported in only lowed the inappropriate splicing of exons 8 and 11, which does not one line of CD21/35Ϫ/Ϫ mice (33, 34), a significant difference in induce frame-shift or a null allele. A similar strategy numbers or frequency of peritoneal B1 cells was not observed in was used to generate a second line of CD21/35Ϫ/Ϫ mice, where this study among the 17 littermate pairs assessed (Fig. 3). This exons encoding SCR 8 of CD21 (or SCR 14 of the CD21/35 pro- discrepancy may be due to the different genetic backgrounds as tein) were targeted (34). An absence of detectable C3 binding by described (62) or the housing of mice used in each study, because FDC or marginal zone B cells has been demonstrated for both lines we generated CD21/35Ϫ/Ϫ littermates through paired breedings of of CD21/35Ϫ/Ϫ mice (33, 66, 70). In addition, both C3- and CD21/ CD19ϩ/ϪCD21/35ϩ/Ϫ littermates or hCD19TGϩ/ϪCD21/35ϩ/Ϫ 35-deficient mice share similar phenotypic characteristics. None- littermates. The frequencies of peritoneal B1 cells were also nor- theless, it remains to be established whether the hypomorphic mal in C3Ϫ/Ϫ mice (Fig. 3) as previously shown for both C3Ϫ/Ϫ CD21/35 molecules expressed at low levels by CD21/35Ϫ/Ϫ mice and C4Ϫ/Ϫ mice (39). Therefore, a decreased frequency of peri- are functionally relevant because there is a threshold effect toneal B1 cells in CD21/35Ϫ/Ϫ mice may be a variable phenotypic for CD21/C3d,g interactions whereby a minimum concentration trait. Nonetheless, CD19Ϫ/Ϫ mice had significantly decreased pe- of cell surface CD21 is necessary to bind C3d,g-containing parti- ripheral B cells and peritoneal B1 cells, increased surface IgM cles (71). expression, and decreased serum Ig levels, regardless of CD21/35 That CD21 expression influences CD19 density on the cell sur- or C3 expression (Figs. 3–5). Likewise, hCD19TGϩ/ϩ mice had face provides another mechanism through which the complement significantly decreased numbers of mature B cells in the bone mar- system may regulate B cell signaling thresholds. Alterations in row and peripheral tissues, increased peritoneal B1 cells, down- CD19 expression are important because CD19 cell surface density regulated surface IgM expression, and increased serum Ig levels, intrinsically regulates Src family protein kinase activity in regardless of CD21/35 or C3 expression (Figs. 3–5). Increased or B cells (14, 72). CD21 engagement may further modulate B cell The Journal of Immunology 3199 activation by cross-linking and further augmenting CD19 function. 22. Tedder, T. F., L. T. Clement, and M. D. Cooper. 1984. Expression of C3d re- Although the identity and biological significance of CD19 ligands ceptors during human B cell differentiation: immunofluorescence analysis with the HB-5 monoclonal . J. Immunol. 133:678. remains to be demonstrated (1, 3), CD21-independent ligands may 23. Moore, M. D., N. R. Cooper, B. F. Tack, and G. R. Nemerow. 1987. Molecular also regulate the intrinsic signaling function of CD19. That CD19 cloning of the cDNA encoding the Epstein-Barr virus/C3d receptor (complement receptor type 2) of human B lymphocytes. Proc. Natl. Acad. Sci. USA 84:9194. can function and regulate B cell signaling thresholds independent 24. Weis, J. J., D. T. Fearon, L. B. Klickstein, W. W. Wong, S. A. Richards, of CD21/35 and C3 expression affirms an independent role for A. de Bruyn Kops, J. A. 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Alternative splicing of Cr2 gene tran- scripts predicts two distinct gene products that share homologous domains with gestions and discussions; Dr. Joel R. Ross for editorial help; and Naomi both human CR2 and CR1. J. Immunol. 144:3581. Hasegawa for technical assistance. 27. Ahearn, J. M., and D. T. Fearon. 1989. Structure and function of the complement receptors, CR1 (CD35) and CR2 (CD21). Adv. Immunol. 46:183. 28. Marchbank, K. J., C. C. Watson, D. F. Ritsema, and V. M. Holers. 2000. Ex- References pression of human (CR2, CD21) in Cr2Ϫ/Ϫ mice restores 1. Tedder, T. F., M. Inaoki, and S. Sato. 1997. The CD19/21 complex regulates humoral immune function. J. Immunol. 165:2354.

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