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ActivationTransduction Independent of Complement CD19 Can Regulate B Lymphocyte Signal

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

http://www.jimmunol.org/content/167/6/3190doi: 10.4049/jimmunol.167.6.3190

2001; 167:3190-3200; ;J Immunol 

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2001 by The American Association of9650 Rockville Pike, Bethesda, MD 20814-3994.The American Association of Immunologists, Inc.,

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CD19 Can Regulate B Lymphocyte Signal TransductionIndependent of Complement Activation1

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

B lymphocytes are critically regulated by signals transduced through the CD19-CD21 cell surface receptor complex, wherecomplement C3d binding to CD21 supplies an already characterized ligand. To determine the extent that CD19 function iscontrolled 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 CD21expression were significantly affected by the loss of CD21 and C3 expression, respectively. Mature B cells from CD21-deficientlittermates had �36% higher cell surface CD19 expression, whereas CD21/35 expression was increased by �45% on B cells fromC3-deficient mice. Negative regulation of CD19 and CD21 expression by CD21 and C3, respectively, may be functionally significantbecause small increases in cell surface CD19 overexpression can predispose to autoimmunity. Otherwise, B cell development andfunction 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 protein at low levels that associated withmouse 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, collectivelydemonstrate that CD19 can regulate B cell signaling thresholds independent of CD21 engagement and complementactivation. The Journal of Immunology, 2001, 167: 3190–3200.

B lymphocyte development and function are critically reg-ulated by signals transduced through the CD19-CD21cell surface receptor complex (1–3). The CD19-CD21

complex is composed of at least four proteins: CD19, CD21 (com-plement receptor 2), CD81, and CD225 (Leu13) (4–7). CD19 is amember of the Ig superfamily expressed exclusively on B cells andfollicular dendritic cells (FDC)3 (8). In B cells, CD19 is expressedby early pre-B cells from the time of heavy chain gene rearrange-ment until plasma cell differentiation (6, 9, 10). CD19 has an�240-aa cytoplasmic domain that is critical for CD19-CD21 com-plex signaling (8, 11, 12). Specifically, CD19 functions as a spe-cialized adapter protein for the amplification of Src family kinasesand as an interaction molecule for multiple signaling pathwayscrucial for modulating intrinsic and Ag receptor-induced signals(13–18). The cytoplasmic domains of human CD19 (hCD19) andmouse CD19 (mCD19) are highly homologous (19). In fact,hCD19 can replace mCD19 function when expressed at the ap-propriate site density in CD19�/� mice (20). Cell surface CD19 ispresent in molar excess of CD21 at all stages of B cell develop-ment, although CD19-CD21 complexes are presumed to represent1:1 complexes (4, 21).

CD21 is expressed on FDCs and mature B cells, with expressionfirst by IgMhighIgDlow transitional B cells (3, 22). CD21 is com-posed of an extracellular domain containing 15 or 16 repeatingstructural elements termed short consensus repeats (SCRs), a trans-membrane region, and a 34-aa cytoplasmic domain (23, 24). CD21and CD35 (complement receptor 1) are alternative splice productsof the sameCr2 gene in mice, but are encoded by different genesin humans (25, 26). In mice, CD35 is generated by the addition ofsix SCRs to the amino-terminal end of the CD21 protein. Well-characterized ligands for CD21 are the iC3b/C3d,g cleavage frag-ments of complement. These C3 cleavage products form covalentbonds with foreign Ags or immune complexes to generate C3d-Agcomplexes that are proposed to bind CD21 and regulate B cellfunction by signaling through the CD19 complex (1, 2, 7). CD35binds C3b and C4b, and serves as a cofactor for the hydrolysis ofC3b-Ag complexes into C3d-Ag complexes (10, 27). This processis important for the processing of Ag-Ab complexes and the finaldeposition of C3d-Ag complexes on the surface of B cells andFDCs through CD21. A recent study indicates that human CD21can physically associate with mCD19, and can restore humoralimmune response in CD21/35�/� mice (28).

Studies using mice that lack or overexpress CD19 indicate thatCD19 and/or CD19-CD21 complexes regulate signal transductionthresholds governing humoral immunity. B cells from CD19�/�

mice are hyporesponsive to a variety of transmembrane signals,which leads to significant defects during the later stages of B cellmaturation, clonal expansion, and differentiation (29–31). By con-trast, transgenic mice that overexpress hCD19 (hCD19TG�/�) arehyperresponsive to transmembrane signals and display severe al-terations during early stages of B cell development, which leads todiminished numbers of B cells in the peripheral pool (9, 29, 32).The development of B1 cells is severely decreased in CD19�/�

mice, whereas there is an increased frequency of B1 cells withinthe peritoneum and spleen of hCD19TG�/� mice (9, 29, 30). In

Department of Immunology, Duke University Medical Center, Durham, NC 27710

Received for publication May 3, 2001. Accepted for publication July 19, 2001.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby markedadvertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by National Institutes of Health Grants CA81776 andCA54464.2 Address correspondence and reprint requests to Dr. Thomas F. Tedder, Departmentof Immunology, Duke University Medical Center, Box 3010, Durham, NC 27710.E-mail address: thomas.tedder@duke.edu3 Abbreviations used in this paper: FDC, follicular dendritic cell; KLH, keyhole lim-pet hemocyanin; hCD19, human CD19; hCD19TG, transgenic mice that overexpresshCD19; mCD19, mouse CD19; SCR, short consensus repeat unit; TD, T cell-depen-dent; TNP-LPS, 2,4,6-trinitrophenol-conjugated LPS.

Copyright © 2001 by The American Association of Immunologists 0022-1767/01/$02.00

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two independent lines of CD21/35�/� mice, lymphocyte develop-ment, phenotypes, and numbers are normal (33, 34). A �40%reduction in the frequency of peritoneal B1 cells has been observedin one line of CD21/35�/� mice (33). Both lines of CD21/35�/�

mice exhibit markedly impaired primary and secondary humoralimmune responses and germinal center formation, especially IgGresponses to T cell-dependent (TD) Ags (33–35). This is not dueto a defect in B cell Ag receptor signaling in CD21/35�/� micebecause their B cells respond normally to IgM and/or CD40 cross-linking (33). Pretreatment of mice with either a CD21/35-specificmAb or a CD21-IgG fusion protein also blocks secondary humoralresponses (36–38). C3�/� mice have a normal phenotype and se-rum Ig levels, whereas mice that lack C4, which is required for C3activation, have decreased IgG1, IgG2a, and IgG3 levels (39, 40).Both C3�/� and C4�/� mice have modest TD immune responseswith defects in germinal center formation. In addition, complementand CD21/35 regulate the elimination of self-reactive B cells be-cause systemic lupus erythematosus-prone C57BL/6lpr/lpr micewith CD21/35 or C4 deficiencies have exacerbated disease andincreased autoantibody production (41, 42). Spontaneous autoim-munity has been observed in C4�/� mice, but not in CD21/35�/�

mice, presumably due to an impaired clearance of immune com-plexes (43).

The exact mechanisms by which complement and complementreceptors affect humoral immune responses remain uncertain. C3dbinding to CD21 supplies an already characterized ligand for theCD19 complex, thereby linking complement activation and B cellfunction. However, CD19 may also have signaling roles and li-gand-binding activities independent of CD21 (1, 8). Alternatively,FDC expression of CD21/35 may be required for the generation ofhumoral immunity. In one study, normal humoral immune re-sponses require CD21/35 expression on FDCs (44). Another studyhas reported that B cell but not FDC expression of complementreceptors is required for humoral immune responses (45). In a thirdstudy, optimal humoral immune responses required a combinationof complement-derived CD21 ligands on FDCs and CD21 on Bcells (46). Furthermore, CD21 may provide an Ag-independentsignal required for the survival of B cells in germinal centers (47).To determine the extent that CD19 function is controlled by com-plement activation, CD21/35�/� and C3�/� mice were generatedthat either lack or overexpress CD19. The phenotypes of thesemice demonstrate that CD19, CD21, and C3 expression are inter-related and may thus form a regulatory loop that influences B cellfunction. In addition, the functional properties of these mice dem-onstrate that CD19 can regulate B cell signaling thresholds inde-pendent of complement activation.

Materials and MethodsMice

CD21/35�/� (129 � C57BL/6), C3�/� (129 � C57BL/6), CD19�/�

(C57BL/6, �6 generations), and hCD19TG�/� (C57BL/6, �12 genera-tions) mice were generated as described (29, 32, 33, 40). Specifically, thehCD19TG�/� mice used were from the TG-1 line, which expresses 2.6-fold higher levels of CD19 (20). CD19�/�CD21/35�/�, CD19�/�C3�/�,hCD19TG�/�CD21/35�/�, and hCD19TG�/�C3�/� littermates were gen-erated through breedings of homozygous single-mutant mice to generateheterozygous offspring at each locus. Heterozygous offspring were crossedto generate littermates homozygous at each locus and wild-type controloffspring. In all cases, results with wild-type littermates from each breedinggroup (CD19�/�, hCD19TG�/�, CD21/35�/�, and C3�/�) were similarand were therefore pooled. Thereby, any potential background genetic ef-fects were distributed throughout the test population without regard to thedisrupted gene loci. All mice used were 2–3 mo of age unless indicatedotherwise, and were housed in a specific pathogen-free barrier facility. Allstudies and procedures were approved by the Animal Care and Use Com-mittee of Duke University.

Antibodies

Abs used in this study included: mouse IgA anti-mCD19 (MB19–1; Ref.9), mouse anti-hCD19 (HB12b; Ref. 48), rat anti-mouse CD21/35 (7E9:IgG2a, 7G6: IgG2b; provided by Dr. T. Kinoshita, Osaka University,Osaka, Japan: Ref. 49), FITC-conjugated anti-mouse CD21/35 (7G6; BDPharMingen, San Diego, CA), PE -conjugated anti-mCD19 (1D3; BDPharMingen), PE-conjugated anti-hCD19 (B4; Coulter, Miami, FL), PE-conjugated anti-CD5 (53-7.3; BD PharMingen), biotinylated or FITC-con-jugated anti-mouse IgM (Southern Biotechnology Associates, Birming-ham, AL), biotinylated or FITC-conjugated anti-B220 (CD45RA, RA3-6B2; provided by Dr. R. Coffman, DNAX Research Institute, Palo Alto,CA), and biotinylated anti-mouse IgD (Southern BiotechnologyAssociates) Abs. Anti-CD21/35 Ab binding (7E9) was visualized usingFITC- or PE-conjugated goat anti-rat IgG (H � L) Abs (Caltag Laborato-ries, Burlingame, CA) diluted to the appropriate concentration for optimalimmunostaining. PE-conjugated streptavidin (Southern Biotechnology As-sociates) was used to reveal biotin-coupled Ab staining.

Immunofluorescence analysis

Single-cell suspensions of lymphocytes from spleen, bone marrow, peri-toneal lavage, and peripheral lymph nodes were isolated before two-colorimmunofluorescence analysis. Leukocytes (0.5–1 � 106) were stained at4°C using predetermined optimal concentrations of Abs for 20 min. Blooderythrocytes were lysed after staining using FACS lysing solution (BDBiosciences, San Jose, CA). Cells with the forward and side light scatterproperties of lymphocytes were analyzed on a FACScan flow cytometer(BD Biosciences) with fluorescence intensity shown on a 4-decade logscale. Fluorescence contours for 5000 cells/sample are shown as 50% logdensity plots. Positive and negative populations of cells were determinedusing unreactive isotype-matched mAbs (Caltag Laboratories) as controlsfor background staining. Background levels of staining were delineatedusing gates positioned to include �98% of the control cells.

Immunization of mice

Two-month-old littermates were immunized i.p. with 50 �g of T cell-independent type 1 Ag, 2,4,6-trinitrophenol-conjugated LPS (TNP-LPS;Sigma, St. Louis, MO) in saline. Other littermates were immunized i.p.with 100 �g of the TD Ag, DNP-keyhole limpet hemocyanin (DNP-KLH;Calbiochem-Novabiochem, La Jolla, CA) in CFA and were boosted 21days later with DNP-KLH in adjuvant. Serum was collected before andafter immunization.

Mouse Ig isotype-specific ELISAs

IgM and IgG1 concentrations in sera were determined by ELISA usingaffinity-purified mouse IgM and IgG1 (Southern Biotechnology Associ-ates) to generate standard curves as described (29). Relative Ig concentra-tions in individual samples were determined by comparing the mean ODvalues obtained for duplicate wells to a semilog standard curve of titratedstandard Ab using linear regression analysis. TNP- and DNP-specific Abtiters of sera were measured as described (31), using 96-well microtiterELISA plates (Costar, Cambridge, MA) coated with 5 �g/ml TNP-BSA(Biosearch Technologies, San Rafael, CA) and DNP-BSA (Calbiochem-Novabiochem). ELISA color development was allowed to progress untilthe wells containing the highest Ab levels reached OD values of �2.0.These OD values were determined to be within the linear range of theELISA using sera over multiple dilutions. Serum IgM and IgG anti-dsDNAlevels were determined by ELISA using 96-well microtiter plates coatedwith 5 �g/ml calf thymus dsDNA (Sigma), as described (9).

Immunoprecipitation and Western blot analysis

Splenic B cells were purified by removing T cells with anti-Thy 1.2 Ab-coated magnetic beads (Dynal, Lake Success, NY). Purified (wild-type,�95%; CD21/35�/�, �93%; hCD19TG�/�, �89% B220�) splenic Bcells were lysed in buffer containing 1% digitonin, 10 mM triethanolamine,150 mM NaCl, 5 mM EDTA, and protease inhibitors (pH 7.8). For im-munoprecipitations, the cell lysates were precleared twice with mouse IgGplus protein G-Sepharose beads (Amersham Pharmacia Biotech, Uppsala,Sweden), followed by incubation with protein G beads plus 7G6 Ab for 3 hat 4°C. CD19 was immunoprecipitated using anti-mCD19 (MB19-1) oranti-hCD19 (HB12b) Abs covalently attached to Affigel 10 beads (Bio-Rad, Hercules, CA). After washing the beads with lysis buffer four times,immunoprecipitates were subjected to SDS-PAGE, with subsequent elec-trophoretic transfer to nitrocellulose membranes. The membranes were in-cubated with goat anti-mouse CD21 polyclonal antisera (D-19 or M-19;Santa Cruz Biotechnology, Santa Cruz, CA), followed by incubation withHRP-conjugated donkey anti-rabbit IgG Abs (Jackson ImmunoResearch

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Laboratories, West Grove, PA). Blots were developed using an ECL kit(Pierce, Rockford, IL).

PCR amplification and sequencing of CD21 transcripts

Cytoplasmic RNA free of DNA contamination was isolated from spleno-cytes of wild-type and CD21/35�/� littermates using a RNeasy Mini kit(Qiagen, Chatsworth, CA) according to the manufacturer’s instructions.Equal amounts of RNA were used for cDNA synthesis and PCR amplifi-cation. The region spanning exons 9/10 in the cDNA (25) was amplifiedusing a sense primer identical with sequence in exon 8 (5�-GGA CAG CTGTTA ATT CTT CTT GTG-3�) and an antisense primer identical with se-quence in exon 11 (5�-TCA TAA GTA TAT CCA GTC AAC TGG-3�) togenerate a 622-bp fragment in wild-type cDNA. The conditions used forPCR amplification were: 94°C for 3 min, then 30 cycles at 94°C for 1 min,58°C for 1 min, followed by 72°C for 1 min. The PCR products wereelectrophoresed and visualized by ethidium bromide staining. AmplifiedPCR products were purified from agarose gels using the QIAquick gelpurification kit (Qiagen) and were sequenced directly in both directionsusing an ABI 377 PRISM DNA sequencer after amplification using thePerkinElmer Dye Terminator Sequencing system with AmpliTaq DNApolymerase and the same primers that were used for the initial PCRamplification.

Statistical analysis

ANOVA was used to analyze the data, with Student’s t test used to deter-mine the level of significance for differences between sample means.

ResultsCD21/35 expression negatively regulates mCD19 expression

Because CD19 and CD21/35 physically associate on the cell sur-face, it was assessed whether loss of CD21/35 expression affectedCD19 expression. As determined by immunofluorescence staining,

CD19 expression on B220high mature bone marrow B cells fromCD21/35�/� mice was significantly increased (19% higher) rela-tive to wild-type littermates (Fig. 1, A and B). CD19 expressionwas similar on immature B220low B cells from CD21/35�/� andwild-type littermates (Fig. 1A), consistent with a lack of or low-level CD21/35 expression by immature bone marrow B cells (50).CD19 expression was also increased on CD21/35�/� B cells fromblood, spleen, and lymph nodes by 16, 36, and 31%, respectively(Fig. 1). By contrast, mCD19 expression by mature B cells frombone marrow, blood, spleen, and lymph nodes of C3�/� mice wasnot significantly different from wild-type levels (Fig. 1B). Thus,CD21/35 expression regulates cell surface CD19 expression levels,whereas C3 deficiency does not affect CD19 expression by themajority of B cells.

Whether CD21/35 or C3 expression affected hCD19 expressionin transgenic mice that overexpress CD19 was also assessed.hCD19 expression levels on B cells from bone marrow or periph-eral tissues of hCD19TG�/� mice were not affected by the absenceof CD21/35 or C3 expression (data not shown). However, B cellsfrom blood, spleen, and lymph nodes of hCD19TG�/� mice ex-pressed mCD19 at 29, 20, and 20% lower levels, respectively, thanB cells from wild-type littermates (Fig. 1), as previously reported(20). In the absence of CD21/35 expression, mCD19 was ex-pressed at wild-type levels in hCD19TG�/� mice in these tissues(Fig. 1). C3 deficiency did not significantly alter mCD19 expres-sion levels in hCD19TG�/� mice. Thus, CD21/35 expression neg-atively influenced endogenous mCD19 expression levels, but didnot influence cell surface hCD19 expression.

FIGURE 1. Cell surface CD21/35 expressioninfluences mCD19 expression by B220� lym-phocytes. A, Single-cell lymphocyte suspensionswere isolated from littermates of each mouse ge-notype and examined simultaneously by two-color immunofluorescence staining with flow cy-tometric analysis. Mouse CD19 expression wasvisualized using PE-conjugated 1D3 mAb. Ver-tical dashed lines in each histogram are providedfor reference. These results are representative ofthose 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 intensitychannel numbers for 1D3-PE staining between Bcells from wild-type and transgenic littermatesof each genotype. Values represent the mean(�SEM) percentage of wild-type expression lev-els obtained from at least five sets of littermatesof each genotype. Mean values significantly dif-ferent from wild-type levels are indicated (�, p �0.05; ��, p � 0.01).

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CD19 expression influences CD21/35 expression

Because CD21/35 expression negatively influences cell surfacemCD19 levels, the effect of CD19 deficiency on CD21/35 expres-sion was assessed. CD21/35 expression levels on blood B cellsfrom CD19�/� mice were 25% lower than on B cells from wild-type littermates (Fig. 2). Nonetheless, CD19 deficiency did notsignificantly affect CD21/35 expression by B cells in spleen orlymph nodes (Fig. 2). Similar results were obtained when usingeither the 7E9 or 7G6 mAbs (data not shown) that bind distinctregions of the CD21/35 molecule (49). Thus, basal mCD19 ex-pression levels do not significantly modulate CD21/35 expressionlevels. By contrast, mature B cells from hCD19TG�/� mice ex-pressed �57% less CD21/35 than wild-type B cells (Fig. 2). ThatCD19 overexpression results in a significant decrease in CD21/35expression may relate to the hyperresponsive status of these Bcells.

C3 expression negatively influences CD21/35 expression

Because C3 cleavage fragments are ligands for CD21/35, the effectof C3 deficiency on CD21/35 expression was assessed. Blood,spleen, and lymph node B cells from C3�/� mice had significantlyincreased CD21/35 expression (46, 33, and 31%, respectively)when compared with wild-type B cells (Fig. 2). Similar resultswere obtained when using either the 7E9 or 7G6 mAbs (data notshown). Similarly, CD19�/� littermates that were C3-deficient hadincreased CD21/35 expression (Fig. 2B). CD21/35 expression washigher on B cells from hCD19TG�/�C3�/� mice thanhCD19TG�/� littermates, although the levels of CD21/35 expres-sion remained below those of wild-type littermates (Fig. 2B). Thus,C3 deficiency resulted in significantly increased CD21/35 expres-sion by mature B cells.

CD19 regulates B cell development independent of CD21/35 orC3 expression

In the current studies using CD19�/�, hCD19TG�/�, CD21/35�/�, and C3�/� littermates, CD19 deficiency and overexpres-sion had significant effects on the development and expansion ofperipheral B cells, whereas CD21/35 and C3 deficiencies had mod-est effects (Fig. 3). In all cases, combined CD19 deficiency oroverexpression with CD21/35 or C3 deficiencies did not have ad-ditive influences on B cell development in the bone marrow, blood,spleen, or peritoneal cavity (Fig. 3 and data not shown). Althoughthe frequency and number of B220lowCD5� B1 cells is signifi-cantly inhibited or augmented by CD19 deficiency or overexpres-sion, respectively, the frequency and number of B220lowCD5� Bcells was not significantly affected by CD21/35 or C3 deficiencies(Fig. 3). Thus, the effects of CD19 deficiency or overexpression onB cell development were not significantly influenced by CD21/35or C3 expression.

CD19 regulates IgM and IgD expression independent of CD21or C3 expression

Consistent with CD19 regulating intrinsic signaling thresholds,mature B cells from CD19-deficient or -overexpressing mice havesignificantly altered surface IgM and IgD levels (Fig. 4). By con-trast, blood, spleen, and lymph node B cells from CD21/35�/� orC3�/� littermates had wild-type levels of IgM and IgD expression(Fig. 4). In all cases, combined CD19 deficiency or overexpressionwith CD21/35 or C3 deficiencies did not have significant additiveinfluences on IgM or IgD expression by B cells from blood, spleen,or lymph nodes (Fig. 4). Therefore, the effects of CD19 deficiencyor CD19 overexpression on B cell Ag receptor expression were not

FIGURE 2. Cell surface mCD19 expression influ-ences CD21/35 expression by B220� lymphocytes. A,CD21/35 expression was assessed as in Fig. 1 usingFITC-conjugated 7G6 anti-CD21/35 mAb. Verticaldashed 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 eachgenotype. B, Relative cell surface CD21/35 densitieswere determined by comparing mean linear fluores-cence intensity channel numbers for 7G6-FITC stain-ing between B cells from wild-type and transgeniclittermates of each genotype. Values represent themean (�SEM) percentage of wild-type expressionlevels obtained from at least five sets of littermates ofeach genotype. Mean values significantly differentfrom wild-type levels are indicated (�, p � 0.05; ��,p � 0.01).

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significantly influenced by CD21/35 or C3 expression. Thus, al-though CD19 expression levels regulate signaling thresholds thatinfluence cell surface IgM and IgD density, the loss of CD21 or C3does not alter thresholds to an extent that affects Ag receptorexpression.

CD19 function dominates during B cell differentiation

The effects of CD21/35 or C3 deficiency on B cell differentiationin CD19�/� and hCD19TG�/� littermates were assessed (Fig.5A). Although serum IgM levels were decreased by 70% inCD19�/� mice and increased by 96% in hCD19TG�/� littermates,CD21/35�/� and C3�/� littermates had wild-type IgM levels(Fig. 5A). Serum IgM levels in CD19�/�CD21/35�/� andCD19�/�C3�/� littermates were similar to those of CD19�/� lit-termates. Likewise, IgM levels of hCD19TG�/�CD21/35�/� andhCD19TG�/�C3�/� littermates were comparable to those ofhCD19TG�/� littermates. CD19�/� and hCD19TG�/� littermateshad 89% decreased and 132% increased IgG1 levels, respectively.Unexpectedly, C3�/� mice had 98% higher IgG1 levels thanCD21/35�/� or wild-type littermates (Fig. 5A). However, combin-ing CD21/35 and C3 deficiencies with alterations in CD19 expres-sion did not significantly alter IgG1 levels beyond the CD19-in-duced changes.

CD19 function dominates during autoimmunity

Because hCD19TG�/� mice produce autoantibodies includinganti-dsDNA Abs (9, 51), the effects of CD21/35 or C3 loss onanti-dsDNA Ab production were assessed in 5-mo-old littermates(Fig. 5B). IgM and IgG anti-dsDNA Abs were increased by 510and 340% in hCD19TG�/� mice compared with wild-type lit-termates, respectively. By contrast, anti-dsDNA Ab levels werenot significantly different in wild-type, CD19�/�, CD21/35�/�,or C3�/� littermates. Anti-dsDNA Ab levels were also compa-rable in hCD19TG�/�, hCD19TG�/�CD21/35�/�, and

hCD19TG�/�C3�/� littermates. Thus, CD21/35 or C3 loss didnot significantly affect autoantibody production inhCD19TG�/� littermates.

CD19 function dominates during humoral immune responses

The influence of CD19, CD21/35, and C3 expression on humoralimmune responses was assessed by immunizing littermates with aT cell-independent type 1 Ag, TNP-LPS, and a TD Ag, DNP-KLH. Following immunization with TNP-LPS, wild-type, CD21/35�/�, and C3�/� littermates generated comparable primary IgMresponses (Fig. 6A). CD19�/� littermates demonstrated modestIgM responses to TNP-LPS that were not significantly affected bythe additional loss of CD21 or C3 expression. Given the signifi-cantly reduced numbers of peripheral B cells in hCD19TG�/�

mice relative to wild-type littermates (Fig. 3), hCD19TG�/� micegenerate significant humoral immune responses (31). IgM re-sponses by CD19TG�/�, hCD19TG�/�CD21/35�/�, andhCD19TG�/�C3�/� littermates were comparable, but elevatedcompared with responses of wild-type littermates.

Following DNP-KLH immunizations, primary and secondaryIgM responses were similar in wild-type, hCD19TG�/�, CD21/35�/�, C3�/�, hCD19TG�/�CD21/35�/�, and hCD19TG�/�

C3�/� littermates (Fig. 6B). By contrast, primary and secondaryIgM responses to DNP-KLH were modest in CD19�/� mice andtheir CD19�/�CD21/35�/� and CD19�/�C3�/� littermates.CD19�/�, CD19�/�CD21/35�/�, and CD19�/�C3�/� littermatesalso showed similar profound defects in their primary and second-ary IgG1 responses to DNP-KLH. Primary IgG1 responses toDNP-KLH were similarly delayed in hCD19TG�/�, CD21/35�/�,and C3�/� littermates (Fig. 6B). These effects were not additivebecause primary IgG1 responses were similar in hCD19TG�/�,hCD19TG�/�CD21/35�/�, and hCD19TG�/�C3�/� litter-mates. Nonetheless, secondary IgG1 responses were compara-ble in hCD19TG� /�, hCD19TG� /�CD21/35� /�, and

FIGURE 3. B cell numbers in littermates with altered CD19, CD21/35, and C3 expression. Single-cell lymphocyte suspensions were isolated fromlittermates of each mouse genotype and examined simultaneously by two-color immunofluorescence staining with flow cytometric analysis and gating asdescribed (9). The histograms indicate mean (�SEM) percentages or numbers of lymphocytes expressing the indicated cell surface markers from eachtissue. B cell numbers were calculated based on the total number of cells harvested from each tissue, except for blood values that indicate numbers of cellsper milliliter. Values represent results from 36 wild-type, 12 CD19�/�, 11 CD19�/�CD21/35�/�, 4 CD19�/�C3�/�, 18 hCD19TG�/�, 9 hCD19TG�/

�CD21/35�/�, 11 hCD19TG�/�C3�/�, 18 CD21/35�/�, and 17 C3�/� littermates. Mean values significantly different from wild-type levels are indicated(�, p � 0.05; ��, p � 0.01).

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hCD19TG�/�C3�/� 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 hCD19TG�/� Bcells

Expression of CD21/35 on hCD19TG B cells selectively affectedmCD19 but not hCD19 expression (Fig. 1). Because we have pre-viously stated that hCD19 can associate with mouse CD21/35 atdetectable levels when expressed in mouse B cells that aremCD19�/� (20), it was assessed whether CD21/35 preferentiallyassociates with mCD19 when both hCD19 and mCD19 are ex-pressed. CD19-associated proteins were immunoprecipitated fromdigitonin-lysed hCD19TG�/� and wild-type B cells, transferred tonitrocellulose, and probed using two different antisera reactivewith either the amino- or carboxyl-terminal regions of CD21. Pre-cipitated mCD19 coimmunoprecipitated CD21/35 proteins of�190, 165 (Fig. 7A, upper arrow), 157, 147, 141, and 120 kDafrom wild-type B cells (Fig. 7A). These proteins were also immu-noprecipitated using the anti-CD21/35 mAb 7G6 (Fig. 7A). The190-kDa band most likely represents CD35, whereas the otherforms of CD21/35 protein are likely to result from the differentialuse of exons by CD21 and CD21/35 protein isoforms as described(25, 26). Although coprecipitated at lower levels, a similar spec-trum of CD21/35 proteins was immunoprecipitated fromhCD19TG B cells using anti-mCD19 mAb when the autoradio-graphs were visualized after further exposure. Reduced levels ofcoprecipitated CD21/35 protein are most likely to reflect reducedCD21/35 expression by hCD19TG B cells (Fig. 2). By contrast,coprecipitation of CD21/35 was never detected with hCD19 im-munoprecipitated from wild-type or hCD19TG�/� B cells, al-though the anti-hCD19 Ab efficiently precipitated hCD19 (data notshown). Coprecipitation of CD21/35 was never detected withhCD19 immunoprecipitated from wild-type or hCD19TG�/� B

FIGURE 5. Serum Ig and anti-dsDNA Ab levels inlittermates with altered CD19, CD21/35, or C3 expres-sion. A, Values represent mean (�SEM) serum Ig lev-els for at least nine littermates of each genotype asdetermined by Ab isotype-specific ELISAs. B, Serumanti-dsDNA Ab levels were determined by ELISAs at5 mo of age. Each histogram represents the mean(�SEM) OD value obtained for sera from at least fivelittermates of each genotype. Mean values significantlydifferent from wild-type levels are indicated (�, p �0.05; ��, p � 0.01).

FIGURE 4. Cell surface IgM and IgD expression levels in littermateswith altered CD19, CD21/35, or C3 expression. Relative cell surface IgMor IgD densities were determined by comparing mean linear fluorescenceintensity channel numbers for immunofluorescence staining between Bcells from wild-type and transgenic littermates of each genotype. Valuesrepresent the mean (�SEM) percentage of wild-type expression levels ob-tained from at least five sets of littermates of each genotype. Mean valuessignificantly different from wild-type levels are indicated (�, p � 0.05; ��,p � 0.01).

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cells even when the autoradiographs for mCD19 immunoprecipi-tations were significantly overexposed. Therefore, CD21/35 mayassociate with hCD19 at detectable levels in the absence ofmCD19 expression, but CD21/35 preferentially associates withmCD19 when both hCD19 and mCD19 are expressed. This sug-gests that hCD19 regulates signaling thresholds in hCD19TG�/�

B cells independent of an association with CD21/35.

CD21/35�/� mice express hypomorphic CD21/35

A surprising observation was that CD19 coprecipitated a numberof proteins from CD21/35�/� B cells, although these proteins wereconsistently 14 kDa smaller than those coprecipitated from wild-type B cells (Fig. 7A, lower arrow). These proteins were CD21because they reacted with two different antisera specific for eitherthe amino-or carboxyl-terminal regions of CD21 and were precip-itated by the 7G6 Ab (Fig. 7A and data not shown). In four dif-ferent immunoprecipitations, the CD21-like proteins were abun-dant and were readily coprecipitated with mCD19 from CD21/35�/� B cells. Thus, CD21/35�/� B cells appear to express ahypomorphic CD21/35 protein product.

The genotype of the CD21/35�/� mice was verified usingSouthern blot analysis (Fig. 7B) as described in the original papercharacterizing these mice (33). Immunofluorescence staining of Bcells from CD21/35�/� mice using two independent anti-CD21/35mAbs verified surface expression of the hypomorphic CD21/35protein (Fig. 7C). When assessed using the 7E9 mAb in two-colorimmunofluorescence staining experiments, overall CD21/35 ex-pression by B220� blood and spleen B cells from CD21/35�/�

mice was 37 � 6% of wild-type levels (n 3, data not shown).When assessed using the 7G6 mAb, blood and spleen B220� Bcells from CD21/35�/� mice expressed CD21/35 at 24 � 2% ofwild-type levels (n 5, data not shown), which was consistentwith the immunoprecipitation results (Fig. 7A). Because CD21/

35�/� mice were generated by targeting exons encoding SCRs9–10 of CD21, cDNA generated from CD21/35�/� B cells wasRT-PCR amplified using a forward primer specific for the exonencoding SCR 8 and a reverse primer specific for SCR 11.CD21/35 transcripts were readily detected in wild-type and CD21/35�/� B cells, although exon 9/10 appeared to be selectivelyspliced out in transcripts from CD21/35�/� mice (Fig. 7D). Se-quence analysis of the PCR products verified that CD21/35�/�

mice generated CD21/35 transcripts splicing out the exon encod-ing SCRs 9–10, but with in-frame coding sequence (Fig. 7E).Thus, CD21/35�/� mice produce hypomorphic CD21/35�/� pro-teins lacking the SCRs encoded by Cr2 exons 9/10 (25).

DiscussionThis study demonstrates that CD19 predominantly regulates B cellsignal transduction independent of complement activation. Thehallmark characteristics of CD19�/� B cells were not significantlyaltered when combined with CD21/35 or C3 deficiencies (Figs.3–6). More important is that hCD19 expression by mouse B cellsresults in CD19 overexpression and a dramatic hyperresponsivephenotype (29). However, the hallmark characteristics of hyperre-sponsive B cells in hCD19TG mice were not significantly alteredwhen combined with CD21/35 or C3 deficiencies (Figs. 3–6).Therefore, the hyperresponsive phenotype of B cells that overex-press CD19 is unlikely to result from increased sensitivity to com-plement activation. In addition, biochemical studies revealed thatlittle if any hCD19 associates with CD21/35 in hCD19TG B cellswhen mCD19 is expressed (Fig. 7A). Thus, hCD19 is not equiv-alent to mCD19 in generating CD19-CD21 complexes in mouse Bcells, although hCD19 expression at wild-type levels can restorenormal function in CD19�/� mice (20). These findings do not rule

FIGURE 6. Humoral immune responses in lit-termates with altered CD19, CD21/35, or C3 ex-pression. Values represent mean (�SEM) serumAb levels in at least five littermates of each ge-notype. A, Mice of each genotype were injectedi.p. with 50 �g of TNP-LPS in saline on day 0and 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. with100 �g of DNP-KLH in CFA on days 0 and 21and bled on days 0, 7, 14, and 28. Relative anti-DNP IgM and IgG1 levels were determined byELISA. Mean values significantly different fromwild-type levels are indicated (�, p � 0.05; ��,p � 0.01).

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out the possibility that complement activation and CD21/35 en-gagement generate transmembrane signals through CD19, but sug-gest that CD19 predominantly regulates signal transductionthrough complement-independent mechanisms.

Cell surface CD21/35 expression levels were significantly af-fected by C3 and CD19 expression. Specifically, CD21/35 expres-sion was increased by 31–46% on peripheral B cells from C3-deficient littermates (Fig. 2). This suggests that ongoing C3d,gfragment generation may chronically engage CD21/35, resulting in

receptor internalization. During inflammatory responses or in pa-tients with systemic autoimmune disease, serum immune com-plexes can be loaded with C3d. In fact, B cells from patients withsystemic lupus erythematosus have decreased CD21 (51–53) andCD19 expression levels (51), presumably due to the continuouspresence of CD21/35 ligands that induce CD21 internalization(54). In lupus-prone MRLlpr/lpr mice, CD21/35 expression levelsare also specifically decreased on B cells, even before the devel-opment of clinical or serological manifestations of disease (50).

FIGURE 7. CD21/35�/� mice express hypomorphic CD21/35. A, Coimmunoprecipitation of CD19 and CD21 from detergent lysates of purified splenicB cells (5 � 107/lane) from wild-type, hCD19TG�/�, and CD21/35�/� littermates. Cell lysates were incubated with the indicated Abs covalently boundto beads. The immunoprecipitated proteins or total cell lysate (2 � 106 cells/lane) were subjected to SDS-PAGE and transferred onto nitrocellulose. Themembrane was incubated with goat anti-mouse CD21 polyclonal antisera (M-19), followed by incubation with HRP-conjugated secondary Ab. Similarresults were obtained in four experiments. B, Southern blot analysis of tail DNA from wild-type, CD21/35�/�, and CD21/35�/� littermates digested withBamHI 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 CD21/35�/� (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 whenthe splenocytes were stained with the primary Ab in the presence of 5% mouse serum or Fc blockade. These results represent those obtained with at least10 littermates examined in separate experiments. D, Cytoplasmic RNA from splenocytes was reverse transcribed and amplified by PCR with primersspecific for CD21/35 exons 8 and 11. The PCR products were electrophoresed and visualized by ethidium bromide staining, with the expected sizesindicated. E, The amplified PCR products described in D were purified from agarose gels and sequenced on both strands.

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The 44–58% decrease in CD21/35 expression (Fig. 2) and 20–29% decrease in mCD19 expression (Fig. 1) by B cells fromhCD19TG�/� mice may also result from increased CD21/35 re-ceptor internalization because these mice produce autoantibodiesthat may activate complement. Furthermore, CD21/35 expressionin autoimmune Lyn�/� mice (55–57) is reduced by 60% (our un-published data). Thus, C3, CD21/35, and CD19 expression may becomponents of a common regulatory loop that influences B cellsignaling thresholds. CD21/35 engagement may result in increasedCD19 turnover, which may limit signal transduction or partiallydesensitize B cells chronically stimulated through the CD19-CD21complex.

CD19 expression levels were significantly influenced byCD21/35 and C3 expression (Fig. 1). CD19 expression levels were16–36% higher on peripheral B cells of CD21/35�/� mice (Fig.1), whereas CD21/35 deficiency did not influence cell surface IgMor IgD expression levels (Fig. 4). Human CD19 expression byhCD19TG B cells was not affected by CD21/35 deficiency, con-sistent with a lack of detectable physical interaction between thesexenogeneic molecules (Fig. 7A). Therefore, CD21/35 may influ-ence mCD19 expression by regulating cell surface complex turn-over. This contrasts with CD81-deficient mice where cell surfaceCD19 expression is reduced by half (58–60). Increasing CD19expression by 16–36% on CD21/35�/� B cells may be function-ally significant because �20% increases in CD19 expression pre-dispose mice to autoimmunity, and similar increases in CD19 ex-pression correlate with autoantibody production in humans (51). Ingeneral, CD19 overexpression increases endogenous levels of ac-tivated Lyn, dysregulates tolerance, and results in autoantibodyproduction (9, 51, 61). This may explain in part why CD21/35deficiency contributes to autoimmunity (41, 42). Normally, CD19expression levels are developmentally regulated and tightly con-trolled (10, 20). In mice, the majority of early B lineage cellsexpress mCD19 at relatively high levels in the bone marrow, witha 2.5-fold increase in expression during the transition from an im-mature B220low to a mature B220high CD21� B cell (9, 25). There-fore, it is interesting that peritoneal B1 cells express lower levelsof CD21 (50), yet express incrementally higher levels of CD19than conventional B cells (9). Thus, regulated CD19 and CD21/35expression levels may balance intrinsic signal transduction thresh-olds and B cell responsiveness to transmembrane signals.

B cell development, surface IgM expression, and serum Ig lev-els were almost normal in CD21/35�/� and C3�/� littermates(Figs. 3–5), as previously described (33, 34, 39, 40). Although adecrease in peritoneal B1 cell numbers has been reported in onlyone line of CD21/35�/� mice (33, 34), a significant difference innumbers or frequency of peritoneal B1 cells was not observed inthis study among the 17 littermate pairs assessed (Fig. 3). Thisdiscrepancy may be due to the different genetic backgrounds asdescribed (62) or the housing of mice used in each study, becausewe generated CD21/35�/� littermates through paired breedings ofCD19�/�CD21/35�/� littermates or hCD19TG�/�CD21/35�/�

littermates. The frequencies of peritoneal B1 cells were also nor-mal in C3�/� mice (Fig. 3) as previously shown for both C3�/�

and C4�/� mice (39). Therefore, a decreased frequency of peri-toneal B1 cells in CD21/35�/� mice may be a variable phenotypictrait. Nonetheless, CD19�/� mice had significantly decreased pe-ripheral B cells and peritoneal B1 cells, increased surface IgMexpression, and decreased serum Ig levels, regardless of CD21/35or C3 expression (Figs. 3–5). Likewise, hCD19TG�/� mice hadsignificantly decreased numbers of mature B cells in the bone mar-row and peripheral tissues, increased peritoneal B1 cells, down-regulated surface IgM expression, and increased serum Ig levels,regardless of CD21/35 or C3 expression (Figs. 3–5). Increased or

down-regulated surface IgM expression is a consequence of im-paired or augmented transmembrane signaling through the B cellAg receptor complex, respectively (1, 63). Each of these results isconsistent with the conclusion that CD19 can function indepen-dently of CD19/CD21 complex expression, although CD21/35uses the CD19 complex to generate transmembrane signalsthrough the CD19 cytoplasmic domain (11).

Primary and secondary humoral immune responses to TD andT-independent Ags were severely compromised in CD19�/� mice,particularly IgG1 responses to TD Ags (Fig. 6), as previously de-scribed (30, 31). By contrast, serum IgG1 responses to a TD Agwere only delayed in CD21/35�/� and C3�/� littermates, andthese mice generated normal primary IgM responses and second-ary responses (Fig. 6). Delayed IgG1 responses to DNP-KLH inCD21/35�/� and C3�/� littermates did not hinder the IgG1 re-sponses of hCD19TG�/� littermates when these genetic alter-ations were combined. Therefore, there were dramatic quantitativeand qualitative differences between the immune responses ofCD19-deficient mice and CD21/35�/� or C3�/� littermates. Al-though CD19 deficiency always has profound effects on humoralimmune responses, TD-immune responses and affinity maturationin CD21/35�/� mice vary depending on the Ag dose and use ofadjuvants (39, 64–66). Thus, alterations in CD19 expression caninfluence Ab production irregardless of CD21/35 or C3 expression.

Although the CD21/35�/� mice used in the current studies havebeen well characterized, these mice expressed low levels of a hy-pomorphic CD21/35�/� molecule (Fig. 7). An appropriate geno-type was verified in CD21/35�/� mice by Southern blot analysis(Fig. 7B), as originally described (33). Cell surface CD21/35 ex-pression was verified using two mAbs that react with differentextracellular regions of the protein, with one Ab binding to theligand-binding site in SCR domains 1–2 (Fig. 7C). PCR and nu-cleotide sequence analysis indicate that CD21/35 transcripts in theCD21/35�/� mice had spliced out exons 9/10 of CD21 (Fig. 7, Dand E), which is the Neor insertion site that generates the targetedmutation (33). Thereby, CD21/35�/� mice produce a 14-kDasmaller cell surface CD21 protein that retains its ability to asso-ciate with mCD19 (Fig. 7A) and retains SCR domains 1 and 2 thatnormally mediate CD21 interactions with ligands (67, 68). Inmany cases, exon length can affect RNA splicing in transcriptsinitiated from the endogenous promoter (69). An artificially largeexon with an inserted selective marker may not be recognized bythe splicing mechanism and may be skipped, thereby deleting themutated exon from the mRNA transcript. With CD21/35, this al-lowed the inappropriate splicing of exons 8 and 11, which does notinduce frame-shift mutations or a null allele. A similar strategywas used to generate a second line of CD21/35�/� mice, whereexons encoding SCR 8 of CD21 (or SCR 14 of the CD21/35 pro-tein) were targeted (34). An absence of detectable C3 binding byFDC or marginal zone B cells has been demonstrated for both linesof CD21/35�/� mice (33, 66, 70). In addition, both C3- and CD21/35-deficient mice share similar phenotypic characteristics. None-theless, it remains to be established whether the hypomorphicCD21/35 molecules expressed at low levels by CD21/35�/� miceare functionally relevant because there is a threshold effectfor CD21/C3d,g interactions whereby a minimum concentrationof cell surface CD21 is necessary to bind C3d,g-containing parti-cles (71).

That CD21 expression influences CD19 density on the cell sur-face provides another mechanism through which the complementsystem may regulate B cell signaling thresholds. Alterations inCD19 expression are important because CD19 cell surface densityintrinsically regulates Src family protein tyrosine kinase activity inB cells (14, 72). CD21 engagement may further modulate B cell

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activation by cross-linking and further augmenting CD19 function.Although the identity and biological significance of CD19 ligandsremains to be demonstrated (1, 3), CD21-independent ligands mayalso regulate the intrinsic signaling function of CD19. That CD19can function and regulate B cell signaling thresholds independentof CD21/35 and C3 expression affirms an independent role forCD19 as an intrinsic regulator of B lymphocyte signal transductionthat may be influenced by complement activation.

AcknowledgmentsWe thank Dr. Michael Caroll for providing CD21/35�/� and C3�/� mice,for helpful suggestions and discussions, and for review of this manuscript;Drs. Garnett Kelsoe, Zhibin Chen, and John Weis for their helpful sug-gestions and discussions; Dr. Joel R. Ross for editorial help; and NaomiHasegawa for technical assistance.

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