Uncoupling CD21 and CD19 of the B-cell coreceptorRobert A. Barringtona,1,2, Thomas J. Schneidera,1,3, Lisa A. Pitchera,1, Thorsten R. Mempelb, Minghe Maa,Natasha S. Bartenevaa,c, and Michael C. Carrollc,d,4

aThe Immune Disease Institute, and the Departments of bMedicine, dPediatrics, and cPathology, Harvard Medical School, Boston, MA 02115

Edited by Douglas T. Fearon, University of Cambridge, Cambridge, United Kingdom, and approved July 7, 2009 (received for review April 8, 2009)

Complement receptors (CRs) CD21 and CD35 form a coreceptor withCD19 and CD81 on murine B cells that when coligated with the B-cellreceptor lowers the threshold of activation by several orders ofmagnitude. This intrinsic signaling role is thought to explain theimpaired humoral immunity of mice bearing deficiency in CRs. How-ever, CRs have additional roles on B cells independent of CD19, suchas transport of C3-coated immune complexes and regulation of C4and C3 convertase. To test whether association of CR with CD19 isnecessary for their intrinsic activation-enhancing role, knockin miceexpressing mutant receptors, Cr2�/�gfp, that bind C3 ligands but donot signal through CD19 were constructed. We found that uncouplingof CR and CD19 significantly diminishes survival of germinal center Bcells and secondary antibody titers. However, B memory is lessimpaired relative to mice bearing a complete deficiency in CRs on Bcells. These findings confirm the importance of interaction of CR andCD19 for coreceptor activity in humoral immunity but identify a rolefor CR in B-cell memory independent of CD19.

B-cell memory � complement receptors � germinal centers �humoral immunity

B cells, like T cells, are regulated in large part by signals from theinnate immune system (1–3). For example, engagement of

complement (C3d)-coated antigen (Ag) by mature cognate B cellsligates the coreceptor (i.e., CD19, CD21, and CD81) and the B-cellreceptor (BCR), enhancing BCR signaling by several orders ofmagnitude (1, 4).

In mice, complement receptors CD21 and CD35 (CRs) areencoded at the Cr2 locus by splicing of message, and they arecoexpressed primarily on B cells and follicular dendritic cells(FDCs) (5, 6). CD35 and CD21 bind similar split products of C3,but in addition CD35 binds C3b and C4b. CD35 also interacts withCD19 to form a B-cell coreceptor (7). Systemic blocking of CR byantibody (8–10) or soluble receptor (11) results in impaired hu-moral immunity. Likewise, mice deficient in the receptors (Cr2�/�)bear impaired B-cell immunity to T-independent (12–14) andT-dependent Ags (15–17), infectious bacteria (18), and viruses (19,20). Studies using chimeric mice expressing CRs on either B cells(16) or FDCs (21, 22) indicate that overall humoral immunity isdependent on the presence of the receptors on both cell types. Thus,intrinsic B-cell signaling by the coreceptor and retention of Ags onFDCs are both important in B-cell immunity.

Recent studies demonstrate that CR expression on B cells isimportant in the transport of immune complexes (ICs). Bloodbornecomplexes of complement-coated Ags are bound rapidly by mar-ginal zone (MZ) B cells and are transported into the splenic follicles(12, 13, 23), and Ag is offloaded to FDCs (24). An analogous rolefor CRs on follicular B cells in peripheral lymph nodes (LNs) wasidentified. ICs draining via afferent lymph are trapped by subcap-sular sinus (SCS) macrophages and ‘‘transferred’’ to noncognate Bcells in the underlying follicular region in a CR-dependent manner(25, 26). The complement-coated ICs are then transported to FDCsin a manner similar to that proposed for MZ B cells. Thus, CRs haveat least two intrinsic roles on B cells: coreceptor on cognate B cells,and transport of ICs by noncognate B cells. A third, less studied rolefor CRs on B cells is complement regulatory activity. All mamma-lian cells express complement regulatory proteins (CRPs), such asDecay Activating Factor (DAF) and membrane cofactor protein

(MCP), which act to protect the host from complement activationon the cell surface (27). In the mouse, DAF and Crry are the majorCRPs expressed on host nucleated cells (28–30); however, CD35also has complement regulatory activity. Thus, its deficiency couldcontribute to a loss of CRP activity that leads to activation ofcomplement and an alteration in the local environment (31).

To determine the functional importance of the interactionbetween CR and CD19, a gene-targeting approach was used togenerate the mutant mouse line Cr2�/�gfp, where the transmem-brane (Tm) and cytoplasmic tail (CyT) of HLA class I � chainwere substituted for the analogous region of CR. Based onstudies in a B-cell line expressing a similar CR-HLA chimericreceptor, it was predicted that mutant B cells would lackfunctional coreceptor signaling but retain the ability to bind andtransport C3d-coated ICs and maintain CRP activity (32).

Characterization of Cr2�/�gfp B cells in vitro and in vivo con-firmed their ability to bind C3d-coated specific Ag without activa-tion of CD19, demonstrating functional uncoupling of CR andCD19. Comparison of humoral responses to T-dependent Ags inchimeric mice bearing WT FDCs and B cells derived from Cr2�/�gfp,Cr2�/�, or WT mice identified an intermediate response by theCr2�/�gfp relative to WT and Cr2�/�, and survival of germinalcenter (GC) B cells was reduced in both the Cr2�/� and Cr2�/�gfp

chimeric mice. These findings confirm the importance of interac-tion of CR and CD19 for coreceptor activity in humoral immunitybut identify additional roles for CRs in maintenance of B-cellmemory.

ResultsGeneration of Cr2�/�gfp Mice. To uncouple CR and CD19, a strategydeveloped by Matsumoto et al. (32) was used to substitute the Tmand CyT regions of HLA class I � chain for the analogous regionof CD21/CD35 (SI Text and Fig. S1 A and B). The mutant receptor,Cr2�/�gfp, developed normally, and CR2 expression, detected byGFP fluorescence, was restricted to FDCs and B cells (Fig. S1C).Moreover, Cr2�/�gfp B cells maintained the capacity to bind C3d(Fig. S1C). Therefore, mutant receptor expression and levels werecomparable to levels of CR in WT mice. Furthermore, the mutantCD21/CD35 receptors were functionally intact with regard tobinding ligand (Fig. S1). B-cell surface levels of CD19 are depen-dent on expression of CD21/CD35, and the levels are increased by�30% in mice deficient in CD21/CD35 (18, 33). To examinewhether surface levels are similarly increased in Cr2�/�gfp, splenic Bcells were isolated and analyzed by flow cytometry. The resultsidentify an increase in cell surface expression of CD19 on B cells

Author contributions: R.A.B., L.A.P., and M.C.C. designed research; R.A.B., T.J.S., L.A.P.,T.R.M., and M.M. performed research; T.J.S., T.R.M., and N.S.B. contributed new reagents/analytic tools; R.A.B., T.J.S., L.A.P., and T.M. analyzed data; and M.C.C. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

1R.A.B., T.J.S., and L.A.P. contributed equally to this work.

2Present address: Department of Microbiology and Immunology, University of SouthAlabama, Mobile, AL 36688.

3Present address: Merrimack Pharmaceuticals, One Kendall Square, Cambridge, MA 02139.

4To whom correspondence should be addressed. E-mail: carroll@idi.harvard.edu.

This article contains supporting information online at www.pnas.org/cgi/content/full/0903477106/DCSupplemental.

14490–14495 � PNAS � August 25, 2009 � vol. 106 � no. 34 www.pnas.org�cgi�doi�10.1073�pnas.0903477106

Dow

nloa

ded

by g

uest

on

Apr

il 24

, 202

1

prepared from both the mutant and deficient mice relative to WT(Fig. S1D). Thus, in the absence of interaction between CD21/CD35 and CD19, the levels of CD19 were increased, as found inCr2�/� mice.

Signaling via Mutant CR. Coligation of CD21 and CD19 withsubthreshold BCR stimulus augments B-cell activation (16, 34, 35).To test functional coreceptor signaling in primary B cells, the threestrains of mice (WT; Cr2�/�, ref. 17; and Cr2�/�gfp) were crossedwith the B6.MD4 line, which bears a classical Ig transgene (Tg)encoding anti-hen egg lysozyme (HEL) antibody (36). Splenocyteswere treated with an optimal amount of anti-IgM or a subthresholdlevel of duck egg lysozyme (DEL) alone or combined with C3d(rDEL-C3d3). MD4 B cells from each mouse strain mobilizedintracellular Ca2� in response to cross-linking with 10 �g/mLanti-IgM (Fig. 1A Top). As reported previously (34), stimulation ofMD4 B cells with DEL in amounts exceeding 100 nmol wassufficient to induce release of intracellular Ca2� independently ofcoreceptor (Fig. 1A Middle). However, response to 1 nmol of DELrequired both coreceptor and BCR signaling, as evidenced by lackof threshold response by WT MD4 B cells (Fig. 1A Middle). MD4B cells from all three lines were treated with 1 nmol of rDEL-C3d3and, as expected, WT MD4 B cells but not Cr2�/� B cells wereresponsive. Notably, the mutant B cells were also unresponsive to1 nmol of rDEL-C3d3, demonstrating that the coreceptor was notintact in the Cr2�/�gfp mice (Fig. 1A Bottom).

As a further test, functional coreceptor activity was also evaluatedin vitro by using a carboxyfluorescein succinimidyl ester (CFSE)proliferation assay. Splenocytes from the three lines of mice werelabeled with CFSE before culture with CD40 antibody and optimalanti-IgM (Fig. 1B). To test coreceptor signaling, mixed tetramers,composed of a suboptimal level of anti-IgM and C3d, and anti-CD40 were cultured with B cells for 3 days (Fig. 1B) (37). WT Bcells proliferated in response to mixed tetramers and anti-CD40, asdemonstrated previously (37), whereas Cr2�/� and Cr2�/�gfp B cellswere unresponsive. Therefore, CR binding of C3d tetramers andsignaling via CD19 are functionally uncoupled in the Cr2�/�gfp Bcells, leading to an impaired proliferation in vitro.

In Vivo Transport of ICs. Previous studies have reported that naive Bcells bind ICs through the CR and transport them into the B-cellfollicle (25, 26). To determine whether transport of ICs into thefollicle is dependent on coupling of CR to CD19, WT, Cr2�/�, andCr2�/�gfp mice were passively immunized with anti-B-phycoerythrin(PE) antibody and 24 h later were injected s.c. in the hind flanks

with 10 �g of B-PE. The draining inguinal LNs were collected 8 hlater and analyzed by FACS. As expected, Cr2�/� B cells boundnegligible PE-ICs. By contrast, both WT and mutant B cells boundappreciable levels of PE-ICs (Fig. 2 A and B). Thus, Cr2�/�gfp Bcells, although uncoupled from CD19, retain the ability to transportPE-ICs into the follicle.

To compare the retention of PE-ICs on FDCs in mice with intactCR/CD19 versus the mutant Cr2�/�gfp receptor, the inguinal LNs ofthe passively immunized mice were collected 24 h after PE injec-tion. Confocal microscopic analysis identified a similar level ofPE-ICs colocalized with FDCs in both WT and Cr2�/�gfp mice (Fig.2C). Together, transfer of PE-ICs by naive B cells to FDCs andretention of PE-ICs on FDCs are independent of CD19.

To examine further the uptake of ICs in real time by FDCs inCr2�/�gfp mice, we took advantage of the endogenous expression ofGFP. Note that GFP levels were not sufficiently intense for imagingin vivo of the mutant follicular B cells, whereas the levels were�10-fold higher on FDCs. Cr2�/�gfp mice were injected in thefootpad with 1 �g of preformed ICs comprising Alexa 633-labeledturkey egg lysozyme (TEL) and rabbit anti-lysozyme. In a previousreport, we found that labeled TEL alone injected in the footpadrapidly filled the SCS of the popliteal LNs and drained into thefollicles via discrete conduits (26). In the current study, TEL–IgGcomplexes were used to activate C3 and enhance uptake on FDCsvia CD21/CD35 receptors. Subsequently, the draining poplitealLNs were surgically exposed and imaged by multiphoton intravitalmicroscopy. TEL Ag (red) colocalized with the FDC dendriticprocesses (green) (Fig. 2D; see also Movie S1). Follicular conduits(identified by second harmonic signals; blue), which were shownrecently to channel small Ags from the SCS into the FDC area,colocalized with the FDCs (26). Thus, the mutant line of mice couldbe used in future studies to track uptake of labeled Ag onto FDCby intravital microscopy and to address whether uptake via CD21/CD35 receptors induces signaling.

Humoral Immunity in CR Mutant Mice. To examine the intrinsic effectof uncoupling of CR and CD19 on B cells in vivo, a bone marrow(BM) transplantation approach was used. The chimeric mice boreCR� FDCs from the WT C57BL/6 recipients, but the B-cellcompartment was derived from WT, Cr2�/�, or Cr2�/�gfp donorBM. BM chimeric mice were immunized with either a high or lowdose of 4-hydroxy-3-nitrophenyl conjugated to keyhole limpethemocyanin (NP-KLH) at 0 and 3 weeks, and antibody responseswere analyzed (Fig. S2). A potential disadvantage of the chimeric

Fig. 1. Functional responses of B cells with uncoupled CD21-CD19 coreceptor. (A) Early activation of Tg B cells stimulated with (Top) anti-IgM, (Middle) DEL, and(Bottom) rDELC3d3 as measured by Ca2� mobilization. B cells from WT (MD4; thick black lines), Cr2-deficient (Cr2�/� MD4; thin black lines), and mutant Cr2 (Cr2�/�gfp

MD4; thick gray lines) mice were loaded with Indo-1 AM. The indicated stimuli were added after collecting baseline for 30 s. Results are representative of twoindependent experiments with at least three mice per genotype. (B) Proliferative responses in B cells from WT (Top), Cr2�/� (Middle), and Cr2�/�gfp (Bottom) afterstimulation with anti-CD40 antibody and either 10 �g of anti-IgM (optimal anti-IgM; Left) or with 1 �g of C3dg tetramers containing suboptimal anti-IgM (Right).

Barrington et al. PNAS � August 25, 2009 � vol. 106 � no. 34 � 14491

IMM

UN

OLO

GY

Dow

nloa

ded

by g

uest

on

Apr

il 24

, 202

1

approach is increased variation among individual mice. However,the overall advantage is that the FDCs are consistently Cr2�/�.

In response to high-dose Ag (50 �g i.v.), both WT and Cr2�/�gfp

mice responded comparably (Fig. 3A Right). Further, the frequencyof NP-specific antibody-secreting cells (BASCs) was similar in theBM of both WT and Cr2�/�gfp (Fig. 3B Right). Although thenumbers of BASCs in the spleens of Cr2�/�gfp mice were dramaticallyhigher than Cr2�/�, they were only about one-half that of WT. Asexpected, Cr2�/� mice failed to generate significant serum antibodytiter to the hapten NP, and they also showed a markedly reducedfrequency of NP-specific BASCs (Fig. 3). Therefore, humoral re-sponses to high-dose soluble Ag were comparable between WT andCr2�/�gfp B cells.

Complement-dependent antibody responses are most apparentwith lower amounts of Ag. Thus, humoral responses were measured10 days after challenge with a low dose of Ag (10 �g i.v.). WTchimeras yielded a significantly greater titer than Cr2�/� chimeras(Fig. 3A Left). Importantly, the IgG anti-NP titers were also reducedin Cr2�/�gfp relative to WT chimeras (Fig. 3A). Further, after 7weeks, the decay in circulating NP-specific antibody was greater inthe Cr2�/� and mutant mice than Cr2� chimeras. Although itcorrelated directly with serum anti-NP IgG titers, the frequency ofBASCs was markedly higher in immunized Cr2� chimeras comparedwith Cr2�/� and Cr2�/�gfp cohorts (Fig. 3B Left). Thus, uncouplingof CR and CD19 substantially reduced humoral immunity relativeto WT, but mutant CRs contributed to an enhanced responserelative to Cr2�/�.

GC Responses in CR Mutant Mice. Given the reduced secondaryresponse to low-dose Ag observed in the Cr2�/� and Cr2�/�gfp mice,GC B cells were compared among the three groups of immunizedmice. Ten days after secondary immunization with low-dose NP-KLH, the numbers of peanut agglutinin (PNA)highB220� B cellswere quantitated. WT, Cr2�/�, and Cr2�/�gfp BM chimeric mice didnot differ statistically (Fig. S3 and Table 1). By contrast, a significantreduction was observed in the number of Cr2�/� NP� B cellsrelative to WT that had undergone class switch recombination(CSR) (IgM�) (Table 1). Similarly, the number ofPNAhighNP�IgM� B cells prepared from Cr2�/�gfp mice was re-duced relative to WT (Fig. 4 A and B, and Table 1). Thus, efficientCSR of the NP� B cells required an intact CR CD19 coreceptor.This could be explained by a requirement for coreceptor signalingfor efficient Ag presentation to cognate T cells (38) and/or forenhanced up-regulation of survival genes, such as Bcl-2, cFLIP, orBcl-xl (39, 40). Interestingly, CSR was more efficient in the Cr2�/�gfp

than Cr2�/� B cells, suggesting that binding of C3-coated Ag (orCRP activity) could also contribute to CSR and survival.

Maturation of GC B cells is suggested to occur stepwise fromIgM�IgD� � IgM� IgD� � IgG� (41). To examine the stage ofdifferentiation in which coreceptor signaling is required, Ag-specific, PNAhigh B cells were analyzed at each of the three stages.Interestingly, no statistically significant difference in the number ofNP�IgM� GC B cells was observed between WT, Cr2�/�, andCr2�/�gfp (Table 1). However, when the NP�IgM� population wasexamined for surface IgD levels, the highest mean fluorescence

Fig. 2. CR-mediated transport and uptake of ICs by B cells and FDCs is independent of CD19. (A) WT, Cr2�/�, and Cr2�/�gfp were passively immunized with rabbitanti-B-PE and 24 h later were injected s.c. in the hind flank with 10 �g of B-PE. In vivo uptake of PE-ICs by naive polyclonal B cells was assessed 8 h after PE injectionbyFACS.Exogenouscellswereaddedduringprocessingtocontrol forexvivocaptureofPE-ICs.Dotplotsarerepresentativeofat least threeLNs,andresultsarecompiledinto averages � SEM in the graph (B). (C) Inguinal LNs were analyzed 24 h after PE injection for the degree of PE-IC deposition on FDCs by confocal microscopy by usinganti-CD35 (blue) to label FDCs. (D) TEL-ICs are stably deposited on motile dendrites of FDCs. Preformed ICs containing 1 �g of Alexa 633-labeled TEL (red) were injectedinto a Cr2�/�gfp mouse, in which FDCs (green) express GFP, and were analyzed 24 h later. Multiphoton intravital microscopy analysis with illumination at 880 nm allowedsuboptimal yet simultaneous excitation of both Alexa 633 and GFP. Second harmonic signals from collagen fibers are shown in blue. Macrophages (Mac) are identifiedby their yellow autofluorescence. The smaller images on the right highlight that TEL remains colocalized (Bottom), with motile FDC dendrites in the highlightedsubregion of the main panel. Times are given in minutes and seconds. (Scale bar: 25 �m.)

14492 � www.pnas.org�cgi�doi�10.1073�pnas.0903477106 Barrington et al.

Dow

nloa

ded

by g

uest

on

Apr

il 24

, 202

1

intensity (MFI) for IgD was consistently NP�IgM� Cr2�/� andCr2�/�gfp B cells (Fig. 4C).

B-Cell Memory in Mutant Mice. GCs serve to produce long-termprotective humoral immunity via production of Ag-specificmemory cells (BMEMs) and BASCs (42, 43). To determine whetherB-cell memory correlates with the block in isotype switchobserved in PNAhigh cells, two approaches were taken usingsplenic B cells from immunized BM chimeric mice: (i) cells wereanalyzed by FACS 7 weeks after challenge with low-dose solubleAg, and the number of NP-binding B cells was quantitated, and(ii) B cells were adoptively transferred along with T cells fromKLH-primed mice into Rag-1�/� mice, and recipients weresubsequently challenged with 50 �g of Ag in adjuvant, andantibody titers were determined (Fig. S4).

GC responses began to dissipate by 3 weeks after Ag encounter.To evaluate the production of BMEM cells, BM chimeric mice wereanalyzed by FACS 7 weeks after immunization with low-dose Ag.As predicted, the number of NP-binding B cells was reduced overthe time period analyzed for all three strains (Table 1). Interestingly,Cr2�/�gfp NP� B cells showed an intermediate number of BMEMcells relative to WT and Cr2�/� (Fig. 5A and Table 1). Thus, anintact coreceptor is important for efficient differentiation of BMEM,but CRs appear to have an additional role or roles.

As an additional measure of B-cell memory, a Rag-1�/� chimericmouse model was used (Fig. S4). Rag-1�/� mice were reconstitutedwith a mixture of hapten-primed splenic B cells and carrier-primedT cells. Rag-1�/� chimeras receiving B cells from immunized WTmice had a mean IgG anti-NP titer significantly greater thanrecipients of Cr2�/� B cells (Fig. 5B). B cells from immunizedCr2�/�gfp mice produced an intermediate response compared withWT and Cr2�/� donor-derived B cells (Fig. 5B). ELISPOT resultswere consistent with antibody titers, because Cr2�/�gfp donorsplenocytes produced an intermediate frequency of IgG, NP-specific BASCs compared with Rag-1�/� chimeras receiving eitherWT or Cr2-deficient B cells (Fig. 5C). Thus, coreceptor signaling isa critical factor in formation and maintenance of long-term BMEM,but CRs appear to play additional role/roles in memory that areindependent of CD19.

DiscussionIn the current study, we introduced a mutation within themurine Cr2 locus to uncouple the functional interaction be-tween complement receptors CD21 and CD35 (CRs) fromCD19 based on earlier studies in a human B-cell line (32). Themutant receptor, Cr2�/�gfp, binds C3 ligands and includes theCD35 domain that has complement regulatory activity (N-terminal domain) but impaired coreceptor signaling. To eval-uate the intrinsic effect on B cells, a BM chimeric approach was

used. Characterization of the Cr2�/�gfp mice identified anintermediate (relative to WT and Cr2�/�) humoral response tolow-dose, T-dependent Ag. The overall reduced antibodyresponse correlated with a partial block at the IgM�IgD� toIgM�IgD� stage within the GC similar to that observed inCr2�/� B cells. Moreover, the mutant line had an impaireddevelopment of long-term BASCs and BMEMs.

These results are consistent with the interpretation of findingsfrom earlier studies in Cr2�/� mice and Cr2�/� BM chimeric micethat coreceptor signaling is critical for efficient humoral immunityto T-dependent Ags (15, 16, 44). Importantly, the new resultsidentify the stage in GC differentiation in which coreceptor signal-ing is required (i.e., IgM�IgD� to IgM�IgD�). Wang and Carter(45) identified a block in differentiation at a similar stage in GC Bcells in CD19�/� and CD19�/� transgenic mice that express mutantCD19 (Y482F/Y513F). In their study, analysis of GC by BrdUlabeling and histology identified arrest in differentiation of mutantB cells in the FDC light zone. This region of the GC is thought topromote clonal selection of B cells and includes cognate T cells aswell as C3d-coated Ag.

One explanation for the signaling requirement of coreceptorwithin the GC light zone could be efficient presentation of Ag to

Fig. 3. Effects of uncoupling CR–CD19 interactions on humoral immune re-sponses. (A) Anti-NP IgG titers after immunization and challenge with 50 �g(Right) and 10 �g (Left) of NP-KLH i.v. Immunized BM chimeric mice were bled atday 0 and then 1 and 7 weeks after boost, and titers were assessed by ELISA (34).(B) Frequencies (per 106 splenocytes) of NP-specific BASCs in WT, Cr2�/�, andCr2�/�gfp miceweredeterminedbyELISPOTafter immunizationwith50�g(Right)and10 �g(Left)ofNP-KLH.Asterisksdenotesignificantdifferences relativetoWT(*, P � 0.02; **, P � 0.04, t test).

Table 1. Comparison of GC and memory B cells isolated from WT, Cr2 mutant, and Cr2�/� mice

ChimerasPNAhighB220�IgM� B cells,

mean � SD

NP�PNAhigh B cells 1–2 weeks afterimmunization, mean � SD

NP� BMEM cells 6–8 weeks afterimmunization, mean � SD, IgM�IgM� IgM�

WT 856,331 � 139,023 273,869 � 141,777 8,157 � 2,050 1,169 � 462Cr2�/�gpf 763,728 � 337,073 439,864 � 167,570 1,784 � 1,074* 656 � 437†

Cr2�/� 663,821 � 332,473 368,816 � 186,873 880 � 1,275‡ Undetectable

Efficient class switch recombination and generation of B-cell memory are dependent on intact CR–CD19 coreceptors. GC B cells were enumerated in chimericmice containing WT-derived FDCs and either WT, Cr2�/�, or Cr2�/�gfp BM-derived B cells after immunization with 10 �g (low dose) of NP-KLH. Total numbers ofGC B cells (second column), NP-specific B cells expressing surface IgM (third column), or NP-specific B cells that had undergone class switch recombination (fourthcolumn) were assessed at 10 days after immunization by multiplying their frequency, as determined by flow cytometry, by the total splenocyte count. At 7 weeksafter immunization, NP-specific memory B cells (BMEMs) were quantitated in WT vs. Cr2�/� and Cr2�/�gfp chimeras (fifth column).

*, P � 0.003.†, P � 0.05.‡, P � 0.001.

Barrington et al. PNAS � August 25, 2009 � vol. 106 � no. 34 � 14493

IMM

UN

OLO

GY

Dow

nloa

ded

by g

uest

on

Apr

il 24

, 202

1

cognate T cells to ensure costimulation and up-regulation ofactivation-induced cytidine deaminase. Pierce and colleagues (46)reported that coupling of C3d to HEL enhanced localization ofBCR and coreceptor (CD19/CD21/CD81) in lipid rafts, resulting inprolonged signaling and presentation of Ag to T cells. The tetra-span protein CD81 interacts with CD19 and facilitates localizationof the coreceptor and BCR in lipid rafts, and this enhances signalingvia Ig � and vav (47). CD81-deficient mice have reduced responsesto certain T-dependent Ags and increased responses to type IIT-independent Ags (48). Moreover, the deficient mice have re-duced numbers of B-1 cells, probably resulting from impairedexpression of CD19 (49). Similarly, CD19�/� mice have a block inGC response to T-dependent Ags, as discussed above, and adeficiency in B-1 cells (50).

This stage of differentiation is also critical for up-regulation ofsurvival factors, such as Bcl-2 and Bcl-xl, and overexpression ofthese survival genes leads to excess survival of low-affinity GC Bcells (39, 40). Thus, cross-linking of coreceptor enhances up-regulation of Bcl-2 (51) and Bcl-xl (52) and would enhance con-tinued B-cell differentiation within the GC.

Not all T-dependent Ags require CR for development of hu-moral immunity, because the nature of the Ag and dose areimportant factors. For example, Cr2�/� mice appear to respond toinfectious vesicular stomatitis virus similarly to WT controls (14).Similarly, Cr2�/� mice immunized with bacteriophage (QB phage)developed an apparently normal GC response, with a frequency ofB memory cells and early antibody titers similar to those of WTcontrols; however, the antibody response failed to persist, as hadbeen observed in responses to haptenated proteins (53). Interest-ingly, it was found that transcription factors Blimp-1 and XBP-1were not efficiently up-regulated by post-GC plasma cells in theCr2�/� mice and that the defect was intrinsic to the B cells.Although the current study did not evaluate expression of Blimp-1and XBP-1, identification of a general block in B-cell differentiationwithin the GC would explain the actual reduction in the number ofAg-specific BASCs and BMEMs.

Although the humoral response of Cr2�/�gfp mice was impairedrelative to WT mice, it was less severe than that of Cr2�/� mice. Thisfinding might be explained by a low level of functional interactionbetween mutant CR and CD19. Although we cannot rule out thispossibility, negligible coreceptor activity was observed by using twosensitive assays in vitro. Moreover, the observation of a similarincrease in expression of CD19 on B-cell surfaces in both theCr2�/�gfp and Cr2�/� mice supports a physical separation betweenCD19 and the mutant receptor. By contrast, functional activityinvolving binding of C3d or transport of C3d-coupled ICs appearednormal in the mutant mice.

The less severe defect in Cr2�/�gfp relative to Cr2�/� micesupports CD19-independent role/roles of CR. For example, mutantB cells retain the ability to bind and transport C3d-coated ICs intothe B-cell follicles. Recent studies highlight the importance of B-celltransport of Ag in both the spleen and peripheral LNs (23, 25, 26).In addition, Shlomchik and Rossbacher demonstrated that bindingof Ag by cognate B cells in Ig-deficient mice could activate C3 onthe cell surface, resulting in coupling of C3b to the specific Ag (54).Moreover, focusing of Ag–C3d complexes on the surface of cognateB cells could enhance ‘‘presentation’’ to neighboring B cells ortransport to FDCs (55). Thus, the responsiveness and differentia-tion of B cells in mutants relative to Cr2�/� could be explained inpart by increased efficiency of cognate B cells in presentation ofC3d-coated Ags to neighboring B cells (or deposition on FDC).

Finally, although a requirement for complement regulatoryactivity by CD35 on B cells has not been reported, it is possible thatthe less impaired response of mutant B cells could be explained inpart by the expression of CR. Thus, complete absence of CR onAg-bindingBcellsmight increase their sensitivity tocomplement injury.By contrast, B cells in WT and mutant mice retain full CRP activity.

In summary, characterization of a novel line of mutant mice inwhich CRs are uncoupled from CD19 on B cells confirms theimportance of coreceptor signaling for efficient response to T-dependent Ags. However, the finding of a less impaired humoral

Fig. 4. Identification of NP-specific B cells in GC from spleens of immunized BMchimeric mice. (A) Representative FACS plots from immunized (10 �g i.v.) BMchimeric mice with WT (Left), Cr2�/� (Middle), and Cr2�/�gfp (Right) B cells. B cellswere gated as described in Fig. S3. (B) Scatter plot showing numbers of NP-binding GC cells (three experiments; n � 6) in WT (black squares), Cr2�/� (opencircles),andCr2�/�gfp (graytriangles) chimeras7–14daysafter rechallengewith10�g of NP5-KLH i.v. (C) MFI for IgD levels on the surface of NP-binding IgM cellswithin GCs of chimeric mice. Shown are representative plots for NP�IgM� fromWT (solid gray), Cr2�/� (dotted black line), and Cr2�/�gfp (solid gray line) BMchimericmice.Meanvaluesare indicatedbybars. Statisticaldifferences (t test)areindicated (*, P � 0.001; **, P � 0.003).

Fig. 5. Generation of BMEMs from BM chimeric mice. (A) Spleens were analyzed7 weeks after Ag challenge for the presence of NP�IgM�

(B220�PNA�IgD�CD3�CD4�CD11c�) BMEM cells from WT (black squares), Cr2�/�

(open circles), and Cr2�/�gfp (gray triangles) chimeras (from three experiments).Statistical analyses are indicated: *, P � 0.035; **, P � 0.05. Total splenic B cellsisolated 7 weeks after boost were adoptively transferred into Rag1�/� recipientsalong with carrier-primed T cells from WT mice. After 2 weeks, Rag1�/� chimeraswere immunized with 50 �g of NP5-KLH in alum, and IgG anti-NP titers (B) andASCs (C)weremeasuredasdescribed.Shownisa summaryfromtwoexperiments;statistical significance is indicated (*, P � 0.03; **, P � 0.04).

14494 � www.pnas.org�cgi�doi�10.1073�pnas.0903477106 Barrington et al.

Dow

nloa

ded

by g

uest

on

Apr

il 24

, 202

1

response relative to Cr2�/� mice suggests a role for CRs indepen-dent of CD19.

Materials and MethodsAntibody Titer. Serum was collected from individual mice, and NP-specific anti-body titers were determined by sandwich ELISA, as described previously (56).

C3d Tetramers. Functional binding of C3d on B cells was confirmed by using theapproach of Henson et al. (57). C3d was kindly provided by David Karp (Universityof Texas Southwestern School of Medicine, Dallas, TX). Mixed tetramers of F(ab)2goat anti-mouse IgM and C3d were prepared as described and were purified bygel filtration (37).

Chimeric Rag1�/� Mice. The generation of chimeric Rag1�/� mice is described inSI Text.

Flow Cytometric Analysis. Antibodies and reagents used for flow cytometry aredescribed in SI Text.

Histology. Histological analyses are described in SI Text.

Immunogens and Immunizations. NP-KLH was used at two doses for i.v. immu-nizations: 10 �g (low dose) or 50 �g (high dose). Mice were analyzed 2 to 3 weeksafter primary immunization and 1 to 6 weeks after boost. The formation of ICs invivo and analysis of IC uptake was described previously (26).

Mice and BM Chimeras. Mice were housed at the Immune Disease Institute (IDI)and Harvard Medical School (HMS) in specific pathogen-free facilities. MD4 HELIg Tg mice were maintained on a C57BL/6 background, with either WT (36),Cr2-deficient (Cr2�/�) (17), or mutant (Cr2�/�gfp) Cr2 locus. A BM chimera ap-proach was used to localize CR mutation to the B-cell population, as describedpreviously (16). All animal procedures were Institutional Animal Care and UseCommittee-approved at IDI and HMS.

ACKNOWLEDGMENTS. We thank Svend Rietdij for help on the Ca2� flux assay,Franziska Schuerpf for assistance with confocal microscopy, and Young-A Kim forassistance with the preparation and analysis of uptake of ICs. We thank Dr. DavidKarp for providing C3d-biotin and Dr. David Isenman (University of Toronto,Toronto, ON, Canada) for purified human C3d. This work was supported byNational Institutes of Health Grants AI39246 and AI40181 (to M.C.C.); 1 F32AR08644 (to T.J.S.); and 2 T32 HL066987 (to R.A.B. and L.A.P.).

1. Dempsey P, Allison M, Akkaraju S, Goodnow C, Fearon D (1996) C3d of complement asa molecular adjuvant: Bridging innate and acquired immunity. Science 271:348–350.

2. Janeway CA (1989) Approaching the asymptote: Evolution and revolution in immu-nology. Cold Spring Harb Symp Quant Biol 54:1–13.

3. Matzinger P (1994) Tolerance, danger, and the extended family. Annu Rev Immunol12:991–1045.

4. Tedder TF, Zhou LJ, Engel P (1994) The CD19/CD21 signal transduction complex of Blymphocytes. Immunol Today 15:437–441.

5. Kinoshita T, Fujita T, Tsunoda R (1991) Expression of Complement Receptors CR1 and CR2on Murine Follicular Dendritic Cells and B Lymphocytes (Elsevier, Amsterdam), p 271.

6. Kurtz CB, O’Toole E, Christensen SM, Weis JH (1990) The murine complement receptorgene family. IV. Alternative splicing of Cr2 gene transcripts predicts two distinct geneproducts that share homologous domains with both human CR2 and CR1. J Immunol144:3581–3591.

7. Kalli KR, Fearon DT (1994) Binding of C3b and C4b by the CR1-like site in murine CR1.J Immunol 152:2899–2903.

8. Heyman B, Wiersma EJ, Kinoshita T (1990) In vivo inhibition of the antibody responseby a complement receptor-specific monoclonal antibody. J Exp Med 172:665–668.

9. Wiersma EJ, Kinoshita T, Heyman B (1991) Inhibition of immunological memory andT-independent humoral responses by monoclonal antibodies specific for murine com-plement receptors. Eur J Immunol 21:2501–2506.

10. Typhronitis G, et al. (1991) Modulation of mouse complement receptors 1 and 2suppresses antibody responses in vivo. J Immunol 147:224–230.

11. Hebell T, Ahearn JM, Fearon DT (1991) Supression of the immune response by a solublecomplement receptor of B lymphocytes. Science 254:102–105.

12. GuinamardR,OkigakiM,SchlessingerJ,RavetchJV(2000)AbsenceofmarginalzoneBcellsin Pyk-2-deficient mice defines their role in the humoral response. Nat Immunol 1:31–36.

13. Pozdnyakova I, Wittung-Stafshede P (2003) Approaching the speed limit for Greek Keybeta-barrel formation: Transition-state movement tunes folding rate of zinc-substituted azurin. Biochim Biophys Acta 1651:1–4.

14. Ochsenbien A, et al. (1999) Protective T cell-independent antiviral antibody responsesare dependent on complement. J Exp Med 190:1165–1174.

15. Fischer MB, et al. (1998) Dependence of germinal center B cells on expression ofCD21/CD35 for survival. Science 280:582–585.

16. Ahearn J, et al. (1996) Disruption of the Cr2 locus results in a reduction in B-1a cells andin an impaired B cell response to T-dependent antigen. Immunity 4:251–262.

17. Molina H, et al. (1996) Markedly impaired humoral immune response in mice deficientin complement receptors 1 and 2. Proc Natl Acad Sci USA 93:3357–3361.

18. Haas KM, et al. (2002) Complement receptors CD21/35 link innate and protectiveimmunity during Streptococcus pneumoniae infection by regulating IgG3 antibodyresponses. Immunity 17:713–723.

19. DaCosta X, et al. (1999) Humoral response to herpes simplex virus is complementdependent. Proc Natl Acad Sci USA 96:12708–12712.

20. Gonzalez SF, Jayasekera JP, Carroll MC (2008) Complement and natural antibody arerequired in the long term memory response to influenza virus. Vaccine 26S:186–193.

21. Fang Y, Xu C, Fu Y, Holers VM, Molina H (1998) Expression of complement receptors 1and 2 on follicular dendritic cells is necessary for the generation of a strong antigen-specific IgG response. J Immunol 160:5273–5279.

22. Barrington RA, Pozdnyakova O, Zafari MR, Benjamin CD, Carroll MC (2002) B lympho-cyte memory: Role of stromal cell complement and FcgammaRIIB receptors. J Exp Med196:1189–1199.

23. Cinamon G, Zachariah MA, Lam OM, Foss FW, Jr, Cyster JG (2008) Follicular shuttling ofmarginal zone B cells facilitates antigen transport. Nat Immunol 9:54–62.

24. Ferguson AR, Youd ME, Corley RB (2004) Marginal zone B cells transport and depositIgM-containing immune complexes onto follicular dendritic cells. Int Immunol16:1411–1422.

25. Phan TG, Grigorova I, Okada T, Cyster JG (2007) Subcapsular encounter and comple-ment-dependent transport of immune complexes by lymph node B cells. Nat Immunol8:992–1000.

26. Roozendaal R, et al. (2009) Conduits mediate transport of low-molecular-weightantigen to lymph node follicles. Immunity 30:264–276.

27. Liszewski MK, Farries TC, Lublin DM, Rooney IA, Atkinson JP (1996) Control of thecomplement system. Adv Immunol 61:201–283.

28. Miwa T, et al. (2002) Deletion of decay-accelerating factor (CD55) exacerbates auto-immune disease development in MRL/lpr mice. Am J Pathol 161:1077–1086.

29. Miwa T, et al. (2007) Decay-accelerating factor ameliorates systemic autoimmunedisease in MRL/lpr mice via both complement-dependent and -independent mecha-nisms. Am J Pathol 170:1258–1266.

30. Xu C, et al. (2000) A critical role for murine complement regulator crry in fetomaternaltolerance. Science 287:498–501.

31. Jacobson AC, Weis JH (2008) Comparative functional evolution of human and mouseCR1 and CR2. J Immunol 181:2953–2959.

32. Matsumoto AK, et al. (1993) Functional dissection of the CD21/CD19/TAPA-1/Leu-13complex of B lymphocytes. J Exp Med 178:1407–1417.

33. Hasegawa M, Fujimoto M, Poe JC, Steeber DA, Tedder TF (2001) CD19 can regulate Blymphocyte signal transduction independent of complement activation. J Immunol167:3190–3200.

34. Barrington RA, et al. (2005) CD21/CD19 coreceptor signaling promotes B cell survivalduring primary immune responses. J Immunol 175:2859–2867.

35. Sato S, Jansen P, Tedder T (1997) CD19 and CD22 expression reciprocally regulatestyrosine phosphorylation of Vav protein during B lymphocyte signaling. Proc Natl AcadSci USA 94:13158–13162.

36. Goodnow CC, et al. (1988) Altered immunoglobulin expression and functional silenc-ing of self-reactive B lymphocytes in transgenic mice. Nature 334:676–682.

37. Lyubchenko T, dal Porto J, Cambier JC, Holers VM (2005) Coligation of the B cellreceptor with complement receptor type 2 (CR2/CD21) using its natural ligand C3dg:Activation without engagement of an inhibitory signaling pathway. J Immunol174:3264–3272.

38. Cherukuri A, Cheng PC, Pierce SK (2001) The role of the CD19/CD21 complex in B cellprocessing and presentation of complement-tagged antigens. J Immunol 167:163–172.

39. Takahashi Y, et al. (1999) Relaxed negative selection in germinal centers and impairedaffinity maturation in bcl-xL transgenic mice. J Exp Med 190:399–410.

40. Smith KG, et al. (2000) bcl-2 transgene expression inhibits apoptosis in the germinalcenter and reveals differences in the selection of memory B cells and bone marrowantibody-forming cells. J Exp Med 191:475–484.

41. Wolniak KL, Shinall SM, Waldschmidt TJ (2004) The germinal center response. Crit RevImmunol 24:39–65.

42. MacLennan I (1994) Germinal centers. Ann Rev Immunol 12:117–139.43. McHeyzer-Williams M, et al. (2003) Helper T-cell-regulated B-cell immunity. Microbes

Infect 5:205–212.44. Croix D, et al. (1996) Antibody response to a T-dependent antigen requires B cell

expression of complement receptors. J Exp Med 183:1857–1864.45. Wang Y, Carter RH (2005) CD19 regulates B cell maturation, proliferation, and positive

selection in the FDC zone of murine splenic germinal centers. Immunity 22:749–761.46. Cherukuri A, Cheng PC, Sohn HW, Pierce SK (2001) The CD19/CD21 complex functions

to prolong B cell antigen receptor signaling from lipid rafts. Immunity 14:169–179.47. Cherukuri A, et al. (2004) The tetraspanin CD81 is necessary for partitioning of

coligated CD19/CD21-B cell antigen receptor complexes into signaling-active lipidrafts. J Immunol 172:370–380.

48. Maecker HT, Levy S (1997) Normal lymphocyte development but delayed humoralimmune response in CD81-null mice. J Exp Med 185:1505–1510.

49. Tsitsikov EN, Gutierrez-Ramos JC, Geha RS (1997) Impaired CD19 expression andsignaling, enhanced antibody response to type II T independent antigen and reductionof B-1 cells in CD81-deficient mice. Proc Natl Acad Sci USA 94:10844–10849.

50. Rickert RC, Rajewsky K, Roes J (1995) Impairment of T-cell-dependent B-cell responsesand B-1 cell development in CD19-deficient mice. Nature 376:352–355.

51. Roberts T, Snow EC (1999) Recruitment of the CD19/CD21 Coreceptor to B cell antigenreceptor is required for antigen-mediated expression of B cl-2 by resting and cyclinghen egg lysozyme transgenic B cells. J Immunol 162:4377–4380.

52. Bonnefoy JY, Henchoz S, Hardie D, Holder MJ, Gordon J (1993) A subset of anti-CD21antibodies promote the rescue of germinal center B cells from apoptosis. Eur J Immunol23:969–972.

53. Gatto D, et al. (2005) Complement receptors regulate differentiation of bone marrowplasma cell precursors expressing transcription factors Blimp-1 and XBP-1. J Exp Med201:993–1005.

54. Rossbacher J, Shlomchik MJ (2003) The B cell receptor itself can activate complementto provide the complement receptor 1/2 ligand required to enhance B cell immuneresponses in vivo. J Exp Med 198:591–602.

55. Rossbacher J, Haberman AM, Neschen S, Khalil A, Shlomchik MJ (2006) Antibody-independent B cell-intrinsic and -extrinsic roles for CD21/35. Eur J Immunol 36:2384–2393.

56. Boes M, et al. (1998) Enhanced B-1 cell development, but impaired IgG antibodyresponses in mice deficient in secreted IgM. J Immunol 160:4776–4787.

57. Henson SE, Smith D, Boackle SA, Holers VM, Karp DR (2001) Generation of recombinanthuman C3dg tetramers for the analysis of CD21 binding and function. J ImmunolMethods 258:97–109.

Barrington et al. PNAS � August 25, 2009 � vol. 106 � no. 34 � 14495

IMM

UN

OLO

GY

Dow

nloa

ded

by g

uest

on

Apr

il 24

, 202

1