FCRL5 exerts binary and compartment-specificinfluence on innate-like B-cell receptor signalingZilu Zhua, Ran Lib, Hao Lic, Tong Zhoub,d, and Randall S. Davisa,b,c,d,1

Departments of aBiochemistry and Molecular Genetics, bMedicine, and cMicrobiology, and dComprehensive Cancer Center, University of Alabama atBirmingham, Birmingham, AL 35294

Edited by Arthur Weiss, University of California, San Francisco, CA, and approved February 22, 2013 (received for review August 31, 2012)

Innate-like splenic marginal zone (MZ) and peritoneal cavity B1 Blymphocytes share critical responsibilities in humoral responsesbut have divergent B-cell receptor (BCR) signaling features. A dis-crete marker of these subsets with tyrosine-based dual regulatorypotential termed “Fc receptor-like 5” (FCRL5) was investigated toexplore this discrepancy. Although FCRL5 repressed the robust BCRactivity that is characteristic of MZ B cells, it had no influence onantigen receptor stimulation that is blunted in peritoneal cavity-derived B1 B cells. The molecular basis for the receptor’s inhibitoryfunction derived from recruitment of the Src homology-2 domain-containing tyrosine phosphatase 1 (SHP-1) to a cytoplasmic immu-noreceptor tyrosine-based inhibitory motif. Surprisingly, mutagen-esis of this docking site unearthed coactivation properties forFCRL5 that were orchestrated by independent association of theLyn Src-family kinase with an intracellular immunoreceptor tyro-sine-based activation motif-like sequence. FCRL5’s unique binaryregulation directly correlated with SHP-1 and Lyn activity, which,like BCR function, differed between MZ and B1 B cells. Thesefindings collectively imply a specialized counterregulatory rolefor FCRL molecules at the intersection of innate and adaptiveimmunity.

Innate-like B-lineage cells positioned at strategic microanatom-ical sites provide the first line of effector defense that bridges

host protection until adaptive mechanisms emerge (1). The lym-phocytes charged with these responsibilities include splenic mar-ginal zone (MZ) B cells harbored in a location optimal for filteringblood-borne antigens and B1-lineage cells that guard the perito-neal (PEC) and pleural body cavities (2–4). Their capacity forbroad neutralization is associated with evolutionarily conserved Igrepertoires, distinct sensitivity to T-cell–independent stimuli, andrapid or spontaneous production of ‘‘natural,’’ polyreactive anti-bodies (5, 6). These features distinguish MZ and B1 B cells fromtheir more abundant B2-lineage counterparts that recirculate andparticipate in T-cell–dependent responses.MZ and B1 B-cell development is strongly influenced by B-cell

receptor (BCR) specificity in concert with the composite array ofsurface and intracellular regulatory proteins that help balanceantigenic responses. Mutations in cluster of differentiation 45(CD45), Bruton’s tyrosine kinase (BTK), or phospholipase Cgamma 2 (PLCγ2) that dampen BCR signaling favor MZ de-velopment and a loss of follicular (FO)B cells (7).However, defectsin negative regulatory components, such as Lyn, Src homology-2(SH2) domain-containing tyrosine phosphatase 1 (SHP-1), orCD22, lead to a loss of MZ B cells, a relative expansion of the B1compartment, and increased susceptibility to autoimmunity (8).Although many other trophic, migratory, and retention factorsinstruct their development and positioning, these signals must beintegrated in the context of BCR signaling which primarily drivesB-cell fate and survival (7, 9–11).Correspondingly, BCR triggeringdiffersmarkedly between these subpopulations.MZBcells exhibitmore robust whole-cell protein tyrosine phosphorylation, calciummobilization, and PLCγ2 and spleen tyrosine kinase (Syk) activa-tion than FO B cells but also are more sensitive to apoptosis (12,13). In contrast, B1 B cells have blunted calciummobilization, NF-κB activation, and proliferation but also may possess relatively

higher rates of apoptosis thanPECB2cells (14–16).Notably, theseproperties do not differ according to CD5 expression, becauseboth the B1a (CD5+) and B1b (CD5−) B-cell subsets respondsimilarly to BCR ligation (17). Although they differ from anergiccells, it remains unclear whether the unique biology of B1 B cells issecondary to chronic antigenic exposure, suppressive signalspresent in the coelomic cavity microenvironment, or other causes(4, 14).An evolutionarily conserved gene family related to the Fc

receptors (FCR) for IgG and IgE, termed “FCR-like” (FCRL), ispreferentially expressed by B cells and encodes transmembraneproteins with tyrosine-based immunoregulatory motifs (18). Al-though Ig binding has been detected recently for two humanmembers (19), antibodies do not appear to associate with otherFCRL proteins. Intriguingly, two other representatives have beenfound to interact withMHC-likemolecules (20, 21). Because of thegrowing clinical relevance of FCRLs in infectious diseases, auto-immunity, malignancies, and immunodeficiencies, several groupshave investigated FCRL signaling function (22–25). In humans,FCRL1 has two immunoreceptor tyrosine-based activation motif(ITAM)-like sequences and enhances BCR-induced calcium mo-bilization and cellular proliferation (26). In contrast, FCRL2–5,which feature one or more consensus immunoreceptor tyrosine-based inhibition motifs (ITIM) as well as ITAM-like sequences, allinhibit BCR activation via recruitment of the SH2 domain-con-taining SHP-1 and/or SHP-2 phosphatases (27–30). We haveshown previously that the FCRL5mouse ortholog discretely marksinnate-like B cells and possesses an ITIM as well as an ITAM-likesequence that differs from the canonical motif (D/EX2–3YXXL/IX6–8YXXL/I), with a glutamic acid residue rather than an ali-phatic residue at the second Y+3 position (31). Although FCRL5inhibits BCR-mediated calcium mobilization in MZ B cells, themolecular basis for this activity and its function in B1 B cellsremains unclear. Furthermore, the conservation of both activatingand inhibitory sequences in FCRL5 and other FCRLs suggeststhey have dual signaling properties, but definitive functional evi-dence for this bifunctionality is lacking. Because of its distinctdistribution and regulatory potential, we investigated the biologicalrole of FCRL5 in these specialized B lymphocytes that have rec-ognized differences in their adaptive signaling capacity.Here we report that FCRL5 has dual modulatory and compart-

mental subset-specific effects on antigen receptor signaling. Uponassociation with the BCR, the ITAM-like and ITIM sequences inFCRL5 are tyrosine phosphorylated and recruit the Lyn Src-familykinase (SFK) and SHP-1 protein tyrosine phosphatase. The non-redundant contributions of these elements to FCRL5’s unique

Author contributions: Z.Z. and R.S.D. designed research; Z.Z., R.L., and H.L. performedresearch; T.Z. generated BAFF transgenic mice; Z.Z. and R.S.D. analyzed data; and Z.Z. andR.S.D. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.1To whom correspondence should be addressed. E-mail: rsdavis@uab.edu.

See Author Summary on page 5289 (volume 110, number 14).

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1215156110/-/DCSupplemental.

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counterregulatory function further revealed that differences inadaptive signaling in MZ and B1 B cells correlate directly with theirintrinsic SHP-1 activity.

ResultsFCRL5 Attenuates MZ but Not B1 BCR Signaling. Although MZ andB1 B cells share many similarities, they differ significantly in an-tigen receptor signaling (13, 14). Consistent with previous work,calcium mobilization among wild-type (WT) C57BL/6 splenic B-cell subsets was most intense in MZ B cells, followed by newlyformed (NF) and FO B cells, whereas PEC B2 B cells demon-strated a stronger response than B1 cells (Fig. 1A). Given theirdissimilar BCR signaling properties, we investigated whetherFCRL5 regulation also might vary in MZ versus B1 B cells. Inaccord with our earlier findings (31), the strong calcium responseobserved in gated MZ B cells after BCR engagement alone wasdiminished significantly by FCRL5 co-crosslinking (Fig. 1B). Incontrast, FCRL5 did not alter this impaired activation cascade inB1 B cells. An analysis of global tyrosine phosphorylation by in-tracellular phospho-specific flow yielded similar results. AlthoughFCRL5 could inhibit this downstream outcome in MZ cells, it hadno influence in B1 B cells which showed less robust whole-celltyrosine phosphorylation (Fig. 1B). Intriguingly, FCRL5 coag-gregation reduced BCR-mediated calcium mobilization in MZ Bcells to the same extent seen in B1 cells stimulated through theBCR alone. Because this blunted activation response in B1 B cellsmight be caused by constitutive association of FCRL5 with theBCR, we independently crosslinked FCRL5 before BCR stimula-

tion. However, sequestering FCRL5 did not affect BCR-mediatedcalcium mobilization in WT B1a B cells (Fig. S1). These data in-dicate that FCRL5 does not constitutively associate with the BCRand that other mechanisms likely account for observed compart-ment-specific differences in its regulatory function.

FCRL5 Inhibits BCR-Mediated Tyrosine Phosphorylation. To define themolecular basis for its differential innate-like B-cell activity, wenext dissected the roles of the FCRL5 cytoplasmic tyrosine-basedmotifs. FcγRIIb/FCRL5 chimeric constructs encoding six differentcytoplasmic mutants as well as full-length FcγRIIb and a vectorcontrol were stably transduced into the A20-IIA1.6 (IgG2aκ)mouse B-cell line that lacks endogenous FCRL5 and FcγRIIbexpression (Fig. 2A) (29). After confirming equivalent levels ofsurface expression, responses following coligation of the variouschimeric proteins with the BCR were compared with antigen re-ceptor ligation alone (Fig. S2 A and B).Because the FCRL5 cytoplasmic tail contains both ITAM-like

and ITIM sequences, we initially examined whether both motifscould be phosphorylated. After treatment with the sodium perva-nadate phosphatase inhibitor, theWT, Y543F, Y556F, Y566F, andY543F/Y556F (FF) chimeras were all tyrosine phosphorylated, butthe Y543F/Y556F/Y566F (FFF) mutant was not (Fig. S3). This lastvariant confirmed the functional inactivity of a fourth cytoplasmictyrosine at position 544 and indicates that FCRL5 tyrosine-basedsignaling is confined to its ITAM-like and ITIM units.We then investigated the impact of the FCRL5 WT and FFF

mutants on BCR-induced whole-cell tyrosine phosphorylation.

Fig. 1. FCRL5 differentially regulates innate-like B-cell signaling. (A) Spleen and PEC leukocytes (1 × 106) from WT C57BL/6 mice were labeled with Indo-1/acetoxymethyl (AM), stained for the indicated markers and biotinylated F(ab’)2 fragments of a rat anti-mouse IgM mAb (SB73a, 10 μg/mL) and were thenrested for 30 min. Calcium mobilization in the gated subsets (MZ: CD19highCD21highCD23low; NF: CD19highCD21lowCD23low; FO: CD19highCD21lowCD23high; B1a:CD19highCD5high; B1b: CD19highCD5lowCD11bhigh; and B2: CD19highCD5lowCD11blow) was analyzed before and after crosslinking with 20 μg/mL of streptavidin(SA) by flow cytometry. Arrows indicate the time of SA administration. (B) Spleen and PEC leukocytes from WT mice were stained as in A and were coun-terstained with biotinylated F(ab’)2 anti-FCRL5 (3B7) followed by SA-phycoerythrin (SA-PE) to define FCRL5 expression (Left). Indo-1/AM-loaded cells werestained for distinguishing surface markers and with biotinylated F(ab’)2-digested mAbs including rat anti-mouse IgM (αIgM, SB73a) and anti-FCRL5 (αFCRL5,3B7) or an isotype control (Ctrl, mouse IgG1κ) (all 10 μg/mL). Ca2+ mobilization was monitored in the MZ- or B1 B-cell–gated subsets before and aftercrosslinking with SA (Center). Whole-cell tyrosine phosphorylation (pTyr) was analyzed in these subpopulations at baseline (NS) and following SA crosslinkingfor 10 min, fixation, permeabilization, and intracellular staining with anti-pTyr (Right). Results are representative of three independent sets of experiments.

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BCR triggering alone induced rapid tyrosine phosphorylation ofmultiple intracellular proteins, including the ERK MAP kinase;however, coligation with the WT chimera greatly reduced theseeffects as a function of time (Fig. 2B). In contrast, global tyrosinephosphorylation and ERK activation were unaffected by FFFcrosslinking. Immunoprecipitation experiments indicated that theWT FCRL5 cytoplasmic tail was tyrosine phosphorylated follow-ing its association with the BCR but not upon BCR ligation alone.Results in this cell line thus were consistent with FCRL5 regula-tion in MZ B cells and demonstrated that its cytoplasmic domaincan inhibit BCR activation in a tyrosine-dependent manner.

FCRL5 ITIM and ITAM-Like Sequences Counterregulate BCR Signaling.We next explored the functional contributions of the individualFCRL5 tyrosines in calcium mobilization assays. BCR ligationalone induced a characteristic wave of calcium flux, but cross-linking with the WT chimeric receptor significantly abrogated thiseffect (Fig. 3A). In fact, the degree of inhibition was even morepotent than that of the FcγRIIb-positive control. The function ofthe ITIM then was examined by disrupting the ITAM-like 543 and556 tyrosines. Coligation with this FF mutant completely shutdown the calcium response. Conversely, mutating the Y566 ITIMresidue unearthed coactivation properties for FCRL5 and resultedin enhanced calcium flux compared with antibody receptor trig-gering alone. Finally, among the ITAM-like tyrosines, the Y543Fmutant entirely blocked calcium signaling, whereas the Y556Fvariant disclosed little difference from that of the WT tail. As ex-pected, the FFF mutant had no influence on BCR-mediated cal-cium flux. Because our results shown in Fig. 2B indicated thatFCRL5 could restrain the MAP kinase cascade, we next decon-structed the behavior of these tyrosines in ERK activation. TheY543F, Y556F, and FF variants, which all retain intact ITIMsequences, inhibited BCR-dependent ERK phosphorylation (Fig.3B). In contrast, the Y566F mutant possessing an unmodifiedITAM-like sequence moderately enhanced ERK activation. ThusFCRL5’s modulation of ERK signaling was similar to its effects oncalcium mobilization. These findings collectively indicate thatFCRL5 has dual regulatory tyrosine-based signaling propertiesthat are balanced and graded. The ITIM (Y566) exerts stronginhibition individually, whereas the ITAM-like (Y543/Y556) se-quence has the inverse effect and can augment downstream BCR-mediated responses. Importantly, within the noncanonical ITAM,Y543 played a critical role, whereas the Y556 residue appeared tobe dispensable. To confirm this finding, YFF and FYF chimericreceptor cell lines also were generated (Fig. S4 A and B). Col-

igation of the YFF mutant with the BCR positively regulatedcalcium mobilization, whereas the FYF variant had no obviouseffect (Fig. S4C). Accordingly, pervanadate treatment demon-strated that the YFF tail could be tyrosine phosphorylated, butthe FYF mutant could not (Fig. S4D). The opposing influence ofthe Y543 ITAM-like and Y566 ITIM residues therefore equipsFCRL5 with tyrosine-based counterregulatory function.

SHP-1 and Lyn Are Recruited to Independent FCRL5 CytoplasmicTyrosines. The cytosolic effector proteins that mediate FCRL5functions then were examined. To define the elements responsiblefor its inhibitory activity, chimeric protein immunoprecipitateswere probed for SHP-1, SHP-2, and SH2 domain-containing ino-sitol phosphatase (SHIP). Except for the FFF mutant, all thereceptors were tyrosine phosphorylated after BCR coengagement(Fig. 4A). Among these candidates, SHP-1 associated with theWT,Y543F, and Y556F single mutants and with the FF mutant but notwith the Y566F or FFF receptors. In contrast, neither SHP-2 norSHIP was coprecipitated with any FCRL5 tail variant. This ITIM-dependent association also was confirmed by SHP-1–specific im-munoprecipitation which identified a low level of constitutiveFCRL5 binding that was enhanced by BCR coligation (Fig. 4B).Putative signaling components responsible for FCRL5’s acti-

vating properties were then assessed. The Syk and PLCγ2 kinasesdid not associate with the FCRL5 cytoplasmic tail (Fig. 4C). Otherkinases, including BTK and PI3K, as well as the adaptor proteinsgrowth factor receptor-bound protein 2 (Grb2) and B-cell linkerprotein (BLNK), also were undetectable in immunoprecipitates.Because Lyn is the predominant SFK expressed in B cells and isrequired for the activating function of Igα/Igβ, CD19, and CD180/RP105 as well as the inhibitory properties of CD5, CD22, CD32,and CD72 (8), we explored whether it associated with FCRL5. Lynindeed coprecipitated with the WT, Y556F, and Y566F chimerasbut not with the Y543F, FF, or FFF mutants. We also determinedwhether Lyn was physiologically active (pY397) or inactive (pY508)(32). Western blotting with a phospho-specific Ab revealed thatthe SFK associated at the Y543 position was in the active state.Thus, after BCR coligation, the FCRL5 Y566 ITIM residue isphosphorylated and coordinates the receptor’s inhibitory activitythrough its association with SHP-1. The ITAM-like Y543 tyrosinealso is phosphorylated but offsets the receptor’s potent ITIM-mediated repression by recruiting the active form of Lyn.

SHP-1 and LynMediate the UniqueDual Functionality of FCRL5 in Innate-Like B Cells. To uncouple its regulation in primary B cells, we nextvalidated FCRL5 function in viable motheaten (mev/mev) mice,

Fig. 2. Coligation of the WT FcγRIIb/FCRL5 chimeric protein with the BCR inhibits whole-cell tyrosine phosphorylation. (A) Schematic illustration of FCRL5cytoplasmic tyrosine-based motifs and FcγRIIb/FCRL5 chimeric constructs as detailed in SI Materials and Methods. (B) WT or FFF transductants (1 × 107) werestimulated with intact (25 μg/mL) or F(ab’)2 (16.6 μg/mL) rabbit anti-mouse IgG for the specified times, and whole-cell lysates (WCL) were immunoblotted withthe indicated Abs and β-actin as a loading control. Lysates were immunoprecipitated with anti-HA and analyzed for pTyr and HA to verify equal loading. Thearrow indicates the position of the chimeric receptor. Results are representative of three independent experiments.

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which possess a point mutation that disables SHP-1 phosphataseactivity (∼20% of normal), and in Lyn-deficient mice (33, 34). Bothmodels develop severe autoimmunity and have a loss of MZ andFO B cells in the spleen as well as B2 cells in the PEC but developa relative expansion of B1a and B1b B cells (Fig. S5A andB) (8, 35).After confirming that FCRL5 is expressed at comparable levels byB1a and B1b B cells from these three strains (Fig. S5B), we ex-amined its regulatory impact on the BCR cascade. In remarkablecontrast to WT cells, where it had no clear influence, FCRL5crosslinking in SHP-1–mutant B1 B cells strongly augmented BCR-mediated calcium mobilization and whole-cell tyrosine phosphor-ylation (Fig. 5 A and B). Intriguingly, the opposite effect was ob-served in Lyn−/− B1 B cells. Here, coligating FCRL5 with the BCRresulted in marked repression of calcium flux and whole-cell ty-rosine phosphorylation. Notably, this outcome resembled obser-vations made for WT splenic MZ and A20-IIA1.6 B cells.Responses were similar in the B1a and B1b subsets. Furthermore,FCRL5 engagement alone in these genetically modified B cellsfailed to regulate calcium flux or tyrosine phosphorylation.These results in mutant mice confirm the inverse contributionsof SHP-1 and Lyn to its function and demonstrate that theirrelative activity has a direct effect on driving FCRL5 biology.These data validate the essential inhibitory role of SHP-1, and anunsuspected nonredundant requirement for Lyn in mediating itsactivating function also was uncovered. These data collectively in-dicate that FCRL5may serve as a cellular sensor for SHP-1 and Lynfunction in innate-like B cells.

To determine whether dual functionality was a feature uniqueto FCRL5, other well-characterized regulatory proteins includingCD5, CD22, CD32, and CD72 were analyzed also. Cell-surfacestaining demonstrated their similar expression levels on PEC B1aB cells derived fromWT, mev/mev, and Lyn−/−mice (Fig. S6). IgMexpression was slightly lower on mev/mev B1a B cells, and IgD wasuniformly dim. In contrast to FCRL5, all four of the receptorstested could inhibit BCR-mediated calcium mobilization in WTB1a B cells, albeit to different extents (Fig. 5C). In SHP-1–mutantB1a B cells, CD22 and CD32 retained inhibitory function, likelythrough their association with the SHIP inositol phosphatase (36,37). However, the dampening capacity of CD5 and CD72 wasabolished, confirming that their inhibitory properties are SHP-1dependent (38, 39). Furthermore, unlike FCRL5, none of thesemolecules acquired enhancing function in SHP-1–mutant B1a Bcells, including CD5, CD22, and CD72, each of which has beenshown to play both positive and negative roles (40–42). Neverthe-less, these receptors could all block BCR calcium signaling inLyn−/− B1a B cells. These data indicate that ITIM tyrosine phos-phorylation, which is required for SHP-1 and SHIP docking, andthe consequent inhibitory function of each of these proteins, in-cluding FCRL5, could be compensated by SFKs other thanLyn thatare coexpressed in these cells. However, the FCRL5 ITAM-likeY543 residue does not appear susceptible to promiscuous SFKactivity in this fashion and instead is primarily Lyn dependent.Taken together, these results indicate that FCRL5 possesses aunique counterregulatory function in B cells that is directly medi-ated by SHP-1 and Lyn.

Fig. 3. FCRL5 counterregulates BCR signaling. (A) Ca2+ mobilization in Fluo-4 AM– and SNARF-1–loaded A20-IIA1.6 transductants (1 × 106) was analyzedbefore and after BCR stimulation alone (black line) or receptor crosslinking (gray line). Arrows indicate the time of addition of stimulating antibodies.(B) Lysates from transductants (5 × 106) left untreated (−) or stimulated with intact (I) or F(ab’)2 fragments (F) of rabbit anti-mouse IgG for 10 min were probedfor total ERK and pERK. Densitometric quantitation of the pERK/ERK ratio for one representative data set is shown, and pooled results from three in-dependent experiments are indicated below. Error bars specify the mean ± SEM; *P < 0.05; **P < 0.01.

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Divergent FCRL5 Function in Innate-Like B Cells Reflects Differences inSHP-1 Activity. To confirm further the association of SHP-1 andLyn with FCRL5, we examined its biochemical properties inprimary cells. Because innate-like B cells represent only a mi-nority of the total B lymphocytes in normal adult mice, we useda human soluble B-cell activating factor (BAFF) transgenic (Tg)mouse model as a source of larger quantities of MZ B cells (Fig.S7A). The resulting human BAFF (hBAFF) Tg mice possesseda phenotype similar to previous established models and de-veloped markedly enlarged spleens compared with WT litter-mate controls (Fig. S7B) (43). As a fundamental B-cell survivalfactor, mice overexpressing soluble hBAFF dramatically ex-panded their total B-cell compartment and MZ B-cell frequency(Fig. S7C). To corroborate these flow cytometric studies, immu-nohistology was performed on the spleens of adult WT andhBAFF Tg mice. Fig. S7D demonstrates that spleens fromhBAFF Tg mice have a substantially expanded MZ outside theMOMA-1+ ring that demarcates metallophilic macrophages po-sitioned in the marginal sinus. Splenic MZ B cells from thesehBAFF Tg mice then were sorted by FACS and used for bio-chemical validation (Fig. S7E). As expected, BCR coligation inpurifiedMZ B cells induced tyrosine phosphorylation of FCRL5and the coincident association of SHP-1 and Lyn (Fig. 6A).These BCR-dependent interactions with the FCRL5 cytoplas-mic tail also were confirmed in PEC B1 B cells derived from

T-cell leukemia/lymphoma 1 (TCL1) Tg mice that havea marked expansion of these cells (Fig. S8) (44).To determine the nature of FCRL5’s disparate function in MZ

and B1 B cells, we compared the signaling properties of thesesubsets. Previous biochemical studies had found that MZ B cellsexpress comparatively more SHP-1 than FO B cells but expresssimilar levels of total Lyn (12). Furthermore, splenic MZ andPEC B1 cells show higher constitutive whole-cell tyrosine phos-phorylation than the conventional B2 cells present in their re-spective microenvironments (12, 45). To examine compartment-specific differences in these variables among these cell typesdirectly, we used a flow cytometry-based analysis for quantitativecomparisons at baseline and in response to different stimuli (Fig.6B). Consistent with previous work, both innate-like B-cell sub-populations had significantly higher constitutive and BCR-induced tyrosine phosphorylation relative to their B2-lineagecounterparts; however, activation was greatest in MZ B cells(Fig. 6 C and D). Correspondingly, basal SHP-1 expression wassignificantly elevated in MZ and B1 B cells compared with con-ventional B2 cells but was twofold greater in MZ than in B1aor B1b B cells (Fig. 6E). Relative SHP-1 abundance also corre-lated strongly with global tyrosine phosphorylation in response topervanadate treatment (Fig. 6F). Although total Lyn expressionwas similar among these subsets, its active and inactive formswere slightly higher in PEC B1 than in B2 cells (Fig. 6G). Alongwith our findings in mutant mice defining the requisite con-tributions of Lyn and SHP-1 to FCRL5’s function, these dataindicate that divergent FCRL5 biology in MZ and B1 B cellscorrelates directly with SHP-1 activity.

FCRL5 Binary Immunoregulation Discloses the Contributions of SHP-1and Lyn to MZ and B1 BCR-Induced Apoptosis. Because BCR stim-ulation markedly induces apoptosis in MZ B cells but has moremodest effects on B1 B-cell viability (13, 16, 46), we next in-vestigated the differential influence of Lyn and SHP-1 on antigenreceptor-mediated survival. Following culture alone, the survivalof WT MZ B cells was much shorter than that of B1 B cellspurified fromWT, mev/mev, or Lyn−/− mice (Fig. S9). AlthoughSHP-1–mutant B1 B cells were more sensitive to spontaneousapoptosis than WT B1 B cells, Lyn−/− B-cell viability was higheroverall (Fig. 7). Thus, in the absence of stimulation, Lyn deficiencyfavored B1 B-cell survival, and SHP-1 deficiency promoted apo-ptosis. We then examined the impact of FCRL5 on BCR-medi-ated survival. Anti-IgM crosslinking markedly increased MZ B-cell apoptosis; however, consistent with the relatively elevatedSHP-1 activity in this subset, FCRL5 coengagement rescueda significant number of these cells. BCR ligation also enhancedapoptosis in WT PEC B1 B cells. But, in contrast to its failure tomodulate calcium or tyrosine phosphorylation, here FCRL5 aug-mented apoptosis. This effect was in line with the comparativelylower SHP-1 levels in these cells and was magnified in mev/mev B1B cells. Conversely, in the absence of Lyn, FCRL5 demonstratedregulation akin to that of MZ B cells and suppressed apoptosis.These findings collectively indicate that compartmental differ-ences in SHP-1 and Lyn activity play a key role in influencing thedifferential adaptive function of innate-like MZ and B1 B cells.

DiscussionIn these studies a distinguishing marker of innate-like B cells wasfound to exert compartment-specific regulatory function. Al-though FCRL5 could inhibit the striking BCR activation typical ofsplenic MZ B cells, it had no obvious influence on this cascadewhich is muted in PEC B1 B cells. These findings led to broaderquestions concerning the molecular basis for FCRL5 function andits significance in these specialized subsets. Although it failed toinitiate downstream responses by itself, FCRL5 as a coreceptorhad a unique dualistic impact on BCR signaling that derived fromits tyrosine-based recruitment of Lyn and SHP-1. The incongruent

Fig. 4. FCRL5 ITIM and ITAM-like sequences recruit SHP-1 and Lyn in-dependently. (A) Lysates from A20-IIA1.6 cells (1 × 107) stimulated with intactrabbit anti-mouse IgG for 10 min (+) or left untreated (−) were immunopre-cipitated with anti-HA and blotted for pTyr, indicated phosphatases, or HAas a loading control. (B) Lysates from the FF and FFF transductants stimu-lated as in A were immunoprecipitated with anti–SHP-1 and probed for HAor SHP-1 as a loading control. (C) Immunoprecipitates from the chimericpanel stimulated as in A were blotted for the indicated kinases.

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biology evident for FCRL5 as well as the BCR in MZ versus B1 Bcells correlated directly with the distinct activity of SHP-1 in thesesubsets. Collectively these findings demonstrate that FCRL5 isa discrete counterregulatory biomarker of innate-like B cells di-rectly coupled to the Lyn–SHP-1 biochemical circuit.Through their direct and indirect associations with the BCR

complex, Lyn and SHP-1 have profound effects on B-cell selec-tion and function (47–49). Deficiency of either of these proteinsleads to a loss of MZ B cells, expansion of the B1 lineage, anda breakdown in peripheral tolerance (33, 35, 50, 51). We foundthat basal calcium influx, whole-cell tyrosine phosphorylation,and SHP-1 levels were significantly higher in MZ B cells than inB1 B cells but that Lyn activity did not vary substantially. Thesedifferences indicate that splenic MZ B cells are more globallyactivated at homeostasis and thus are primed for stronger signaltransduction once triggered. Given its governing effects on BCRsignaling strength (52), the effector responsibilities of these cells,and their developmental loss in its absence, SHP-1 likely is up-regulated to raise the triggering threshold and offset the pre-activation induced by tonic BCR and other environmental signalsthat these lymphocytes experience in the splenic MZ niche (7,53). Dampening their preamplified resting state by means ofelevated phosphatase activity not only would suspend BCRactivation but also would heighten their overall potential forexplosive responsiveness once this restriction is lifted. The re-lationship between SHP-1 expression, preactivation status, and

BCR responsivity also was apparent in the other splenic sub-populations, and recent work by the DeFranco group has dem-onstrated that increased signaling sensitivity among transitionalB2 cells declines in a maturation-dependent fashion as the Lyn–SHP-1 pathway becomes active (54). As a distinctive bifunctionalsubstrate in this circuit, FCRL5 inhibitory function in MZ B cellsreflects more elevated SHP-1 than Lyn activity. A pivotal role forSHP-1 was also substantiated by findings that neither total Lynabundance nor its physiologic state differed dramatically amongsplenic subsets. Thus compensatory SHP-1 up-regulation appearscritical for buffering MZ B-cell excitation and fate. Although whatmodulates its expression remains unclear, factors originating fromthe MZ milieu likely contribute given the extreme sensitivity ofMZ B cells to spontaneous apoptosis ex vivo and the markedelevation of SHP-1 in this subset.B1 B cells were less constitutively activated than splenic MZ B

cells; however, their global tyrosine phosphorylation and SHP-1and Lyn activity were slightly higher than in PEC B2 cells. Thesefeatures, along with their attenuated BCR responsiveness, againhighlight their unconventional biology. The inverse functionalityof FCRL5 in mev/mev and Lyn−/− B1 B cells but relative indolencein WT cells indicates the SHP-1-Lyn circuit is more balanced inthis subset at homeostasis. Under these conditions, Lyn binding tothe FCRL5 ITAM-like Y543 residue could be stoichiometricallycompensated by SHP-1 recruitment to the FCRL5 Y566 ITIM.Importantly, lower SHP-1 activity would yield relatively higher

Fig. 5. FCRL5 has unique SHP-1– and Lyn-dependent dual functionality in B1 B cells. (A and B) PEC cells isolated from mev/mev and Lyn−/− mice were stainedand stimulated as in Fig. 1, and Ca2+ mobilization and intracellular pTyr were analyzed in the gated subsets. (C) PEC cells from the indicated mice werepreloaded with Indo-1/AM and blocked with anti-CD16/32 (2.4G2) before staining with discriminating surface markers, biotinylated F(ab’)2 rat anti-mouse IgM(SB73a), and intact CD5, CD22, CD32, CD72 or isotype-matched control mAbs (all at 10 μg/mL). Ca2+ mobilization in gated B1a cells was monitored before andafter the addition of SA (20 μg/mL). Arrows indicate the time of SA administration. The data are representative of at least two independent experiments.

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Lyn kinase function. Elevated Lyn activity was apparent in theaugmentation of FCRL5-induced apoptosis in mev/mev as com-pared with WT lymphocytes. Diminished SHP-1 activity also mightbe related to elevated basal levels of phosphorylated ERK (55, 56).Despite its enigmatic source of stimulation in B1 B cells, ERK

activation in response to agonistic T-cell receptor ligation canmodify SFK activity in a feedback loop that blocks SHP-1 function(57). Perhaps additional signals native to the coelomic cavitiestrigger Toll-like and/or cytokine receptors and commandeer B1 B-cell function. Sustained BCR triggering in combination with these

Fig. 6. FCRL5 function in innate-like B cells correlates directly with SHP-1 activity. (A) MZ B cells (5 × 106) sorted from hBAFF Tg mice were washed in serum-freeRPMI medium 1640 and rested for 2 h. Cells were then lysed directly or stained with biotinylated F(ab’)2 mAb fragments specific for IgM (SB73a) and FCRL5 (3B7)or an isotype control and were crosslinked with SA for 10 min at 37 °C. WCL or anti-FCRL5 (9D10) immunoprecipitates were immunoblotted for the indicatedproteins. Blots were stripped and reprobed with anti-FCRL5 (5-3B2) as a loading control. (B) Whole-cell protein tyrosine phosphorylation in the specified B-cellsubsets, discriminated by surface staining WT splenocytes with anti-CD19, CD21, and CD23 or PEC leukocytes with anti-CD19, CD5, and CD11b, was determinedat homeostasis, after stimulation with a biotinylated F(ab’)2 rat anti-mouse IgM mAb (SB73a) crosslinked with SA or after pervanadate treatment for 10 min.Cells were stained intracellularly with anti-pTyr or an isotype control mAb before analysis by flow cytometry. (C) Quantitative comparisons of constitutive pTyramong the indicated B-cell subsets. (D) Relative whole-cell protein pTyr in response to anti-IgM stimulation. (E) Comparison of basal SHP-1 abundance de-termined by intracellular staining. (F) Whole-cell protein pTyr in response to pervanadate treatment. (G) Assessment of total as well as active (Y397) or inactive(Y508) states of Lyn analyzed by intracellular staining with phospho-specific antibodies. Data in C–G are expressed as mean ± SEM for three independentexperiments, and isotype control staining represents combined analysis from the six subsets. *P < 0.05; **P < 0.01; ***P < 0.001.

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stimuli may down-modulate adaptive signaling as well as SHP-1expression, leading to their exhaustion, as seen for T cells in chronicinflammatory states (58).The possession of both functional ITAM-like and ITIM sequences

distinguishes FCRL5 from other coreceptors. These experimentsshowed that CD5, CD22, CD32, and CD72 could each inhibit BCR-mediated calcium mobilization in WT and Lyn-deficient B1 B cellsbut, unlike FCRL5, failed to acquire activation properties in theabsence of SHP-1. Relative to FCRL5’s balanced function in WTcells, mev/mev B1 B cells validated the bimodal qualities of FCRL5and also indirectly exposed Lyn’s preferential affinity for the ITAM-like Y543 residue. The lack of SFK redundancy at this location issuggestive of sequence-specific characteristics that distinctly accom-modate Lyn there. Although the molecular features conferring SFKtyrosine phosphorylation at the Y566 ITIM appear more in-discriminate, phosphatase docking was restricted to SHP-1. Thus,although Lyn and SHP-1 can concomitantly associate with FCRL5,their relative cytosolic activity balances this receptor’s overall func-tion. As a scaffold that directs their tandem positioning, it is possiblethat Lyn occupancy at Y543 could promote processive phosphory-lation at the Y566 ITIM (59). Lyn-dependent transphosphorylation,which has been shown for CD19, would maintain FCRL5 binaryfunction at balance unless quantitative differences or other in-tracellular factors compromised Lyn and/or SHP-1 recruitment tothese respective sites (60). Conversely, SHP-1 conceivably could in-terfere intramolecularly with Lyn’s activity. Lyn also imbues FCRL5with properties analogous to other activation receptors such as Igα,Igβ, CD19, andCD180/RP105 (8).When altered or untethered fromSHP-1 countersuppression, this Lyn-driven cascade potentially couldfuel FCRL5-mediated B-cell pathogenesis.Very recent studies by the Colonna group identified that human

FCRL4 and FCRL5 bind heat-aggregated IgA and IgG (19), butthe endogenous ligands for other B-cell–expressed FCRLs, in-cluding mouse FCRL5, have yet to be elucidated. Notably, twoadditional members interact with MHC-like proteins. In humansFCRL6, expressed by cytotoxic CD8+ T and natural killer cells,interacts with MHC class II; whereas in mice an immunoevasintermed “orthopoxvirus MHC class I-like protein” was identified asan FCRL5 ligand (20, 21). The consequences of these interactionsare under investigation; however, their associations have in-triguing implications for these receptors in mechanisms of toler-ance induction. Furthermore, the signaling characteristics definedhere for FCRL5 have relevance for other FCRL proteins. Thus farthese molecules have been found to inhibit BCR signaling gen-erally, but their conserved cytoplasmic motifs along with evidencefrom mutagenesis studies has revealed subtle hints of their possi-ble bifunctionality (27, 28). Thus, unearthing the FCRL5–Lyn

relationship raises the likelihood that other FCRLs possess similaractivation features.One underlying question relates to the integration of FCRL5

signaling with its discrete expression by MZ and B1 B cells. Giventheir front-line access to potential pathogens, a hallmark aspect ofthese lymphocytes is their preferential responsiveness to innateagonists and T-cell–independent antigenic stimulation. Accord-ingly, recent work demonstrated fascinating regulatory potentialfor FCRL4 in human B cells. Despite its possession of a tyrosine-based switch motif and two ITIMs that inhibit adaptive BCRfunction via SHP-1 and SHP-2 binding (29), Sohn et al. haveshown that FCRL4 also can enhance innate Toll-like receptor9-dependent signaling (61). Its variable influence on these pathwayswas transmitted in the absence of direct FCRL4 engagement, in-dicating that its expression alone maymodulate B-cell responsivitydifferentially to adaptive or innate stimuli. The identification ofthese features in another FCRL, along with the ability of FCRL5to recruit the active form of Lyn shown here, strongly indicatesthat these molecules are important modifiers of B-cell adaptiveand innate signaling. Moreover, in addition to its constitutive ex-pression by MZ and B1 B cells, FCRL5’s role as an innate regu-lator also is endorsed by its detection on subsets of B2 B cellsthat vary among mice and its sensitivity to induction by innatestimuli such as LPS (31). Importantly, Lyn also is required forCD180/RP-105 TLR activation in response to LPS binding (62).Thus, future studies of mouse deficiency models should betterclarify FCRL5’s roles in these two facets of B-cell biology.Finally, our clearer understanding of FCRL5 function provides

insight into the possible pathogenic importance of these moleculesin disorders with which they already have been widely associated.For example, FCRL2 and FCRL3 are expressed by a putative MZeffector memory population that circulates in human blood as wellas by chronic lymphocytic leukemia B cells that are believed tooriginate from this subset (22, 63). In addition to this lymphopro-liferative disorder, FCRL3 has been linked tomultiple autoimmunediseases including rheumatoid arthritis (25), and FCRL4 is associ-ated with dysfunctional humoral responses found in HIV- andmalaria-infected patients (24, 64). These observations underscorethe probability that FCRL proteins participate in perturbed im-munity. The current findings therefore provide insight into thefundamental regulatory contributionsof theFCRL family innormaland pathogenic B-cell function.

Materials and MethodsDetails of the mice strains used and their maintenance are described in SIMaterials and Methods. Similarly, the construction of chimeric receptors, theretroviral transduction methodology, and the cell lines and antibodies used

Fig. 7. FCRL5 reveals opposing roles for SHP-1 and Lyn in MZ- and B1 BCR-mediated survival. Splenic MZ or PEC B1 B cells (1 × 105) purified from the indicatedmice were stained with biotinylated F(ab’)2 mAbs specific for IgM (SB73a) and FCRL5 (3B7) or an isotype control, crosslinked with SA, and plated for culture intriplicate. Apoptosis was determined for MZ B cells at 12 h and for B1 B cells at 24 h by annexin V and propidium iodide staining. Total apoptotic cells includeboth the early (annexin V+PI−) and late fractions (annexin V+PI+). Data are shown as mean ± SEM for three independent experiments. **P < 0.01; ***P < 0.001.

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are outlined there. Also included in SI Materials and Methods are the pro-tocols for immunoblotting, coimmunoprecipitation, calcium mobilization,intracellular-staining, phospho-flow analysis, histologic staining, confocalmicroscopic imaging, apoptosis assays, and statistical analysis.

ACKNOWLEDGMENTS. We thank John Kearney and Peter Burrows fortechnical assistance and critical reading of the manuscript; Goetz R. A.Ehrhardt at the University of Toronto for providing materials and experttechnical help; Larry Gartland and Marion Spell in the University of

Alabama at Birmingham (UAB) Center for AIDS Research (NationalInstitutes of Health Grant AI027767) for cell-sorting assistance; CarloCroce at Ohio State University for the gift of TCL1 Tg mice; and CliffordLowell at the University of California, San Francisco for the contributionof Lyn-KO mice and suggestions concerning the manuscript. Confocalimaging was carried out at the UAB Arthritis and Musculoskeletal DiseaseCenter High-Resolution Imaging Facility (Grant P30 AR48311). This workwas supported in part by funding from the National Institutes of Health(Grant AI067467) and the American Cancer Society (Grant RSG 08-232-01).

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