Unique Ligand-Binding Property of the Human IgM Fc Receptor This information is current as of April 4, 2015.

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J Immunol 2015; 194:1975-1982; Prepublished online 19 January 2015; doi: 10.4049/jimmunol.1401866 http://www.jimmunol.org/content/194/4/1975

http://www.jimmunol.org/content/suppl/2015/01/19/jimmunol.140186 6.DCSupplemental.html This article cites 45 articles, 21 of which you can access for free at: http://www.jimmunol.org/content/194/4/1975.full#ref-list-1 Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscriptions Submit copyright permission requests at: http://www.aai.org/ji/copyright.html Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/cgi/alerts/etoc

The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 9650 Rockville Pike, Bethesda, MD 20814-3994. Copyright © 2015 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606.

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Supplementary Material

Kazuhito Honjo, Yoshiki Kubagawa, John F. Kearney and Hiromi Kubagawa

The Journal of Immunology

Unique Ligand-Binding Property of the Human IgM Fc Receptor Kazuhito Honjo,* Yoshiki Kubagawa,* John F. Kearney,† and Hiromi Kubagawa*,1

A

ntibodies have dual binding activity: to Ag via their N-terminal variable regions, and to effector molecules such as FcRs via their C-terminal constant regions. FcRs are expressed by many different cell types, and their interaction with Abs can initiate a broad spectrum of effector functions essential in host defense. These functions include phagocytosis of Ab-coated microbes, lysosomal degradation of endocytosed immune complexes, Ab-dependent cell-mediated cytotoxicity, secretion of cytokines and chemokines, release of potent inflammatory mediators, regulation of Ab production by B cells, survival of plasma cells, and presentation of degradable as well as nondegradable Ags (1–7). These diverse functions depend on the Ab isotype and the cell type expressing the FcR. Structurally and functionally diverse FcRs, namely FcR for IgG (FcgRI/CD64, FcgRII/CD32, FcgRIII/CD16, FcgRIV, FcRn), IgE (FcεRI, FcεRII/CD23), IgA (FcaR/CD89), and both polymeric IgA and IgM (Fca/mR/CD351), have been characterized extensively at both the protein and genetic levels (1–5, 8–10). It has long been a puzzle why an FcR for IgM (FcmR), the first Ig isotype to appear during phylogeny, ontogeny, and the immune

*Division of Laboratory Medicine, Department of Pathology, University of Alabama at Birmingham, Birmingham, AL 35294; and †Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL 35294 1

Current address: Deutsches Rheuma-Forschungszentrum Berlin, Berlin, Germany.

Received for publication July 22, 2014. Accepted for publication December 16, 2014. This work was supported in part by National Institutes of Health/National Institute of Allergy and Infectious Diseases Grants AI4782-36 (to J.F.K.) and R21AI094625 (to H.K.). Address correspondence and reprint requests to Dr. Hiromi Kubagawa, Department of Pathology, University of Alabama at Birmingham, 506 Shelby, 1825 University Boulevard, Birmingham, AL 35294-2182. E-mail address: [email protected] The online version of this article contains supplemental material. Abbreviations used in this article: 7-AAD, 7-aminoactinomycin D; [Ca2+]i, intracellular Ca2+ concentration; FAIM3, Fas apoptosis inhibitory molecule 3; NP, nitrophenyl; WT, wild-type. Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00 www.jimmunol.org/cgi/doi/10.4049/jimmunol.1401866

response, has defied identification, despite extensive biochemical evidence of IgM Fc–binding proteins accumulated over decades (11–13). We previously successfully identified a cDNA encoding an authentic FcmR from cDNA libraries of human B-lineage cells using a functional cloning strategy (14). FCMR is a single-copy gene located on chromosome 1q32.2, adjacent to two other IgMbinding receptor genes, Fca/mR and polymeric Ig receptor. The predicted FcmR is a transmembrane protein that consists of a single V-set Ig-like domain responsible for Fcm binding, an additional extracellular region with no known domain structure, a transmembrane segment containing a charged His residue, and a relatively long cytoplasmic tail (118 aa) containing three conserved Tyr and five conserved Ser residues. FcmR binds pentameric IgM with a surprisingly high avidity of ∼10 nM as determined by Scatchard plot analysis, with the assumption of a 1:1 stoichiometry of FcmR to IgM ligand. Upon ligation of FcmR with IgM ligands, both Tyr and Ser residues in the cytoplasmic tail are phosphorylated (14), and receptors are rapidly internalized into lysosomal compartments (15). Unlike other FcRs, the expression of FcmR is restricted to lymphocytes: B, T, and NK cells (14, 16). This suggests potentially distinct functions of FcmR as compared with other FcRs that are mainly expressed by myeloid cells. Alternatively, the FcmR was initially designated as Fas apoptotic inhibitory molecule 3 (FAIM3), because coligation of Fas and FcmR/FAIM3 with an agonistic IgM anti-Fas mAb prevented Fas-mediated apoptosis (17). Unlike the effect of IgM anti-Fas mAb, however, ligation of Fas with an agonistic IgG mAb induced apoptosis irrespective of the expression of FcmR/FAIM3 (14, 16, 18). Notably, coligation of Fas and FcmR/FAIM3 with the corresponding mouse IgG mAbs plus a secondary reagent [e.g., F(ab9)2 fragments of anti-mouse g Ab] had no demonstrable effects on the IgG anti-Fas mAb-induced apoptosis (14). This suggests that the antiapoptotic activity of FcmR/FAIM3 depends on usage of the IgM anti-Fas mAb, and not on physical proximity of two receptors induced by artificial coligation. However, because the FcmR-mediated inhibition of the IgM anti-Fas mAb-

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The IgM Fc receptor (FcmR) is the newest FcR, and coligation of FcmR and Fas/CD95 on Jurkat cells with agonistic IgM anti-Fas mAb was shown to inhibit Fas-induced apoptosis. The ligand-binding activity of human FcmR was further examined. FcmRmediated protection from apoptosis was partially blocked by addition of 104 molar excess of IgM or its soluble immune complexes, but it could be inhibited by addition of 10-fold excess of IgM anti-CD2 mAb. This suggests that FcmR binds more efficiently to the Fc portion of IgM reactive with plasma-membrane proteins than to the Fc portion of IgM in solution. The former interaction occurred in cis on the same cell surface, but not in trans between neighboring cells. This cis engagement of FcmR resulted in modulation of Ca2+ mobilization via CD2 on Jurkat cells or BCRs on blood B cells upon cross-linkage with the corresponding IgM mAbs. Several functional changes were observed with FcmR mutants: 1) significant increase in IgM ligand binding in the cytoplasmic tail-deletion mutant, 2) enhanced cap formation in FcmR upon IgM binding at 4˚C with a point mutation of the transmembrane His to Phe, and 3) less protective activity of FcmR in IgM anti-Fas mAb-mediated apoptosis assays with a point mutation of the membrane-proximal Tyr to Phe. These findings show the importance of the cis engagement of FcmR and its critical role in receptor function. Hence, FcmR on B, T, and NK cells may modulate the function of surface proteins recognized by natural or immune IgM Abs on the shared membrane cell surface. The Journal of Immunology, 2015, 194: 1975–1982.

1976 induced apoptosis is a reproducible and well-defined function, we employed this antiapoptotic assay in the present study as one of the readouts to explore the mode of action of FcmR. In this study, we have defined the unique ligand-binding properties of FcmR and the key residues involved in its receptor function.

Materials and Methods Cells

Immunofluorescence analysis Flow cytometric analysis of cell surface FcmR on transductants was performed by using biotin-labeled, receptor-specific mAb (HM14, g1k isotype) and isotype-matched control mAb, along with PE-labeled streptavidin as described (14). For IgM binding, cells were incubated with biotin-labeled mouse myeloma IgM (TEPC183) at 1 mg/ml or PBS, washed, and then incubated with PE-labeled streptavidin. Stained cells were examined by an Accuri C6 flow cytometer (BD Biosciences), and flow cytometric data were analyzed with FlowJo software (Tree Star).

Apoptosis assay Apoptosis assays were performed essentially the same as described before (14). In brief, FcmR+ or control Jurkat cells were cultured for 15 h with or without agonistic IgM anti-Fas mAb (CH11, 10 ng/ml; Millipore), along with various concentrations of inhibitors: 1) IgM mAbs specific for nitrophenyl (NP) hapten (B1-8) (19) or a1-3 dextran (clone 1-21) (20), 2) their soluble immune complexes with NP25-coupled BSA (molecular mass of ∼70 kDa; Biosearch Technologies) or a1-3 dextran (molecular mass of ∼1000 kDa), 3) FcmR-blocking (HM7) or nonblocking (HM3) mAbs (both g2bk), and 4) IgM mAb specific for the human CD2 (C373) or Jurkat TCR (C305) (21). The latter two mAbs were provided by Dr. Arthur Weiss (Howard Hughes Medical Institute, University of California, San Francisco) and were purified from ascites by Sephacryl S300 gel filtration, and the remainders were purified by Ag-coupled affinity columns. For soluble immune complexes, IgM anti-NP and IgM anti-dextran mAbs and the corresponding Ags were premixed to reach a final concentration in the cultures of 104 or 105 ng/ml for IgM mAbs with different molar ratios of Ab/Ag (1:10 to 1:1022). Apoptotic cells were detected by staining with 7-aminoactinomycin D (7-AAD) and allophycocyanin-labeled annexin V (BD Biosciences) as described (14). For mixed cell cultures, FcmR+ or control Jurkat cells were cocultured with 10-fold excess number of FcmR+ BW5147 cells in the presence or absence of CH11 anti-Fas mAb, followed by cell surface staining with PE–anti-mouse CD3 mAb (145-2C11; BD Biosciences). Apoptosis was assessed for Jurkat cell populations by excluding CD3+ live BW5147 cells.

Calcium mobilization A mixture containing equal numbers of FcmR+ and WT Jurkat cells preloaded with 5 mM Indo-1/AM (Molecular Probes), a Ca2+-sensitive dye, was stimulated by various concentrations of IgM mAbs specific for CD2 or TCR or by 1 mM ionomycin as a positive control. The intracellular Ca2+ concentrations ([Ca2+]i) in both cell populations were simultaneously assessed by the ratio of emitted fluorescence of bound Ca2+ at 405 nm and free Ca2+ at 485 nm using an LSR II flow cytometer (BD Biosciences). In some experiments, an equal mixture of control GFP+ and WT Jurkat cells was also assessed similarly as controls. For blood B cells, MACS-enriched B cells (.95%) were loaded with Fluo-4/AM (0.25 mM; Molecular Probes), rested, and then treated with a mixture of mouse IgM anti-human k mAb (END-5C1, 10 mg/ml) and mouse IgG2b anti-FcmR mAb (HM7 or HM3, 100 mg/ml). Changes in Fluo-4 fluorescence in the labeled cells following treatment were measured over real time with an Accuri C6 flow cytometer and analyzed by FlowJo software as described (22).

FcmR mutants FcmR cDNA constructs with point mutations (H253F, Y315F, Y366F, or Y385F) were generated by a QuickChange site-directed mutagenesis kit (Stratagene) using nonmutated FcmR cDNA as a template DNA and a set of primers: 1) 59-GAAGGCCAAGGATTTTTCATCCTGATCCCGACCAT-39 and 59-ATGGTCGGGATCAGGATGAAAAATCCTTGCCCTTC-39 for H253F, 2) 59-CTCCCAAAACAACATCTTCAGCGCCTGCC-39 and 59-GGCAGGCGCTGAAGATGTTGTTTTGGGAG-39 for Y315F, 3) 59-GACCAGCTGTGAATTTGTGAGCCTCTACCACCAG-39 and 59-CTGGTGGTAGAGGCTCACAAATTCACAGCTGGTC-39 for Y366F, and 4) 59-GGACAGTGATTCAGATGACTTCATCAATGTTCCTG-39 and 59-CAGGAACATTGATGAAGTCATCTGAATCACTGTCC-39 for Y385F (underlining indicates mutation sites), according to the manufacturer’s recommendation. For FcmR cytoplasmic tail deletion, an FcmR cDNA construct with a deletion of most of the cytoplasmic tail (A281–A390; DCy) but with an inclusion of the posttransmembrane basic aa-rich region (K273–K280) was generated by PCR using PrimeStar HS DNA polymerase (TaKaRa), FcmR cDNA as a template DNA, and a set of primers: 59-GAATTCTAGAAGGGACAATGGACT-39 and 59-GCGGCCGCCTATTTCCTCCTTTCAACGGCC-39 (italics indicate EcoRI and NotI sites, and underlining indicates translation start and stop sites). The amplified PCR products of the expected size were gel purified and subcloned into the ZeroBlunt TOPO vector (Invitrogen). All plasmid DNAs encoding FcmR mutants were digested with appropriate restriction enzymes, gel purified, and ligated into the pMX-PIE retroviral expression vector as described (14). After confirming the correct sequences with the expected mutation of the resultant cDNAs at both strands of DNA, they were transfected into 293T-A packaging cell line before transducing Jurkat cells as described (14). GFP+ transductants were enriched by FACS before IgM binding, apoptosis, and Ca2+ mobilization analyses.

Epifluorescence microscopic analysis Viable cells were incubated for 20 min on ice with Alexa Fluor 555–labeled, affinity-purified mouse monoclonal IgM (MD4 anti-hen egg lysozyme mAb) at 10 mg/ml in PBS with or without 0.1% NaN3, washed in PBS/1% FCS/0.1% NaN3, and cytocentrifuged onto glass slides prior to being airdried and fixed in 95% ethanol/5% glacial acetic acid at 220˚C. Some aliquots of cells were resuspended in warmed RPMI 1640/10% FCS and incubated for an additional 10 min at 37˚C before cytocentrifugation. The stained cells were examined under a Leica/Leitz DMRB microscope. Images were acquired with a Hamamatsu C4742 video camera and processed with Openlab software.

Results Interaction of FcmR with soluble IgM immune complexes versus IgM bound to membrane components via the Fab region In our previous studies apoptosis-prone human Jurkat cells stably expressing FcmR were shown to be protected from Fas/CD95mediated apoptosis when ligated with an agonistic IgM anti-Fas mAb (CH11), but not when ligated with an agonistic IgG anti-Fas mAb or Fas ligand (14, 18). Notably, coligation of FcmR and Fas with the corresponding IgG mAbs plus a common secondary reagent could not prevent IgG anti-Fas mAb-induced apoptosis (14). To determine whether this apoptosis protection mediated by FcmR is affected by IgM ligands, IgM mAbs specific for NP (clone B1-8) or a1-3 dextran (clone 1-21) and their immune complexes were initially used as inhibitors in the apoptosis assays. As expected, addition of IgM anti-Fas mAb at 10 ng/ml induced robust apoptosis of control GFP+ cells, but not GFP+/FcmR+ cells as determined by visualizing early (annexin V+/7-AAD2) and late (Annexin V+/7-AAD+) apoptotic cells (Fig. 1A, upper second column). The addition of both types of IgM mAbs at 104 ng/ml (upper third column; not shown for anti-dextran mAb) or their soluble immune complexes (upper fourth and lower first columns) did not affect the FcmR-mediated protection, suggesting the inability of such IgM molecules to block the interaction of the Fc portion of IgM Fas mAb with FcmR. The failure to inhibit protection was observed with wide ranges of Ab/Ag molar ratios (Fig. 1B, Supplemental Fig. 1). However, addition of both types of IgM at 105 ng/ml (Fig. 1B, Supplemental Fig. 1) and their immune complexes with (Fig. 1A, lower second column, 1B, Supplemental

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The human leukemic T cell (Jurkat) and mouse thymoma (BW5147) lines, which stably expressed either only GFP as a control or both GFP and human FcmR, were prepared by retrovirus-mediated transduction as previously described (14) and were maintained in media containing puromycin at 1.5 and 0.75 mg/ml, respectively. BW5147 cells stably expressing human FcmR (without GFP) and wild-type (WT) Jurkat or BW5147 cells were maintained in media only as described (14). For Ca2+ mobilization, mononuclear cells isolated from healthy individuals by Ficoll-Hypaque density gradient centrifugation were enriched for B cells using a B cell isolation kit II (Miltenyi Biotec) with MACS. The frequency of CD19+ B cells in the resultant enriched fractions was .95% as determined by flow cytometry. This study involving human subjects was approved by the institutional review board of the University of Alabama at Birmingham.

THE CIS ENGAGEMENT OF FcmR

The Journal of Immunology

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FIGURE 1. Role of FcmR in IgM anti-Fas mAb–induced apoptosis. (A) Representative flow cytometric profiles. Jurkat cells stably expressing GFP only (GFP) as a control or both GFP and human FcmR (GFP/FcmR) were cultured with (+) or without (2) agonistic IgM anti-human Fas mAb (clone CH11, 10 ng/ml) in the presence or absence of the inhibitors with the indicated concentrations (ng/ml) in parentheses: IgM anti-NP mAb (B1-8) or its soluble complexes with NP25-BSA, IgM anti-dextran mAb (1-21, not shown) or its soluble complexes with dextran, IgM anti-CD2 mAb (C373), and IgG2b blocking (bl; HM7) or nonblocking (nb; HM3) anti-FcmR mAb. The Ab/Ag molar ratios were 1:1021 and 1:1022 in the upper fourth and fifth columns and 1:1 and 1:1021 in the lower first and second columns. After culture, cells were stained with allophycocyanin–annexin V and 7-AAD before identification of early apoptotic (annexin V+/7-AAD2), late apoptotic (annexin V+/7-AAD+), and dead (annexin V2/7-AAD+) cells by flow cytometric analysis. Numbers in each quadrant indicate percentages of cells. (B) Effects of IgM and its immune complexes on FcmR-mediated antiapoptotic activity. GFP+ (open columns) or GFP+/FcmR+ (filled columns) cells were cultured with IgM anti-Fas mAb (10 ng/ml) in the presence of the indicated concentrations (ng/ml) of IgM anti-NP mAb alone or with NP25-BSA as indicated for Ab/Ag molar ratios. Apoptotic (early and late) cells were similarly assessed and percentage relative apoptosis was determined as follows: 100 3 [(% anti-Fas–induced apoptotic cells with or without inhibitors in GFP+ or FcmR+/GFP+ cell line) 2 (% spontaneous apoptotic cells in the corresponding cell line)] 4 [(% anti-Fas mAb–induced apoptotic cells without inhibitors in GFP+ cell line) 2 (% spontaneous apoptotic cells in GFP+ cell line)]. Results are shown as means 6 1 SD from three to five independent experiments. The dotted line indicates percentage apoptosis of FcmR+/GFP+ cells in the presence of IgM anti-Fas mAb without inhibitors. (C) Efficient inhibition of IgM anti-CD2 mAb in FcmR-mediated antiapoptotic activity. GFP+ (open columns) or GFP+/FcmR+ (filled columns) cells were similarly cultured with the indicated doses (ng/ml) of IgM anti-CD2 mAb along with IgM anti-Fas mAb (10 ng/ml) before assessment of apoptotic cells. Results are shown as means 6 1 SD from three independent experiments. The dotted line indicates percentage apoptosis of FcmR+/GFP+ cells in the presence of IgM anti-Fas mAb without IgM anti-CD2 mAb. Results using a Student t test comparison of antiFas mAb–induced apoptosis in the absence and presence of inhibitors are indicated as *p , 0.05, **p , 0.01, and ***p , 0.001.

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Cis engagement of FcmR with IgM anti-Fas mAb on the same cell surface To determine whether the engagement of FcmR with IgM attached to plasma membrane occurs in trans or cis (see Fig. 2A), we performed mixed cell cultures of FcmR+ (or control) Jurkat cells

FIGURE 2. Cis engagement of FcmR. (A) Theoretical FcmR-mediated protection in IgM anti-Fas mAb– induced apoptosis. In theory, human Fas receptor (three red vertical lines) on Jurkat cells (light yellow-filled circles) is recognized by CH11 IgM anti-human Fas mAb (purple Y; x5 indicates pentamer) and its Fc portion binds FcmR (blue ovals) in cis on the same cells or in trans between neighboring cells, thereby inhibiting Fas-induced apoptosis. Addition of 10-fold excess of mouse thymoma line (BW5147) expressing human FcmR (white circles) to the apoptosis assay of Jurkat cells is predicted to inhibit the trans (two-headed solidline arrow), but not cis (two-headed broken-line arrow), interaction of FcmR with the CH11 IgM anti-Fas mAb. (B) Mixed cell cultures. FcmR+ (lower) or control (upper) Jurkat cells were cocultured with 10-fold excess number of human FcmR+ BW5147 cells (of mouse origin) in the presence or absence of CH11 anti-Fas mAb. Cells were stained with PE-labeled anti-mouse CD3 mAb prior to assessment of apoptosis in Jurkat cell populations by excluding CD3+ live BW5147 cells. The surface levels of FcmR on BW5147 cells were higher than those on Jurkat cells. Result representative of three independent experiments.

plus a 10-fold excess of mouse BW5147 cells stably expressing human FcmR. When the Fc portion of IgM anti-Fas mAb binds FcmR in trans between the excess of cocultured FcmR+ BW5147 cells, then FcmR+ Jurkat cells will become apoptotic. Alternatively, when the Fc portion of IgM anti-Fas mAb must bind FcmR in cis on the same cell surface to perform this protective function, FcmR+ Jurkat cells will still be protected against IgM anti-Fas mAb–mediated apoptosis even in the presence of a 10-fold excess number of FcmR+ BW5147 cells. As shown in Fig. 2B, addition of excessive FcmR+ BW5147 cells did not affect the FcmR-mediated protection from apoptosis (second versus fourth column), suggesting predominance of the protective mechanism involving a cis interaction of the Fc portion of IgM anti-Fas mAb with FcmR on the same cell surface. Addition of excessive FcmR+ BW5147 cells did not spontaneously induce apoptosis of either of FcmR+ or control Jurkat cells in the absence of anti-Fas mAb (third column). Collectively, these findings show that although FcmR binds soluble IgM pentamers at a high avidity (14), FcmR binds more efficiently to the Fc portion of IgM Ab when it reacts with a membrane component on the same cell surface. This finding implies that FcmR can modulate the functional activity of lymphocyte cell surface receptors or molecules recognized by either natural or immune IgM Abs. Calcium mobilization by coligation of FcmR with CD2 or BCR by IgM mAbs To explore this possibility, we compared Ca2+ mobilization in cells with a single ligation of CD2 or TCR versus the coligation of both FcmR and CD2 or TCR. An equal mixture of WT and FcmR+ Jurkat cells was preloaded with the Ca2+ dye Indo-1/AM and simultaneously stimulated by various concentrations of IgM mAbs specific for CD2 or TCR. Although it has been well documented that induction of CD2-mediated Ca2+ mobilization in T cells requires ligation of CD2 with two different IgG mAbs (23–25), the IgM anti-CD2 mAb (C373) alone clearly induced Ca2+ mobilization (Fig. 3A). Notably, the rise in [Ca2+]i occurred signifi-

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Fig. 1) partially but significantly blocked the FcmR-mediated protection. All three IgM preparations (CH11, B1-8, and 1-21) were found to bind equally to FcmR+ cells (not shown), ruling out the possibility that the CH11 IgM has a unique posttranslational modification to bind more efficiently the FcmR compared with others. In contrast, addition of FcmR-blocking IgG2b mAb (HM7), but not FcmR nonblocking IgG2b mAb (HM3), at 104 ng/ml specifically inhibited the interaction of the Fc portion of IgM Fas mAb with FcmR, thus permitting the FcmR+ cells to become apoptotic (Fig. 1A, lower fourth and fifth columns). These findings indicate that soluble IgM immune complexes are not good competitors in the interaction of FcmR with the Fc portion of IgM antiFas mAb, although FcmR is shown by Scatchard plot analysis to bind pentameric IgM with a surprisingly high avidity of ∼10 nM (14). This also suggests the possibility that FcmR may bind more efficiently to the Fc portion of IgM, which is attached to plasma membranes via its Fab region, than to the Fc portion of free IgM or its immune complexes in solution. To test this hypothesis, we employed additional IgM mAbs reactive with CD2 or TCR on the surface of Jurkat cells as potential competitors for the interaction of IgM Fas mAb with FcmR. Addition of IgM anti-CD2 mAb (C373) at 103 ng/ml efficiently inhibited FcmR-mediated protection, thereby permitting the FcmR+ cells to undergo apoptosis (see Fig. 1A, lower third column, 1C). The anti-CD2 mAb alone did not induce apoptosis of either of cell types (not shown). Similar results were also obtained with an IgM anti-TCR mAb (C305, not shown). Thus, these findings suggest that FcmR binds more efficiently to IgM mAbs, which attach to cell surface components via their Fab regions, than to IgM immune complexes in solution.

THE CIS ENGAGEMENT OF FcmR

The Journal of Immunology

Key residues in the FcmR transmembrane and cytoplasmic tail responsible for receptor functions In FcRs and in other paired Ig-like receptors, it is generally observed that when the ligand-binding a-chain has a short cytoplasmic tail with no signal-transmitting potential, then it contains a charged residue in the transmembrane segment that facilitates noncovalent association with another transmembrane protein– containing ITAMs. This association allows for the transmission of activating signals to cells as seen in FcgRI, FcgRIII, FcaR, and FcεRI. Alternatively, when the ligand-binding a-chain has a conventional hydrophobic transmembrane segment, then it usually has a long ITAM-containing cytoplasmic tail (26). This recruits the phosphatase SHIP upon phosphorylation to attenuate the signals as in FcgRIIB. However, FcmR is unusual in having both features: a charged His residue (H253) in the transmembrane region and a long cytoplasmic tail containing three conserved Tyr (Y315, Y366, Y385) and five conserved Ser residues, features that have been conserved in multiple species (see Fig. 4A) (14). These characteristics suggest a dual signaling ability of FcmR: one from a potential adaptor protein noncovalently associating with the FcmR via the H253 residue, and the other from its own Tyr and/or Ser residues in the cytoplasmic tail.

To determine which part of the FcmR molecule is responsible for these potential functions, we made FcmR cDNA constructs with point mutations (H253F, Y315F, Y366F, or Y385F) or a deletion of most of the cytoplasmic tail (A281–A390; DCy) and expressed them in Jurkat T cells. After enriching GFP+ cells by FACS, the surface expression of FcmR as determined by reactivity with receptor-specific mAbs was comparable among the nonmutant and mutant FcmR transductants (Fig. 4B), suggesting that any introduced mutation does not affect the surface expression of FcmR on Jurkat cells. Remarkably, however, the IgM ligand-binding activity was significantly increased by the DCy mutant (Fig. 4C, 4D), suggesting the influence of the FcmR cytoplasmic tail on its ectodomain-mediated ligand binding. Next, the fate of IgM ligands after binding to FcmR on these transductants was examined by epifluorescence microscopy. IgM binding at 4˚C in the presence of NaN3, an inhibitor of energydependent contraction of actin filament, was observed in a weak patchy staining pattern at the peripheral rim of transductants regardless of their mutations. However, when incubated at 4˚C without NaN3, distinct IgM binding, marked by a more intense and localized or capping pattern, was observed with the H253F mutant (Fig. 4Eb) as compared with all others except for the DCy mutant, which exhibited a more broadly localized staining pattern (Fig. 4Ea for nonmutated). This suggests an important role of the His253 residue in the anchoring of FcmR in the transmembrane. After washing and incubating at 37˚C for 10 min without NaN3, the IgM/FcmR complexes were internalized and localized in intracellular locations, presumably endosomal or lysosomal compartments, in the FcmR nonmutated (Fig. 4Ec), H253F (Fig. 4Ed), and Y315F transductants (not shown), but not in other mutants (Y366F, Y385F, and DCy, not shown). These results are consistent with previous findings reported by others that two C-terminal– conserved Tyr residues were involved in receptor-mediated endocytosis (15). The FcmR-mediated protection from apoptosis was significantly diminished in the FcmR Y315F and DCy mutants, as the frequencies of apoptotic cells in these mutants were indistinguishable from those in the control transductant (Fig. 4F). Other FcmR mutants (H253F, Y366F, and Y385F) still had significant protective activity compared with controls (GFP+), but the extent of their protective activity was significantly lower than that of the nonmutated FcmR. These findings suggest that the membraneproximal Tyr residue (Y315) is largely responsible for, but other Tyr residues also contribute minimally to, the FcmR-mediated protection. Notably, the rapid rise in IgM anti-CD2 mAb– induced [Ca2+]i observed in the FcmR nonmutant was abolished by all of the FcmR mutants, suggesting that both the cytoplasmic tail including all three Tyr residues and the transmembrane His residue are involved in signaling mediated by coligation of CD2 and FcmR.

Discussion FIGURE 3. Ca2+ mobilization by IgM mAbs against CD2 or Ig k. (A) An equal mixture of WT (GFP2, blue lines) and FcmR+/GFP+ (red lines) Jurkat cells preloaded with Ca2+ dye Indo-1/AM were stimulated with IgM anti-CD2 (C373) mAb at 10 mg/ml or by 1 mM ionomycin at the time point indicated by arrows. The [Ca2+]i levels were assessed by the 405/485 nm fluorescence ratio in each viable cell population using an LSR II flow cytometer. This is a representative result from four independent experiments. (B) Fluo-4–loaded blood B cells were treated with IgM anti-human k mAb (END-5C1, 10 mg/ml) in the absence (red line) or presence of IgG2b, FcmR-blocking HM7 (blue line), or nonblocking HM3 (green line) mAb (100 mg/ml) at the time point indicated by an arrow. The [Ca2+]i levels were assessed by fluorescence intensity during a 5-min period. Representative result from three independent experiments.

The present study is focused on the IgM ligand-binding property of FcmR, and several remarkable features of the FcmR have been identified. First, although FcmR binds soluble pentameric IgM at a relatively high avidity of ∼10 nM (14), FcmR actually interacts more efficiently with the Fc portion of IgM Abs when bound to plasma membranes via their Ag-binding Fab regions, and this interaction occurs in cis on the same cell surface. This conclusion is based on the functional assays rather than actual ligand-binding assessments. Second, the Ca2+ mobilization induced by crosslinkage of CD2 or BCRs with IgM mAbs differs between the ligation of CD2 or BCR alone and the coligation of FcmR and CD2 or BCR. Third, several remarkable functional changes are observed

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cantly faster in FcmR+ cells than in WT cells when ligated with the anti-CD2 mAb at three different concentrations (3, 10, and 30 mg/ml, data at 3 and 30 mg/ml are not shown). A similar difference, but to a much less extent, was also observed with the crosslinkage of TCR with IgM mAb (C305) (not shown). In contrast, the ionomycin-induced [Ca2+]i rise occurred at the same time in both cell types. These findings suggest that the coligation of FcmR with CD2 or TCR induces a more rapid increase in [Ca2+]i than does the ligation of CD2 or TCR alone. Because we found that freshly prepared blood T cells, unlike Jurkat cells, were not activated by the C373 anti-CD2 mAb (not shown), we switched to using a mitogenic IgM anti-k mAb (END5C1) and examined Ca2+ mobilization induced by BCR crosslinkage in the presence of FcmR-blocking (HM7) or nonblocking (HM3) mAbs. As shown in Fig. 2B, IgM anti-k mAb–induced Ca2+ mobilization was indistinguishable in the absence or presence of FcmR nonblocking mAb (HM3). In contrast, the presence of FcmR-blocking mAb (HM7) diminished the IgM anti-k mAb–induced Ca2+ mobilization, suggesting that FcmR provides a stimulatory signal upon BCR cross-linkage with IgM mAbs.

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THE CIS ENGAGEMENT OF FcmR

with FcmR mutants: 1) increased IgM ligand binding by the FcmR DCy mutant, 2) enhanced cap formation of the FcmR H253F mutant even at 4˚C upon IgM ligand binding, and 3) less protective activity of the FcmR Y315F mutant in IgM anti-Fas mAb–mediated apoptosis. The preferential interaction of FcmR with IgM attached to plasma membrane via its Fab region was initially notified in our previous reconcilement of antiapoptotic activity of Toso, the original designation given to FcmR (14, 17). In the present study, we have defined more quantitatively the ligand-binding property of FcmR. The interaction of FcmR with the Fc portion of IgM antiFas mAb in Jurkat cells was not inhibited by addition of 103-fold excess of IgM mAbs or their soluble immune complexes, but it was blocked by addition of 104-fold excess of IgM mAbs or im-

mune complexes, 103-fold excess of FcmR-blocking mAb (HM7, g2bk isotype), or 10-fold excess of IgM anti-CD2 or anti-TCR mAb binding to the Jurkat cells. The interaction of FcmR with the Fc of IgM anti-Fas mAb occurred in cis on the same cell surface and not in trans between neighboring cells, as evidenced by the fact that adding 10-fold excess of FcmR+ BW5147 cells (of mouse origin) did not affect FcmR-mediated protection from IgM antiFas mAb–induced apoptosis. The preferential cis engagement of FcmR is thus distinct from the trans engagement of FcgRIIb, an inhibitory FcgR, in death receptor–mediated apoptosis. The interaction of agonistic IgG mAbs against death receptors, including Fas/CD95, with FcgRIIb is essential for the death receptor–mediated apoptosis and occurs in trans, but not in cis (27–29). The cis engagement of FcmR is significant in that FcmR has the po-

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FIGURE 4. Effects of FcmR mutations on receptor function. (A) Predicted protein structure of FcmR. The human FcmR cDNA encodes type I transmembrane protein that consists of a single V-set Ig-like domain (oval shape), an additional extracellular region with no known structure, a transmembrane segment (between two thick lines) containing a charged His residue (d), and a relatively long cytoplasmic tail containing three conserved Tyr residues (s). Point mutations are indicated and the extent of the deletion of the cytoplasmic tail is shown by the black bracket. Hatch marks indicate exon boundaries in the FCMR gene. (B–E) Cell surface FcmR levels and IgM ligand binding. Jurkat cells stably expressing only GFP (Cont.) or both GFP and FcmR of nonmutated (NM) or indicated mutations (H253F, Y315F, Y366F, Y385F or DCy) were assessed for cell surface levels of FcmR determined by HM14 mAb (B) and for IgM ligand-binding activity (C). The mean fluorescence intensity (MFI) of FcmR levels was determined as MFI of anti-FcmR mAb minus MFI of control mA (B) and the MFI of IgM binding as MFI of IgM minus MFI of PBS (C). Results are shown as the MFI 6 1 SD from three to five independent experiments. *p , 0.05 when compared with nonmutated (NM) FcmR. (D) Representative flow cytometric profiles. FcmR NM (blue) and DCy (red) cells were stained with biotin-HM14 anti-FcmR (open) or control (shaded) mAb for cell surface expression of FcmR (left) and with biotin-IgM myeloma (open) or PBS (shaded) for ligand binding (right). Because profiles with isotype-matched mAb or PBS were the same between FcmR NM and DCy cells, only one shaded profile is shown. (E) Representative epifluorescence microscopic images. The FcmR NM (Ea and Ec) and H253F (Eb and Ed) cells were incubated with Alexa Fluor 555–IgM (without NaN3) on ice (Ea and Eb), washed, and then incubated for additional 10 min at 37˚C (Ec and Ed) before cytocentrifugation. Fluorescence images were combined with phase-contrast cell images. Scale bars, 10 mm. (F) FcmR-mediated protection from apoptosis. FcmR NM or mutant and control Jurkat cells were incubated with CH11 anti-Fas mAb as described in the Fig. 1 legend, and the frequencies of early and late apoptotic cells are plotted as means 6 1 SD from three independent experiments. *p , 0.05, **p , 0.01, *** p , 0.001 when compared with FcmR-/GFP+ control cells.

The Journal of Immunology

of interest, given the unique sequence around the Y315 residue: P·R·S/T·Q·N·N·I/V·Y·S/T·A·C·P·R·R·A·R323 (bold type indicates conserved amino acids). This does not match any known Tyr-based signaling motifs. The mechanism of Toso/FcmRmediated protection from apoptosis was suggested to result from potentiation of the cellular FLIP, a master antiapoptotic regulator (17), or alternatively by prevention of the internalization of Fas, an important step for apoptosis signaling (44, 45), owing to simultaneous cross-linkage of both Fas and FcmR with IgM Fas mAb (16). With regard to the latter, it is noteworthy that the coligation of FcmR with CD2 or TCR induced a more rapid increase in intracellular Ca2+ levels than in the ligation of CD2 or TCR alone. In conclusion, FcmR, the newest member of the FcR family, interacts more efficiently with the Fc portion of the IgM Abs, which are bound to plasma membranes via their Ag-binding Fab region, as compared with the Fc portion of IgM Abs or their immune complexes in solution. The former interaction occurs in cis on the same cell surface and not in trans between neighboring cells. This strongly implies that FcmR expressed on B, T, and NK cells may have a potential to modulate the function of target Ags or receptors, which are recognized by natural or immune IgM Abs, on the same cell surface. 308

Acknowledgments We thank Dr. Arthur Weiss for the gift of IgM mAbs specific for CD2 (C373) or Jurkat TCR (C305) and for valuable suggestions, Dr. Peter Burrows for critical reading of the manuscript, Enid Keyser for FACS sorting, and Marion Spell for Ca2+ mobilization.

Disclosures The authors have no financial conflicts of interest.

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tential to modulate the functional activity of lymphocyte surface receptors on the same cell when they are recognized by natural or immune IgM Abs. The physiological relevance of this cis engagement may be related to unique features of the IgM ligand and its receptor. In idiotype network theory, BCRs (or their secreted products) are proposed to be directed toward the variable regions of other BCRs or their Ig molecules. Two thirds of the newly generated B cells in bone marrow are now known to react with self-antigens such as dsDNA, ssDNA, insulin, and LPS (30). According to our estimates, approximately a fourth of the IgMcontaining culture supernatants from 96 different EBV-transformed neonatal B cell lines react with lymphocyte surface components (H. Kubagawa, K. Honjo, and Y. Kubagawa, unpublished observations). IgM anti–lymphocyte Abs are often present in individuals with autoimmune diseases or chronic viral infections, such as HIV-1, and recognize many different surface Ags (e.g., CD45, CD175/Tn, CD3ε, CD4, chemokine receptors, sphingosine-1-phosphate receptor 1), and some of those Abs regulate T cell–mediated inflammatory responses in vitro (31–41). Unlike FcRs for switched Ig isotypes, the expression of FcmR is restricted to lymphocytes, namely, B, T, and NK cells (14). Therefore, it is highly probable that FcmR on B, T, and NK cells preferentially binds the Fc portion of IgM Ab reactive with their cell surface Ags or receptors, thereby modulating the function of their target Ags or receptors. Supporting this idea, the CD2-mediated Ca2+ mobilization after cross-linkage with IgM mAb was clearly faster in FcmR+ than control Jurkat cells. The Ca2+ mobilization induced by cross-linkage of BCR on blood B cells with IgM anti-k mAb was significantly different in the presence of an FcmR blocker (HM7) versus an FcmR nonblocker (HM3). The HM7 blocker diminished BCR-mediated Ca2+ mobilization, whereas the HM3 nonblocker did not. Unlike the coligation of FcmR and Fas receptor, FcmR provides a stimulatory signal upon coligation with CD2 or BCR. Mutational analyses of FcmR revealed several remarkable alterations in its function. Whereas nonmutated and mutant FcmR transductants expressed comparable levels of cell surface FcmR as judged by receptor-specific mAbs, the IgM ligand-binding activity was significantly increased in the DCy mutant. This suggests either the formation of oligomeric FcmR due to its presumably mobile nature within the plasma membrane or the inside–out regulation of ligand-binding activity of FcmR by its cytoplasmic tail. Given the precedent of inside–out signaling regulation in the integrin family of cell surface adhesion molecules (42) and the FcaR (43), both possibilities are equally conceivable and remain to be elucidated. When the fate of IgM-bound FcmR was examined by immunofluorescence microscopy, enhanced cap formation was clearly observed with the H253F mutant even at 4˚C without NaN3 as compared with FcmR nonmutated or other mutant transductants, except the DCy mutant, which displayed a more broadly localized staining pattern. This finding suggests a functional role of His253 residue in the anchoring of FcmR in the transmembrane. Although we have not yet identified a membrane protein noncovalently associating with the ligand-binding chain of FcmR through its H253, there are several possible candidates, including the ITAMcontaining CD3z, FcεRIg, or DAP12, the PI3K-recruiting DAP10, or the unknown 40-kDa protein (p40), which was often coprecipitated with the 60-kDa FcmR in our previous analyses (14). Notably, unlike other multichain FcRs, the FcmR H253F mutant presented on the cell surface without a potentially associated membrane protein. The findings that the Y315F and DCy mutants had no significant protective activity against IgM anti-Fas mAb–induced apoptosis is

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THE CIS ENGAGEMENT OF FcmR