Cutting Edge: FcR-Like 6 Is an MHC Class II Daniel M. Schreeder, John P. Cannon, Jiongru Wu, Ran Li, Mikhail A. Shakhmatov and Randall S. Davis This information is current as of September 29, 2021. J Immunol 2010; 185:23-27; Prepublished online 2 June 2010; doi: 10.4049/jimmunol.1000832 http://www.jimmunol.org/content/185/1/23 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2010 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Cutting Edge: FcR-Like 6 Is an MHC Class II Receptor †,‡ x x x Daniel M. Schreeder,* Johnx { P. Cannon, Jiongru Wu, Ran Li, Mikhail A. Shakhmatov, andRandallS.Davis*, , Receptors for the Fc portion of Ig have been extensively being phosphorylated and recruiting the Src homology 2- characterized and are known to regulate humoral re- domain containing phosphatase 2 (7, 8). sponses, but members of the closely related FcR-like Despite the many similarities between the FcR and FCRL (FCRL)familyhavenotbeenfoundtobindIg,andtodate, families, no FCRL has been shown to bind Ig, and thus ligands no ligand has been identified for any FCRL. Using a cell- for these receptors remain unknown. In this study, we report basedGFPreportersystemandarecombinantFcchimeric that the MHC class II molecule HLA-DR is a ligand for hu- protein, we show that human FCRL6, a receptor selec- man FCRL6. Using a cell-based reporter system, FCRL6 li- gand reactivity was found to be restricted to APCs, and the tively expressedbycytotoxicTandNK cells,directlybinds Downloaded from HLA-DR,an MHC class II molecule. Given the similarity development of a panel of blocking Abs facilitated the identi- among constant regions of Ig and MHC molecules, these fication of HLA-DR as the interacting partner. This associ- findings suggest that representatives of the FcR and FCRL ation was further confirmed using HLA-DR transductants for FCRL6-specific induction assays and selective binding of multigene families may have independently evolved to a soluble FCRL6-Fc chimeric molecule. engage two ancestral elements fundamental to adaptive immunity. This discovery may offer new insight into Materials and Methods http://www.jimmunol.org/ the interaction between cytotoxic and APCs Cells and may have important implications for better under- 43-1 FCRL6z cells were generated as previously described (9). Briefly, the FCRL6 standing HLA disease susceptibility and pathogen- extracellular region was PCR amplified from full-length cDNA with the KOD esis. The Journal of Immunology, 2010, 185: 23–27. polymerase (Novagen/EMD Chemicals, Gibbstown, NJ) using the following SfiI- flanked primers: forward 59-TAT AGG CCA TTA TGG CCG AAC TGT CTG GCT GTA C-39 and reverse 59-TAT AGG CCA CCG CGG CCA GTG AAC AAG ACT TG-39, and inserted into the pEF.CD3z backbone to generate a he characterization in recent years of multiple recep- lentiviral vector plasmid termed pEF.FCRL6z. Vector packaging, transduction, tor-gene families with activating or inhibitory poten- and transductant sorting was performed as previously described (9). by guest on September 29, 2021 a b tial has disclosed that the is integrated Full-length HLA-DR and HLA-DR 1 cDNAs were amplified from T human PBL cells and the SUDHL6 cell line, respectively. The DRb3 with a remarkable number of regulatory mechanisms to bal- (DRB3*01010201), DRb4 (DRB4*01030101), and DRb5 (DRB5*010101) ance effector responses. The classical FcRs for IgG and IgE allele cDNAs were purchased from Open Biosystems (Huntsville, AL). HLA- are located on human chromosome 1q21-23 and play funda- DRa (DRA*0101), HLA-DRb1 (DRB1*040101), and DRb3-5 cDNAs were subcloned into the pMX-PIE retroviral vector, which contains the GFP gene, mental roles in both positive and negative immune regulation and used to transduce BW5147 mouse T cells as described previously (10). (1–3). The discovery of an extended family of FcR-like (FCRL) Doubly transduced HLA-DRa+b1 and b3-5 cells were sorted with a PE- genes (FCRL1–6) positioned in the same human chromosomal labeled anti–HLA-DR mAb (Sigma-Aldrich, St. Louis, MO); singly transduced a b region has uncovered unexpected diversity at this locus (4–6). In HLA-DR and HLA-DR 1 lines were sorted for GFP. Human tonsil and spleen samples were obtained from the University of addition to their genomic linkage and similar genetic organiza- Alabama at Birmingham (UAB) Tissue Procurement program (Birmingham, tion, FCRL1–6 share many features with the FCRs, including AL). Blood specimens were obtained from healthy adult volunteers with related extracellular Ig-like domains and cytoplasmic tyrosine- Institutional Review Board approval following informed consent according to the Declaration of Helsinki. Mononuclear cells were isolated as detailed based signaling capability of their encoded type I transmem- previously (8). Dendritic cells were generated from FACS-sorted brane protein products (6). Of these, human FCRL6 is dis- as described (11). tinctly expressed by cytotoxic T and NK cells, is upregulated on expanded populations of terminally differentiated CD8+ 43-1 experiments T cells in patients with HIV and chronic lymphocytic For Ab stimulation experiments, Immulon 96-well high-binding plates leukemia, and possesses a cytoplasmic ITIM that is capable of (Thermo Fisher Scientific, Waltham, MA) were coated with Abs for 1 h at

x { *Department of Microbiology, Department of Medicine, and Department of Bio- Address correspondence and reprint requests to Dr. Randall S. Davis, University of chemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, Alabama at Birmingham, 1825 University Boulevad, SHEL402 Birmingham, AL AL 35294; †Department of Pediatrics, University of South Florida College of Medicine, 35294-2182. E-mail address: [email protected] St. Petersburg, FL 33701; and ‡H. Lee Moffitt Cancer Center and Research Institute, The online version of this article contains supplemental material. Tampa, FL 33612 Abbreviations used in this paper: FCRL, FcR-like; UAB, University of Alabama at Received for publication March 15, 2010. Accepted for publication April 28, 2010. Birmingham. This work was supported in part by National Institutes of Health Grants AI055638, AI067467, and CA131656, the Dana Foundation Program in Human Immunology, Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 and the Cancer Research Institute (to R.S.D.). D.M.S. received support from National Institutes of Health Training Grant AI007051 and University of Alabama at Birming- ham Medical Scientist Training Program Grant T32 GM008361. www.jimmunol.org/cgi/doi/10.4049/jimmunol.1000832 24 CUTTING EDGE: FCRL6 IS AN MHC CLASS II RECEPTOR room temperature and plated with 3 3 104 43-1 cells for 18 h. For coculture experiments, primary cells were added at a 5:1 ratio and dendritic cells and cell lines at a 2:1 ratio with 43-1 reporter cell lines. In blocking experiments, 30 ml of hybridoma supernatant or purified Ab diluted in culture media was added to stimulator cells prior to plating the 43-1 cells. After coculture, cells were stained with anti-mouse CD5 PE (Southern Biotechnology Associates, Birmingham, AL) to delineate the 43-1 fraction and then analyzed by flow cytometry. To distinguish between the 43-1 and BW5147 cell lines, BW5147 cells were fluorescently labeled with the PKH26 labeling kit (Sigma-Aldrich) prior to coculture. Abs FCRL6 ligand mAbs were generated by immunizing BALB/c mice with the SUDHL6 and Mino cell lines according to established techniques (12). All procedures were approved by the UAB Institutional Animal Care and Use Committee. Hybridoma supernatants were screened for reactivity with SUDHL6 and their ability to block 43-1 FCRL6z cell GFP induction by SUDHL6. Hybridomas were subcloned by limiting dilution and isotyped using the Southern Biotechnology Associates clonotyping system. The resulting mAbs are moIgG1k (L2-1E2), moIgG3k (L2-3F7, L2-3H2), and moIgMk (L2-1A8, L2-2A6, L2-2B1, L2-3C5, L2-3C6, L2-4C2) isotypes. The anti-FCRL6–specific mAbs 7B7 and 1D8 (both moIgG1k) were generated as FIGURE 1. Development of a GFP-inducible system to detect FCRL6 Downloaded from previously described (8), and Fab digests were prepared using the ImmunoPure engagement. A, 43-1 cells transduced with the FCRL6z reporter construct IgG1 Fab Preparation kit (Pierce, Rockford, IL). The following additional Abs were stained with the anti-FCRL6 7B7 mAb (black line) or with an isotype- were used: control hamster IgG (anti-KLH) (BD Biosciences, San Jose, CA), matched control (gray shade). B, Untransduced 43-1 control (gray columns) b HLA-DR clone DA2 (Santa Cruz Biotechnology, Santa Cruz, CA), control or 43-1 FCRL6z cells (black columns) were stimulated at 5 mg/ml with the mouse IgG Fab fragments (Rockland Immunochemicals, Gilbertsville, PA), indicated plate-bound Abs for 18 h, and GFP induction was assayed by flow and the hamster anti-mouse CD3 clone 145-2C11 (a gift from Dr. Chandar 6 z Raman, UAB). cytometry. Columns represent the mean SD; n =3.C, FCRL6 cells were

stimulated as in B with different concentrations of plate-bound anti-FCRL6 http://www.jimmunol.org/ Fc chimeric proteins mAb. The percentage of GFP+ 43-1 cells is indicated in each histogram and demarcated by horizontal bars. Fc soluble recombinant proteins were generated by modifying the pRB1- Fcmut plasmid (a gift of Dr. Roberto Biassoni, Gaslini Institute, Genoa, Italy), which contains a cDNA encoding the CH2-3 Fc portion of the human IgG1 z Hc (IGHG1) with 3 aa residues mutated (L234A, L235A, and G237A) to FCRL6 cells (Fig. 1B). The response to mAb stimulation was abrogate FcR binding. The FCRL6-Fc and control-Fc constructs were gen- dose dependent, such that a larger percentage of 43-1 cells erated by inserting the endogenous FCRL6 leader and ectodomain sequence expressed GFP when exposed to higher concentrations of or a murine Ig k leader sequence, respectively, upstream of the CH2 portion of IGHG1. stimulating Ab (Fig. 1C).

Final cDNA constructs were subcloned into the pShuttle-CMV adenovirus by guest on September 29, 2021 transfer vector (Quantum Biotechnologies, Laval, Quebec, Canada) for trans- FCRL6 interacts with a surface molecule on APCs fection and expression in HEK293 cells. Soluble protein containing superna- tants were purified over protein A-sepharose columns (GE Healthcare, Having established a functional readout for FCRL6 engage- Piscataway, NJ) before SDS-PAGE and analysis by Coomassie staining ment, we began investigating different lymphoid tissues to (Bio-Rad, Hercules, CA) and Western blot. explore the cellular distribution of the FCRL6 ligand(s). In For staining, Fc proteins were incubated with Alexa-Fluor 647-labeled pro- tein A (Invitrogen, Carlsbad, CA) at a 2.5:1 molar ratio for 30 min at room coculture experiments, mononuclear cells isolated from blood, temperature, then diluted and used to stain BW5147 transductants at a tonsil, and spleen were found to induce GFP expression in 200 nM concentration for 1 h at 37˚C. FCRL6z cells but not in control 43-1 cells (Fig. 2A). To ascer- tain which cell type(s) was responsible for this reactivity, distinct Results and Discussion leukocyte populations were sorted from blood samples and Development of a GFP-inducible system to detect FCRL6 engagement assayed for FCRL6-dependent GFP induction. The putative To assay functional engagement between FCRL6 and potential FCRL6 ligand was found only on cells with Ag-presenting capa- ligand sources, a reporter cell line was engineered. A chimeric bility, namely, B cells and monocytes, but not on resting CD4+ molecule termed FCRL6z, which possesses the three FCRL6 T cells, CD8+ T cells, or granulocytes (Fig. 2B). - Ig-like extracellular domains in frame with an uncharged hydro- derived dendritic cells were also found to evoke a robust response phobic transmembrane region and the ITAM-containing cyto- (Fig. 2C). plasmic portion of mouse CD3z, was transduced into a murine To further define FCRL6 ligand expression and facilitate its hybridoma, 43-1, which harbors an inducible GFP gene eventual cloning, representative human cell lines were ana- downstream of three tandem NFAT transcription factor bind- lyzed. Coculture with EBV-immortalized B lymphoblastoid ing sites (9, 13). The rationale behind this approach was that an (721.221), diffuse large B lymphoma (SUDHL6), and mantle encounter between FCRL6 and its ligand should initiate a B cell lymphoma-derived (Mino) cell lines activated FCRL6z CD3z-driven activation cascade, resulting in NFAT nuclear cells, whereas no detectable response was observed following translocation and GFP expression. coculture with Jurkat T cells or the U937 and K562 myeloid cell Following transduction of the chimeric construct into lines (Fig. 2D). Conditioned media from SUDHL6-cultured 43-1 cells, FCRL6z surface expression was verified by staining cells failed to stimulate GFP, suggesting that the interacting with an FCRL6-specific Ab (Fig. 1A). The 43-1 cells have an counterpart is a cell surface-associated molecule (data not intact TCR complex (13); therefore, stimulation of parental shown). The FCRL6 specificity in this reporter system was or FCRL6z cells with mAbs to mouse CD3 resulted in potent supported by several lines of evidence. Induction by SUDHL6 GFP induction by both cell lines, whereas stimulation with an in coculture assays cells was blocked by both intact and mono- FCRL6-specific mAb (7B7) only induced GFP expression in meric Fab fragments of an FCRL6-specific mAb (1D8), as well The Journal of Immunology 25

pelling evolutionary observations that provided useful clues for predicting its possible nature.

HLA-DR is an FCRL6 ligand Striking phylogenetic relationships are evident between Ig and MHC C regions (14–19). Thus, the finding that an FcR/FCRL family member recognized a surface determinant common to APCs prompted us to consider that the ligand for FCRL6 could be MHC class II. To test this hypothesis, we first performed competition assays with 43-1 FCRL6z cells utilizing an anti–HLA-DRb mAb (clone DA2). Addition of this reagent in coculture experiments with SUDHL6 cells Downloaded from http://www.jimmunol.org/

FIGURE 2. FCRL6 interacts with a molecule expressed by APCs. FCRL6z

(black columns) or untransduced 43-1 control cells (gray columns) were cocul- by guest on September 29, 2021 tured with mononuclear cells isolated from the indicated tissues (A); FACS- sorted blood CD4+ T cells (CD3+CD4+), CD8+ T cells (CD3+CD8+), B cells (CD19+), monocytes, or granulocytes (both defined by light scatter) (B); mono- cyte-derived dendritic cells (C); or the indicated human cell lines (D). After 18 h, 43-1 cell GFP expression was analyzed by flow cytometry. 43-1 control cells cultured alone (None) were employed to determine background GFP levels. E, FCRL6z cells were cocultured with the SUDHL6 B cell line either alone (No treatment) or in the presence of the indicated blocking reagents and assayed for GFP expression. Columns represent the mean 6 SD; n $ 3. as by the addition of a soluble chimeric FCRL6-Fc fusion pro- tein but not a control Fc-only recombinant molecule consisting of the CH2–CH3 domains of human IgG1 (Fig. 2E). Further- more, a mouse FCRL1z 43-1 transductant that only differs from human FCRL6z cells in its extracellular region failed to respond following coculture with 721.221 or SUDHL6 B cell lines (Supplemental Fig. 1). FIGURE 3. FCRL6 directly binds to HLA-DR. A, FCRL6z cells were A panel of mAbs obstructs binding between FCRL6 and its ligand cocultured for 18 h with SUDHL6 B cells either alone (No treatment) or in the presence of an HLA-DRb–specific mAb (clone DA2) and assayed for As a tool to molecularly characterize the unknown FCRL6 GFP expression. B, FCRL6z (black columns) or untransduced 43-1 control ligand, we developed a panel of ligand-reactive mAbs. Hybrid- cells (gray columns) were cultured for 18 h either alone (None) or in the oma supernatants from mice immunized with the SUDHL6 presence of parental BW5147 cells, BW cells singly transduced with HLA- 2 and Mino B cell lines were tested for mAbs that would both DRa or HLA-DRb1 (both surface HLA-DR ), or doubly transduced with + react with SUDHL6 cells by flow cytometry and suppress HLA-DRa and b1 (surface HLA-DR ) and analyzed for GFP expression. 6 SUDHL6-induced GFP expression by FCRL6z cells. This Columns represent the mean SD; n =3.C, rFCRL6-Fc (black line) or a control Fc-only protein (gray shaded) was complexed with Alexa Fluor 647- strategy led to the isolation of nine mAb-secreting clones that labeled protein A and used to stain the indicated BW5147 cells transduced fulfilled both criteria (Supplemental Fig. 2A,2B). However, with the following HLA-DR alleles (DRA*0101, DRB1*040101, DRB3* prior to utilizing these mAbs in approaches to identify the 01010201, DRB4*01030101, and DRB5*010101). Histograms are represen- FCRL6 ligand biochemically, we were struck by several com- tative of three independent experiments. 26 CUTTING EDGE: FCRL6 IS AN MHC CLASS II RECEPTOR indeed abrogated FCRL6z GFP induction (Fig. 3A). A series autologous dendritic cells, a mechanism proposed to regulate of BW5147 murine thymoma cell transductants expressing adaptive immune responses (24–27). What influence, if any, HLA-DRa and/or HLA-DRb1 was then generated and em- the FCRL6–MHC class II interaction may have in this cross ployed in coculture reporter assays. Surprisingly, HLA-DRa+ talk is subject to further investigation. FCRL6 contains a cyto- b1 BW cells (surface HLA-DR+), but not parental or sin- plasmic domain with a consensus ITIM motif that is capable of 2 gly transduced control cells (surface HLA-DR ), triggered phosphorylation and Src homology 2-domain containing FCRL6z GFP expression (Fig. 3B). Importantly, the panel of phosphatase 2 recruitment (7, 8). It is therefore tempting to FCRL6 ligand-reactive mAbs all selectively stained the HLA- speculate that FCRL6 may function as an inhibitory receptor DRa+b1 transductants, and all but the L2-1E2 clone were for MHC class II. However, studies using mAbs to ligate capable of blocking FCRL6z activation when added to cocul- FCRL6 have failed to demonstrate convincing inhibitory func- ture experiments with the HLA-DRa+b1 BW cells (Supple- tion (7, 8), and definitive functional studies for this receptor mental Fig. 3A,3B). await further exploration. MHC class II is both extremely polymorphic and polygenic. The discovery of this interaction raises many questions. For Therefore,weexploredthepotentialofFCRL6tobindHLA-DR example, do other FCRL family members bind MHC mole- heterodimers composed of different DRb-chains. HLA-DRb3, cules, and is FCRL6 capable of recognizing other MHC class II b4, and b5 were each stably transduced into BW HLA-DRa isotypes? The data shown in this study demonstrate that FCRL6 cells and used for coculture experiments with 43-1 cells. Inter- is capable of binding HLA-DR molecules composed of distinct estingly, although HLA-DRa+b1, HLA-DRa+b4, and HLA- DRb subunits; future studies addressing whether FCRL6 dif- Downloaded from DRa+b5 cells invoked moderate to high levels of activation in ferentially binds MHC class II allelic variants may provide the 43-1 system, HLA-DRa+b3 cells induced much lower insight into the molecular mechanisms underlying HLA- levels of GFP expression (Supplemental Fig. 4). To indepen- based disease associations. dently verify the FCRL6–HLA-DR interaction, we then tested the ability of a recombinant soluble FCRL6-Fc fusion protein to stain the panel of HLA-DR transductants. Multimeric Acknowledgments http://www.jimmunol.org/ We thank G. Larry Gartland for excellent assistance with cell sorting; Michael FCRL6-Fc selectively bound to HLA-DRa+b1, HLA-DRa+ b a b O. Alberti for help in generating 43-1 transductants; Takashi Saito, Kerry S. 4, and HLA-DR + 5 BW cells and to a lesser extent HLA- Campbell, Louis M. Staudt, Josef T. Prchal, and Eric Vivier for donating cell DRa+b3 transductants, but not to control cells (Fig. 3C). These lines; and Paul A. Goepfert, Olusimidele T. Akinsiku, Steffanie Sabaj, Peter D. results demonstrate that FCRL6 is capable of binding MHC Burrows, Allan J. Zajac, and Gary W. Litman for discussion and critical reading class II molecules and suggest that it may have differential of the manuscript. affinity for HLA-DR that varies according to its HLA-DR b -chain composition. Disclosures by guest on September 29, 2021 Concluding remarks R.S.D. is a coinventor on U.S. Patent 7,317,087, which covers the FCRL6 molecule. In addition, R.S.D., D.M.S., and J.P.C. are coinventors on pend- In summary, these findings clearly indicate that MHC class II ing patent applications. is an FCRL6 ligand. Phylogenetic analyses suggest that the nonpolymorphic C region domains of IgH isotypes and the membrane-proximal MHC class I a3 and MHC class II a2 References b b 1. Ravetch, J. V., and J. P. Kinet. 1991. Fc receptors. Annu. Rev. Immunol. 9: 457– and 2 domains, as well as 2-microglobulin, arose from a 492. common ancestral C1-set Ig-like domain (14–19). Given the 2. Dae¨ron, M. 1997. biology. Annu. Rev. Immunol. 15: 203–234. 3. Nimmerjahn, F., and J. V. Ravetch. 2008. 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