Cells Expression and Function of CD300 in NK

Expression and Function of CD300 in NK Cells Dikla Lankry, Hrvoje Simic, Yair Klieger, Francesca Levi-Schaffer, Stipan Jonjic and Ofer Mandelboim This information is current as of September 27, 2021. J Immunol 2010; 185:2877-2886; Prepublished online 23 July 2010; doi: 10.4049/jimmunol.0903347 http://www.jimmunol.org/content/185/5/2877 Downloaded from

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References This article cites 44 articles, 13 of which you can access for free at: http://www.jimmunol.org/ http://www.jimmunol.org/content/185/5/2877.full#ref-list-1

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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. The Journal of Immunology

Expression and Function of CD300 in NK Cells

Dikla Lankry,* Hrvoje Simic,† Yair Klieger,* Francesca Levi-Schaffer,‡ Stipan Jonjic,† and Ofer Mandelboim*

The killing activity of NK cells is regulated by signals derived from inhibitory and activating NK cell receptors, including the CD300 family of proteins. CD300a was reported to be expressed on all NK cells and to deliver an inhibitory signal upon binding to a yet unknown ligand/s. The CD300a protein contains four ITIMs and is highly homologous to CD300c. Little is known about the function and distribution of these two receptors and the identity of their ligand/s. In this article, we show that CD300a is indeed an inhibitory receptor expressed by human NK cells, but surprisingly, we show that not all NK clones are inhibited in a CD300a- dependent manner. We demonstrate, using a panel of 13 new anti-CD300a and CD300c Abs that we generated, that CD300a and CD300c are indistinguishable on the surface of NK cells. Using mutational-analysis survey, we show that tyrosine 267 located in the third ITIM motif of the CD300a protein is important for the inhibitory function of CD300a. The Journal of Immunology, 2010, 185: 2877–2886. Downloaded from

atural killer cells are bone marrow-derived lymphocytes The KIR gene family is divided, based on functional activity, that make up 5–15% of the PBLs (1, 2). NK cells are into inhibitory and stimulating KIRs (17, 18). Products of in- N able to kill a broad spectrum of pathogens and tumors hibitory KIR genes are characterized by long cytoplasmic tails without prior speciﬁc stimulation (3–5), although recent evidence featuring ITIMs that, upon engagement, transmit inhibitory sig-

suggests that NK cells possess some adaptive properties. The NK nals. In contrast, stimulating KIRs have short cytoplasmic tails http://www.jimmunol.org/ cytotoxicity is controlled by multiple activating and inhibitory sur- lacking ITIMs but instead they carry a positively charged amino face receptors and intracellular signal-transduction molecules (6– acid in the transmembrane region, which provides a docking site 9). In humans, the major NK-triggering receptors include the for the activating adaptor molecule DAP12 (17, 18). In addition to NKp80, NKG2D, CD16, and the natural cytotoxic receptors, which MHC class I binding receptors, other inhibitory receptors also include NKp46, NKp44, and NKp30 (3, 6, 10–13). exist. However, interestingly, as opposed to the class I inhibitory Inhibition of NK cell activity is mediated mainly by the two receptors, the inhibitory receptors that do not bind MHC class I major families of MHC-specific inhibitory receptors, which include are sometimes expressed on all NK cells (9). the Ig superfamily receptors (killer Ig-related receptor [KIR] and The CD300 proteins are a family of leukocyte membrane reg- leukocyte Ig-like receptor) and the C-type lectin (CD94/NKG2A) ulatory molecules that modulate leukocyte responses. There are by guest on September 27, 2021 receptor superfamily (14–16). The different inhibitory receptors seven members in the family, named CD300a–g (19). The CD300a show diverse specificity and can discriminate between different and CD300c receptors, which are the subject of this work, are cell class I MHC proteins. An important feature of all inhibitory NK surface glycoproteins that are encoded by independent, but closely receptors that interact with MHC class I is that they are expressed linked, genes on human chromosome 17q 22–24. Both molecules in a variegated fashion so that each NK cell expresses multiple are members of the Ig superfamily and each contains a single V- receptors in a complex combinatorial repertoire (16). like Ig domain, a membrane proximal region, a transmembrane region, and a cytoplasmic region. The Ig domains of the CD300a and CD300c molecules show 80% identity at the amino acid level *Lautenberg Center for General and Tumor Immunology, BioMedical Research In- (20–23). The CD300a molecule has a long cytoplasmic tail of 100 stitute, Hadassah Medical School and ‡Department of Pharmacology and Experimen- tal Therapeutics, Faculty of Medicine, School of Pharmacy, Hebrew University, aa. This region contains four tyrosines, three of which are found Jerusalem, Israel; and †Department of Histology and Embryology, Center for Pro- within the ITIM consensus sequences (20, 21). The CD300a cy- teomics, Faculty of Medicine, University of Rijeka, Rijeka, Croatia toplasmic region also contains a di-leucine motif that is associated Received for publication October 15, 2009. Accepted for publication June 24, 2010. with endocytosis and delivery to lysosomes (20, 22). The CD300c This work was supported by Israeli Science Foundation and The Israeli Science Foun- molecule has a proline-rich membrane proximal region, a trans- dation (Morasha) Grants, a Croatia Israel Research Grant, a MOST-DKFZ Research Grant, European Consortium Grant MRTN-CT-2005, the Rosetrees Trust, Israel Cancer membrane region containing a charged glutamic acid residue, and Association Grant 20100003, the Association for International Cancer Research, a Cro- a short cytoplasmic tail of only 18 aa. atia-Israel Joint Research Grant (all to O.M.), and European Union Grant FP7- The CD300a and CD300c receptors demonstrate several unique REGPOT-2008-1 (to S.J.). O.M is a Crown Professor of Molecular Immunology. characteristics. First, like the KIR gene family, they share a high Address correspondence and reprint requests to Dr. Ofer Mandelboim, The Lauten- berg Center for General and Tumor Immunology, Hadassah Medical School, Hebrew degree of homology but might have different functions (23), University, P.O. Box 12272, Jerusalem, Israel or Dr. Stipan Jonjic, Department of perhaps similar to the pairwised receptors (17, 18). Second, most Histology and Embryology Center for Proteomics, Faculty of Medicine, University of of the inhibitory receptors expressed by NK cells have one or two Rijeka, Brace Branchetta 2051 000, Rijeka, Croatia. E-mail addresses: oferm@ekmd. huji.ac.il or [email protected] ITIM motifs at the cytoplasmic tail, with the exception of leu- The online version of this article contains supplemental material. kocyte Ig-like receptor 1 and CD300a, which have four ITIM Abbreviations used in this paper: Bulk NK, bulk NK cultures; HA, hemagglutinin; INH, motifs (20). Thus, it will be interesting to investigate which of inhibited; KIR, killer Ig-related receptor; MIX, mixed population of inhibited and the ITIMs are important for CD300a function. Third, none of the noninhibited clones; NK Fresh, freshly isolated NK cells; non-INH, noninhibited; activating receptors identified contains a charged glutamic acid WT, wild-type. residue in the transmembrane, suggesting that CD300c delivers Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 its activating signals via signaling proteins other than those pre- www.jimmunol.org/cgi/doi/10.4049/jimmunol.0903347 2878 EXPRESSION AND FUNCTION OF CD300 IN NK CELLS viously observed or that CD300c is not an activating receptor. (including the last seven nucleotides of human CD300c extracellular por- Finally, the ligands for both receptors are still unknown. tion) and 39-CC GAA TTC TTA GCG AGG GGC CAG GGT CTG (in- In this study, we investigated the activity of CD300a. We dem- cluding an EcoRI restriction site). The two amplified fragments were mixed, and PCR was performed with the 59 HindIII primer and the 39 onstrate that CD300a is actually functional only on a subset of NK EcoRI primer for the generation of the CD300c z construct. To insert clones and that CD300a and CD300c are indistinguishable on the HA tag, the following primers were used: 59HindIII and 39-AGC the cell surface, and we identified a critical ITIM that is important GTA ATC TGG AAC ATC GTA TGG GTA AGG AAA ATA GCC TGG for CD300a function. GAC AAG (including the HA tag fragment) and 59-TAC CCA TAC GAT GTT CCA GAT TAC GCT CTG AGC CAC CCC ATG ACC GTG (including the HA tag fragment) and 3EcoRI. The CD300c HA z construct Materials and Methods was cloned into a pcDNA3 expression vector (Invitrogen, Carlsbad, CA) Abs and cells and stably transfected into BW cells. The following mouse anti-human Abs were used: anti-CD300a/c (P192) Ig-fusion proteins (20), anti-CD300a/c (MEM-260, Cedarlane Laboratories, Hornby, Ontario, Canada), anti-MHCI (W6/32), anti-KIR2DL1 (HP3E4), anti-KIR2DL3 The Ig fusion proteins CD300a-Ig, CD300c-Ig, and D1-Ig were generated as (CD158b, Beckman Coulter, Fullerton, CA), anti-CD16 (B73.1.1), anti- described previously (28). Briefly, the sequence encoding the extracellular CD99 (12E7; a kind gift from A. Bernard, Hopital de L’Archet, Nice, France), portion of the receptor was amplified by PCR from cDNA isolated from anti-hemagglutinin (HA) tag (12C45), anti-2B4 (CD244 clone C1.7, Beck- human NK clones. The primers used for the PCR of the extracellular man Coulter), and anti-CD3 (T3D). To generate the anti-CD300a and CD300c portion of CD300a/c were CD300a: 59-CCC AAG CTT GCC GCC ACC mAbs, BALB/c mice were injected s.c. with the fusion proteins in CFA. Two ATG TGG CTG CCT TGG GCT CTG (including a HindIII restriction site) weeks later, mice were boosted with the same protein in IFA. After an addi- and 39-GA AG ATC TGA GTT CAC CAC CTC CTC AGT (including tional 2 wk, the sera of immunized mice were screened for the Ab titer against a BglII restriction site); CD300c: 59-CCC AAG CTT GCC GCC ACC Downloaded from the immunogen using ELISA. The best responders were additionally boosted ATG ACT GCC AGG GCC TGG GCC (including a HindIII restriction i.p. with fusion protein dissolved in PBS. Three days later, spleen cells were site) and 39-GA AG ATC TCG GAC ATT GCT GAA CAG GGA (in- collected, and after lysis of erythrocytes, they were fused with SP2/0 mye- cluding a BglII restriction site). These PCR-generated fragments were loma cells in a 1:1 ratio. The cells were seeded on 96-well tissue-culture plates cloned into a mammalian expression vector containing the Fc portion of in 20% RPMI 1640 medium with hypoxanthine, aminopterine, and thymi- a human IgG1. The constructs were transfected into COS-7 cells, and the dine. The cultures were fed every 4 d; after 12 d, each well was screened for protein produced was purified using a protein G column. Abs reactive against fusion proteins in ELISA. Positive mother wells were http://www.jimmunol.org/ expanded and cloned. Redirected killing assay The cell lines used in the current study were mouse BW cells, a murine thymoma cell line that lacks TCR expression; YTS cells, a subline of the YT The inhibitory activity of CD300a in NK cells was assessed in a redirected killing assay, in which NK line cells, NK clones, or various YTS cells were NK leukemia cell line (24) transfected with the ecotropic murine retrovirus 35 receptor alone (YTS/eco) or the ecotropic receptor; and CD300a: wild-type cocultured with S-labeled P815 (murine mastocytoma) target cells that (WT) or mutated receptor (YTS CD300a or YTS Y231A, YTS Y255A, were precoated with 0.2 mg/well various mAbs: anti-CD16, anti-CD99, YTS Y267A, and YTS Y293A) (25). anti-KIR2DL3, or anti-CD300 Abs. The E:T ratio was 3:1 or 5:1 for NK line/clones cells or YTS cells, respectively. In all presented cytotoxicity Isolation of human NK cells assays, the spontaneous release was ,20% of maximal release. The level of cytotoxicity was determined as previously described (29). Primary NK cells were isolated from PBLs using the human NK cell iso- by guest on September 27, 2021 lation kit and the autoMACS instrument (Miltenyi Biotec, Auburn, CA). To PCR and quantitative real-time PCR obtain NK clones, freshly separated NK cells were diluted in NK medium, based on the number of cells desired per well, and seeded in 96U plates. NK The sequence encoding the CD300a/c receptor was amplified by PCR from clones were collected and split every 2–3 d in a 1:2 ratio. The validation of cDNA isolated from human NK clones. The primers used for the PCR of the NK cell phenotype was performed by FACS staining using the mouse anti- CD300a/c receptor were CD300a: 59-CCG GAA TTC GCC GCC ACC human Abs anti-CD56 (BD Biosciences) and anti-CD3 (BioLegend, San ATG TGG CTG CCT TGG GCT CTG (including an EcoRI restriction site) Diego, CA) or anti-NKp46 (R&D Systems, Minneapolis, MN). and 39-TT CC GCG GCC GCT ATG GCC GAC GTC GAC CTA TGT CTT CCT TAT CAC ACT (including an SalI restriction site); CD300c: 59- YTS and BW transfectants CCG GAA TTC GCC GCC ACC ATG ACT GCC AGG GCC TGG GCC (including an EcoRI restriction site) and 39-TT CC GCG GCC GCT ATG The cDNA of CD300a used for transfection into YTS/eco cells was GCC GAC GTC GAC CTA CTG GTT CTC ACC CTT GGG (including an amplified by PCR from cDNA isolated from human NK clones. The SalI restriction site). primers used for the PCR of the CD300a were 59-CCG GAA TTC GCC For quantitative real-time PCR analysis, total RNA was isolated with Per- GCC ACC ATG TGG CTG CCT TGG GCT CTG (including an EcoRI fectPure RNA Cultured cell Kit (5 PRIME), and cDNA was generated using restriction site) and 39-TT CC GCG GCC GCT ATG GCC GAC GTC the SuperScript II kit (Invitrogen). DNA was amplified with specific primers: GAC CTA TGT CTT CCT TAT CAC ACT (including an SalI restriction CD300a: 59-GCA TTG TTG CTG CTT CTG TTG and 39-CAC CAG CTT site). The tyrosine residues located in positions 231, 255, 267, and 293 of TGA TCC ATT TCTG; CD300c: 59-ACA CCC TGG CTC CCT GTTC and the cytoplasmic tail of the CD300a protein were mutated to alanine by 39-CTA GAG CTT CTC TGA GGT CTG TTC AC. Real-time PCR was two-step PCR amplification using the following primers: 39-CTC CAG performed with a DyNAmo SYBR Green qPCR kit (Invitrogen) on an ABI ATT TGC GGC GTG CAG CTC, 39-AGT GCT GGC TTC CAC CTC PRISM 7500 Real-Time PCR system (Applied Biosystems, Foster City, CA). CAC CTC, 39-GAACTTCACGCTGCCTCGGTGGTG,59-GAG 9 CTG CAC GCC GCA AAT CTG GAG, 5 -GAG GTG GAG GTG Western blot and immunoprecipitation GAA GCC AGC ACT, and 59-GAA CTT CAC GCT GCC TCG GTG GTG. For Y293A, one-step PCR amplification was done using the fol- CD300a/c detection. NK cells (20M cell) were washed four times with cold lowing primer: 59-TTCCGCGGCCGCTATGGCCGACGTCGAC PBS containing 1 mM MgCl2 and 0.1 mM CaCl2. For immunoprecipitation CTA TGT CTT CTT TAT CAC ACT GGC ATC TGA. The WT or with anti-CD300a/c (CD300c Hybridoma #9), the cells were biotinylated mutated CD300a constructs were cloned into a pBABE-puro retrovirus with EZ-Link Sulfo-NHS-LC-Biotin (Pierce, Rockford, IL) for 30 min at vector, and YTS cells were transfected with cDNA encoding for the 4˚C and washed four times with cold PBS. After washing, cells were lysed CD300a protein, as previously described (26, 27). with lysis buffer (PBS containing 150 mM NaCl, 50 mM Tris [pH 7.6], The generation of chimeric CD300c HA z cDNA used for transfection 0.5% Nonidet P-40, 9 mM iodoacetamide, 5 mM EDTA, 1 mM PMSF, into BW cells was performed by two-step PCR amplification using cDNA 1:100 aprotinin). Cell lysates were then immunoprecipitated overnight at isolated from human NK clones. The primers used for the PCR of the 4˚C with Protein-G plus beads (Pierce) conjugated with 100 ml anti- extracellular portion of CD300c were: 59-CCC AAG CTT GCC GCC CD300a/c (CD300c Hybridoma #9). The immunoprecipitates were washed ACC ATG ACT GCC AGG GCC TGG GCC (including a HindIII restric- with lysis buffer, and the proteins were eluted by using IgG elution buffer tion site) and 39-GTA GCA GAG GCG GAC ATT GCT GAA CAG GGA (Pierce). All samples were loaded on SDS-PAGE and transferred to nitro- (including the first seven nucleotides of mouse z-chain transmembrane cellulose membrane. The blotted proteins were visualized by streptavidin portion). The mouse z-chain was amplified by PCR using the following HRP (Jackson ImmunoResearch Laboratories, West Grove, PA) using ECL primers: 59-AAT GTC CGC CTC TGC TAC TTG CTA GAT GGA detection (Biological Industries, Bet-haemek, Israel). The Journal of Immunology 2879

CD300a phosphorylation PBLs and NK cells from healthy donors and analyzed them for CD300a expression by using a commercial mAb P192, pre- NK cells (20M) were washed with TBS and activated with 500 mM H2O2 for 30 s and 5 min incubation at 37˚C. Control cells were not exposed to sumably specific to CD300a. Indeed, as previously reported, H2O2. Following incubation, cells were washed three times with cold a subset of PBLs expressed CD300a, and all NK cells tested, ei- TBS-PI (containing phosphate inhibitors). After washing, cells were lysed ther freshly isolated or bulk NK cell cultures, which were grown in with lysis buffer (TBS containing 0.5% Triton X-100, 50 mM Tris (pH 7.4), the presence of IL-2 (NK bulk), expressed the CD300a receptor 150 mM NaCl, and phosphatase and protease inhibitors). Cell lysates were then loaded on SDS-PAGE and transferred to nitrocellulose membrane. The (Fig. 1A). C1R cells were used as a positive control and were blotted phosphorylated proteins were visualized by anti-phosphotyrosine also stained with the anti-CD300a mAb P192 (Fig. 1A). To test (4G10; Upstate Biotechnology, Lake Placid, NY) after incubation over- whether the uniform expression of CD300a was also observed in night at 4˚C, goat anti-mouse biotin for 2 h at room temperature and various NK clones derived from different donors, we isolated NK streptavidin HRP (Jackson ImmunoResearch Laboratories), using ECL detection (Biological Industries). clones from two different healthy donors and analyzed them for CD300a expression. As can be seen in Fig. 1B and 1C, all NK Internalization assay clones expressed CD300a, in contrast to KIR2DL1, which is Cells were resuspended at 105 in 100 ml RPMI 1640/10% FCS and in- expressed by only some NK clones. Similar results were observed cubated on ice for 30 min with 5 ml anti-CD300a/c (CD300c Hybridoma in all NK clones that we tested (.100). Thus, all NK cells tested #9) mAb. Cells were then washed three times with RPMI 1640/10% FCS expressed CD300a, as detected by staining with the P192 mAb. and incubated at 37˚C in 5% CO2. After various incubation times, aliquots of cells were removed, fixed by the addition of FACS medium (0.1% azide/ We next tested the functionality of CD300a. Primary bulk 1% BSA/PBS), and kept on ice. After an appropriate number of aliquots NK cell cultures were incubated with P815 cells, which were had been taken, cells were labeled with an excess of FITC-labeled goat precoated with anti-CD16 mAb (to induce killing), in the presence Downloaded from anti-mouse IgG for 30 min and subsequently washed in FACS medium or absence of mAb P192, anti-CD99 (which was used as a negative before analysis by flow cytometry. control), and anti-KIR2DL3 (which was used as a positive control) (Fig. 1D). As previously reported, the coincubation of NK cells Results with the anti-CD16 and anti-CD300a/c mAb resulted in an inhi- CD300a expression and function on NK cells bition of killing compared with the activation by anti-CD16 mAb

It was shown previously that CD300a is an inhibitory receptor (20, alone (Fig. 1D). However, surprisingly, despite the observations http://www.jimmunol.org/ 21). However, it is still unknown whether CD300a is an inhibitory that CD300a contains four ITIMs and is expressed on all NK cells, receptor for all NK clones. Therefore, we initially tested whether the CD300a-mediated inhibition seemed to be moderate compared CD300a is expressed by all NK cells. For that purpose, we isolated with the inhibition mediated by the other NK receptors (e.g., by guest on September 27, 2021

FIGURE 1. CD300a expression and function. A, Flow-cytometric analysis of Bulk NK, NK Fresh, PBLs, and CIR cells. B and C, Flow-cytometric analysis of Bulk NK and NK clones obtained from two healthy donors. Staining was performed with 5 ml/well anti-CD300a/c mAb (P192) and 100 ml anti-

KIR2DL1 (HP3E4 Hybridoma) (black line). Background (shaded graph) is the staining with FITC-conjugated F(ab9)2 goat anti-mouse IgG only. D, Redirected experiments were performed with 0.1 mg/well anti-CD16 (B73.1), 0.1 mg/well anti-CD99 (12E7), 0.1 mg/well anti-KIR2DL3 (GL183b), and 5 ml/well anti-CD300a/c (P192). The E:T ratio was 3:1. The horizontal line represents the inhibition observed when the CD300a receptor was cross-linked with CD16. pp # 0.05. Representative result of four separate experiments. Bulk NK, bulk NK cultures; NK Fresh, freshly isolated NK cells. 2880 EXPRESSION AND FUNCTION OF CD300 IN NK CELLS

KIR2DL3; Fig. 1D), and it was not observed in all NK clones against NK cells. Using this strategy, we obtained four new anti- (data not shown). CD300a mAbs (CD300a Hybridoma #1–4) and nine new anti- CD300c mAbs (CD300c Hybridoma #6–14). Importantly, none of CD300a and CD300c are indistinguishable on the cell surface the mAbs generated, including the P192 mAb, could discriminate Because the CD300a inhibition observed was weak (Fig. 1D) and between CD300a and CD300c, and all Abs recognized all of the NK was not observed in all NK clones (data not shown), we hypoth- clones and bulk NK cultures tested (Fig. 3). Although it was pre- esized that CD300a is not expressed by all NK cells and that the viously demonstrated that the anti-CMRF35 mAb recognizes the anti-CD300a mAb (P192) used by us in the above experiments short protein (CD300c) and the long protein (CD300a) (31–33), and by other investigators (20, 30), cross-reacted with another the question of whether this observation is restricted to this mAb protein. Indeed, the CD300c, which was suggested to be a putative or is a general phenomenon is still open. In this study, we show that activating receptor, showed high similarity to CD300a at the DNA such cross-reactivity is also observed with a large panel of Abs, as and protein levels (Fig. 2A). The expression pattern of CD300c well as the commercial mAb P192. and its precise activity are still unknown. To test whether the P192 mAb cross-reacts with CD300a and CD300c, we constructed sev- NK clones exhibit different phenotypes upon CD300 cross- eral reagents: YTS cells expressing CD300a and BW cells expres- linking sing CD300c in-frame with mouse z-chain. Because we did not As demonstrated above, and even by using a large panel of new have an Ab for CD300c, the CD300c protein was tagged with HA, mAbs that we generated, we could not distinguish between CD300a and its expression was conﬁrmed with anti-HA mAb. Despite the and CD300c on the cell surface of various cells. In addition, the use of several plasmids (including lenti vectors and other expres- inhibition of CD300a was moderate compared with KIR inhibition Downloaded from sion vectors) and several attempts, we could not express CD300a z (Fig. 1D). Thus, we next examined whether there would be a dif- in BW cells. However, we were able to express the full-length ference in NK inhibition upon CD300a/c cross-linking of different CD300a in BW cells and, thus, we assumed that the CD300a long NK clones. Indeed, as shown in Fig. 4 and in Table I, only a small cytoplasmic tail is required for stable expression of the protein on portion of the NK clones tested was inhibited by CD300a/c cross- the cell surface. linking, whereas the majority of the clones tested could not be

In addition, we generated two reagents in which we fused the inhibited by the cross linking of CD300a/c (Fig. 4 and Table I). http://www.jimmunol.org/ extracellular portion of CD300a and CD300c to human IgG1 and Although the percentages of the inhibitory clones vary between produced these fusion proteins in COS-7 cells. Equipped with these different donors, our conclusion, which is deduced from experi- reagents, we now generated new mAbs directed against CD300a ments performed in several donors, as well as with several differ- and CD300c. Mice were injected with these two proteins, and ent anti-CD300a/c hybridomas, is that most of the NK clones are the emerging hybridomas were tested against the YTS and the not inhibited by CD300a/c cross-linking (Table I). Interestingly, BW cells expressing CD300a and CD300c, respectively, as well as the CD16-mediated killing of clones that were not inhibited was by guest on September 27, 2021

FIGURE 2. A, CD300a and CD300c share a high degree of homology. Sequence alignment of CD300a and CD300c using the Clustalw program. B, Schematic presentation of the CD300a and CD300c receptors. The Journal of Immunology 2881 Downloaded from http://www.jimmunol.org/

FIGURE 3. CD300a and CD300c are indistinguishable on the cell surface. FACS analysis of bulk NK cultures (A), YTS cells expressing CD300a (B), and BW cells expressing CD300c fused to HA9z(C). Staining is represented by the black line. Background (shaded graph) is the staining with FITC-conjugated

F(ab9)2 goat anti-mouse IgG only. Representative re-

sult of three separate experiments. by guest on September 27, 2021

not enhanced by the CD300 cross-linking (Fig. 4), and cross- receptor. As expected, all clones tested expressed CD300a/c; how- linking of CD300 alone on these clones did not induce killing, ever, because of the mAb cross-reactivity, it is not clear whether suggesting that CD300c might not be an activating NK cell CD300a, CD300c, or both are present on the cell surface (Fig. 4). 2882 EXPRESSION AND FUNCTION OF CD300 IN NK CELLS Downloaded from http://www.jimmunol.org/

FIGURE 4. Not all NK clones are inhibited following CD300 cross-linking. A and C, Redirected killing assays. Various NK clones were cross-linked with no mAb, 0.1 mg/well of anti-CD16, 5 ml of anti-CD300a/c (CD300a Hybridoma #1), or with mAb combinations. p # 0.03 for all inhibited NK clones. The E:T ratio was 3:1. B and D, FACS analysis of NK clones, stained with 5 ml anti-CD300 (CD300a Hybridoma #1, black line). Background (gray by guest on September 27, 2021 shading) is the staining with FITC-conjugated F(ab9)2 goat anti-mouse IgG only. Representative result of three separate experiments.

In an attempt to better characterize the inhibited versus non- CD300a and CD300c mRNA or protein. As can be seen in Fig. 5, inhibited clones, we also stained the inhibited and the noninhibited all NK clones and bulk NK cell cultures tested, irrespectively of clones with the anti-CD300a/c hybridoma (CD300c Hybridoma #9) whether they were inhibited, express CD300a and CD300c tran- and compared it with the commercial Ab MEM-260, which is as- scripts, detected by PCR (Fig. 5A) or by real-time PCR (Fig. 5B, sumed to recognize the CD300a protein with high afﬁnity. No sig- 5C). Because the CD300c protein is shorter than CD300a (Fig. 2), niﬁcant differences were detected among the various clones by we also performed immunoprecipitation experiments to assay using both Abs (Supplemental Fig. 1), indicating that at this stage, whether clones that were not inhibited would express the short there are no Abs able to discriminate between the various NK form of CD300c and clones that were inhibited would express the clones with regard to the expression of CD300a and CD300c. long form of CD300a. In agreement with the PCR and the real- We next tried to evaluate whether there is a correlation between time PCR results, all clones tested expressed the long and short the functional outcome of a particular clone and the presence of forms of CD300 (Fig. 5D). In general, all NK cells tested con-

Table I. Percentage of NK clones that undergo inhibition following anti-CD300 cross-linking

Inhibited Noninhibited Total Inhibited Noninhibited Experiment Donor NK Clones (n) NK Clones (n) NK Clones (n) NK Clones (%) NK Clones (%) 1 1 2 30 32 6.25 93.75 2 1 3 29 32 9.38 90.625 3 1 4 28 32 12.50 87.5 4 2 8 24 32 25.00 75 5 2 9 23 32 28.13 71.88 6 2 2 30 32 6.25 93.75 7 2 7 57 64 10.94 89.06 8 2 8 48 56 14.29 85.71 Sum: 43 269 312 13.78 86.22 Summary of inhibited NK clones of eight experiments from two different donors. Inhibited NK clones represent 6–14% of the total NK clones tested. Percentage was calculated as follows: (sum of number of inhibited/noninhibited NK clones/sum of NK clones) 3 100. Experiments were performed with the following anti-CD300a/c hybridomas: Experiments 1 and 5, CD300a Hybridoma #1; Experiments 2 and 3, CD300a Hybridoma #1 and #4; Experiment 6, CD300a Hybridoma #2 and #13; Experiment 7,: CD300a Hybridoma #2; and Experiment 8, CD300c Hybridoma #9. The Journal of Immunology 2883 Downloaded from

FIGURE 5. All clones tested express the mRNA and the protein of CD300a and CD300c. RT-PCR (A) and real-time RT-PCR analysis (B) were performed on mRNA extracted from different NK clones. NK clones were divided into four groups: INH, non-INH, MIX, and NK bulk. Relative mRNA

abundance is shown as a percentage of the level of hypoxanthine phosphoribosyltransferase mRNA (encoding hypoxanthine guanine phosphoribosyl- http://www.jimmunol.org/ transferase), and error bars (SD) are derived from triplicates. D, All NK clones, INH and non-INH, express CD300a and CD300c proteins. The cells were biotinylated, and cell lysates were immunoprecipitated overnight at 4˚C with Protein-G plus beads (Pierce) conjugated with 100 ml anti-CD300a/c. The blotted proteins were visualized by streptavidin-HRP using ECL. Representative of two independent experiments. INH, inhibited; non-INH, noninhibited; MIX, mixed population of inhibited and noninhibited clones; NK bulk, bulk NK cultures.

tained lower levels of CD300c and although it seems as if some inhibitory signal transduction, we inserted a point mutation in each differences could be observed among the various cells with regard ITIM, changing tyrosine to alanine. Once we constructed 15 to CD300c expression, they were not significant. CD300a proteins, each with a different ITIM mutation (Fig. 6), we by guest on September 27, 2021 A general defect in the inhibitory capacity of these clones might expressed each of the proteins in the YTS cell line and performed explain why inhibition was not observed in some of the clones. a redirected killing assay. All proteins were expressed at similar Therefore, we isolated noninhibitory clones that express other levels on the cell surface, except from the CD300a protein, in which inhibitory receptors, such as KIR2DL1 and KIR2DL3 (Supple- all four ITIMs were mutated (Fig. 6A), suggesting that the ITIMs mental Fig. 2), and redirected the killing of these clones by using are probably needed for efficient cell surface expression of CD300a. anti-CD300a/c Abs, as well as the anti-KIR2DL1 or -KIR2DL3 This result might also explain the lack of expression of CD300a z mAb. Because the triggering of the inhibitory receptors, together on the surface of BW cells, because it lacks the tail and trans- with CD16, resulted in efficient inhibition in these clones, whereas membrane of CD300a. To test this hypothesis, we performed in- no inhibition was observed with CD300a/c (Supplemental Fig. 2), ternalization experiments in which the quadruple CD300a mutant, we concluded that noninhibited clones are normal functional clones CD300c, and the parental CD300a-expressing YTS cells were stained with regard to NK inhibition. with anti-CD300a, and the spontaneous internalization rate was mea- To further substantiate this point, we also tested the general sured over time. As can be seen in Supplemental Fig. 4, the internal- phosphorylation pattern of the inhibitory versus the noninhibitory ization rate of the quadruple CD300a mutant and that of CD300c was clones by treating them with H2O2. As can be seen in Supplemental more rapid compared with the parental CD300a-expressing cells, Fig. 3, there was no major difference in the phosphorylation pattern suggesting that the ITIM motifs are important for the stable expres- between the clones, further indicating that the noninhibitory clones sion of CD300a on the cell surface. are normal (Supplemental Fig. 3). We next assessed the importance of each of the ITIM motifs in Thus, it seems that CD300 activity is quite complex and that, the inhibition mediated by CD300a. Although single-point muta- despite the observations that different clones express CD300a and tion in a single ITIM had no effect on the CD300a-mediated inhi- CD300c transcripts and proteins, some are functionally inhibited bition, double or triple mutation involving the third ITIM, Y267, by anti-CD300a/c, whereas others are not. significantly reduced the CD300a-mediated inhibition (Fig. 6). Combinations of double mutation, which did not include the third The third ITIM motif of CD300a is important for its inhibitory ITIM, Y267, did not affect the CD300a inhibition, whereas triple function mutations significantly downregulated the receptor’s inhibitory A common feature of the inhibitory receptors is the presence of one function. In the triple mutant, reduced inhibitory activity of or more ITIMs in their cytoplasmic tail. The cytoplasmic portion of CD300a was observed even when the third ITIM was not mutated CD300a contains three motifs (LHYANL, VEYSTV,and LHYASV), (Fig. 6B, triple mutation Y231A + Y255A + Y293A), albeit, to which fits with the consensus sequence of a classical ITIM (I/V/ a lower extent compared with other triple mutations that included L/S xYxx L/V) and a fourth nonclassical motif (SDYSVI) (20, 21). the third ITIM. Finally, mutation in all four ITIMs completely To study which of the four ITIMs is most important for the abolished the inhibition caused by the CD300a receptor. These 2884 EXPRESSION AND FUNCTION OF CD300 IN NK CELLS Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 6. Mutation of the CD300a ITIM motifs causes downregulation of the receptor’s inhibition. A, Flow-cytometric analysis of the different CD300a mutants stained with 5 ml of anti-CD300a/c mAb (CD300a Hybridoma #1) (open graph). Background (shaded graph) is the staining with FITC- conjugated F(ab9)2 goat anti-mouse IgG only. Representative result of three separate experiments. B, Redirected killing assay of YTS cells expressing the different mutants and YTS CD300a (WT). CD300a receptor cross-linking was performed with 0.1mg/well anti-2B4 and 5 ml of anti-CD300a/c (CD300a Hybridoma #1). The E:T ratio was 5:1. pp # 0.03; ppp # 0.00005. Representative result of three separate experiments. C, Table summarizing the various mutations used. results indicate that all four ITIMs are probably involved in the all NK clones; however, not all NK clones could deliver an inhib- CD300a-mediated inhibition and that the third ITIM is the most itory signal, probably due to the presence of CD300c. Indeed, crucial ITIM involved in this inhibition. CD300a and CD300c share a high degree of homology (23). Such a high degree of homology is a reminiscent of paired receptors, such Discussion as KIR2DL and KIR2DS receptors. These two receptors, perhaps NK cell activation results from a balance of opposing signals that, similarly to the CD300a and CD300c pair, share a high degree of on the one hand, activate NK cells, allowing them to eradicate sequence resemblance but differ in function; KIR2DL is an inhib- tumor or virally infected cells, and, on the other hand, inhibit the itory receptor, whereas KIR2DS is a coactivating receptor (17, 18). NK cells, to prevent killing of self (34, 35). The NK inhibitory In contrast, although the function of CD300a as an inhibitory re- receptors, which recognize MHC class I proteins, are the most ceptor is well established [this study and (19, 20)], the function of studied NK cell receptors with regard to NK cell inhibition; CD300c is yet to be determined. Furthermore, unlike other activat- however, in recent years, it became apparent that MHC class ing KIR receptors that contain a positively charged amino acid in I-independent inhibitory receptors also exist (9, 36, 37). the transmembrane region, which provides a docking site for the The CD300a and CD300c receptors of the CD300 family belong activating adapter molecules (17, 18), CD300c has a negative to the Ig super-family of proteins. Little is known about their cell- charged amino acid (glutamic acid) in the transmembrane region, surface expression phenotype, their functionality, and the identity and its associated partner/s remain to be revealed (19, 22). of their ligand/s. In this study, we tried to shed new light on the We show that although transcripts and proteins of CD300a and involvement of these two proteins in the regulation of human CD300c are found within a particular clone, some NK clones are NK cell activity. CD300a has been identified as a surface molecule inhibited, whereas others are not. Furthermore, we show that the expressed on all NK cells (20, 21), displaying an inhibitory func- noninhibitory clones are normal with regard to NK inhibition tion upon binding to its unknown non-MHC class I ligand (20, (i.e., the triggering of other inhibitory receptors on these clones 21). As we showed in this article, CD300a might be expressed on leads to NK cell inhibition). The different function of the various The Journal of Immunology 2885 clones could be explained in several ways. Because there are no 8. Biassoni, R., C. Cantoni, D. Marras, J. Giron-Michel, M. Falco, L. Moretta, and N. Dimasi. 2003. Human natural killer cell receptors: insights into their mAbs that could discriminate between CD300a and CD300c, it is molecular function and structure. J. Cell. Mol. Med. 7: 376–387. possible that, on the cell surface, the expression of these proteins is 9. Lebbink, R. J., and L. Meyaard. 2007. Non-MHC ligands for inhibitory immune differentially controlled or modified. It is also possible that one receptors: novel insights and implications for immune regulation. Mol. Immunol. 44: 2153–2164. protein antagonizes the activity of the other and that the activity of 10. Raulet, D. H. 2003. Roles of the NKG2D immunoreceptor and its ligands. Nat. CD300a is more dominant in the inhibited clones. Although un- Rev. Immunol. 3: 781–790. likely, it is possible that the expression of CD300c in NK cells is 11. Pessino, A., S. Sivori, C. Bottino, A. Malaspina, L. Morelli, L. Moretta, R. Biassoni, and A. Moretta. 1998. Molecular cloning of NKp46: a novel irrelevant to the inhibitory activity of CD300a. Finally, it might be member of the immunoglobulin superfamily involved in triggering of natural possible that the activity of CD300a is somehow dependent upon cytotoxicity. J. Exp. Med. 188: 953–960. CD300c. Clearly, further research needs to be performed with this 12. Cantoni, C., C. Bottino, M. Vitale, A. Pessino, R. Augugliaro, A. Malaspina, S. Parolini, L. Moretta, A. Moretta, and R. Biassoni. 1999. NKp44, a triggering regard, including the generation of mAbs that could discriminate receptor involved in tumor cell lysis by activated human natural killer cells, is between the ectodomains of the two receptors. a novel member of the immunoglobulin superfamily. J. Exp. Med. 189: 787–796. 13. Pende, D., S. Parolini, A. Pessino, S. Sivori, R. Augugliaro, L. Morelli, E. Marcenaro, Indeed, because CD300a and CD300c are indistinguishable on L. Accame A. Malaspina, R. Biassoni, et al. 1999. Identification and molecular the cells surface, many questions still need to be answered, for characterization of NKp30, a novel triggering receptor involved in natural cy- example: what is the expression pattern of CD300a and CD300c totoxicity mediated by human natural killer cells. J. Exp. Med. 190: 1505–1516. 14. Long, E. O. 1999. Regulation of immune responses through inhibitory receptors. receptors on NK cells? Does it change following NK activation? Annu. Rev. Immunol. 17: 875–904. Can it be influenced by cytokines? What is the function of CD300c? 15. Lo´pez-Botet, M., and T. Belloń. 1999. Natural killer cell activation and All of these questions cannot be precisely answered until specific inhibition by receptors for MHC class I. Curr. Opin. Immunol. 11: 301–307. 16. Natarajan, K., N. Dimasi, J. Wang, R. A. Mariuzza, and D. H. Margulies. 2002. mAbs for CD300a and CD300c are developed. Structure and function of natural killer cell receptors: multiple molecular sol- Downloaded from The CD300a receptor is expressed on NK cells, as well as on utions to self, nonself discrimination. Annu. Rev. Immunol. 20: 853–885. many other immune cells, such as plasmacytoid dendritic cells (38), 17. Bashirova, A. A., M. P. Martin, D. W. McVicar, and M. Carrington. 2006. The killer immunoglobulin-like receptor gene cluster: tuning the genome for defense. T cells (39), neutrophils (40), mast cells (30, 41), and others (42, Annu. Rev. Genomics Hum. Genet. 7: 277–300. 43). Different studies evaluated the CD300a activity in immune 18. Uhrberg, M. 2005. The KIR gene family: life in the fast lane of evolution. Eur. J. a Immunol. 35: 10–15. regulation. CD300a Ab cross-linking resulted in reduced TNF- 19. Clark, G. J., X. Ju, C. Tate, and D. N. Hart. 2009. The CD300 family of mol- secretion and increased IFN-g secretion in plasmacytoid dendritic ecules are evolutionarily significant regulators of leukocyte functions. Trends http://www.jimmunol.org/ cells (38), CD300a affected T cell proliferation and IFN-g secre- Immunol. 30: 209–217. 20. Cantoni, C., C. Bottino, R. Augugliaro, L. Morelli, E. Marcenaro, R. Castriconi, tion (39), coactivation of CD300a and CD32a inhibited CD32a- M. Vitale, D. Pende, S. Sivori, R. Millo, et al. 1999. Molecular and functional mediated signaling in neutrophils (40), and mAb neutralization of characterization of IRp60, a member of the immunoglobulin superfamily that LMIR1 (the mouse CD300a homolog) in BALB/c mice led to a sig- functions as an inhibitory receptor in human NK cells. Eur. J. Immunol. 29: 3148–3159. nificant augmentation of inflammatory mediators and eosinophilic 21. Clark, G. J., B. J. Green, and D. N. Hart. 2000. The CMRF-35H gene structure infiltration (30). In addition, a genetic link was established between predicts for an independently expressed member of an ITIM/ITAM pair of the CD300a and CD300c chromosomal location, 17q25, and psori- molecules localized to human chromosome 17. Tissue Antigens 55: 101–109. 22. Clark, G. J., B. Cooper, S. Fitzpatrick, B. J. Green, and D. N. Hart. 2001. The asis, which may link CD300a and CD300c as part of the dis- gene encoding the immunoregulatory signaling molecule CMRF-35A localized ease mediators (44). In view of the observasions that CD300a and to human chromosome 17 in close proximity to other members of the CMRF-35 by guest on September 27, 2021 CD300c are so far indistinguishable, these works should be reeval- family. Tissue Antigens 57: 415–423. 23. Clark, G. J., S. Fitzpatrick, B. Kuo, C. Modra, L. Jamriska, and D. N. Hart. 2002. uated because some of the effects seen might be due to the activation CMRF-35A, CMRF-35H: potential new CD. J. Biol. Regul. Homeost. 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Supplementary 1: Inhibited and non-inhibited NK clones are stained by MEM-

260 antibody. Flow cytometry analysis of different inhibited (INH) and none inhibited (non INH) NK clones stained with 5μl of anti CD300a/c (CD300c

Hybridoma #9) (black line) 0.1μg/μl anti CD300a/c antibody MEM-260 (dark gray line). Background (filled bright gray histogram) is the staining with FITC-conjugated

F(ab')2 goat anti-mouse IgG only. *P≤0.03, Representative of two independent experiments.

Supplementary 2: The function of additional inhibitory receptors in the noninhibited NK clones (A) Redirected experiments were performed with 0.1μg/well anti CD16 (B73.1), 0.1μg/well anti CD99 (12E7), 0.1μg/well anti KIR2DL3

(GL183b), 100μl anti KIR2DL1 (HP3E4 Hybridoma) and 5µl/well anti CD300a/c

(anti CD300c Hybridoma #9). The E:T ratio was 3:1. The horizontal line represents the activation observed when the CD16 receptor was cross linked, *P≤0.05.

Representative result out of 3 separated experiments. (B) Flow cytometry analysis and summarizing tables of the different receptors expressed by each non-inhibited NK clone. Cells were stained for different receptors (black line): anti CD16, anti

KIR2DL1 or KIR2DL3, anti CD300a/c and CD99. Background (filled gray histogram) is the staining with FITC-conjugated F(ab')2 goat anti-mouse IgG only.

Supplementary figure 3: Phosphorylation pattern of the various NK clones. The

Phosphorilation pattern of inhibited and non-inhibited NK clones following activation with 500mM H2O2 for different time periods: no H2O2, 30sec and 5min.

Phosphorylated proteins were detected with the anti- phosphotyrosine (4G10) mAb.

Representative of two independent experiments.

Supplementary 4: Spontaneous internalization. Internalization assays were performed with YTS CD300a, YTS CD300a mutated in all 4 ITIM motifs and YTS

CD300c. Cells were incubated in 37°C with 5μl of the anti CD300a/c (CD300c

Hybridoma #9) for 0min and 60min, fixed and levels of protein expression on the cell surface were examined by Flow cytometry analysis. Representative of 3 independent experiments, *P≤0.01,**P≤0.003.

2 Supplementary 1 **

Supplementary 2 INH Non INH 500mM 500mM 500mM 500mM 500mM 500mM 500mM 500mM H2O2, 5min H2O2, H2O2, 5min H2O2, No H2O2 H2O2, 30sec H2O2, No H2O2 H2O2, 30sec H2O2,

250

150

100 75

60kDa CD300a 50

Supplementary 3 *

Supplementary 4