Chain Γ Associates with Fc Receptor an Activating ILT Receptor (ILT1

Cutting Edge: Human Myeloid Cells Express an Activating ILT Receptor (ILT1) That Associates with Fc Receptor γ-Chain

This information is current as Hideo Nakajima, Jacqueline Samaridis, Lena Angman and of September 30, 2021. Marco Colonna J Immunol 1999; 162:5-8; ; http://www.jimmunol.org/content/162/1/5 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 © 1999 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. ●

Cutting Edge: Human Myeloid Cells Express an Activating ILT Receptor (ILT1) That Associates with Fc Receptor ␥-Chain1

Hideo Nakajima, Jacqueline Samaridis, Lena Angman, and Marco Colonna2

main (2, 7). Transmembrane domains with a basic residue (lysine Ig-like transcripts (ILTs) encode cell surface receptors ex- or arginine) followed by short cytoplasmic domains are shared by Downloaded from pressed on myeloid and lymphoid cells that are structurally activating NK cell receptors for MHC class I molecules (9) and and functionally related to killer cell inhibitory receptors. One Fc␣R (10). To transduce signals, activating NK cell receptors and ILT, designated ILT1, contains a short cytoplasmic domain Fc␣R associate with distinct tyrosine-based activating motif-con- that lacks sequence motifs implicated in signal transduction. taining proteins, called DAP12 (11–13) and Fc⑀RI␥ (14, 15) re- Its function is unknown. Similar short cytoplasmic domains spectively. Because of these structural similarities, it seems most

have been observed in activating NK cell receptors and Fc␣R, likely that ILT receptors with short cytoplasmic tails activate cells http://www.jimmunol.org/ which transduce stimulatory signals via associated DAP12 and and use an associated protein to transduce stimulatory signals. Fc⑀RI␥ proteins, respectively. Here we show that ILT1 recep- To verify this hypothesis, we have generated a speciﬁc mAb tor is selectively expressed on myeloid cells, functions as an for one of these receptors, designated ILT1 (2), and have ex- activating receptor, and associates with Fc⑀RI␥ rather than amined its biological function, signaling properties, and bio- DAP12. The Journal of Immunology, 1999, 162: 5–8. chemical composition.

Materials and Methods mmunoglobulin-like transcripts (ILTs)3 encode Ig superfam-

Cells and transfections by guest on September 30, 2021 ily receptors structurally and functionally related to killer cell inhibitory receptors. ILTs are expressed on lymphoid and/or Monocytes were prepared from PBMCs by percoll gradient density cen- I trifugation. Dendritic cells (DC) were cultured from purified monocytes in on myeloid cells, and some of them are specific for MHC class I 4 RPMI 1640 and 10% FCS supplemented with granulocyte-macrophage molecules (see Ref. 1 for review). ILTs can be classified by dif- CSF and IL-4 as previously described (3). Macrophages were obtained by fering transmembrane and cytoplasmic domains (2). One subset of culturing monocytes for 10 days in RPMI 1640 and 20% human serum ILT receptors displays long cytoplasmic domains containing im- supplemented with 1 ng/ml macrophage CSF. ILT1, Fc⑀RI␥, and DAP12 munoreceptor tyrosine-based inhibitory motifs (ITIMs). These re- cDNAs were subcloned into pCMV-FLAG-1 (Kodak, Rochester, NY) and expressed as amino-terminal FLAG peptide fusion proteins in rat baso- ceptors mediate inhibition of cell activation by recruiting protein philic leukemia (RBL) cells (ILT1/FLAG), in mouse mastocytoma P815 tyrosine phosphatase SHP-1 (3–8). Another subset of ILT recep- cells (ILT1/FLAG) and 293 cells (Fc⑀RI␥/FLAG, DAP12/FLAG). Full- tors is characterized by a short cytoplasmic domain that lacks se- length ILT1 cDNA was subcloned into pcDNA3 (Invitrogen, San Diego, quence motifs implicated in signal transduction as well as a single CA) and expressed into 293 cells. Transient and stable transfections were basic arginine residue within the hydrophobic transmembrane do- performed as previously described (8). Cell surface expression of transfected cDNAs was determined by FACS analysis.

Production of anti-ILT1 receptor mAb Basel Institute for Immunology, Basel, Switzerland Received for publication September 1, 1998. Accepted for publication November Wistar rats were immunized with ILT1/FLAG-transfected RBL cells. Anti- 2, 1998. ILT1 mAb 135 (rat IgG2a) was selected by ﬂow cytometry for staining ILT1/FLAG-transfected RBL cells, as compared with ILT2, ILT3, ILT4, The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance ILT5 (3, 5, 8) and LIR6a (7) transfectants or untransfected cells (data not with 18 U.S.C. Section 1734 solely to indicate this fact. shown). 1 The Basel Institute for Immunology was founded and is supported by Hoffmann-La Serotonin release Roche Ltd., Basel, Switzerland. 2 Address correspondence and reprint requests to Dr. Marco Colonna, Basel Institute ILT1/FLAG-transfected and untransfected RBL cells were pulsed with for Immunology, 487 Grenzacherstrasse, CH-4005 Basel, Switzerland. E-mail ad- [3H]hydroxytryptamine (NEN, Boston, MA) as described (5), and incu- dress: [email protected] bated for 30 min on ice with either one of the following Abs: mouse 3 Abbreviations used in this paper: ILT, Ig-like transcript; ITIM, immunoreceptor anti-2,4-dinitrophenyl (DNP) IgE (Sigma, St. Louis, MO), anti-FLAG M2 tyrosine-based inhibitory motif; Fc␣R, Fc receptor for IgA; Fc⑀RI, Fc receptor for mAb, or control mouse IgG (Southern Biotechnology Associates, Birming- IgE; Fc⑀RI␥, ␥-chain of the Fc receptor for IgE; DC, dendritic cells; RBL, rat baso- ham, AL). Cells were then incubated in RPMI 1640 and 5% FCS for 1 h Ј philic leukemia at 37°C. Cross-linking Ab (goat F(ab )2 anti-mouse IgG (Southern Bio- ␮ 4 ILT receptors and closely related members of the same receptor family are also technology Associates), 10 g/ml) was also added to cells previously in- designated leukocyte Ig-like receptors (LIRs) and monocyte/macrophage inhibitory cubated with anti-FLAG M2 mAb and control mouse IgG. Serotonin re- receptors (MIRs) (see Ref. 1 for review). lease was measured as described (5).

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FIGURE 1. ILT1 receptor is preferentially expressed on myeloid lin- FIGURE 2. ILT1 receptor is an ϳ69-kDa glycoprotein. Left panel, Pe- 8 125 eage cells. Whole leukocytes were stained with control mAb PB493 (rat ripheral monocytes (2 ϫ 10 ) were surface labeled with I and precipi- IgG2a) (A) or anti-ILT1 135 mAb (B), followed by FITC-conjugated goat tated with anti-ILT1 mAb 135. Precipitates were treated with or without anti-rat IgG, and analyzed by flow cytometry. SSC high, intermediate, and N-glycosidase F for 18 h and analyzed by standard SDS-PAGE under re- low populations correspond to granulocytes, monocytes, and lymphocytes ducing conditions. After deglycosylation, the molecular mass of ILT1 re- respectively. Anti-ILT1 mAb 135 stains DC (C) and macrophages (D) ceptor is reduced from ϳ69 kDa to ϳ49 kDa, which corresponds to the http://www.jimmunol.org/ cultured from monocytes (shaded profiles). Thin profiles illustrate staining predicted molecular mass of ILT1 receptor. Right panel, Proteins with the with isotypic control PB493. same electrophoretic mobility were immunoprecipitated from ILT1/FLAG- transfected P815 cells using anti-FLAG M2 mAb.

Intracellular calcium measurement ϳ Cells were loaded with Indo-1 AM dye (Sigma, St. Louis, MO) and ana- cipitated a prominent protein of 69 kDa under reducing condi- lyzed on a flow cytofluorometer as described (5). After a baseline was tions, and of ϳ49 kDa after N-deglycosylation of the immunopre- acquired for at least 30 s, 100 ␮l of culture supernatants of either anti-ILT1 cipitates (Fig. 2). A protein with identical electrophoretic mobility mAb 135 or an isotype-matched control mAb (PB493, rat IgG2a, anti- was immunoprecipitated from 125I-surface labeled ILT1/FLAG- by guest on September 30, 2021 mouse pre-B cells, kindly provided by Antonius Rolink, Basel, Switzer- ␮ Ј transfected P815 cells with the anti-FLAG peptide M2 mAb, thus land) and 10 g of a cross-linking Ab (goat F(ab )2 anti-rat IgG Fc (The Jackson Laboratory, Bar Harbor, ME)) were added to the cells, and anal- confirming the specificity of mAb 135 for ILT1 (Fig. 2). ysis was allowed to continue up to 512 s. ILT1 receptor triggers cell activation in transfected RBL cells Immunoprecipitations and immunoblottings and monocytes ILT1 receptor was immunoprecipitated from 125I-surface labeled human The capacity of the ILT1 receptor to mediate cell activation was 125 monocytes with mAb 135 and from I-surface labeled ILT1/FLAG-trans- initially assessed by testing whether it can trigger serotonin release fected P815 cells with anti-FLAG M2 mAb (16). Immunoprecipitates were analyzed by standard SDS-PAGE. In immunoblotting experiments, ILT1 re- in ILT1/FLAG-transfected RBL cells. As shown in Fig. 3, sero- ceptor was immunoprecipitated from monocytes and 293 transfectants with tonin secretion was clearly stimulated by cross-linking of ILT1 mAb 135 and from ILT1/FLAG-transfected P815 with anti-FLAG M2 mAb. using anti-FLAG M2 mAb followed by a second step Ab. The Fc⑀RI␥/FLAG and DAP12/FLAG were also immunoprecipitated with anti- amount of serotonin released was about 50% of the release ob- FLAG M2 mAb. Cells were lysed in 1% digitonin buffer (16). Immunopre- served in control experiments, in which ILT1/FLAG-transfected cipitates were separated on one-dimensional or two-dimensional (nonreducing ⑀ vs reducing) SDS-PAGE, transferred to nitrocellulose membranes, and immu- RBL cells were triggered through the Fc RI with mouse IgE. noblotted with anti-Fc⑀RI␥ (kindly provided by Francois Letourneur, Lyon, These results indicate that ILT1 receptor can trigger cell activation France) or with anti-DAP12 (kindly provided by Kerry S. Campbell, Phila- upon Ab ligation and, importantly, suggest that RBL cells have all delphia, PA) rabbit antisera as previously described (16). the components of the ILT1 signaling apparatus. (Interestingly, mAb 135 was less efficient than anti-FLAG M2 mAb in triggering Results serotonin release of ILT1/FLAG-transfected RBL cells (data not ϳ ILT1 receptor is an 69-kDa cell surface glycoprotein shown). It is possible that, upon expression in RBL cells, ILT1 preferentially expressed on myeloid lineage cells receptor undergoes posttranslational modifications that inhibit pro- In whole blood leukocytes, anti-ILT1 mAb 135 stained all mono- ductive binding with the specific mAb 135.) cytes (Fig. 1, A and B), and, to a lesser extent, all granulocytes To further explore the stimulatory function of ILT1 receptor, we (Fig. 1, A and B). ILT1 receptor was also expressed on DC and tested whether ILT1 receptor can trigger Ca2ϩ mobilization in mono- macrophages derived from purified monocytes under appropriate cytes and in ILT1/FLAG-transfected cells. As shown in Fig. 4, cross- culture conditions (Fig. 1, C and D). Only a very small percentage linking of ILT1 using mAb 135 induced significant intracellular cal- of peripheral NK cells (Ͻ1% of total lymphocytes) expressed cium mobilization in monocytes (Fig. 4A), in ILT1/FLAG-transfected ILT1 receptor in the blood donors tested (n ϭ 20) (data not P815 cells (Fig. 4C), and ILT1/FLAG-transfected RBL cells (data not shown). However, it cannot be excluded that, in some donors, NK shown), whereas no changes were detected with control Abs (Fig. 4, cells expressing ILT1 receptor may represent a higher percentage B and D). Moreover, mAb 135 did not trigger calcium mobilization in of total lymphocytes. From monocytes, the mAb 135 immunopre- untransfected P815 cells (data not shown). These results demonstrate The Journal of Immunology 7 Downloaded from

FIGURE 3. ILT1 receptor triggers serotonin release in ILT1/FLAG- transfected RBL cells. ILT1/FLAG-transfected and untransfected RBL cells were incubated with anti-2,4-dinitrophenyl (DNP) IgE, anti-FLAG M2 mAb, or control mouse IgG on ice for 30 min. Cells were washed,

Ј http://www.jimmunol.org/ resuspended in 5% FCS and RPMI 1640, and incubated 1 h. Goat F(ab )2 anti-mouse IgG was also added to cells incubated with M2 mAb and mouse IgG as a cross-linker at the final concentration of 10 ␮g/ml. Serotonin release is expressed as a percentage of maximum release. that ILT1 receptor mediates positive signaling in myeloid cells and that mouse P815 cells, like rat RBL cells, express the signal transduction components necessary for ILT1 receptor to main- tain its signaling capacity. FIGURE 5. Upper panels,Fc⑀RI␥ facilitates cell surface expression of by guest on September 30, 2021 ⑀ ␥ ILT1. 293 cells were transfected with ILT1 cDNA alone (A) or in combination ILT1 receptor associates with Fc RI with either Fc⑀RI␥-FLAG (B) or DAP12-FLAG (C). Cell surface expression To determine whether ILT1 receptor associates with either Fc⑀RI␥ of ILT1 was determined by FACS analysis using mAb 135 followed by FITC- or DAP12, which serve as signaling partners for Fc␣R and acti- conjugated goat anti-rat IgG. Levels on negative controls (stained with PB493 followed by second step Ab) fell within the lower quadrants. Expression of Fc⑀RI␥-FLAG and DAP-FLAG in 293-transfected cells was similar, as determined by FACS analysis with anti-FLAG M2 mAb (data not shown). Cen- tral panels, ILT1 receptor is associated with Fc⑀RI␥ chain. Peripheral monocytes (2 ϫ 108) were lysed in 1% digitonin and immunoprecipitated with anti-ILT1 mAb 135 (D) or control rat IgG2a PB493 (F). Immunoprecipitates were separated by two-dimensional SDS-PAGE (nonreducing/reducing) and immunoblotted with rabbit anti-Fc⑀RI␥ antiserum. Immunoprecipitates from ILT1/FLAG-transfected (E) and untransfected P815 cells (G) with anti-FLAG M2 mAb were also immunoblotted with anti-Fc⑀RI␥ antiserum. The 10-kDa spots below the diagonal marked with an arrow in D and E represent Fc⑀RI␥ chain homodimers. Lower panels, ILT1 receptor does not associate with DAP12 in monocytes (H) and in Fc receptor negative 293 cells triply transfected with ILT1-, DAP12/FLAG-, and Fc⑀RI␥/FLAG-encoding cDNAs (J). ILT1 receptor associates with Fc⑀RI␥ in monocytes (L) and in Fc receptor- negative 293-transfected cells (N). Monocytes were immunoprecipitated with anti-ILT1 mAb 135 (H and L) or control mAb PB493 (I and M). 293 triple transfectants were immunoprecipitated with anti-ILT1 mAb 135 (J and N)or with anti-FLAG M2 mAb (K and O) to control expression of DAP12/FLAG and Fc⑀RI␥/FLAG cDNAs. Immunoprecipitates were immunoblotted with anti- DAP12 or with anti-Fc⑀RI␥ antisera as noted. Electrophoretic mobilities of FIGURE 4. ILT1 receptor induces intracellular Ca2ϩ mobilization. Fc⑀RI␥-FLAG (N and O) and Fc⑀RI␥ (L) are slightly different (bracket). Cross-linking of ILT1 receptor by mAb 135 triggers significant Ca2ϩ mobilization in monocytes (A) and in ILT1/FLAG-transfected P815 cells (C) as compared with monocytes and ILT1/FLAG-transfected P815 cells incubated with isotypic control mAb PB493 (B and D). Cytoplasmic calcium vating NK cell receptors, we first measured ILT1 cell surface ex- levels were monitored in individual cells by measuring 405/525 spectral pression after transfection of 293 cells with ILT1 cDNA alone emission ratio of loaded Indo-1 dye by flow cytometry. After establishing (Fig. 5A) or in combination with cDNAs encoding either Fc⑀RI␥ a baseline, analysis was paused for mAb and/or cross-linker addition, as or DAP12 as amino-terminal FLAG peptide fusion proteins (Fig. indicated by the gaps in the dot plots. 5, B and C). As shown in Fig. 5B, 293 transfectants expressed 8 CUTTING EDGE

ILT1 receptor at significant levels only in the presence of Fc⑀RI␥/ tional association of Fc␣R and Fc⑀RI␥ (15). Interestingly, no FLAG, suggesting that Fc⑀RI␥ associates with ILT1 receptor and association was detected between ILT1 receptor and the newly is critical for ILT1 cell surface expression. To directly demonstrate discovered signal transduction molecule DAP12, which is ex- this interaction, ILT1 receptor was immunoprecipitated from pressed in NK cells and myeloid cells. Thus, DAP12 may associate monocytes, analyzed by two-dimensional SDS-PAGE (nonreduc- with yet another receptor on myeloid cells. ing/reducing), and immunoblotted to test for Fc⑀RI␥ association. This analysis confirmed that ILT1 is associated with a 10-kDa Acknowledgments molecule reactive with anti-Fc⑀RI␥ antiserum. Fc⑀RI␥ was associated with ILT1 as a disulfide-linked homodimer, as demonstrated We thank Annette Pickert, Stephan Meyer, and Mark Dessing (Basel In- stitute for Immunology) for expert technical advice in FACS and Ca2ϩ by migration below the diagonal (Fig. 5D). Murine Fc⑀RI␥ was mobilization analyses; and Kerry S. Campbell (Fox Chase Cancer Center, also associated with ILT1/FLAG immunoprecipitated from ILT1- Philadelphia, PA), Luca Bolliger, Marina Cella, Susan Gilfillan, and Raul transfected P815 cells using the anti-FLAG M2 mAb (Fig. 5E). In Torres (Basel Institute for Immunology) for reviewing the manuscript. control experiments, Fc⑀RI␥ was not immunoprecipitated either from monocytes using an isotype-matched control Ab (Fig. 5F)or References from untransfected P815 cells using the anti-FLAG M2 mAb (Fig. 1. Yokoyama, W. M. 1997. What goes up must come down: the emerging spectrum 5G). Both of these results strongly suggest that the association of of inhibitory receptors. J. Exp. Med. 186:1803. ILT1 receptor with Fc⑀RI␥ is not due to nonspecific coprecipita- 2. Samaridis, J., and M. Colonna. 1996. Cloning of novel immunoglobulin super- tion of Fc receptors for IgG. Lack of association with DAP12 was family receptors expressed on human myeloid and lymphoid cells: structural

evidence for new stimulatory and inhibitory pathways. Eur. J. Immunol. 27:660. Downloaded from demonstrated by performing an anti-DAP12 immunoblot on ILT1 3. Cella, M., C. Dohring, J. Samaridis, M. Dessing, M. Brockhaus, immunoprecipitates obtained either from monocytes (Fig. 5H)or A. Lanzavecchia, and M. Colonna. 1997. A novel inhibitory receptor (ILT3) from 293 cells triply transfected with ILT1-, DAP12/FLAG-, and expressed on monocytes, macrophages, and dendritic cells involved in antigen ⑀ ␥ ⑀ ␥ processing. J. Exp. Med. 185:1743. Fc RI /FLAG-encoding cDNAs (Fig. 5J). A control anti-Fc RI 4. Cosman, D., N. Fanger, L. Borges, M. Kubin, W. Chin, L. Peterson, M. L. Hsu. immunoblot on these immunoprecipitates again detected Fc⑀RI␥ 1997. A novel immunoglobulin superfamily receptor for cellular and viral MHC (Fig. 5, L and N). Since 293 cells are Fc receptor-negative, this class I molecules. Immunity 7:273. 5. Colonna, M., F. Navarro, T. Bellon, M. Llano, P. Garcia, J. Samaridis, experiment confirmed that coprecipitation of Fc⑀RI␥ with ILT1 L. Angman, M. Cella, and M. Lopez-Botet. 1997. A common inhibitory receptor http://www.jimmunol.org/ receptor is due to a specific interaction rather than to coprecipita- for major histocompatibility complex class I molecules on human lymphoid and tion of Fc receptors for IgG. myelomonocytic cells. J. Exp. Med. 186:1809. 6. Wagtmann, N., S. Rojo, E. Eichler, H. Mohrenweiser, and E. O. Long. 1997. A new human gene complex encoding the killer cell inhibitory receptors and related Discussion monocyte/macrophage receptors. Curr. Biol. 7:615. 7. Borges, L., M. L. Hsu, N. Fanger, M. Kubin, and D. Cosman. 1997. A family of Our results demonstrate that ILT1 receptor is an activating cell human lymphoid and myeloid Ig-like receptors, some of which bind to MHC class I molecules. J. Immunol. 159:5192. surface glycoprotein preferentially expressed on myeloid cells that 8. Colonna, M., J. Samaridis, M. Cella, L. Angman, R. Allen, C. OЈCallaghan, 2ϩ triggers intracellular Ca mobilization in monocytes and trans- R. Dunbar, G. Ogg, V. Cerundolo, and A. Rolink. 1998. Human myelomonocytic cells express an inhibitory receptor for classical and nonclassical MHC class I fected cells, as well as release of serotonin in transfected RBL by guest on September 30, 2021 molecules. J. Immunol. 160:3096. cells. The physiological functions regulated by ILT1 receptor in 9. Lanier, L. L. 1997. Natural killer cells: from no receptors to too many. Immunity myeloid cells are yet to be defined. It may control oxidative burst, 6:371. production of cytokines (such as IL1, TNF-␣ and IL12), or induce 10. Maliszewski, C. R., C. J. March, M. A. Schoenborn, S. Gimpel, and L. Shen. 1990. Expression cloning of a human Fc receptor for IgA. J. Exp. Med. 172:1665. expression of costimulatory molecules. Engagement of ILT1 re- 11. Lanier, L. L., B. Corliss, J. Wu, C. Leong, and J. H. Phillips. 1998. Immunore- ceptor with its ligand may be crucial to elicit significant physio- ceptor DAP12 bearing a tyrosine-based activation motif is involved in activating logical responses. In preliminary studies, several soluble HLA NK cells. Nature 391:703. 12. Lanier, L. L., B. Corliss, J. Wu, and J. H. Phillips. 1998. Association of DAP12 class I tetramers did not bind to ILT1-transfected cells (data not with activating CD94/NKG2C NK cell receptors. Immunity 8:693. shown). We are evaluating the possibility that ILT1 is a recep- 13. Smith, K. M., J. Wu, A. B. Bakker, J. H. Phillips, and L. L. Lanier. 1998. Ly-49D and Ly-49H associate with mouse DAP12 and form activating receptors. J. Im- tor for MHC class I-related molecules such as CD1, MR1, and munol. 161:7. MIC (17). 14. Pfefferkorn, L. C., and G. R. Yeaman. 1994. Association of IgA-Fc receptors Biochemical analysis has revealed that Fc⑀RI␥ is associated (Fc␣R) with Fc⑀RI ␥2 subunits in U937 cells. 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