NK-Activating Receptor KIR2DS4 MHC Class I-Independent

MHC Class I-Independent Recognition of NK-Activating Receptor KIR2DS4 Gil Katz, Roi Gazit, Tal I. Arnon, Tsufit Gonen-Gross, Gabi Tarcic, Gal Markel, Raizy Gruda, Hagit Achdout, Olga This information is current as Drize, Sharon Merims and Ofer Mandelboim of September 29, 2021. J Immunol 2004; 173:1819-1825; ; doi: 10.4049/jimmunol.173.3.1819 http://www.jimmunol.org/content/173/3/1819 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 © 2004 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

MHC Class I-Independent Recognition of NK-Activating Receptor KIR2DS41

Gil Katz,* Roi Gazit,* Tal I. Arnon,* Tsuﬁt Gonen-Gross,* Gabi Tarcic,* Gal Markel,* Raizy Gruda,* Hagit Achdout,* Olga Drize,† Sharon Merims,† and Ofer Mandelboim2*

Natural killer cells are capable of killing tumor and virus-infected cells. This killing is mediated primarily via the natural cytotoxicity receptors, including NKp46, NKp44, NKp30, and by the NKG2D receptor. Killer cell Ig-like receptors (KIRs) are mainly involved in inhibiting NK killing (inhibitory KIRs) via interaction with MHC class I molecules. Some KIRs, however, have been found to enhance NK killing when interacting with MHC class I molecules (activating KIRs). We have previously demonstrated that KIR2DS4, an activating KIR, recognizes the HLA-Cw4 protein. The interaction observed was weak and highly restricted to HLA-Cw4 only. These ﬁndings prompted us to check whether KIR2DS4 might have additional ligand(s). In this study, we show that KIR2DS4 is able to also interact with a non-class I MHC protein expressed on melanoma cell lines and on a primary Downloaded from melanoma. This interaction is shown to be both speciﬁc and functional. Importantly, site-directed mutagenesis analysis reveals that the amino acid residues involved in the recognition of this novel ligand are different from those interacting with HLA-Cw4. These results may shed new light on the function of activating KIRs and their relevance in NK biology. The Journal of Immunology, 2004, 173: 1819Ð1825.

atural killer cells are non-T, non-B lymphocytes that are In addition to the inhibitory KIR2Ds, activating KIR2Ds also http://www.jimmunol.org/ stained positively for CD56 and are negative for CD3. exist, which activate NK killing after interacting with HLA-C- N They comprise between 5 and 10% of PBL. NK cells expressing cells (15, 20). These activating KIR2Ds resemble the recognize and eliminate tumor and virus-infected cells (1). This inhibitory KIR2Ds in their extracellular domains, but contain a killing is mediated via the natural cytotoxicity receptors, including charged residue in their transmembrane domain (Lys) and also NKp46, NKp44, NKp30, and the NKG2D receptor (2, 3). In ad- lack any known motifs in their short cytoplasmic tail (21). The dition, NK cells also express inhibitory receptors that inhibit NK activating receptors are associated with the signal transduction activity (4–10). protein DAP12, which contains the ITAM motif in its cytoplasmic The inhibition of the killing mediated by NK cells is mostly domain (22). The ligands that are recognized by the activating achieved via the interactions of MHC class I proteins with inhib- KIR2Ds are only poorly defined. by guest on September 29, 2021 itory killer cell Ig-like receptors (KIRs).3 The inhibitory KIRs con- The KIR2DS4, an activating KIR2D, is a unique molecule. It sist of either two (2D) or three (3D) extracellular Ig-like domains contains a mixture of residues from both group 1 and 2 KIR2Ds. and interact with HLA-C or HLA-B molecules, respectively (4– For example, KFN, instead of MFN or KFK in positions 44–46, it 8). The inhibitory signal is transduced by ITIM motif, located in also expresses its own unique residues (23). We have recently the long cytoplasmic tail of the receptor (6, 8). Once phosphory- shown that KIR2DS4 interacts with HLA-Cw4, but not with HLA- lated, these ITIMs recruit Src homology 2-containing protein ty- Cw6 (24). The observed interaction was of low affinity, compared rosine phosphatase 1 and 2 (11, 12). By means of phosphorylation, with that of KIR2DL1, an inhibitory KIR2D (24). We suggested the recruited phosphatases become activated and interfere with NK that the presence of group 2 residues in positions 67–70 (GPMM activity. instead of SRMT) weakens the interaction between KIR2DS4 and The inhibitory KIR2Ds can be divided into two subgroups: HLA-Cw4. We further demonstrated that the W14 residue of groups 1 and 2, interacting either with Lys80 or Asn80 of the HLA-Cw4 is critical for the recognition by KIR2DS4 (24). HLA-C molecules, respectively (13, 14). Group 1 is characterized These results were puzzling, first because KIR2DS4 could only by the presence of MFN and SRMT residues in positions 44–46 interact in a very restricted specificity, and second because the and 67–70, respectively. In group 2, the KFK and GPMM residues interaction was of low affinity. We, therefore, investigated whether are located in these positions (15–19). the KIR2DS4 might have an additional ligand(s). In this study, we demonstrate binding of KIR2DS4 to a non-class I MHC protein, *Lautenberg Center for General and Tumor Immunology, Hadassah Medical School, expressed on melanoma cell lines and on a primary melanoma. The Hebrew University, Jerusalem, Israel; and †Sharett Institute of Oncology, Hadassah interaction observed was also functional, resulting in NK-mediated Medical Center, Jerusalem, Israel killing of the target cells expressing this, yet unidentified ligand. Received for publication February 17, 2004. Accepted for publication May 26, 2004. 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 with 18 U.S.C. Section 1734 solely to indicate this fact. Materials and Methods 1 O.M. is supported by research grants from the Israel Cancer Research Fund, the Cells and mAb Israel Science Foundation, and the Israel Cancer Association (20042031-B). The cell lines used in this work are the human B lymphoblastoid cell line 2 Address correspondence and reprint requests to Dr. Ofer Mandelboim, Lautenberg 721.221 and other human melanoma lines, all of which are MHC class I Center for General and Tumor Immunology, Hadassah Medical School, Jerusalem negative. Transfection of 721.221 cells with the cDNA of various class I 91120, Israel. E-mail address: [email protected] MHC molecules was performed by electroporation, as previously described 3 Abbreviation used in this paper: KIR, killer cell Ig-like receptor. (13). Primary NK cells (NK clones) were isolated from PBL using the

human NK cell isolation kit and the autoMACS instrument (Miltenyi Bio- tec, Auburn, CA). mAb used in this work are mAb W6/32, directed against MHC class I molecules; mAb HP3E4 (a kind gift from M. Lopez-Botet, DCEXS Uni- versitat Pompeu Fabra, Barcelona, Spain), directed against KIR2DL1, KIR2DS1, and KIR2DS4 (24–26); and mAb EB6 (Immunotech, West- brook, ME), directed against KIR2DL1 and KIR2DS1 (24, 25). The anti- CD99 mAb 12E7 (a kind gift from A. Bernard, Hoˆpital de L’Archet, Nice, France) was used as a control. Cytotoxic assays The cytotoxic activity of primary NK cells against the various targets was assessed in 5-h 35S release assays, as previously described (13). In experiments in which mAb were included, the final mAb concentration was 5 ␮g/ml or 1/2 dilutions in cases in which the mAb were used as tissue culture supernatants. Ig fusion proteins The Ig fusion proteins used in this work are CD99-Ig, KIR2DL1-Ig, and KIR2DS4-Ig (wild type and mutants) (24). Briefly, the sequence encoding the extracellular portion of the receptor was amplified by PCR from cDNA

isolated from human NK clones. These PCR-generated fragments were Downloaded from cloned into a mammalian expression vector, containing the Fc portion of human IgG1. The construct was transfected into COS-7 cells, and the protein produced was puriﬁed using protein G column. Point mutations in the cDNA coding for the KIR2DS4 were performed by PCR. Flow cytometry Cells were stained either with mAb or Ig fusion proteins. Second reagents Ј http://www.jimmunol.org/ were FITC-conjugated F(ab )2 goat anti-mouse IgG (ICN Biomedicals, Ј Aurora, OH) and PE-conjugated F(ab )2 goat anti-human IgG (Jackson ImmunoResearch Laboratories, West Grove, PA), directed against mAb and Ig fusion proteins, respectively. mAbs were used at a ﬁnal concentration of 3 ␮g/ml, and Ig fusion proteins at 100 ␮g/ml. The staining proce- dure was as follows: 30,000 cells were washed once in FACS medium (1ϫ ␮ PBS, 0.5% BSA, 0.05% NaN3) and then incubated in 100 l of FACS medium containing either mAb or Ig fusion proteins for 1 or2honice (4°C), respectively. Incubations were performed in 96 U-shaped plates (Nunc, Roskilde, Denmark). Cells were then washed twice in FACS me- FIGURE 1. Binding of KIR2DS4-Ig to MHC class I-negative cells. Left

dium and incubated on ice for 1 h with the appropriate second reagents. panel, Different melanomas (indicated in the figure) were stained with by guest on September 29, 2021 Following the incubation, cells were washed twice, resuspended in 200 ␮l anti-MHC class I mAb W6/32 (gray line). Background (black line) is the of FACS medium, and analyzed on a FACSCalibur (BD Biosciences, San staining with FITC-conjugated F(abЈ) goat anti-mouse IgG only. Right ␮ 2 Jose, CA). For blocking experiments, Ig fusion proteins (1 g) were co- panel, The same cells were incubated with or without various Ig fusions: ␮ ␮ incubated with mAb (4 g)for4honice(inafinal volume of 100 lof no protein (thin line), CD99-Ig (dotted line), KIR2DL1-Ig (dashed line), FACS medium), before the incubation with the cells. In cases in which KIR2DS4-Ig (bold line), followed by staining with PE-conjugated F(abЈ) proteolysis was performed, cells were washed once in RPMI 1640 (Sigma- 2 Aldrich, Rehovot, Israel) and incubated for 30 min at 37°C in the presence goat anti-human IgG. The figure shows one representative experiment of of increasing concentrations of proteinase K (Sigma-Aldrich). Cells were four performed. then washed three times in RPMI 1640 supplemented with 10% heat-in- activated FBS (HyClone, Logan, UT) and stained, as described. microglobulin protein (27). As 1106mel cells were stained with Results KIR2DS4-Ig, we tested whether additional MHC class I-negative KIR2DS4-Ig binds MHC class I-negative cells melanomas 1074mel, 1259mel (27, 28), and FO-1 (29) would also We have previously shown that the binding of KIR2DS4-Ig to be recognized by KIR2DS4-Ig. In addition, we obtained 13 pri- class I MHC molecules is of low affinity compared with that of mary melanomas, directly derived from patients and screened for KIR2DL1-Ig, and also further demonstrated that this interaction is the expression of MHC class I. One of these melanomas, desig- highly restricted, i.e., KIR2DS4-Ig binds HLA-Cw4 only (24). nated M-24, was found to be MHC class I negative. As can be seen We, therefore, speculated that KIR2DS4 might have an additional in Fig. 1, left panel, none of the melanoma cells tested expressed ligand(s). To test this hypothesis, various cell lines were stained MHC class I molecules. Importantly, binding of KIR2DS4-Ig to all with KIR2DS4-Ig (HeLa, HT-29, SW-480, Jeg3, SJSA-1, melanomas was observed (Fig. 1, right panel). The levels, how- 1106mel, HUVEC, COS-7, RPMI 8866, 721.221, Daudi, YTS, ever, of KIR2DS4-Ig staining were variable. Relatively weak P815). Indeed, among the cell lines tested, the MHC class I-neg- staining was observed to 1074mel and M-24 cells, moderate stain- ative melanoma 1106mel (27, 28) (Fig. 1, left panel) was found to ing was observed to 1259mel and 1106mel cells, and the FO-1 bind the KIR2DS4-Ig, whereas little or no binding of either cells were strongly recognized by KIR2DS4-Ig (Fig. 1, right KIR2DL1-Ig or CD99-Ig was observed (Fig. 1, right panel). The panel). Thus, additional ligand(s) for KIR2DS4, other then MHC 1106mel cells were derived from a metastatic melanoma specimen class I molecules (HLA-Cw4), exists on the various melanomas. obtained from a patient who had undergone tumor-infiltrating lym- phocyte-based therapy (27). The immunotherapy protocol resulted KIR2DS4 is involved in the killing of 1106mel cells in a more aggressive form of the disease that eventually caused To test whether the specific KIR2DS4 binding observed to patient death. When screened for cell surface expression, 1106mel 1106mel cells is functional, NK killing assays were performed. We cells were shown to be devoid of MHC class I molecules. This selected the 1106mel for further investigation due to several rea- ␤ deficiency was found to be due to the apparent absence of 2- sons: 1106mel cells are shown to be moderately killed by NK cells, The Journal of Immunology 1821 compared with 1259mel cells (28), and it is therefore possible that cells. As can be seen in Fig. 2, middle panel, the killing of 221/ the blocking of one activating receptor will result in a significant Cw4 cells was enhanced by all four clones compared with the effect. The staining of KIR2DS4 was higher on 1106mel cells killing of 721.221 cells (shown is one representative clone). This compared with 1074mel or M-24 cells. Finally, there was a back- increased killing resulted from KIR2DS4 interaction with HLA- ground staining of KIR2DL1 on FO-1 that might influence the Cw4, as it was blocked by the HP3E4 mAb. We next assayed the activity of inhibitory receptors in an NK killing assay. same clones against 1106mel cells that were found to bind the We have previously shown that the expression of KIR2DS4 on KIR2DS4-Ig (Fig. 1, right panel). Importantly, the killing of NK cells can be distinguished by the dim EB6 and the bright 1106mel cells was markedly reduced when the HP3E4 mAb was HP3E4 mAb staining (24). These results were confirmed by the included in the assays, whereas the control mAb 12E7 had no Sixth International Leukocyte Typing Workshop (www.ncbi.nlm. effect (shown is one representative clone in Fig. 2, lower panel). nih.gov/prow/guide/682373991_g.htm), and by several publica- These results suggest that the binding of KIR2DS4 to 1106mel tions (25, 26). Moreover, the KIR2DS4 cDNA was cloned from cells is functional and that 1106mel cells express a yet unidentified NK clones that were EB6 dim and HP3E4 bright (data not shown). ligand for KIR2DS4. We, therefore, stained Ͼ300 NK clones derived from different As an additional control for the experiment, we also assayed the individuals with both mAb. Four clones that met with the above same target cells against KIR2DL1-positive NK clones. NK clones criteria were identified, and a representative clone is shown in Fig. positive for KIR2DL1 were identified based on the similar staining 2, upper panel. of both EB6 and HP3E4 mAb (a representative clone is shown in To verify that we indeed isolated KIR2DS4-positive clones, we Fig. 3, upper panel). As previously reported (24), such clones were then assayed the clones in killing against 721.221 and 221/Cw4 inhibited by 221/Cw4 cells, and this inhibition could be reversed Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 2. KIR2DS4 is important in killing of 1106mel cells. FIGURE 3. KIR2DL1 has no effect on the killing of 1106mel cells. KIR2DS4-expressing NK clones were tested against target cells, with or KIR2DL1-expressing NK clones were tested against target cells, with or without a preincubation with various mAb (HP3E4 or 12E7). A represen- without a preincubation with various mAb (HP3E4 or 12E7). A representative KIR2DS4 NK clone (upper panel) is shown assayed against 721.221 tative KIR2DL1 NK clone (upper panel) is shown assayed against 721.221 and 221/Cw4 cells (middle panel) or against 1106mel cells (lower panel). and 221/Cw4 cells (middle panel) or against 1106mel cells (lower panel). The E:T ratio was 2.5:1 in 721.221 experiments and 10:1 in 1106mel The E:T ratio was 2.5:1 in 721.221 experiments and 10:1 in 1106mel experiments. One representative experiment is shown of two performed. experiments. One representative experiment is shown of two performed. 1822 NOVEL LIGAND FOR KIR2DS4 using the HP3E4 mAb (Fig. 3, middle panel). In contrast, however, to the enhanced killing of 1106mel cells observed with the KIR2DS4-positive clones, no difference in the percentages of killing of 1106mel cells was observed when the KIR2DL1-positive clones (preincubated with or without the various mAb) were used (Fig. 3, lower panel). The killing of 1106mel cells was consistently higher when KIR2DS4Ϫ NK clones were used. We do not as yet know what reasons account for this phenomenon. Comparison of NK-activating receptors such as NKp46 and NKG2D revealed no apparent differences between KIR2DS4Ϫ and KIR2DS4ϩ NK clones (data not shown). It might be that additional, yet unidentified NK-activating receptors are primarily expressed on KIR2DS4Ϫ NK clones only. To further confirm the functional results, we used the HP3E4 mAb to block the KIR2DS4-Ig binding to 1106mel cells. KIR2DS4-Ig was coincubated with the HP3E4 mAb and then incubated with 1106mel cells. As expected, a significant reduction in the KIR2DS4-Ig binding was observed after incubation with

HP3E4 (Fig. 4). No effect was observed when other fusion proteins Downloaded from such as CD99-Ig and KIR2DL1-Ig were preincubated with HP3E4 (Fig. 4). Preincubation with the control 12E7 mAb had no effect on KIR2DS4-Ig binding to 1106mel cells (data not shown). Different amino acid residues control KIR2DS4 binding to 1106mel and 221/Cw4 cells http://www.jimmunol.org/ The binding of KIR2DS4 to HLA-Cw4 is unique. It is of low affinity and of limited specificity (24). To characterize the amino acid residues involved in the binding of KIR2DS4 to HLA-Cw4 and to the unknown tumor ligand, we performed an extensive site- directed mutagenesis of potential contact residues. The amino acid residues that were selected for mutagenesis are those that were previously shown to participate either in KIR2DL1 or KIR2DL2 binding to their ligands (residues 44–45, 67–72, 184) (15–19) or those that are in proximity to such residues and differ between by guest on September 29, 2021 KIR2DS4 and KIR2DL1 (residues 50 and 190) or KIR2DL2 (residue 46) (24) (Fig. 5A). Residues 44–46, 50, and 67–72 are within the membrane-distant domain D1, and residues 184 and 190 are located in the membrane-proximal domain D2 (18, 19) (Fig. 5B). Site-directed mutagenesis was performed by PCR, and the various FIGURE 4. KIR2DS4-Ig binding to 1106mel cells is blocked by mAb cDNA were sequenced, cloned in frame with the human IgG1, and HP3E4. Upper panel, 1106mel cells were incubated with or without var- produced in COS-7 cells, as previously described (24). As shown ious Ig fusions: no protein (thin line), CD99-Ig (dotted line), KIR2DL1-Ig in Fig. 6, the binding of KIR2DS4-Ig to 221/Cw4 cells was abro- (dashed line), KIR2DS4-Ig (bold line), followed by staining with PE-con- Ј gated when residues 44, 50, and 190, which are present in jugated F(ab )2 goat anti-human IgG. Lower panel, The various Ig fusions KIR2DL2, were replaced by the KIR2DL1 residues (K44M, were preincubated with mAb HP3E4 and then used as above. Vertical line H50R, N190K) or when the unique KIR2DS4 residues were re- indicates border of background staining (no protein). The figure shows one representative experiment of three performed. placed by residues present in both KIR2DL1 and KIR2DL2 (PV71,72QD, A184S). Mutating residues 67–70 that are present in KIR2DL2 to the corresponding residues in KIR2DL1 (GPMM67– 70SRMT) resulted in an enhanced binding of KIR2DS4-Ig to 221/ 184, and 190 differently affected the binding of KIR2DS4-Ig to Cw4 cells, as well as to 221/Cw6 cells (which are not bound by the 1106mel cells compared with 221/Cw4 cells. Mutating residues 50 intact KIRD2S4-Ig) (Fig. 6). Thus, these particular residues deter- (H50R) and 190 (N190K) had little or no effect on the binding of mine the fine specificity of KIR2DS4 to HLA-Cw4 only. Mutating KIR2DS4-Ig to 1106mel cells (Fig. 7), while mutating the same residue 46 present in KIR2DL1 to the corresponding residue in residues abrogated the binding of KIR2DS4-Ig to 221/Cw4 cells KIR2DL2 (N46K) slightly increased the binding of KIR2DS4-Ig (Fig. 6). Mutating residues 67–70 (GPMM67–70SRMT) prevented to 221/Cw4 cells, while, remarkably, it had a dramatic effect in KIR2DS4-Ig binding to 1106mel cells (Fig. 7), while the same facilitating KIR2DS4-Ig binding to 221/Cw3 cells (which, like mutation resulted in enhanced binding of KIR2DS4-Ig to 221/Cw4 221/Cw6 cells, are not bound by the intact KIR2DS4-Ig) (Fig. 6). and 221/Cw6 cells (Fig. 6). Inversely, mutating residue 184 The binding of the various KIR2DS4-Ig mutants was then as- (A184S) increased KIR2DS4-Ig binding to 1106mel cells (Fig. 7), sayed on 1106mel cells (Fig. 7). Similar to the results obtained while eliminating binding to 221/Cw4 cells (Fig. 6). Thus, we with 221/Cw4 cells, the binding of KIR2DS4-Ig to 1106mel cells show in this study the molecular mechanisms controlling the was abolished when residues 44 (K44M) and 71–72 (PV71,72QD) unique and specific recognition of HLA-Cw4 by KIR2DS4. In were mutated. Also, a slight increase in the binding of addition, we show that KIR2DS4 uses different amino acid resi- KIR2DS4-Ig to 1106mel cells was observed when mutating residues to recognize two different ligands: class I MHC ligand, HLA- due 46 (N46K). In contrast, however, mutating residues 50, 67–70, Cw4, and an as yet unknown, non-class I MHC ligand. The Journal of Immunology 1823

FIGURE 5. Location of key residues for the KIR2D receptors. A, Sequence alignment of the inhibitory receptors KIR2DL1 and KIR2DL2, and of the activating receptor KIR2DS4. Conserved positions are indicated by dashes. The sequences are shown for two regions (positions 40–79 and 180–193). The indicated dots separate between the two regions. The KIR2DS4 residues that were selected for site-directed mutagenesis are shown in bold. B,An␣-carbon diagram of the KIR2DS4 receptor. The residues that might be involved in the Downloaded from recognition of HLA-Cw4 and 1106mel cells are highlighted in gray. Domains D1 and D2 are indicated. This backbone modeling of the KIR2DS4 receptor was generated using Geno3D Web Server and the Rasmol 2.6 software. http://www.jimmunol.org/

The 1106mel ligand is sensitive to proteolysis therefore considered a nonspecific protease (30). Treatment of by guest on September 29, 2021 The nature of the 1106mel ligand can be versatile. It can be a lipid, 1106mel cells with proteinase K eliminated the binding of a carbohydrate, a protein, or a combination of all of these, i.e., a KIR2DS4-Ig in a dose-dependent manner (Fig. 8). Thus, the novel glycolipid, a proteoglycan, etc. Despite several attempts to immu- ligand for KIR2DS4 that is expressed on the surface of 1106mel noprecipitate the 1106mel ligand using the KIR2DS4-Ig reagent, cells is most probably a protein. no specific protein band was observed (data not shown). Therefore, to obtain more general information regarding the nature of the Discussion 1106mel ligand, we used the proteinase K enzyme, a highly reac- Several reasons may exist to explain the role of activating KIRs. 1) tive serine protease that cleaves peptide bonds at the carboxylic Allele-specific down-regulation: the particular class I MHC pro- sides of aliphatic, aromatic, or hydrophobic amino acids, and tein that interacts with a given inhibitory KIR is down-regulated,

FIGURE 6. Identification of the critical residues in KIR2DS4 that are important for the interaction with HLA-Cw4. The figure shows the median fluorescence intensity (MFI) of the binding of various Ig fusion proteins (wild type and mutants) to 721.221 cells and 721.221 cells expressing different HLA-C proteins, as indicated. Background (Bg) is the staining of the same cells with PE- Ј conjugated F(ab )2 goat anti-human IgG only. The figure shows one representative experiment of six performed. 1824 NOVEL LIGAND FOR KIR2DS4

FIGURE 7. Identification of the critical residues in KIR2DS4 that are important for the interaction with 1106mel cells. The figure shows the median fluorescence intensity (MFI) of the binding of various Ig fusion proteins (wild type and mutants) to 721.221 and 1106mel cells. Background (Bg) is the staining of the same cells with PE-conjugated Ј F(ab )2 goat anti-human IgG only. The figure shows one representative experiment of six performed. Downloaded from while the same cells still express the particular class I MHC pro- Importantly, different residues control the binding of KIR2DS4 tein that interacts with the activating KIR. In such a situation, to both the MHC class I ligand, HLA-Cw4, and the ligand present killing of these abnormal cells would be enhanced. 2) Peptide- on 1106mel cells (Figs. 5–7). Although few residues had the same specific interactions: it is possible that some, as of yet unidentified effect when mutated, i.e., decreased binding of KIR2DS4-Ig to http://www.jimmunol.org/ peptides will enhance the binding of activating KIRs in certain both 221/Cw4 and 1106mel cells (residues 44, 71–72) or slightly viral spread or tumor transformation, resulting in enhanced killing increased binding (residue 46), the majority of the critical residues of these hazardous cells. However, no distinct observations were (7 of 11) had a different effect on KIR2DS4-Ig binding to both found to support these explanations. Additionally, it is still difficult ligands. Residues 50, 184, and 190 were critical in the to understand the very restricted specificity of KIR2DS4, an acti- KIR2DS4-Ig binding to 221/Cw4 cells, but either had no effect on vating KIR, toward HLA-Cw4 only (24). the binding to 1106mel cells (residues 50 and 190) or even en- We, therefore, speculated that the activating KIRs might interact hanced the binding (residue 184). The opposite effect is seen when with non-class I MHC ligands. Indeed, we show in this study that mutating residues 67–70 (GPMM67–70SRMT). These residues

KIR2DS4-Ig, a soluble form of the activating receptor KIR2DS4, enhanced KIR2DS4-Ig binding to 221/Cw4 cells, while decreasing by guest on September 29, 2021 binds to various MHC class I-negative melanoma cells. This bind- the binding to 1106mel cells. We, therefore, suggest that residues ing was specific and functional (Figs. 1–4). Supporting our obser- 50, 184, and 190 are crucial to the specific interaction of KIR2DS4 vation, a recent report (31) demonstrated that subjects with acti- with HLA-Cw4, and that residues 67–70 are important in the spe- vating KIR2DS1 and/or KIR2DS2 genes are susceptible to cific recognition of the non-class I MHC ligand. Furthermore, in developing psoriatic arthritis, but only when HLA ligands for their agreement with other publications (15, 17), mutating residue 46 homologous inhibitory receptors, KIR2DL1 and KIR2DL2/3, are (within the context of residues 44–46) or residues 67–70 to the missing, thus suggesting that other, non-class I MHC ligands corresponding residues in KIR2DL2 (N46K) or KIR2DL1 might interact with these activating KIRs. (GPMM67–70SRMT) partially restored the capability of KIR2DS4 to bind HLA-Cw3 or HLA-Cw4/HLA-Cw6 molecules, respectively (Fig. 6). These results provide a molecular basis to explain the highly restricted manner in which KIR2DS4 binds its class I MHC ligand, HLA-Cw4. Finally, it seems as if residues 67–70 might play a dual role: first, they weaken the interaction with HLA-Cw4, while preventing the binding to HLA-Cw6; second, they control the specific binding to the non-class I MHC ligand present on 1106mel cells. In an attempt to further characterize the 1106mel ligand for KIR2DS4, we treated the cells with proteinase K, a nonspecific protease. As was observed, such treatment reduced the KIR2DS4-Ig binding in a dose-dependent manner (Fig. 8). It, therefore, might be deduced that the newly detected ligand is a protein. Despite numerous trials, we were not able to immunopre- cipitate a specific protein from 1106mel cells using the KIR2DS4-Ig as the precipitating agent (data not shown). This could be either because the interactions between KIR2DS4 and the FIGURE 8. The novel ligand for KIR2DS4 is probably a protein. The unknown ligand are too weak to permit precipitations or because 1106mel cells were subjected to increasing concentrations of proteinase K (0–2 mg/ml), followed by staining either with KIR2DS4-Ig or KIR2DL1- the ligand is expressed on 1106mel cells in low levels (Fig. 1, right Ј panel). Ig. Background (Bg) is the staining with PE-conjugated F(ab )2 goat anti- human IgG only. MFI, median fluorescence intensity. Figure shows one The reasons that activating KIRs are expressed on NK cells are representative experiment of two performed. not completely understood. The results presented in this work The Journal of Immunology 1825 bring a new perspective as to the function of the activating KIR. It 15. Biassoni, R., A. Pessino, A. Malaspina, C. Cantoni, C. Bottino, S. Sivori, would be interesting to test whether the activating KIRs might also L. Moretta, and A. Moretta. 1997. Role of amino acid position 70 in the binding affinity of p50.1 and p58.1 receptors for HLA-Cw4 molecules. Eur. J. Immunol. recognize some viral ligands, and whether infection of a particular 27:3095. population would result in the acquisition of a particular 16. Winter, C. C., J. E. Gumperz, P. Parham, E. O. Long, and N. Wagtmann. 1998. activating KIR. Direct binding and functional transfer of NK cell inhibitory receptors reveal novel patterns of HLA-C allotype recognition. J. 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