Recognition of HLA-Cw4 but Not HLA-Cw6 by the NK Cell Receptor Killer Cell Ig-Like Receptor Two-Domain Short Tail Number 4

Gil Katz, Gal Markel, Sa’ar Mizrahi, Tal I. Arnon and Ofer Mandelboim
J Immunol June 15, 2001, 166 (12) 7260-7267; DOI: https://doi.org/10.4049/jimmunol.166.12.7260

Abstract

NK cells are cytotoxic to virus-infected and tumor cells that have lost surface expression of class I MHC proteins. Target cell expression of class I MHC proteins inhibits NK cytotoxicity through binding to inhibitory NK receptors. In contrast, a similar family of activating NK receptors, characterized by the presence of a charged residue in their transmembrane portion and a truncated cytoplasmic tail, augment lysis by NK cells when ligated by an appropriate class I MHC protein. However, the class I MHC specificity of many of these activating NK receptors is still unknown. Here, we show enhanced lysis of HLA-Cw4 but not HLA-Cw6-expressing cells, by a subset of NK clones. This subset may express killer cell Ig-like receptor two-domain short tail number 4 (KIR2DS4), as suggested by staining with various mAb. It is still possible, however, that these clones may express receptors other than KIR2DS4 that might recognize HLA-Cw4. Binding of KIR2DS4-Ig fusion protein to cells expressing HLA-Cw4 but not to those expressing HLA-Cw6 was also observed. The binding of KIR2DS4-Ig to HLA-Cw4 is weaker than that of killer cell Ig-like receptor two-domain long tail number 1 (KIR2DL1)-Ig fusion protein; however, such weak recognition is capable of inhibiting lysis by an NK transfectant expressing a chimeric molecule of KIR2DS4 fused to the transmembrane and cytoplasmic portion of KIR2DL1. Residue α14 is shown to be important in the KIR2DS4 binding to HLA-Cw4. Implications of the role of the activating NK receptors in immunosurveillance are discussed.

Natural killer cells that belong to the innate immunity system, as well as CTL that are part of the acquired immune response, are able to destroy foreign or infected tissue (1). In contrast to CTL, which are activated in the presence of class I MHC molecules and an appropriate specific peptide, one well-defined function of NK cells is the lysis of target cells deficient in expression of MHC class I proteins (2, 3).

Recognition of polymorphic determinants on HLA molecules by human NK cell-inhibitory receptors is mediated by three types of class I MHC-binding receptors (reviewed in Ref. 4). The Ig-like transcript 2 receptor, a member of the Ig-like transcript family of receptors (5), is able to bind to distinct class I MHC protein and to deliver an inhibitory signal (6). The C-type lectin complex CD94/NKG2A can deliver an inhibitory signal on binding to HLA-E (7, 8). Finally, members of the Ig superfamily of receptors, in particular the 58-kDa and 70-kDa NK-inhibitory receptors, containing two and three domains, respectively, can inhibit NK cell cytotoxicity when binding to the appropriate class I MHC molecules (9, 10, 11). In contrast, related 50-kDa receptors associated with DAP12 (12) augment NK cell cytotoxicity (13) and T cell proliferation (14). However, the class I MHC molecules recognized by many of these activating NK receptors and functions of activating NK receptors are currently poorly defined. Due to the possible expression of a multitude of NK receptors on any given NK cell, it is difficult to define the class I MHC-binding specificity of any given NK receptor using cellular assays with peripheral NK cells. One example of such difficulty is the analysis of the binding of killer cell Ig-like receptor two-domain short tail number 4 (KIR2DS4,3 also known as CL 39 or NKAT8) to class I MHC molecules. The expression of KIR2DS4, as well as other NK receptors, was reported on a CD4+ T cell clone (14). Enhanced proliferation was observed when this T cell clone (named TANK-1) was incubated with superantigen-coated 721.221 cells expressing Cw4 or Cw7 class I MHC proteins, but not with Cw3 or Cw6 (14). In contrast, KIR2DS4 has been postulated to interact with HLA-Cw3 (15), but again, this suggestion was based on cellular assays using NK cells expressing many NK receptors.

Here, we show binding of the KIR2DS4-Ig to HLA-Cw4, but not to HLA-Cw6, although both alleles belong to the same allotype-specific group characterized by the presence of K80 (16). We demonstrate that the W residue at position 14 of HLA-Cw4 is important in the binding of KIR2DS4 to HLA-Cw4. The binding of KIR2DS4-Ig to HLA-Cw4 was weak compared with the killer cell Ig-like receptor two-domain long tail number 1 (KIR2DL1)-Ig binding to the same HLA-C molecules. However, it was still functionally significant in that it was sufficient to inhibit the lysis of Cw4-expressing target cells by YTS cells (an NK tumor line) expressing the chimeric protein of KIR2DS4 fused to the transmembrane and cytoplasmic tail portion of KIR2DL1 (YTS/KIR2DS4*) and to cause enhancement of lysis by NK cells expressing KIR2DS4.

Materials and Methods

Cells and mAb

The cell lines used in this work are the class I MHC-negative human cell line 721.221 (17) and the YTS NK tumor line (a kind gift from Dr. Eshhar, The Weizmann Institute, Rehovot, Israel). Point mutation in HLA-Cw4 and HLA-Cw6 cDNA was performed by PCR. Transfection of 721.221 cells with the cDNA of various class I MHC molecules (wild-type and mutants) was performed as previously described (16). NK cells (lines and clones) were isolated from PBL using the human NK cell isolation kit and the autoMACS instrument (Miltenyi Biotec, Auburn, CA). NK cells were grown in culture as previously described (16).

mAb used in this work were mAb W632, G46-2.6, and HP1F7 directed against class I MHC molecules. mAb HP3E4 (18) and EB6 (Immunotech, Westbrook, ME) are both directed against KIR2DL1R. The HP3E4 and HP1F7 mAb were a kind gift from M. Lopez-Botet (Hospital de la Princesa, Madrid, Spain). The anti-CD99 mAb 12E7, used as a control, was a kind gift from A. Bernard (Hôpital de L’Archet, Nice, France).

Cytotoxic assays

The cytotoxic activity of YTS cells and NK cells against the various targets was assessed in 5-h 35S release assays as described previously (16). In experiments where mAb were included, the final mAb concentration was 10 μg/ml, or 1/2 dilution in cases where the mAb were used as tissue culture supernatants. In all presented cytotoxic assays, the spontaneous release was <15% of maximal release.

Ig fusion proteins

The generation of KIR2DL1-Ig and CD99-Ig fusion proteins was previously described (19). The sequence encoding the extracellular portion of KIR2DS4 protein was amplified by PCR from cDNA isolated from human NK clones. The KIR2DS4 specific primers were: 5′-primer (including HindIII site and Kozak sequence), 5′-CCCAAGCTTGGGGCCGCCACCATGTCGCTCATGGTCATCATC-3′; 3′-primer, (including BamHI site), 5′-CGGGATCCAGAACATGTAGGTGTCTGGG-3′. These PCR-generated fragments were cloned into the mammalian expression vector containing the Fc portion of human IgG1 as previously described (19). Sequencing of the constructs revealed that cDNA of all Ig fusion proteins were in frame with the human Fc genomic DNA and were identical with the reported sequences. The production of CD99-Ig, KIR2DL1-Ig, and KIR2DS4-Ig fusion proteins and the FACS staining procedure were previously described (19). All Ig fusion proteins used in this work run as a single protein band on nonreduced SDS-PAGE. The Ig fusion proteins were routinely tested on SDS-PAGE for protein degradation.

Generation of YTS cells transfected with the chimeric protein containing the extracellular portion of KIR2DS4 fused to the transmembrane and cytoplasmic tail portion of KIR2DL1

For the expression of KIR2DS4 with the transmembrane and cytoplasmic tail portion of KIR2DL1, plasmid constructs were prepared that replaced an EcoRI-AgeI fragment of the KIR2DL1 cDNA with that of the KIR2DS4 cDNA. Sequencing of the constructs revealed that KIR2DL1 cDNA was in frame with the KIR2DS4 cDNA and was identical with the reported sequences. YTS cells were transfected with the cDNA encoding for the chimeric KIR2DS4/KIR2DL1 (KIR2DS4*) protein, as previously described (20, 21).

Results

721.221/Cw4 cells are protected from lysis by YTS cells expressing KIR2DS4* chimeric protein

cDNA encoding for KIR2DS4 was amplified by PCR from peripheral NK clones and cloned into the mammalian expression vector containing the Fc portion of human IgG1 to produce the KIR2DS4-Ig fusion protein. KIR2DS4-Ig was then used to stain 721.221 cells transfected with various class I MHC proteins. Strikingly, specific KIR2DS4-Ig binding was observed only to cells expressing the Cw4 class I MHC protein (data not shown). No binding was observed to 721.221 transfected either with Cw3 or Cw7, while little or no binding was observed to 721.221 cells transfected either with HLA-Cw6 or with HLA-Cw6 in which the cysteine at position 309 was replaced by tryptophan (Cw6/C309W) (Ref. 22 and data not shown). The KIR2DL1 binds to both Cw4 and Cw6 class I MHC molecules (reviewed in Ref. 4). Indeed, efficient binding of KIR2DL1-Ig, which was about 20 times higher than that of KIR2DS4-Ig binding, was observed to 721.221/Cw4 cells (data not shown). The expression of all class I MHC molecules on the surface of 721.221 cells measured by W632 binding was similar (data not shown). KIR2DS4-Ig fusion protein did not bind to 721.221 expressing HLA-A alleles (HLA-A2) or HLA-B alleles (B7, B8, B44, B2702, B2705, B58, and B73) or to 721.221 cells expressing nonclassical class I MHC proteins (HLA-E, HLA-G, and CD1d; data not shown).

Two main conclusions can be drawn from the above experiments. 1) The KIR2DS4-Ig protein specifically binds to 721.221/Cw4 but not to 721.221/Cw6 cells and thus can discriminate between the two alleles that belong to the same allotype group characterized by the presence of K80 (16). 2) The binding of KIR2DS4-Ig to 721.221/Cw4 cells is much weaker than that of KIR2DL1-Ig. Thus, the question arises as to whether the low level of binding between KIR2DS4 and 721.221/Cw4 is sufficient to induce cellular response. An NK tumor line, YTS, was infected with the cDNA encoding for KIR2DS4, KIR2DS2, and CD16 using the retroviral vector pBABE as previously described (20). Although surface expression of all receptors was detected using the mAb HP3E4, GL183, and 3G8 for KIR2DS4, KIR2DS2, and CD16, respectively, none of the transfectants was able to induce lysis in redirected killing assays against P815 cells using appropriate mAb (data not shown). This may be because the signaling pathway, which these receptors use to enhance NK cytotoxicity, is impaired in YTS cells. In contrast, inhibition of lysis of 721.221/Cw4 and Cw6 cells was observed when YTS infected with cDNA encoding for KIR2DL1 (YTS/KIR2DL1) were used (Ref. 20 and Figs. 2⇓ and 3⇓), thus indicating that the inhibitory signal cascade is functioning in YTS cells.

The functional differences between the inhibitory and activating NK receptors are made possible due to the different transmembrane and cytoplasmic tail portions that exist between the two receptors (reviewed in Ref. 4 . A chimeric cDNA construct was therefore made that included the extracellular portion of KIR2DS4 cDNA fused in frame to the transmembrane and cytoplasmic tail portion of KIR2DL1 (KIR2DS4*). cDNA were inserted into the retroviral vector pBABE and transfected into the YTS cell line as previously described (20, 24). Six different transfectants were obtained expressing the KIR2DS4* chimeric protein. All transfectants were positively stained with HP3E4 mAb, but not with EB6 mAb (two representative clones, 1 and 2, are seen in Fig. 1⇓). In contrast, YTS/KIR2DL1 cells were positively stained with both mAb (Fig. 1⇓). No staining was observed to YTS or to YTS/MOCK cells (Fig. 1⇓). Thus, mAb HP3E4 can recognize the extracellular portion of KIR2DS4.

FIGURE 1.
FIGURE 1.

Expression of KIR2DS4* on YTS cells. YTS cells were infected with the cDNA encoding for the extracellular portion of KIR2DS4 fused to the transmembrane and cytoplasmic tail portion of that of KIR2DL1 (KIR2DS4*; see Materials and Methods) as previously described (20 ,21 ). Six independent clones were obtained, of which two (clones 1 and 2) are shown. YTS/MOCK cells are YTS cells transfected with the pBABE plasmid. Controls were the same cells stained with FITC-conjugated goat anti-mouse Abs only. Values show one representative experiment of three performed. FL1-H, Fluorescence.

We next tested whether incubation of YTS/KIR2DS4* with 721.221/Cw4 cells will result in inhibition of lysis. YTS/MOCK, YTS/KIR2DL1, and YTS/KIR2DS4* were incubated with [35S]methionine-labeled target cells in 5-h killing assays (Fig. 2⇓). YTS/MOCK lysed all target cells tested to the same extent (Fig. 2⇓, top). Efficient inhibition of YTS/KIR2DL1 lysis was observed when cells were incubated with either 721.221/Cw4 or 721.221/Cw6 cells in all E:T ratios tested (Fig. 2⇓, middle). In contrast, inhibition of YTS/KIR2DS4* was observed only when target cells expressing HLA-Cw4 were used (Fig. 2⇓, bottom). Correlating to the weak staining of 221/Cw4 by KIR2DS4-Ig compared to KIR2DL1-Ig (Fig. 6⇓), the inhibition of YTS/KIR2DS4* was weaker than that observed with YTS/KIR2DL1 (Fig. 2⇓). The results presented above suggest that the interaction between the KIR2DS4* and the HLA-Cw4 molecules is sufficient to transmit the inhibitory signal. To test whether this inhibition is indeed the result of the binding of the extracellular portion of KIR2DS4 to the HLA-Cw4, mAb HP3E4, shown to bind KIR2DS4 in Fig. 1⇑, was used to block the KIR2DS4 protein binding to HLA-Cw4. HP3E4 was indeed able to reverse the inhibition mediated by 721.221/Cw4 cells, whereas a control mAb 12E7 had no effect (Fig. 3⇓).

FIGURE 2.
FIGURE 2.

721.221 cells expressing the Cw4 class I MHC protein are protected from lysis by YTS/KIR2DS4* cells. Cells were labeled with [35S]methionine and incubated with YTS/MOCK, with YTS/KIR2DL1, or with YTS/KIR2DS4* #2, for a 5-h killing assay. Similar results were obtained when YTS/KIR2DS4* #1 cells were used. Values show one representative experiment of three performed.

FIGURE 3.
FIGURE 3.

Reversal of the inhibition of YTS killing by the HP3E4 mAb. YTS cells (YTS/MOCK, YTS/KIR2DL1, or YTS/KIR2DS4* #2) were incubated with the control anti-CD99 mAb (12E7) or with anti-KIR2DS4/anti-KIR2DL1 mAb HP3E4. Next, cells were incubated with various target cells that are indicated in the figure. The E:T ratio was 2.5:1. When no mAb were included in the assays, the lysis of all targets was similar to that observed with mAb 12E7. Similar results were obtained when YTS/KIR2DS4* #1 cells were used. The figure shows one representative experiment of three performed.

Lysis of 721.221 cells expressing the Cw4 class I MHC protein is enhanced by NK clones expressing the KIR2DS4

The functional relevance of the above observation suggesting a specific interaction between KIR2DS4 and HLA-Cw4 was tested by using NK clones derived from various healthy donors. NK clones were generated as described in Materials and Methods. Two hundred fifty independent NK clones were tested in killing experiments against various class I MHC-transfected target cells. Three killing phenotypes were observed with regard to the killing of 721.221/Cw4 cells, of which three representatives are shown in Fig. 4⇓. NK clone 3 was stained equally brightly with mAb EB6 and HP3E4. This clone probably expresses KIR2DS1, because activation of lysis was observed against 721.221/Cw4 and 721.221/Cw6 cells, and both EB6 and HP3E4 mAb blocked the enhancement of lysis (Fig. 4⇓, top). Among the 250 NK clones tested, 5 (2%) were stained brightly with mAb HP3E4 and dimly with mAb EB6 (representative NK clone 6 is seen in Fig. 4⇓, middle). This unique staining suggests the existence of KIR2DS4 (Fig. 1⇑). Indeed, enhancement of lysis of 721.221/Cw4 cells only was observed when cells were incubated with all five clones mentioned. The addition of HP3E4 mAb blocked this enhancement, whereas EB6 or 12E7 mAb had no effect (representative NK clone 6 is seen in Fig. 4⇓, middle). This is in agreement with our above observation suggesting that the ligand for KIR2DS4 is the HLA-Cw4 only. Finally, inhibition of lysis was observed when 721.221/Cw4 and 721.221/Cw6 cells were tested against NK clone 52. As expected, this clone was stained equally brightly with EB6 and HPE4 mAb (Fig. 4⇓, bottom), suggesting the existence of the KIR2DL1.

FIGURE 4.
FIGURE 4.

Different lysis phenotypes of different NK clones incubated with 721.221 cells expressing HLA-Cw4. NK clones were prepared from PBL of various donors as described in Materials and Methods. About 250 independent NK clones were obtained, of which 3 representatives (clones 3, 6, and 52) are shown. Cells were stained (three left panels) with no mAb, the EB6 mAb (dotted line) or with HP3E4 mAb (bold line). Control were the same cells stained with FITC-conjugated goat anti-mouse Abs only (plain line). The E:T ratio was 2.5:1. NK killing assays (three right panels) were performed as described in Materials and Methods. Values show one representative experiment of two performed. FL1-H, Fluorescence.

The W residue at position 14 of HLA-Cw4 is important in the binding to KIR2DS4

It has been demonstrated that peptides bound in the groove can influence recognition by NK receptors (23, 24, 25). Thus, the binding of KIR2DS4 to HLA-Cw4, but not -Cw6 could be the result of the influence of the repertoire of peptides bound by -Cw4 vs -Cw6. All four HLA-C molecules (Cw3, Cw4, Cw6, and Cw7) analyzed here exhibit related peptide motifs, although each allelic product shows individual characteristics in their fine specificity of peptide binding (26). Alternatively, and perhaps more likely, given the limited effect of peptides on NK recognition (23, 24), it is possible that KIR2DS4 binds to the class I MHC proteins on a site that is different from the one identified for the inhibitory NK receptors KIR2DL1 and KIR2DL2 (16, 27, 28). Indeed, the KIR2DS4* inhibition could not be blocked with any of the anti-HLA mAb tested including HP1F7, G46-2.6, W632, and W632 F(ab′)2 (data not shown). Moreover, comparison of the amino acid sequences of all known HLA-C proteins characterized by the presence of K80 revealed the existence of a pair of residues that can be found in HLA-Cw4 only (S11 and W14; Fig. 5⇓A). The location of both residues is shown in Fig. 5⇓C. Although S11 can also be found in the sequence of other HLA-C alleles that belong to the other allotype-specific group, characterized by the presence of N80 (e.g., Cw0101, Cw0102, Cw1402, and Cw1403), W14 is unique to HLA-Cw4 (Fig. 5⇓A).

FIGURE 5.
FIGURE 5.

Location of the key residues for the KIR receptors and for HLA-C proteins. A, Sequence alignment of some of the HLA-C proteins that belong to group 1 (16 ), Cw*0201, Cw*0401, Cw*0501, and Cw*0602. Conserved positions are indicated by dashes. The sequences are shown for positions 1–93 and 140–163. Bold indicates the putative binding site on HLA-C (28 ). B, Sequence alignment of the inhibitory receptors KIR2DL1, KIR2DL2, and of the activating receptor KIR2DS4. Conserved positions are indicated by dashes. The sequences are shown for two regions (positions 1–82 and 100–193). Bold indicates the putative binding site for binding to HLA-C (28 ). C, Location of the contact residues (•) on HLA-C that affect its recognition by KIR2DL2 (28 ). Embedded Image, Residues α11 and α14 that might be involved in the KIR2DS4 recognition. The residues are highlighted on an α-carbon diagram of a class I MHC molecule viewed from the top (35 ).

The involvement of S11 and W14 of HLA-Cw4 in the binding to KIR2DS4 was therefore tested using site-directed mutagenesis experiments in which residues S11 and W14 were replaced with the corresponding residues found in HLA-Cw6 (Cw4 S11A and Cw4 W14R). Mutations were performed by PCR, sequenced, and transfected into 721.221 cells. The expression of both Cw4 S11A and Cw4 W14R was assayed by staining with W632 mAb and was similar to that observed with the wild-type HLA-Cw4 (see legends to Fig. 6⇓). Whereas binding of the KIR2DL1-Ig fusion was observed to all HLA-Cw4 proteins (wild-type and mutants), binding of KIR2DS4-Ig was observed to 721.221/Cw4 and 721.221/Cw4 S11A but not to 721.221/Cw4 W14R (Fig. 6⇓), thus suggesting that residue W14 is important in the KIR2DS4 binding to HLA-Cw4. To further demonstrate the importance of residue W14 in the binding to HLA-Cw4, we mutated by PCR residue R14 in HLA-Cw6 cDNA to W (Cw6 R14W). cDNA was sequenced and transfected into 721.221 cells. Whereas KIR2DL1-Ig fusion protein binds all HLA-Cw4 and HLA-Cw6 expressing cells, including wild types and mutants, binding of KIR2DS4-Ig was observed only to cells expressing HLA-C alleles containing the W14 residue, including the 721.221/Cw6 R14W cells (Fig. 6⇓).

FIGURE 6.
FIGURE 6.

Specific binding of KIR2DS4-Ig is dependent on W14 residue. 721.221 cells and 721.221 cells expressing various HLA-C proteins (wild type and mutants) were stained either with the control CD99-Ig (plain line) KIR2DS4-Ig (dotted line) or with KIR2DL1-Ig (gray line) as previously described (19 ). The staining of the same cells with PE-conjugated anti human Fc Abs was similar to that observed with the CD99-Ig staining. The median fluorescence intensities for the various class I MHC transfectants stained with W632 were 433, 226, 835, 162, 226, and 500 for 721.221 cells transfected with HLA-Cw3, -Cw4, -Cw6, -Cw4/S11A, -Cw4/W14R, and -Cw6/R14W, respectively. FL2-H, Fluorescence.

The functional relevance of the above observation, suggesting the involvement of W14 in the binding to KIR2DS4, was tested by using NK clones derived from various healthy donors. NK clones that were activated by HLA-Cw4 but not by HLA-Cw6 and that stained brightly with HP3E4 mAb (data not shown) were tested against various transfectants. One representative clone is shown in Fig. 7⇓A. Enhancement of lysis was observed with 721.221/Cw4 cells, but not with 721.221, 721.221/Cw3, and 721.221/Cw6 transfectants (Fig. 7⇓A). In agreement with the FACS staining results, mutations in residue 14 affect the lysis of the target cells. Enhancement of lysis was not observed against target cells expressing HLA-C alleles containing the R14 residue, i.e., 721.221/Cw4 W14R, 721.221/Cw6 transfectant (these cells do not bind KIR2DS4-Ig (Fig. 6⇑)). In contrast, activation of lysis was observed when 721.221/Cw4, 721.221/Cw6 R14W, or 721.221/Cw4 S11A cells were used (all cells bind KIR2DS4-Ig (Figs. 6⇑ and 7⇓A)). In all targets in which activation of lysis was observed, the activation could be blocked with HP3E4 mAb but not with EB6 mAb or 12E7 mAb, suggesting the involvement of KIR2DS4 (Fig. 7⇓A). Similar results were obtained when the YTS/KIR2DS4* cells were used as effector cells. Inhibition of YTS/KIR2DS4* lysis was observed when cells were incubated with 721.221/Cw4, 721.221/Cw4 S11A, and 721.221/Cw6 R14W, whereas incubation with 721.221, 721.221/Cw3, 721.221/Cw6, and 721.221/Cw4 W14R had no effect (Fig. 7⇓B). Abolishment of the inhibition was observed when the YTS/KIR2DS4* cells were incubated with HP3E4 mAb, but not with EB6 mAb or 12E7 mAb.

FIGURE 7.
FIGURE 7.

Mutation in residue 14 affects the lysis by NK cells expressing KIR2DS4. A, Lysis of different target cells by NK clone; B lysis of various targets by YTS/KIR2DS4* cells. The E:T ratio was 2.5:1. NK lysis experiments were performed as described in Materials and Methods. Values show one representative experiment of two performed.

Discussion

Three main conclusions can be drawn from our results. First, KIR2DS4 can bind to cells expressing HLA-Cw4 but not to cells expressing -Cw6 class I MHC molecules (Fig. 6⇑) and that binding is dependent on residue W14 (Figs. 6⇑ and 7⇑). This is in agreement with previous results demonstrating no binding of soluble KIR2DS4 to either Cw6 or Cw7 class I MHC proteins (29). This surprising result is particularly interesting because the key residues which determine the binding site for KIR2DL1 and KIR2DL2 on the HLA-C (Refs. 16, 27, 28 and Fig. 5⇑, A and C) are identical in both HLA-Cw4 and -Cw6. It thus suggests that KIR2DS4 binds to HLA-Cw4 on a binding site that is different from the binding site to KIR2DL1. Indeed, changing of the W14 residue in Cw4 to R abolished recognition by KIR2DS4-Ig but not by KIR2DL1-Ig (Fig. 6⇑). The second conclusion is that the binding of KIR2DS4-Ig to Cw4 is much weaker than that observed with KIR2DL1-Ig (Figs. 2⇑ and 6⇑). Few experiments have been reported analyzing the binding of activating NK receptors to class I MHC proteins. A soluble form of the activating NK receptor molecule EB6ActI (KIR2DS1) bound very weakly to HLA-Cw4-expressing cells (30). The molecular basis for the lack of binding of CL 49 (KIR2DS2) to various class I-transfected cells was localized to the presence of tyrosine in position 45 (29, 31). Phenylalanine occupies this position in all other two-domain-activating or inhibitory receptors. The same authors (31) could not detect binding of KIR2DS4 to any of the class I transfectants tested, including Cw4. This might be due to the lower concentration of KIR2DS4-Ig used to stain the transfectants, i.e., 50 μg/ml in Ref. 31 vs 100 μg/ml in this work, as well as to a higher cell-protein ratio that could have been used that might be sufficient to detect binding of KIR2DL1-Ig to Cw4, but not enough to detect binding of KIR2DS4-Ig to the same cells. Indeed, in the current work, binding of KIR2DS4-Ig to 721.221/Cw4 cells is diminished at 12.5 μg/ml, whereas binding of KIR2DL1-Ig to the same cells is still detected even at a concentration as low as 1.5 μg/ml (data not shown).

The molecular basis for the unique binding of KIR2DS4 to HLA-Cw4 and not -Cw3, -Cw6, or -Cw7, is unclear. The K residue at position 44 of KIR2DL2 was reported to be critical for the binding of this receptor to HLA-Cw3 (28, 32). KIR2DS4 contains the KFN amino acid residues at positions 44–46 (Fig. 5⇑B), which is a hybrid of the amino acids found in the same position in KIR2DL1 (MFN) and KIR2DL2 (KFK). Molecular modeling of KIR2DS4 receptor based on the crystal structure of KIR2DL1 predicted that the surface potential of KIR2DS4 is dramatically different from that of KIR2DL2 (29). Moreover, the mAb EB6, which recognizes an epitope important for the binding of KIR2DL1 to HLA-Cw4 (33), does not recognize KIR2DS4 (Fig. 1⇑). This suggests that the site on these two NK receptors involved in the binding to HLA-C is distinct. Residues at positions 67–70, GPMM, found in KIR2DL2, which binds to HLA-Cw3, can also be found in KIR2DS4 (Fig. 5⇑B). Some of these residues were also reported to be important in the HLA-C binding (28, 30, 32). Thus, of seven amino acid residues that are found in the two regions of KIR sequences reported to be important in HLA-Cw4 binding (30, 31, 32), only three, namely, F45, N46, and M69, are found in KIR2DS4. This might account for the weak interaction observed between KIR2DS4 and HLA-Cw4 (Figs. 2⇑ and 6⇑). In addition, the crystal structure of KIR2DL2 in complex with HLA-Cw3 was recently solved (28). Sixteen contact residues were identified in the KIR2DL2 protein interacting with HLA-Cw3 (Ref. 28 and Fig. 5⇑B). Among these residues, three were unique for KIR2DS4 only, namely, P71, V72, and A184. Site-directed mutagenesis analysis of these residues would determine their involvement in the KIR2DS4 binding to HLA-Cw4.

The third conclusion is that the low binding affinity between the activating NK receptor KIR2DS4 and HLA-Cw4 is likely to be sufficient for the function of this receptor ( Figs. 2–4⇑⇑⇑ and 7). Accumulating evidence suggests that activating NK receptors can function on binding to various class I molecules. A correlation between the expression of p50 receptor stained with EB6 mAb and enhanced cytotoxicity against Cw4-expressing target cells was observed (13). Enhanced proliferation was observed when a T cell clone (TANK-1) positive for KIR2DS4 expression was incubated with superantigen-coated 721.221 expressing Cw4 or Cw7 class I MHC proteins, but not with Cw3 or Cw6 (14). This last observation (14) seems to be in contrast with the results presented here demonstrating binding of KIR2DS4 to HLA-Cw4 only. However, as the field developed and more sequence information became available, a later analysis using additional PCR primers revealed that the TANK-1 clone expresses mRNA for other NK receptors (29). Using YTS cells transfected with KIRD2S4 and KIR2DS4-Ig protein, it is now clear that KIR2DS4 binds to HLA-Cw4-expressing cells only.

Finally, a model for the interplay of NK-activating and inhibitory receptors can be speculated. Inhibitory receptors bind with higher affinity to MHC class I proteins but have a higher threshold for the number of MHC molecules required for triggering signaling, whereas the activating receptors bind with lower affinity and have a lower threshold for triggering signaling. Thus, on recognition of normal MHC expression, signaling through the inhibitory receptors dominates, whereas on recognition of reduced class I MHC expression, e.g. in viral-infected cells, the activating NK receptors dominate the NK cell response. Alternatively, it might be that the activating NK receptors function only in cases where allele-specific down-regulation can be observed, e.g., after HIV infection (20) or tumor spread (reviewed in Ref. 34). In these situations, after down-regulation of HLA molecules for which specific inhibitory NK receptors are present, activating NK receptors may be triggered via other HLA molecules remaining on the cell surface.

Acknowledgments

We thank Dr. Lopez-Botet (Hospital de la Princesa, Madrid, Spain) and Dr. A. Bernard (Hopital de L’Archet, Nice, France) for the kind gift of mAb used in this work.

Footnotes

  • ↵1 This research was supported by the United States–Israel Science Foundation (BSF) and by the joint German–Israeli research program.

  • ↵2 Address correspondence to Dr. Ofer Mandelboim, Lautenberg Center for General and Tumor Immunology, Hadassah Medical School, Jerusalem 91120, Israel. E-mail address: oferman{at}md2.huji.ac.il

  • 3 Abbreviation used in this paper: KIR2DS4, killer cell Ig-like receptor two-domain short tail number 4; KIR2DL1, killer cell Ig-like receptor two-domain long tail number 1. Other moieties are similarly abbreviated.

  • Received June 1, 2000.
  • Accepted April 13, 2001.

References

  1. ↵
    Trinchieri, G.. 1989. Biology of natural killer cells. Adv. Immunol. 47: 187
    OpenUrlCrossRefPubMed
  2. ↵
    Parham, P.. 1997. NK cells, MHC class I antigens and missing self. Immunol. Rev. 155: 5
    OpenUrlCrossRefPubMed
  3. ↵
    Ljunggren, H. G., K. Karre. 1990. In search of the “missing self”: MHC molecules and NK recognition. Immunol. Today 11: 237
    OpenUrlCrossRefPubMed
  4. ↵
    Long, E. O.. 1999. Regulation of immune responses through inhibitory receptors. Annu. Rev. Immunol. 17: 875
    OpenUrlCrossRefPubMed
  5. ↵
    Colonna, M., H. Nakajima, F. Navarro, M. Lopez-Botet. 1999. A novel family of Ig-like receptors for HLA class I molecules that modulate function of lymphoid and myeloid cells. J. Leukocyte Biol. 66: 375
    OpenUrlAbstract
  6. ↵
    Colonna, M., F. Navarro, T. Bellon, M. Llano, P. Garcia, J. Samaridis, L. Angman, M. Cella, M. Lopez-Botet. 1997. A common inhibitory receptor for major histocompatibility complex class I molecules on human lymphoid and myelomonocytic cells. J. Exp. Med. 186: 1809
    OpenUrlAbstract/FREE Full Text
  7. ↵
    Braud, V. M., D. S. Allan, C. A. O’Callaghan, K. Soderstrom, A. D’Andrea, G. S. Ogg, S. Lazetic, N. T. Young, J. I. Bell, J. H. Phillips, L. L. Lanier, A. J. McMichael. 1998. HLA-E binds to natural killer cell receptors CD94/NKG2A, B and C. Nature 391: 795
    OpenUrlCrossRefPubMed
  8. ↵
    Braud, V. M., A. J. McMichael. 1999. Regulation of NK cell functions through interaction of the CD94/NKG2 receptors with the nonclassical class I molecule HLA-E. Curr. Top. Microbiol. Immunol. 244: 85
    OpenUrlPubMed
  9. ↵
    Colonna, M., J. Samaridis. 1995. Cloning of immunoglobulin-superfamily members associated with HLA-C and HLA-B recognition by human natural killer cells. Science. 268: 405
    OpenUrlAbstract/FREE Full Text
  10. ↵
    Wagtmann, N., S. Rajagopalan, C. C. Winter, M. Peruzzi, E. O. Long. 1995. Killer cell inhibitory receptors specific for HLA-C and HLA-B identified by direct binding and by functional transfer. Immunity 3: 801
    OpenUrlCrossRefPubMed
  11. ↵
    D’Andrea, A., C. Chang, K. Franz-Bacon, T. McClanahan, J. H. Phillips, L. L. Lanier. 1995. Molecular cloning of NKB1. A natural killer cell receptor for HLA-B allotypes. J. Immunol. 155: 2306
    OpenUrlAbstract
  12. ↵
    Lanier, L. L., B. C. Corliss, J. Wu, C. Leong, J. H. Phillips. 1998. Immunoreceptor DAP12 bearing a tyrosine-based activation motif is involved in activating NK cells. Nature 391: 703
    OpenUrlCrossRefPubMed
  13. ↵
    Moretta, A., S. Sivori, M. Vitale, D. Pende, L. Morelli, R. Augugliaro, C. Bottino, L. Moretta. 1995. Existence of both inhibitory (p58) and activatory (p50) receptors for HLA-C molecules in human natural killer cells. J. Exp. Med. 182: 875
    OpenUrlAbstract/FREE Full Text
  14. ↵
    Mandelboim, O., D. M. Davis, H. T. Reyburn, M. Vales-Gomez, E. G. Sheu, L. Pazmany, J. L. Strominger. 1996. Enhancement of class II-restricted T cell responses by costimulatory NK receptors for class I MHC proteins. Science 274: 2097
    OpenUrlAbstract/FREE Full Text
  15. ↵
    Campbell, K. S., M. Cella, M. Carretero, M. Lopez-Botet, M. Colonna. 1998. Signaling through human killer cell activating receptors triggers tyrosine phosphorylation of an associated protein complex. Eur. J. Immunol. 28: 599
    OpenUrlCrossRefPubMed
  16. ↵
    Mandelboim, O., H. T. Reyburn, M. Vales-Gomez, L. Pazmany, M. Colonna, G. Borsellino, J. L. Strominger. 1996. Protection from lysis by natural killer cells of group 1 and 2 specificity is mediated by residue 80 in human histocompatibility leukocyte antigen C alleles and also occurs with empty major histocompatibility complex molecules. J. Exp. Med. 184: 913
    OpenUrlAbstract/FREE Full Text
  17. ↵
    Shimizu, Y., R. DeMars. 1989. Production of human cells expressing individual transferred HLA-A, -B, -C genes using an HLA-A, -B, -C null human cell line. J. Immunol. 142: 3320
    OpenUrlAbstract
  18. ↵
    Melero, I., A. Salmeron, M. A. Balboa, J. Aramburu, M. Lopez-Botet. 1994. Tyrosine kinase-dependent activation of human NK cell functions upon stimulation through a 58-kDa surface antigen selectively expressed on discrete subsets of NK cells and T lymphocytes. J. Immunol. 152: 1662
    OpenUrlAbstract
  19. ↵
    Mandelboim, O., P. Malik, D. M. Davis, C. H. Jo, J. E. Boyson, J. L. Strominger. 1999. Human CD16 as a lysis receptor mediating direct natural killer cell cytotoxicity. Proc. Natl. Acad. Sci. USA 96: 5640
    OpenUrlAbstract/FREE Full Text
  20. ↵
    Cohen, G. B., R. T. Gandhi, D. M. Davis, O. Mandelboim, B. K. Chen, J. L. Strominger, D. Baltimore. 1999. The selective downregulation of class I major histocompatibility complex proteins by HIV-1 protects HIV-infected cells from NK cells. Immunity 10: 661
    OpenUrlCrossRefPubMed
  21. ↵
    Davis, D. M., I. Chiu, M. Fassett, G. B. Cohen, O. Mandelboim, J. L. Strominger. 1999. The human natural killer cell immune synapse. Proc. Natl. Acad. Sci. USA 96: 15062
    OpenUrlAbstract/FREE Full Text
  22. ↵
    Davis, D. M., O. Mandelboim, I. Luque, E. Baba, J. Boyson, J. L. Strominger. 1999. The transmembrane sequence of human histocompatibility leukocyte antigen (HLA)-C as a determinant in inhibition of a subset of natural killer cells. J. Exp. Med. 189: 1265
    OpenUrlAbstract/FREE Full Text
  23. ↵
    Mandelboim, O., S. B. Wilson, M. Vales-Gomez, H. T. Reyburn, J. L. Strominger. 1997. Self and viral peptides can initiate lysis by autologous natural killer cells. Proc. Natl. Acad. Sci. USA 94: 4604
    OpenUrlAbstract/FREE Full Text
  24. ↵
    Peruzzi, M., N. Wagtmann, E. O. Long. 1996. A p70 killer cell inhibitory receptor specific for several HLA-B allotypes discriminates among peptides bound to HLA-B*2705. J. Exp. Med. 184: 1585
    OpenUrlAbstract/FREE Full Text
  25. ↵
    Rajagopalan, S., E. O. Long. 1997. The direct binding of a p58 killer cell inhibitory receptor to human histocompatibility leukocyte antigen (HLA)-Cw4 exhibits peptide selectivity. J. Exp. Med. 185: 1523
    OpenUrlAbstract/FREE Full Text
  26. ↵
    Falk, K., O. Rotzschke, B. Grahovac, D. Schendel, S. Stevanovic, V. Gnau, G. Jung, J. L. Strominger, H. G. Rammensee. 1993. Allele-specific peptide ligand motifs of HLA-C molecules. Proc. Natl. Acad. Sci. USA 90: 12005
    OpenUrlAbstract/FREE Full Text
  27. ↵
    Mandelboim, O., H. T. Reyburn, E. G. Sheu, M. Vales-Gomez, D. M. Davis, L. Pazmany, J. L. Strominger. 1997. The binding site of NK receptors on HLA-C molecules. Immunity 6: 341
    OpenUrlCrossRefPubMed
  28. ↵
    Boyington, J. C., S. A. Motyka, P. Schuck, A. G. Brooks, P. D. Sun. 2000. Crystal structure of an NK cell immunoglobulin-like receptor in complex with its class I MHC ligand. Nature. 405: 537
    OpenUrlCrossRefPubMed
  29. ↵
    Vales-Gomez, M., H. T. Reyburn, R. A. Erskine, J. L. Strominger. 1998. Differential binding to HLA-C of p50-activating and p58-inhibitory natural killer cell receptors. Proc. Natl. Acad. Sci. USA 95: 14326
    OpenUrlAbstract/FREE Full Text
  30. ↵
    Biassoni, R., A. Pessino, A. Malaspina, C. Cantoni, C. Bottino, S. Sivori, L. Moretta, 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. 27: 3095
    OpenUrlCrossRefPubMed
  31. ↵
    Winter, C. C., J. E. Gumperz, P. Parham, E. O. Long, N. Wagtmann. 1998. Direct binding and functional transfer of NK cell inhibitory receptors reveal novel patterns of HLA-C allotype recognition. J. Immunol. 161: 571
    OpenUrlAbstract/FREE Full Text
  32. ↵
    Winter, C. C., E. O. Long. 1997. A single amino acid in the p58 killer cell inhibitory receptor controls the ability of natural killer cells to discriminate between the two groups of HLA-C allotypes. J. Immunol. 158: 4026
    OpenUrlAbstract
  33. ↵
    Moretta, A., M. Vitale, C. Bottino, A. M. Orengo, L. Morelli, R. Augugliaro, M. Barbaresi, E. Ciccone, L. Moretta. 1993. P58 molecules as putative receptors for major histocompatibility complex (MHC) class I molecules in human natural killer (NK) cells. Anti-p58 antibodies reconstitute lysis of MHC class I-protected cells in NK clones displaying different specificities. J. Exp. Med. 178: 597
    OpenUrlAbstract/FREE Full Text
  34. ↵
    Eisenbach, L.. 1996. Curing metastases? Gene and peptide therapy. Curr. Top. Microbiol. Immunol. 213: 85
    OpenUrlPubMed
  35. ↵
    Bjorkman, P., M. Saper, B. Samraoui, W. Bennett, J. L. Strominger, D. Wiley. 1987. Structure of the human class I histocompatibility antigen HLA-A2. Nature 329: 506
    OpenUrlCrossRefPubMed
Back to top

In this issue

Print
Download PDF
Article Alerts
Email Article
Citation Tools

Jump to section