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Keywords:

  • Cytotoxicity;
  • Human NK cells;
  • Killer immunoglobulin-like receptors

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and discussion
  5. Concluding remarks
  6. Materials and methods
  7. Acknowledgements
  8. Appendix

In this study, after immunization with NK cells from a KIR2DS5+ donor and screening on cell transfectants expressing different members of the killer immunoglobulin-like receptor (KIR) family, we generated a mAb, DF200, reacting with several KIR2D receptors including KIR2DL1/L2/L3, KIR2DS1/S2 and KIR2DS5. By the analysis of peripheral blood NK cells and in vitro derived NK cell clones, we have demonstrated for the first time that KIR2DS5 is expressed at the cell surface in discrete subsets of NK cells and, after DF200 mAb-mediated engagement, can induce both cytotoxicity and cytokine release. Using co-transfection and co-immunoprecipitation, we found that KIR2DS5 associates with the DAP12 signaling polypeptide. Finally, soluble KIR2DS5-Fc fusion protein does not bind to cell transfectants expressing different HLA-C alleles, suggesting that, if KIR2DS5 does recognize HLA-C molecules, this may only occur in the presence of certain peptides.

Abbreviation:
KIR:

Killer immunoglobulin-like receptors

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and discussion
  5. Concluding remarks
  6. Materials and methods
  7. Acknowledgements
  8. Appendix

NK cell function is regulated by an array of surface receptors transducing either inhibitory or activating signals 1. While the activating receptors include an heterogeneous group of surface molecules with disparate and still partially unknown specificities, the inhibitory receptors include a family of strictly homologous surface molecules referred as killer Ig-like receptors (KIR) recognizing unique patterns of HLA class I alleles 25 as well as the NKG2A/CD94 heterodimer specific for the non classical HLA-E alleles 6, 7. The susceptibility to the NK cell-mediated lysis is commonly determined by the type and the number of ligands (for either inhibitory or activating NK receptors) expressed by a given cell target 1. In cells undergoing viral infection or tumor transformation alterations of HLA class I molecules that include either the whole HLA class I phenotype, or selected alleles are frequently observed 1, 8, 9. Detection of even a single HLA class I allele down-modulation appears to be crucial to the strategy whereby NK cells recognize and eliminate pathological cells 1, 5, 9. That is why, in different species, evolution has converged towards the generation of a large family of homologous receptors (KIR in humans and Ly49 in mice) with peculiar MHC specificities 2. Their clonal distribution would allow the generation of a repertoire of NK cells capable of surveying the perturbation of almost all MHC alleles independently.

An additional role in checking alteration of HLA class I phenotype in pathological cells may be sustained by activating KIR 2, 10, 11. Convincing evidence has indicated that at least one of these activating receptors, KIR2DS1 recognizes HLA-C alleles characterized by Lys at position 80 12, 13. In the presence of HLA/inhibitory KIR interaction the engagement of activating KIR may be ineffective. However, when target cells selectively down-regulate HLA alleles recognized by inhibitory KIR, the engagement of activating KIR would result in NK cell activation. The simultaneous expression of activating and inhibitory KIR may therefore represent a strategy whereby NK cells can recognize and kill pathological cells uniquely by detecting changes in their HLA alleles repertoire.

Currently, due to the lack of specific reagents, not all the receptors encoded by the known KIR genes have been demonstrated to be expressed and functional in NK cells.

Here we describe a newly generated mAb (DF200) specific for an epitope shared by most KIR2D including KIR2DL1, KIR2DL2, KIR2DL3, KIR2DS1, KIR2DS2 and KIR2DS5. Using DF200 mAb, we identified and characterized, for the first time, the receptor encoded by the previously described KIR2DS5 gene.

Results and discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and discussion
  5. Concluding remarks
  6. Materials and methods
  7. Acknowledgements
  8. Appendix

Isolation of a mAb recognizing KIR2D molecules including KIR2DS5

Mice were immunized with a polyclonal NK cell line derived from a healthy individual bearing the KIR2DS5 gene (donor D, Fig. 1A). To isolate KIR2DS5-specific mAb, HEK-293T cells transiently transfected with plasmid coding for the above KIR, were used in immunofluorescence staining assays and cytofluorimetric analysis. By this approach a mAb (termed DF200, IgG1), which reacted with KIR2DS5+ cell transfectant, was identified (Fig. 1B).

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Figure 1. DF200 mAb recognizes KIR2DS5, a KIR receptor that associates with DAP12. (A) KIR genotype of the analyzed donors was analyzed by SSP-PCR. Gray and white indicate the presence or the absence of the indicated genes respectively. (B) KIR2DS5 HEK-293T cell transfectants were stained with DF200 mAb and analyzed by flow cytometry. Dotted profile represents cells stained with second reagent only. (C) HEK-293T cells expressing the indicated KIR were stained with DF200 mAb (left) or KIR specific mAb (right). Dotted profiles indicate staining with second reagent only. (D) HEK-293T cells co-transfected with KIR2DS5 and Flag-DAP12 were stained with anti-Flag FITC (M2) and/or DF200 mAb followed by goat anti-mouse PE-conjugated isotype-specific second reagent and analyzed by one- or two-color cytofluorimetric analysis. Dotted profiles indicate staining with second reagent only. (E) HEK-293T cells either untransfected or co-transfected with KIR2DS5 and Flag-DAP12 (KIR2DS5/DAP12) were immunoprecipitated as indicated, analyzed in 8% (under nonreducing conditions, left panel) or 14% (under reducing conditions, right panel) SDS-PAGE and probed with DF200 or M2 (anti-Flag) mAb. Ig light chains (IgL) are indicated. Molecular weight markers (given in kDa) are indicated on the left.

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To gain further information, the reactivity of the mAb was assessed on a panel of HEK-293T cells transfected with different KIR family members known to be expressed on NK cells. DF200 mAb recognized various KIR2D molecules with the exception of KIR2DS4, KIR2DL4 (Fig. 1C) and members of the KIR3D family (not shown). Notably, none of the known KIR-specific mAb analyzed (including GL183, EB6b, ECM41, FES172, Z27, Q66, XA141, and Y249) stained KIR2DS5 transfected cells (not shown). Therefore, DF200 mAb, besides recognizing different KIR2D molecules, displays the unique capability to react with the KIR2DS5 receptor. Importantly, these data provide evidence, for the first time, that the KIR2DS5-encoded protein can be expressed at the cell surface.

KIR2DS5 associates with DAP12

The predicted KIR2DS5 receptor is characterized by two Ig domains of the D1-D2 type, a short cytoplasmic tail and a positive charged transmembrane (TM) portion 14. We therefore analyzed whether it associates with DAP12, a small polypeptide characterized by a negatively charged TM portion and a cytoplasmic tail containing an ITAM, which associates with other activating KIR 15. We co-transfected HEK-293T cells with KIR2DS5 and a Flag epitope tagged DAP12 molecule. Both KIR2DS5 (see Fig. 1B) and KIR2DS5/DAP12 (Fig. 1D) cell transfectants were brightly stained by DF200 mAb. This suggests that this receptor can be efficiently expressed at the cell surface in the absence of DAP12. On the other hand, double fluorescence analysis indicated that, within transfected cells, those displaying high surface expression of KIR2DS5 receptor also expressed high levels of DAP12 molecule (Fig. 1D, right panel). Moreover, western blot analysis showed co-immunoprecipitation of the receptor and DAP12 in double cell transfectants (Fig. 1E).

KIR2DS5 is expressed on a discrete subset of peripheral blood NK cells

We assessed the reactivity of DF200 mAb on purified NK cell populations derived from two healthy individuals: donor K typed as KIR2DS5+ and donor M as KIR2DS5 (their complete KIR genotype is shown in Fig. 1A). The staining profile of DF200 mAb was compared in double fluorescence analysis to that of a pool of mAb specific for either KIR2DL1/S1 or KIR2DL2/L3/S2. Analysis of KIR2DS5+ donor NK cells (Fig. 2A, left panel) revealed that DF200 mAb, besides staining KIR2DL1/S1+ and KIR2DL2/L3/S2+ NK cells (i.e., double-positive cells, upper right quadrant), also allowed the identification of a distinct KIR2DS5+ cell subset (i.e., single DF200+ cells, lower right quadrant). On the other hand, no single DF200+ cells could be detected in the KIR2DS5 individual (Fig. 2A, right panel). These results were confirmed by the comparative analysis of additional KIR2DS5+and KIR2DS5 donors. These data indicate that the combined use of DF200 together with different anti-KIR mAb may facilitate distinguishing haplotypes B donors carrying the KIR2DS5 gene by cytofluorimetric analysis 11, 16, 17.

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Figure 2. Analysis of the surface expression and function of the KIR2DS5 molecule on polyclonal and clonal NK cell populations. (A) Peripheral blood NK cells from the representative donors K (KIR2DS5+) and M (KIR2DS5) were stained with DF200 mAb followed by PE-conjugated isotype-specific second reagent and with a pool of mAb (specific for KIR2DL1/S1 or KIR2DL2/L3/S2) followed by FITC-conjugated isotype-specific second reagents. (B) (right) The cytolytic activity of three representative NK cell clones (K1, K2 and K3) was evaluated in a redirected killing assay either in the absence or in the presence of DF200 mAb or mAb-specific for KIR2DL1/S1 or KIR2DL2/L3/S2 molecules; (left) the KIR surface phenotype of the three clones was assessed by immunofluorescence and cytofluorimetric analysis. White profiles indicate cells incubated with the second reagent alone. (C) RT-PCR analysis of KIR2DS1/S2/S3/S5 transcripts in the K1 clone. A polyclonal NK cell population whose genotype included all the KIR2DS genes analyzed was used as positive control (CTR+). Size markers (MW, given in bp) are indicated on the left. (D) Supernatant from the representative KIR2DL-KIR2DS5+ (K1) and KIR2DL+KIR2DS5 (M4) NK cell clones stimulated with the indicated mAb were analyzed for IFN-γ content by specific ELISA. Anti-CD56 mAb was used as negative control.

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DF200 mAb triggers cytotoxicity and IFN-γ production in KIR2DS5+ NK cells

To assess the actual function of KIR2DS5, we derived NK cell clones from the above KIR2DS5+ donors, analyzed their KIR surface phenotype and then evaluated the KIR function in a redirected killing assay against the P815 FcγR+ target cell line. All of the single DF200+ (i.e., KIR2DL1/S1 KIR2DL2/L3/S2 KIR2DS5+) NK clones were induced by DF200 mAb to kill P815 target cells (see the representative clone K1 in Fig. 2B), thus indicating that KIR2DS5 engagement triggers NK cell function. As expected, anti-KIR2DL1/S1 EB6b mAb or anti-KIR2DL2/3/S2 GL183 mAb were ineffective on such clones. On the other hand, in clones KIR2DL1/S1+ or KIR2DL2/3/S2+, the effect of DF200 mAb paralleled that of EB6b or GL183 mAb, either when they were reacting with an activating (Fig. 2B clones K2 and K3) or an inhibitory (not shown) KIR. To confirm the presence of KIR2DS5 transcript the single KIR2DS5+ clone K1 was also analyzed at the mRNA level. As shown in Fig. 2C, among different KIR2DS this clone expressed KIR2DS5 transcript only.

In addition to cytotoxicity, cytokine production is another critical effector function of NK cells. Therefore, using plate-bound mAb, we assessed the ability of KIR2DS5 to elicit IFN-γ production in NK cells. DF200 mAb selectively induced IFN-γ production by single KIR2DS5+ clones (including the representative clone K1, Fig. 2D), while it was unable to stimulate the DF200+ NK clones expressing KIR2DL1/2/3 but not KIR2DS5 (such as clone M4). All the clones analyzed, however, when stimulated with antibodies against the NKp46 triggering NK receptor, produced large amounts of IFN-γ; thus indicating that both KIR2DS5+ and KIR2DS5 NK clones displayed similar capability to produce cytokines in response to triggering stimuli. Thus, KIR2DS5 functions as a receptor that in NK cells triggers both cytotoxicity and IFN-γ release.

Assessment of the reactivity of KIR2DS5 with HLA class I molecules

We generated a soluble KIR2DS5-Fc fusion protein and assessed its reactivity on the 721.221 B cell line transfected with different HLA-C alleles. KIR2DL1-Fc and KIR2DS1-Fc fusion proteins were used as positive controls. All the chimeric receptors were recognized by their respective anti-KIR-specific mAb (DF200 and EB6b) in ELISA assays (not shown), suggesting that chimeric proteins were folded properly. As shown in Fig. 3, KIR2DS5-Fc, similar to KIR2DS2-Fc (not shown), did not stain any of the HLA-C transfectants analyzed. On the other hand, in line with previous reports, the KIR2DL1-Fc receptor strongly reacted with HLA-Cw4 and HLA-Cw6 cell transfectants, while KIR2DS1-Fc proteins reacted strongly with cells transfected with the HLA-Cw6 allele and weakly with those transfected with HLA-Cw4 12, 13. The lack of KIR2DS5-Fc staining was not due to different HLA class I surface densities, as the various transfectants did not differ significantly in HLA expression (see first column in Fig. 3). Based on these data we conclude that, under our experimental conditions, KIR2DS5, similar to other KIR2DS receptors including KIR2DS2 and KIR2DS4, does not seem to recognize the HLA-C alleles evaluated in these experiments.

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Figure 3. Reactivity of KIR2DS5 soluble receptor on HLA-C 721.221 transfectants. HLA-Cw3, -Cw4, -Cw6 or -Cw7 721.221 transfected cells were stained with an anti-HLA-class I mAb (aHLA class I) and with equal concentrations of KIR2DL1-, KIR2DS1- and KIR2DS5-Fc soluble receptors, followed by PE-conjugated isotype specific goat anti-mouse or goat anti-human second reagents. Cells were analyzed by flow cytometry. White profiles indicate staining with second reagent only.

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Concluding remarks

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and discussion
  5. Concluding remarks
  6. Materials and methods
  7. Acknowledgements
  8. Appendix

In this study we have characterized the expression and function of the KIR2DS5 molecule, a member of the KIR family that, so far, has not yet been described at the protein level. Although, similar to other activating KIR, its HLA class I specificity could not be assigned, the demonstration that the KIR2DS5 is a surface receptor clonally distributed within the NK cell population that activates NK cell function represents an important contribution to the current studies aimed at evaluating the role of activating KIR in hematopoietic transplants as well as in the control of tumors or infectious diseases.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and discussion
  5. Concluding remarks
  6. Materials and methods
  7. Acknowledgements
  8. Appendix

mAb

The following mAb were produced in our lab: BAB281 (anti-NKp46), c218 (anti-CD56), A6–17 (anti-HLA class I), EB6b and XA141 (anti-KIR2DL1/S1), GL183 and Y249 (anti-KIR2DL2/L3/S2), FES172 (anti-KIR2DS4), Z27 (anti-KIR3DL1/S1), and Q66 (anti-KIR3DL2).

Polyclonal or clonal NK cell populations

PBL were derived from healthy donors by Ficoll-Hypaque gradients and depletion of plastic-adherent cells. Purified NK cells were obtained by negative selection using the NK cell isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany). Purified NK cells were cultured on irradiated feeder cells in the presence of 100U/mL rIL-2 (Proleukin, Chiron Corp., Emeryville, USA) and 1.5 ng/mL PHA (Gibco Ltd, Paisley, Scotland) to obtain polyclonal NK cell populations or, after limiting dilution, NK cell clones.

Flow cytofluorimetric analysis

Cells stained with the appropriate mAb followed by PE- or FITC-conjugated isotype-specific goat anti-mouse second reagent or with Fc soluble receptors followed by PE-conjugated goat anti-human antibodies (Southern Biotechnology Associated, Birmingham, AL), were analyzed by one- or two-color cytofluorimetric analysis (FACSCalibur, Becton Dickinson, Mountain View, CA) as previously described 13.

KIR genes profile and RT-PCR analysis

The KIR genes profile was performed using Olerup SSP KIR genotyping kit (GenoVision, Saltsjoebaden, Sweden). The RNA extracted from K1 NK cell clone was analyzed for the presence/absence of KIR2DS1/S2/S3 and S5 transcripts using RT-PCR protocols. The specific sets of primers have been previously described 16.

Cell transfectants

HEK-293T cells were transfected with KIR2DL1-, KIR2DL2-, KIR2DL3-, Flag-KIR2DL4-, KIR2DS1-, KIR2DS2-, KIR2DS4-, and KIR2DS5-pcDNA 3.1 plasmids using non-liposomal FuGene-6 reagent (Roche, Monza, Italy). KIR2DS5 coding plasmid was kindly provided by C. Vilches (Hospital Universitario, Puerta de Hierro, Madrid, Spain). The 721.221 cell line transfected with HLA-Cw3 or Cw4 18 and with HLA-Cw6 or -Cw7 (kindly provided by C. Falk, National Center for Tumor Diseases and Institute for Immunology, University Heidelberg, Germany) were used in this study.

Biochemical analysis of the KIR2DS5/DAP12 receptor complex

HEK-293T cell transfectants (50 × 106 cells) were lysed in 1% digitonin and immunoprecipitated with Sepharose CNBr (GE-Healthcare, Easton Tpke Fairfield, CT)-coupled DF200 mAb or Sepharose-PA-CL4B (GE-Healthcare)-coupled M2 (anti-Flag, Sigma-Aldrich, St. Louis, MO) mAb. Samples were analyzed in discontinuous SDS-PAGE, transferred to Immobilon P (Millipore Corp, Bedford, MA) and probed with the Biotin- (Pierce, Rockford, IL) labeled DF200 mAb or the anti-Flag mAb followed by anti-mouse second reagent (DAKO A/S, Denmark). The Western Lightning Chemiluminescence Kit (Perkin Elmer, Boston, MA) was used for detection.

Cytolytic assays and cytokine analysis

For the redirected killing experiments, NK cells were tested for cytolytic activity in a 4-h 51Cr-release assay against the FcγR+ P815 target cell line (E/T ratio: 1/1). mAb were added in the test at 0.5 μg/mL. For the evaluation of the cytokine production 5 × 104 NK cells were cultured overnight in a plastic well (96-well microtiter plate) coated or not with 10 μg/mL of the indicated mAb. The culture supernatants were then analyzed for the presence of IFN-γ using ELISA kits from BioSource International (Camarillo, CA).

Production of KIR soluble receptors

Constructs encoding KIR2DL1-, KIR2DS1- and KIR2DS5-Fc soluble receptors were obtained keeping in frame the cDNA coding for the first 245 aa of the indicated KIR with the cDNA coding for Fc portion of human IgG1 using pRB1vector. These KIR-Fc/pRB1 plasmids were transiently transfected into HEK-293T and transfected cells were cultured using DMEM supplemented with 10% ultra-low IgG FBS (Gibco BRL). Soluble chimeric proteins were purified from the supernatant by protein A-Sepharose affinity column (Amersham, Biosciences) and protein concentrations were determined using Bio-Rad protein assay. SDS-PAGE and silver staining established the purity of the soluble receptors.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and discussion
  5. Concluding remarks
  6. Materials and methods
  7. Acknowledgements
  8. Appendix

This work was supported by grants awarded by Associazione Italiana per la Ricerca sul Cancro (A.I.R.C.), Istituto Superiore di Sanità (I.S.S.), Ministero della Salute (Ricerca Finalizzata Ministeriale 2005 “Caratterizzazione delle proprietà di immunomodulazione delle cellule staminali mesenchimali e possible applicazione nel trattamento delle malattie autoimmuni”), Ministero dell'Università e della Ricerca Scientifica e Tecnologica (M.I.U.R. - PRIN 2005, project 2005063024_004), FIRB-MIUR project-RBNE017B4, European Union FP6, LSHB-CT-2004–503319-AlloStem (the European Commission is not liable for any use that may be made of the information contained), Fondazione Compagnia di San Paolo, Torino, Italy, Fondazione CARIGE and Fondazione CARIPLO.

  • 1

    WILEY-VCH

  • 2

    WILEY-VCH

  • 3

    WILEY-VCH

  • 1
    Moretta, L., Bottino, C., Pende, D., Vitale, M., Mingari, M. C. and Moretta, A., Different checkpoints in human NK-cell activation. Trends Immunol. 2004 25: 670676.
  • 2
    Vilches, C. and Parham, P., KIR: Diverse, rapidly evolving receptors of innate and adaptive immunity. Annu. Rev. Immunol. 2002 20: 217251.
  • 3
    Colonna, M. and Samaridis, J., Cloning of immunoglobulin-superfamily members associated with HLA-C and HLA-B recognition by human natural killer cells. Science 1995 268: 405408.
  • 4
    Wagtmann, N., Biassoni, R., Cantoni, C., Verdiani, S., Malnati, M. S., Vitale, M., Bottino, C. et al., Molecular clones of the p58 NK cell receptor reveale immunoglobulin-related molecules with diversity in both the extra- and intracellular domains. Immunity 1995 2: 439449.
  • 5
    Long, E. O., Regulation of immune responses through inhibitory receptors. Annu. Rev. Immunol. 1999 17: 875904.
  • 6
    Lee, N., Llano, M., Carretero, M., Ishitani, A., Navarro, F., Lòpez-Botet, M. and Geraghty, D. E., HLA-E is a major ligand for natural killer inhibitory receptor CD94/NKG2A. Proc. Natl. Acad. Sci. USA 1998 95: 51995204.
  • 7
    Braud, V. M., Allan, D. S., O'Callaghan, C. A., Soedersrroem, K., D'Andrea, A., Ogg, G. S., Lazetic, S. et al., HLA-E binds to natural killer cell receptor CD94/NKG2A, B and C. Nature 1998 391: 795799.
  • 8
    Gumá, M., Budt, M., Sáez, A., Brckalo, T., Hengel, H., Angulo, A. and López-Botet, M., Expansion of CD94/NKG2C+ NK cells in response to human cytomegalovirus-infected fibroblasts. Blood 2006 107: 36243631.
  • 9
    Garrido, F., Ruiz-Cabello, F., Cabrera, T., Pérez-Villar, J. J., López-Botet, M., Duggan-Keen, M. and Stern, P. L., Implications for immunosurveillance of altered HLA class I phenotypes in human tumours. Immunol. Today 1997 18: 8995.
  • 10
    Bottino, C., Sivori, S., Vitale, M., Cantoni, C., Falco, M., Pende, D., Morelli, L. et al., A novel surface molecule homologous to the p58/p50 family of receptors is selectively expressed on a subset of human natural killer cells and induces both triggering of cell functions and proliferation. Eur. J. Immunol. 1996 26: 18161824.
  • 11
    Parham, P., MHC class I molecules and KIRs in human history, health and survival. Nat. Rev. Immunol. 2005 5: 201214.
  • 12
    Stewart, C. A., Laugier-Anfossi, F., Vély, F., Saulquin, X., Riedmuller, J., Tisserant, A., Gauthier, L. et al., Recognition of peptide–MHC class I complexes by activating killer immunoglobulin-like receptors. Proc. Natl. Acad. Sci. USA 2005 102: 1322413229.
  • 13
    Biassoni, R., Pessino, A., Malaspina, A., Cantoni, C., Bottino, C., Sivori, S., Moretta, L. and Moretta, A., Role of amino acid position 70 in the binding affinity of p50.1 and p58.1 receptors for HLA-Cw4 molecules. Eur. J. Immunol. 1997 27: 30953099.
  • 14
    Doehring, C., Samaridis, J. and Colonna, M., Alternatively spliced forms of human killer inhibitory receptors. Immunogenetics 1996 44: 227230.
  • 15
    Lanier, L. L., Corliss, B. C., Wu, J., Leong, C. and Phillips, J. H., Immunoreceptor DAP12 bearing a tyrosine-based activation motif is involved in activating NK cells. Nature 1998 391: 703707.
  • 16
    Uhrberg, M., Parham, P. and Wernet, P., Definition of gene content for nine common group B haplotypes of the Caucasoid population: KIR haplotypes contain between seven and eleven KIR genes. Immunogenetics 2002 54: 221229.
  • 17
    Hsu, K. C., Xiao-Rong, L., Selvakumar, A., Mickelson, E., O'Reilly, R. J. and Dupont, B., Killer Ig-like receptor haplotype analysis by gene content: Evidence for genomic diversity with a minimum of six basic framework haplotypes, each with multiple subsets. J. Immunol. 2002 169: 51185129.
  • 18
    Biassoni, R., Falco, M., Cambiaggi, A., Costa, P., Verdiani, S., Pende, D., Conte, R. et al., Amino acid substitutions can influence the natural killer (NK)-mediated recognition of HLA-C molecules. Role of serine-77 and lysine-80 in the target cell protection from lysis mediated by “group 2” or “group 1” NK clones. J. Exp. Med. 1995 182: 605609.