Discrimination between the main activating and inhibitory killer cell immunoglobulin-like receptor positive natural killer cell subsets using newly characterized monoclonal antibodies

Abstract

Natural killer (NK) cells are key components of the innate anti-viral and anti-tumour immune responses. NK cell function is regulated by the interaction of killer cell immunoglobulin-like receptors (KIR) with human leucocyte antigen (HLA) class I molecules. In this study, we report on the generation of KIR-specific antibodies allowing for discrimination between activating and inhibitory KIR. For this purpose, BALB/c mice were immunized with human KIR2DS2 recombinant protein. The precise specificity of KIR2DS2-specific clones was determined on KIR-transfected BW cells and KIR-genotyped NK cells. When used in combination with EB6 (KIR2DL1/2DS1) or GL183 (KIR2DL2/2DL3/2DS2), two KIR-specific monoclonal antibodies (mAbs), 8C11 (specific for KIR2DL1/2DL2/2DL3/2DS2) and 1F12 (specific for KIR2DL3/2DS2), discriminated activating KIR2DS1 (8C11⁻ EB6⁺) from inhibitory KIR2DL1 (8C11⁺ GL183⁻) and KIR2DL2 (1F12⁻ GL183⁺), while excluding the main HLA-Cw-specific KIR. Using these mAbs, KIR2DS1 was shown to be expressed on the surface of NK cells from all individuals genotyped as KIR2DS1⁺ (n = 23). Moreover, KIR2DS1 and KIR2DL1 were independently expressed on NK cells. We also determined the amino acid position recognized by the 8C11 and 1F12 mAbs, which revealed that some KIR2DL1 allele-encoded proteins are not recognized by 8C11. Because most available anti-KIR mAbs recognize both inhibitory and activating forms of KIR, these newly characterized antibodies should help assess the expression of activating and inhibitory KIR and their functional relevance to NK biology.

Introduction

Natural killer (NK) cells constitute a first line of cellular defence against virus-infected or transformed cells, while maintaining tolerance to healthy cells. The function of NK cells is specifically regulated by a balance between numerous inhibitory and activating cellular receptors. Killer immunoglobulin-like receptors (KIR) are the main family of human leucocyte antigen (HLA) class I specific receptors present on NK cells. The interaction between inhibitory KIR on NK cells and their HLA ligands leads to the generation of a negative signal, which inhibits their cytolytic activity and so prevents target cell lysis. Down-regulation of surface HLA class I molecules resulting from cell transformation or viral infection prevents inhibitory KIR engagement by their specific HLA ligands, which then enables NK cell activation. The NK cells can be further triggered upon engagement of activating KIR by tumour or viral-induced ligands. Even though KIR are mainly expressed on NK cells, they are also expressed on a small proportion of memory T lymphocytes where they are thought to regulate cell function.^1–4

As the HLA complex is highly polymorphic, the KIR gene family presents both haplotypic polymorphism (up to 16 genes and pseudogenes) and allelic polymorphism (up to 63 alleles for KIR3DL1) (IPD-KIR sequence database, release 2.0.1; http://www.ebi.ac.uk/ipd/kir). The KIR are characterized by the presence of two or three extracytoplasmic immunoglobulin-like domains (KIR2D or KIR3D). Inhibitory KIR transduce the inhibitory signal through the phosphorylation of immunoreceptor tyrosine-based inhibition motifs (ITIM) inserted in their long cytoplasmic tail (KIR2DL or KIR3DL). KIR2DL1 and KIR2DL2/2DL3 recognize HLA-Cw molecules carrying respectively a lysine (called C2 group) or asparagine (called C1 group) at amino acid position 80.⁵ KIR2DL2 and KIR2DL3 segregate as alleles of one locus that is referred to as KIR2DL2/3.⁶ KIR3DL1 recognizes the Bw4 serological epitope (IALT, TALR or TLLR in amino acid positions 77–80) present in one-third of HLA-B molecules⁷ and a small number of HLA-A molecules (A23, A24 and A32).⁸ KIR3DL2 on the other hand binds HLA-A3 and HLA-A11 allotypes⁷ and HLA-B27 homodimers.^9,10

Besides the inhibitory KIR, NK cells can also express activating KIR which are characterized by a short cytoplasmic tail (KIR2DS or KIR3DS) with a lysine residue that is necessary for pairing with KARAP/DAP12 polypeptide. The cytoplasmic tail contains immunoreceptor tyrosine activation motifs (ITAM) that are involved in the transduction of triggering signals. While inhibitory KIR allelic polymorphism is extensive, the number of activating KIR alleles is limited. For example, only seven KIR2DS3, 10 KIR2DS2 and 12 KIR2DS1 alleles have been identified (IPD-KIR sequence database, release 2.0.1; http://www.ebi.ac.uk/ipd/kir), with only one predominant allele.¹¹ Some of these triggering KIR molecules display a high degree of homology with the corresponding inhibitory KIR and accordingly, have been shown to recognize the same HLA class I ligands in some instances.^12,13 However, the expression modalities and specificity of activating KIR are still unclear.

Genetic KIR polymorphism and clonal expression of KIR lead to considerable heterogeneity within the NK cell population. Nevertheless, not all theoretically distinct NK cell subsets are represented and the presence of a KIR gene is not systematically associated with the cell surface expression of the corresponding protein. Moreover, given the high degree of amino acid sequence homology between different KIR, most KIR-specific monoclonal antibodies (mAbs) recognize epitopes shared not only by two or more inhibitory KIR but also by their activating counterpart. As a result of the limited polymorphism in the extracellular part of membrane KIR2DS2 and KIR2DL2/2DL3 molecules, no antibody was generated specifically against the extracellular part of KIR2DS2. In the same way, available mAbs do not discriminate between inhibitory KIR2DL1 and activating KIR2DS1, both of which are recognized by the EB6 mAb. Consequently, the expression and function of activating KIR have not been investigated in detail at the protein level.

Here, we describe the specificity of two new KIR-specific mAbs (8C11 and 1F12) generated by immunizing mice with soluble KIR2DS2 protein. The combination of these mAbs with commercially available mAbs enabled us to discriminate KIR2DS1, KIR2DL1 and KIR2DL2, while excluding the main HLA-Cw-specific KIR. These reagents allowed us to assess the expression of KIR2DS1, KIR2DL1 and KIR2DL2 in a large cohort of KIR and HLA genotyped individuals (n = 52). These new KIR-specific mAbs constitute useful tools to study the phenotype of KIR-expressing NK cells and to better understand the functional implication of these individual KIR.

Materials and methods

Cells

Peripheral blood mononuclear cells (PBMC) were isolated from the blood of healthy adult volunteers by gradient centrifugation on Ficoll–Hypaque (Lymphoprep, Axis-Shield, PoC AS, Oslo, Norway). All blood donors were recruited at the Blood Transfusion Centre (Nantes, France) after obtaining informed consent from all donors.

The BW5147 mouse thymoma (BW) cell line was transduced to express one KIR (KIR2DS1, -2DS2, -2DS3, -2DS4, -2DL1, -2DL3 or -3DS1) and the green fluorescent protein (GFP) reporter gene (provided by E. Vivier, Centre d’Immunologie de Marseille-Luminy, France). The RBL-DAP12⁺ 2DS2⁺ and untransfected rat basophil leukaemia (RBL) cells were obtained from E. Vivier.

Immunization of mice

BALB/c mice were immunized by intra-peritoneal injection of 50 μg of soluble KIR2DS2 protein as described previously.¹⁴ A first immunization was performed with complete Freund’s adjuvant and four additional immunizations were performed with incomplete Freund’s adjuvant. Blood samples were collected from mice before the first injection and 3–5 days after subsequent injection. The immune response was monitored by enzyme-linked immunosorbent assay (ELISA) and antibody titres were determined as the inverse of the dilution that gave optical density (OD) values just above 0·2 (corresponding to three times the background signal).

Screening for KIR2DS2-reactive wells and cloning

The stimulated spleen cells were harvested and mixed at a 5 : 1 ratio with the Sp2/O-AG14 murine myeloma cell line. Fusion was performed by the polyethylene glycol method, as previously described.¹⁵ Hybridomas that secreted antibodies which significantly bound the coated soluble KIR2DS2 protein (OD > 0·5 by ELISA) were amplified to produce 1 ml of culture supernatants in 24-well culture plates. Hybridomas of interest were subcloned threefold by limiting dilution.

Production, purification, immunoglobulin subclass determination and labelling of mAbs

The antibodies were purified from culture supernatants by affinity chromatography on immobilized protein A. Isotypes were determined by ELISA using mouse mAb isotyping reagent (ISO2, Sigma-Aldrich, Steinheim, Germany), according to the manufacturer’s recommendations. Purified mAbs (∼1 mg/ml in phosphate-buffered saline) were labelled with fluorescein isothiocyanate (FITC) for 2 hr at room temperature using FITC (Sigma, dissolved to 5 mg/ml with dimethyl sulphoxide just before use) with a FITC : mAb concentration ratio of 100 : 1. Excess dye was removed by dialysis against phosphate-buffered saline through a 10 000 molecular weight cut-off membrane (Pierce, Rockford, IL).

KIR genotyping

Genomic DNA was extracted from PBMC using a classical salting-out method.¹⁶ The KIR genes were typed using the polymerase chain reaction-sequence-specific primer (PCR-SSP) method using a KIR genotyping SSP kit (Dynal Biotech, Invitrogen, Compiègne, France). Primer sets amplified the alleles described by the international nomenclature committee of the World Health Organization¹⁷ corresponding to the KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL4, KIR2DL5, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4 and KIR1D allele, KIR2DS5, KIR3DL1, KIR3DL2, KIR3DL3, KIR3DS1, KIR2DP1, KIR3DP1 and KIR3DP1 variant (3DP1v). Genomic PCR was performed as recommended by the manufacturer and as previously described.¹⁸

Flow cytometry analysis

Cell surface phenotypes were determined by three- or four-colour flow cytometry using the following mouse anti-human mAbs: phycoerythrin-conjugated (-PE) anti-KIR2DL1/2DS1 (EB6), anti-KIR2DL2/2DL3/2DS2-PE (GL183), anti-KIR3DL1/3DS1 (Z27) (Beckman Coulter, Immunotech, Marseille, France), anti-KIR2DS4 (FES172), peridinin chrlorophyll protein-conjugated anti-CD3 (SK7) and allophycocyanin-conjugated anti-CD56 (B159) (BD Biosciences). Cells were also stained with a corresponding isotype-matched control mAb (BD Biosciences, San Jose, CA). Data were collected using a FACSCalibur (BD Biosciences) and analysed using flowjo 5.7 software (TreeStar, Ashland, OR).

Amplification and sequencing of KIR2DL1 transcripts

Total cellular RNA was prepared from 5 × 10⁶ PBMC using TRIzol (Invitrogen, Paisley, UK). First-strand complementary DNA was synthesized from 1 μg RNA, using Moloney murine leukaemia virus reverse transcriptase (Invitrogen, Carlsbad, CA) at 37° for 50 min. One PCR primer pair was used, including KIR2DL1.331G(F) (5′-ACTCACTCCCCCTATCAGG-3′), described by Shilling et al.¹⁹ as the forward primer and KIR2DL1.724C(R) (5′-CAGAATGTGCAGGTGTCG-3′) as reverse primer, amplifying a 0·4-kilobase fragment. Amplifications were performed as previously described²⁰ in a GeneAmp PCR System 9700 thermal cycler (Applied Biosystems, Foster City, CA). The PCR products were purified using the QIAquick PCR purification kit (Qiagen, Hilden, Germany) and then sequenced using the BigDye Terminator v1.1 Cycle Sequencing kit (Applied Biosystems, Foster City, CA) with the primers described above. Sequencing products were detected using an ABI Prism 310 Genetic Analyzer (Applied Biosystems, Courtaboeuf, France). Sequence data files were analysed using assign-SBT software (Conexio Genomics, Applecross, Australia) with IPD-KIR database version 2.0.0 (January 2008).

Results

Generation of KIR2DS2-specific mAb-secreting hybridoma clones

To generate KIR2DS2-specific mAbs, BALB/c mice were immunized with soluble KIR2DS2 protein. Supernatants from 895 hybridoma colonies were screened by flow cytometry using RBL-DAP12⁺ KIR2DS2⁺ and untransfected RBL. Fifty-nine colonies, which produced antibody that bound to RBL-DAP12⁺ KIR2DS2⁺ transfectants but not untransfected RBL cells were subcloned two or three times, and seven KIR2DS2-specific hybridomas (1A6, 1F12, 4A8, 5F4, 6F1, 8C11 and 9H7) were selected for further characterization.

Characterization of the KIR specificity of the generated mAbs using BW-KIR cell lines

To define the other KIR specificities of the selected mAbs, we used a panel of BW-KIR transduced cells expressing KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DL1, KIR2DL3 or KIR3DS1. The KIR expression by the transduced BW was checked by flow cytometry using the KIR-specific EB6 (anti-KIR2DL1/2DS1), GL183 (anti-KIR2DL2/2DL3/2DS2) and FSE175 (anti-KIR2DS4) mAbs (Fig. 1a). Consequently, the BW-KIR2DL1 and BW-KIR2DS1 cell lines stained positive with the EB6 mAb, the BW-KIR2DL3 and BW-KIR2DS2 cell lines stained positive with the GL183 mAb and the BW-KIR2DS4 cell line stained positive with the FSE175 mAb. We failed to detect surface expression of KIR3DS1 on BW-KIR3DS1 transduced cells, using the available KIR3DL1/3DS1-specific Z27 mAb (data not shown). However, as for all of the BW-KIR cell lines, the BW-KIR3DS1 cells expressed GFP, the reporter gene associated with the KIR gene indicating that the BW cell line was well transduced (Fig. 1b). The negative control BW cell line did not stain positive with any of the studied mAbs. All seven selected KIR2DS2-specific mAbs also bound to BW-KIR2DL3 cell lines, as illustrated for the five mAbs assessed (1F12, 5F4, 1A6, 8C11 and 4A8) (Fig. 1b). The 1F12 and 4A8 mAbs did not recognize any of the BW-KIR cell lines studied, except the BW-KIR2DS2 and BW-KIR2DL3 cell lines (Fig. 1b). Interestingly, the 8C11 mAb recognized the BW-KIR2DS2, BW-KIR2DL3 and BW-KIR2DL1 but not the BW-KIR2DS1 cell lines. In contrast, the 5F4 and 1A6 mAbs recognized the BW-KIR2DS2, BW-KIR2DL3 and BW-KIR2DS1 but not the BW-KIR2DL1 cell lines. Finally 6F1 and 9H7 mAbs recognized all the BW-KIR cell lines, except for the BW-KIR3DS1 and BW-KIR2DS3 cell lines (data not shown). Five KIR-specific mAbs (1A6, 1F12, 4A8, 5F4 and 8C11) were selected for further studies.

Figure 1.

Definition of three groups of KIR2DS2-specific monoclonal antibodies (mAbs) following killer immunoglobulin-like receptor (KIR) recognition profile (a) Expression of KIR by BW-KIR2DL1, BW-KIR2DS1, BW-KIR2DL3, BW-KIR2DS2 and BW-KIR2DS4 was evaluated by flow cytometry using commercialized mAbs (EB6 specific for KIR2DL1/2DS1, GL183 specific for KIR2DL2/2DL3/2DS2 and FSE175 specific for KIR2DS4). An isotype-matched control staining is shown in grey in each histogram. (b) Staining of the KIR-transfected BW cell lines with generated mAbs (5F4, 1A6, 8C11, 4A8, 1F12) in parallel with immunoglobulin G control mAb.

Characterization of the KIR specificity of the generated mAbs in KIR genotyped individuals

The fine specificity of 1A6, 1F12, 4A8, 5F4 and 8C11 mAbs was studied by four-colour flow cytometry. We checked that the studied mAbs did not recognize other molecules besides the KIR2DL1/2DL2/2DL3, 2DS1/2DS2/2DS4. To this end, we assessed binding of FITC-labelled mAbs to NK cells from KIR genotyped individuals. The KIR genotypes of 13 individuals herein are summarized in Table 1. Figure 2(a) illustrates the staining of NK cells defined as CD3⁻ CD56⁺ cells with each tested mAb in combination with commercial EB6-PE (KIR2DL1/2DS1) and GL183-PE (KIR2DL2/2DL3, 2DS2) from the individual D1 who did not have the KIR3DL1 and KIR2DS4 genes. The 1F12, 4A8, 8C11 and 1A6 mAbs did not recognize any NK cell population that was not recognized by the EB6 and GL183 mAbs. However, the 5F4 mAb stained the EB6⁻ GL183⁻ NK cell population, showing that this mAb recognized another cell surface molecule (data not shown). Figure 2(b) illustrates the staining patterns obtained with individual D2 who possesses the KIR3DL1 and KIR2DS4 genes. In this case, the mAb staining was performed in combination with a mix of PE-coupled EB6, GL183, Z27 and FSE175 commercial mAbs. The flow cytometry results confirmed that 1F12, 4A8, 8C11 and 1A6 did not recognize any molecule other than the KIR2DL1/2DL2/2DL3/2DS2/2DS4/3DL1/3DS1.

Table 1.

The killer immunoglobulin-like receptor (KIR) genotypes of individuals presented in the study

	KIR typing
	2DL1	2DL2	2DL3	2DL4	2DL5	3DL1	3DL2	3DL3	2DS1	2DS2	2DS3	2DS4	2DS5	3DS1
D1	+	+	+	+	+	−	+	+	+	+	+	−	+	+
D2	+	+	+	+	−	+	+	+	−	+	−	+	−	−
D3	+	+	+	+	−	+	+	+	−	+	−	+	−	−
D4	+	−	+	+	−	+	+	+	−	−	−	+	−	−
D5	+	−	+	+	+	+	+	+	+	−	+	+	+	+
D6	−	+	−	+	+	+	+	+	+	+	−	+	+	+
D7	−	+	−	+	−	+	+	+	−	+	−	+	−	−
D8	+	+	+	+	+	−	+	+	+	+	+	−	+	+
D9	+	−	+	+	+	+	+	+	+	−	−	+	+	+
D10	+	+	+	+	−	+	+	+	−	+	−	+	−	−
D11	+	−	+	+	−	+	+	+	−	−	−	+	−	−
D12	+	+	−	+	+	+	+	+	−	+	+	+	−	+
D13	+	+	+	+	+	+	+	+	−	+	+	+	+	−

Hereditary haemochromatosis-positive blood donors are indicated in bold.

Figure 2.

Characterization of the killer immunoglobulin-like receptor (KIR) specificity of the generated monoclonal antibodies (mAbs) in KIR genotyped individuals. Peripheral blood mononuclear cells (PBMC) were stained with CD3- and CD56-specific mAbs to discriminate natural killer (NK) cells (CD3⁻ CD56⁺) by four-colour flow cytometry. (a) Combined use of the generated mAbs with a mix of EB6 and GL183 mAbs from KIR genotyped D1 individual and (b) combined use of the generated mAbs with a mix of EB6, GL183, Z27 and FSE175 mAbs from KIR genotyped D2 individual.

Assessment of KIR2DL2 recognition by the generated mAbs

The previous results (Fig. 1b) did not allow us to determine whether 1F12, 4A8, 8C11 and 1A6 mAbs recognized the inhibitory KIR2DL2. To test this, we assessed the specificity of these FITC-labelled mAbs in combination with only KIR2DL2/2DL3/2DS2-specific GL183-PE mAb by four-colour flow cytometry on NK (CD3⁻ CD56⁺) cells from KIR genotyped individuals who possessed or lacked the KIR2DL2 gene (Fig. 3a). Because all GL183⁺ cells were stained by the 8C11 and 1A6 mAbs in KIR2DL2⁺ individuals, these antibodies recognized the KIR2DL2. However, the 1F12 mAb recognized only a fraction of GL183⁺ NK cells from a KIR2DS2/2DL2/2DL3⁺ individual (D3), whereas it stained all GL183⁺ cells from a KIR2DL2/2DS2⁻ KIR2DL3⁺ individual (D4). Hence, the 1F12 mAb was specific for KIR2DS2 and KIR2DL3 but not for KIR2DL2. The 4A8 mAb showed a staining pattern different from that of 1F12 in combination with GL183, even though both mAbs recognized the KIR2DS2 and KIR2DL3 (Fig. 3a). The 4A8 mAb presumably recognized an epitope close to the GL183 epitope because it blocked GL183 binding (Fig. 3a). These results were extended to a larger panel of different KIR2DL2-positive (n = 38) or negative (n = 23) genotyped individuals (data not shown). Expression of the KIR2DL2/2DL3 ligands (i.e. group 1 HLA-Cw alleles: C1) did not affect the frequency of the KIR2DL2⁺ KIR2DL3/2DS2⁻ NK cell population (Fig. 3b), which was very similar in C1C1 (n = 9), C1C2 (n = 19) and C2C2 (n = 10) individuals (Fig. 3c). However, the frequency of KIR2DL2⁺ KIR2DL3/2DS2⁻ NK cells was significantly higher in KIR2DL3⁻ individuals (n = 12) in comparison with KIR2DL3⁺ individuals (n = 26) (P < 0·0004) (Fig. 3c).

Figure 3.

All the studied monoclonal antibodies (mAbs) recognize KIR2DL2 except the 1F12 mAb. Peripheral blood mononuclear cells were stained with CD3- and CD56-specific mAbs to discriminate natural killer (NK) cells (CD3⁻ CD56⁺) by four-colour flow cytometry. (a) Combined use of the generated mAbs with the KIR2DL2/2DL3/2DS2-specific GL183 mAb from the KIR2DL2/2DL3/2DS2⁺ genotyped D3 individual and from the KIR2DL2/2DS2⁻ KIR2DL3⁺ genotyped D4 individual. The boxes define the NK cell population only stained by GL183 determining the studied mAbs which do not recognize KIR2DL2. Results are representative of 26 KIR2DL2/2DL3/2DS2⁺ individuals and 12 KIR2DL2/2DS2⁻ KIR2DL3⁺ individuals (b) Density plot illustrating the KIR2DL2⁺ KIR2DL3/2DS2⁻ NK cell subset. (c) Frequency of KIR2DL2⁺ KIR2DL3/2DS2⁻ NK cell subset in C1C1 (n = 9), C1C2 (n = 19) and C2C2 (n = 10) groups. (d) Frequency of KIR2DL2⁺ KIR2DL3/2DS2⁻ NK cell subset in KIR2DL3⁺ group (n = 26) and KIR2DL3⁻ group (n = 12), P < 0·0004 (Student’s t-test).

Discrimination of KIR2DS1⁺ and KIR2DL1⁺ NK cell subsets

Because 8C11 did not recognize the BW-KIR2DS1 cell line, we tested this mAb in combination with EB6 (KIR2DS1, KIR2DL1) to target KIR2DS1 expression, and with GL183 (KIR2DL2, KIR2DL3 and KIR2DS2) to target KIR2DL1 expression, from four different KIR2DL1/2DS1-genotyped individuals (D4, D5, D6 and D7). As illustrated in Fig. 4(a), while all EB6⁺ NK cells from KIR2DS1⁻ individuals were stained by 8C11 mAb, only a fraction of EB6⁺ cells from KIR2DS1⁺ donors was stained by this mAb, indicating recognition of KIR2DL1 but not KIR2DS1. The frequency of KIR2DS1⁺ KIR2DL1/2DL2/2DL3/2DS2⁻ NK cells (EB6⁺ 8C11⁻) was then assessed in a large panel of KIR2DS1-genotyped individuals (n = 23). The frequency of (EB6⁺ 8C11⁻) NK cells ranged from 1% to 14·65% of NK cells, with a mean of 6·25% (data not shown). No significant difference in KIR2DS1⁺ KIR2DL1/2DL2/2DL3/2DS2⁻ NK cell frequencies was observed when comparing C1C1 (n = 6), C1C2 (n = 11) or C2C2 (n = 6) individuals, with even a trend towards an increased frequency in the C2C2 group (Fig. 4b). In parallel, we assessed KIR2DL1⁺ KIR2DL2/2DL3/2DS2⁻ (GL183⁻ 8C11⁺) NK cell frequencies in a large panel of KIR-genotyped individuals (23 KIR2DS1⁺ and 28 KIR2DS1⁻ individuals). The mean frequency of KIR2DL1⁺ KIR2DL2/2DL3/2DS2⁻ (GL183⁻ 8C11⁺) was not significantly lower in KIR2DS1⁺ individuals than in KIR2DS1⁻ individuals (Fig. 4c). However, the mean frequency of KIR2DL1⁺ KIR2DL2/2DL3/2DS2⁻ cells was significantly higher in the C2C2 group than in the C1C2 group (P = 0·038) (Fig. 4d). Because this cell frequency in C1C1 individuals was not significantly lower than in the C2C2 group, it is difficult to explain the significant difference of frequencies observed between C1C2 and C2C2 groups only by the expression of the C2 ligand. Interestingly, when taking into account the KIR2DS1 gene, a trend towards a lower frequency of KIR2DL1⁺ KIR2DL2/2DL3/2DS2⁻ cells was observed in the KIR2DS1⁺ C1C2 group compared with the KIR2DS1⁻ C1C2 group (P = 0·07) (Fig. 4e).

Figure 4.

Discrimination of KIR2DS1⁺ and KIR2DL1⁺ natural killer (NK) cell subsets. Peripheral blood mononuclear cells were stained with CD3- and CD56-specific monoclonal antibodies (mAbs) to discriminate NK cells (CD3⁻ CD56⁺) by four-colour flow cytometry. (a) Combined use of 8C11 with EB6 or GL183 mAb to target KIR2DS1⁺ KIR2DL1/2DL2/2DL3/2DS2⁻ NK cells and KIR2DL1⁺ KIR2DL2/2DL3/2DS1/2DS2⁻ NK cell subsets respectively from four KIR2DL1/2DS1 genotyped individuals (D4, D5, D6 and D7). The EB6⁺8C11⁻ boxes define the KIR2DS1⁺ KIR2DL2/DL3/2DS2⁻ NK cells and the GL183⁻ 8C11⁺ define the KIR2DL1⁺ KIR2DL2/2DL3^– NK cells (b) Frequency of KIR2DS1⁺ KIR2DL1/2DL2/2DL3/2DS2⁻ NK cell subsets in C1C1 (n = 6), C1C2 (n = 11) and C2C2 (n = 6) groups. (c) Frequency of KIR2DL1⁺ KIR2DL2/2DL3/2DS1/2DS2⁻ NK cell subsets in KIR2DS1⁺ individuals (•) (n = 23) and KIR2DS1⁻ individuals (○) (n = 28), (d) in C1C1 (n = 11), C1C2 (n = 20) and C2C2 (n = 19) groups. (e) Frequency of KIR2DL1⁺ KIR2DL2/2DL3/2DS1/2DS2⁻ NK cells in C1C1 (n = 11), C1C2 (n = 21) and C2C2 (n = 19) individual groups harbouring (•) or not (○) the KIR2DS1 gene.

The combined use of 1A6 (KIR2DL2/2DL3, 2DS1/2DS2) with EB6 (KIR2DS1, KIR2DL1) enabled specific assessment of KIR2DL1 expression, whereas its combined use with GL183 (KIR2DL2, KIR2DL3 and KIR2DS2) targeted KIR2DS1 expression (data not shown).

Expression of KIR2DL1, KIR2DL2 and KIR2DS1 on different lymphocyte populations

The NK KIR-phenotyping analysis was initially performed by flow cytometry on gated CD3⁻ CD56⁺ cells. We next analysed the KIR phenotype on CD56⁻ CD3⁺ and CD56⁺ CD3⁺ T lymphocytes. In this way, we identified a KIR2DL2⁺ KIR2DL3/2DS2⁻ subset (1F12⁻ GL183⁺) not only within the NK cell population but also within the CD56⁻ and CD56⁺ T lymphocyte compartment, as illustrated for the KIR2DL2⁺ D8 individual (Fig. 5a). As demonstrated previously in Fig. 3(d) for NK cells, the frequency of the KIR2DL2⁺ KIR2DL3/2DS2⁻ subset in CD56⁻ T lymphocytes was higher in KIR2DL3⁻ compared with KIR2DL3⁺ individuals (data not shown). A control staining is also indicated for the KIR2DL2⁻ D9 individual (Fig. 5a). The KIR2DL1⁺ KIR2DL2/2DL3/2DS2⁻ (8C11⁺ GL183⁻) subset was observed not only in the NK cell compartment, but also in both CD56⁻ and CD56⁺ T lymphocyte populations (8C11⁺ GL183⁻), as illustrated for the KIR2DL1⁺ D11 individual (Fig. 5b). A control staining is also indicated for the KIR2DL1⁻ D6 individual. Even though we were able to detect a KIR2DS1⁺ KIR2DL1/2DL2/2DL3/2DS2⁻ NK cell population (8C11⁻ EB6⁺), the frequency of KIR2DS1⁺ expression on T lymphocytes (CD56⁻ or CD56⁺) was low or absent, as illustrated in Fig. 5(c) for the KIR2DS1⁺ D8 individual. Again, a control staining is also provided for the KIR2DS1⁻ D10 individual. In general, the frequency of KIR⁺ CD56⁺ T cells was more constant than the frequency of KIR⁺ cells in the CD56⁻ T-cell population. For the KIR2DS1⁺ D9 individual who displayed 14·5% of KIR2DS1⁺ KIR2DL1/2DL2/2DL3/2DS2⁻ NK cells (EB6⁺8C11⁻), activating KIR2DS1 was mainly expressed on the CD56^dim NK cell population (19·1% versus 0·5% for the CD56^bright NK cells) (Fig. 5d).

Figure 5.

Analysis of the distribution of KIR2DL1, KIR2DL2 and KIR2DS1 on natural killer (NK) cells, CD56⁻ and CD56⁺ T lymphocytes. Peripheral blood mononuclear cells were stained with CD3- and CD56-specific monoclonal antibodies (mAbs) to discriminate NK cells (CD3⁻ CD56⁺), CD56⁻ T lymphocytes (CD3⁺ CD56⁻) and CD56⁺ CD3⁺ T lymphocytes (CD3⁺ CD56⁺) by four-colour flow cytometry. (a) Combined use of 1F12 with GL183 to target KIR2DL2⁺ KIR2DL3/2DS2⁻ NK cells from KIR2DL2⁺ D8 and KIR2DL2⁻ D9 individuals. (b) Combined use of 8C11 with GL183 to target KIR2DL1⁺ KIR2DL2/2DL3/2DS1/2DS2⁻ NK cells from KIR2DL1⁺ D11 and KIR2DL1⁻ D6 individuals. (c) Combined use of 8C11 with EB6 to target KIR2DS1⁺ KIR2DL1/2DL2/2DL3/2DS2⁻ NK cells from KIR2DS1⁺ D8 and KIR2DS1⁻ D10 individuals. (d) Staining of NK cells (CD3⁻ CD56⁺) with the combination of 8C11 with EB6 on total cells, CD56^dim and CD56^bright NK cells.

Allelic KIR2DL1 variant recognition by the 8C11 and 1A6 mAbs

Figure 6(a) shows a typical 8C11/EB6 staining for the KIR2DS1⁻ D11 individual lacking EB6⁺ 8C11⁻ cells. However, for some individuals (D12 and D13 in Fig. 6a), despite lacking the KIR2DS1 gene, an EB6⁺ 8C11⁻ cell population was detected, suggesting that 8C11 does not recognize all the allelic KIR2DL1 molecules recognized by EB6. In fact, for the KIR2DL1 genotyped D12 individual, an 8C11⁺ GL183⁻ cell population (corresponding to KIR2DL1 expression) was detected (Fig. 6a). Taking into account the KIR specificity of 8C11 and the alignment of KIR sequences published in the IPD-KIR sequence database (release 2.0.0; http://www.ebi.ac.uk/ipd/kir), it was possible, by deduction, to identify the recognition site of 8C11 (Fig. 6b). Position 154 (in amino acids) was the unique site potentially recognized by 8C11, with a proline (P¹⁵⁴) for all KIR2DS2, KIR2DL1/2DL2 and 2DL3 but with a threonine (T¹⁵⁴) for the KIR2DS1. However, in this way, it was also possible to postulate that 8C11 does not recognize KIR2DL1*004, *007 or *010 allele-encoded molecules, as mentioned in Fig. 6(b). To check this hypothesis, we sequenced KIR2DL1 alleles from different KIR2DL1⁺ genotyped individuals (n = 11) corresponding to three groups of D11, D12 and D13 individuals (Fig. 6c). Individual D12 had two KIR2DL1 alleles coding for a molecule with a threonine (T) in position 154 (T¹⁵⁴). Based on our hypothesis, 8C11 does not recognize the KIR2DL1 molecule with a T¹⁵⁴, which explains the staining of D12 NK cells with 8C11, characterized by the absence of an 8C11⁺ GL183⁻ cell population (KIR2DL1⁺) and the presence of an EB6⁺ 8C11⁻ cell population in individuals lacking the KIR2DS1 gene (Fig. 6a). Similarly, the D13 individual had one KIR2DL1 allele coding for a molecule with a threonine (T) in position 154 (T¹⁵⁴) and a KIR2DL1 allele coding for a molecule with a proline (P) at position 154 (P¹⁵⁴) (Fig. 6c). For this individual, therefore, 8C11 recognized the KIR2DL1 molecule with a P¹⁵⁴, which was consistent with the presence of an 8C11⁺ GL183⁻ cell population. However, in this individual, 8C11 did not recognize the KIR2DL1 molecule with a T¹⁵⁴, which explained the EB6⁺ 8C11⁻ cell population in the absence of the KIR2DS1 gene (Fig. 6a). On the other hand, individual D11, who presented a widespread phenotypic profile without an EB6⁺ 8C11⁻ cell population and with an 8C11⁺ GL183⁻ cell population, had two KIR2DL1 alleles coding for a KIR2DL1 with P¹⁵⁴, which was recognized by the 8C11 mAb.

Figure 6.

8C11 does not recognize all KIR2DL1 allele-encoded molecules. (a) Peripheral blood mononuclear cells were stained with CD3- and CD56-specific monoclonal antibodies (mAbs) to target natural killer (NK) cells (CD3⁻ CD56⁺) and the combined use of 8C11 with EB6 or GL183 to target KIR2DS1⁺ KIR2DL1/2DL2/2DL3/2DS2⁻ NK cells and KIR2DL1⁺ KIR2DL2/2DL3/2DS1/2DS2⁻ NK cell subsets respectively from three different KIR2DS1⁻ KIR2DL1⁺ genotyped individuals (D11, D12 and D13). (b) Codon at position 154 for all referenced KIR2DS2, KIR2DL2, KIR2DL3, KIR2DS1 and KIR2DL1 alleles. P¹⁵⁴ is indicated in bold on grey background. (c) Electrophoretograms obtained for D11, D12 and D13 individuals are presented above the aligned nucleotide sequences of KIR2DL1 alleles around the position 523. Polymorphic position is boxed on the electrophoretograms and indicated in bold on grey background in all KIR2DL1 allele sequences. M = A + C. D11, D12 and D13 are representative of seven, two and two respectively of 11 studied individuals.

For some individuals (D10, D12 and D13), despite lacking the KIR2DS1 gene (Fig. S1a), an 1A6⁺ GL183⁻ cell population was detected, suggesting that 1A6 recognize some allelic KIR2DL1 molecules. The alignment of KIR sequences enabled us to identify the recognition site for 1A6. The unique site, which is common for KIR2DL2/2DL3/2DS2 and KIR2DS1 and some KIR2DL1 alleles, is a proline at position 114 (P¹¹⁴). In this case, KIR2DL1*001, *002, *004, *007, *008 and *010 molecules can be recognized by 1A6 in accordance with the alignment of KIR sequences published in the IPD-KIR database (release 2.0.0; http://www.ebi.ac.uk/ipd/kir) (Fig. S1b). To check this hypothesis, we analysed the sequence of KIR2DL1 alleles determined from different KIR2DL1⁺-genotyped individuals (n = 11) in position 404 (Fig. S1c). Hence, individual D11 had two KIR2DL1 alleles coding for a molecule with a leucine (L) in position 114 (L¹¹⁴). D10 and D12 individuals had two KIR2DL1 alleles coding for a molecule with a proline (P) in position 114 (P¹¹⁴) and individual D13 had one KIR2DL1 allele coding for a molecule with a proline (P) in position 114 (P¹¹⁴) and one KIR2DL1 allele coding for a molecule with a leucine (L) at position 114 (L¹¹⁴). To conclude, KIR2DL1 encoded by KIR2DL1*001, *002 and *008 alleles are recognized by 8C11 and 1A6, KIR2DL1 encoded by KIR2DL1*003, *005, *006 and *009 alleles are recognized only by 8C11 and KIR2DL1 encoded by KIR2DL1*004, *007 and *010 are recognized only by 1A6.

1F12 mAb recognizes only the KIR2DS2 and KIR2DL3

The alignment of KIR sequences enabled us to identify the recognition site for 1F12. The unique site, which is common for KIR2DS2 and KIR2DL3 but different for KIR2DL2, is a proline at position 16 (P¹⁶) (Fig. 7a). In this case, molecules KIR2DL1*001, *002, *008 and the molecule KIR2DL2*004 can be recognized by 1F12 in accordance with the KIR sequence alignment performed using the IPD-KIR sequence database (release 2.0.0; http://www.ebi.ac.uk/ipd/kir). However, as could be expected, 1F12 could not recognize the KIR2DL2*004-encoded molecule because this molecule has been described as a non-functional KIR that is not expressed on the cell surface. Moreover, the flow cytometry study performed on 51 studied individuals (28 KIR2DL2⁺ and 23 KIR2DL2⁻ donors) did not reveal an 1F12⁺ GL183⁻ cell population. We sequenced KIR2DL1 alleles for different KIR2DL1⁺ individuals (n = 11) in the region of three nucleotides (404, 523 and 549) leading to the discrimination of the KIR2DL1*001, *002 and *008 alleles (Fig. 7b). Three of the 11 individuals studied carried KIR2DL1*001, *002 or *008 alleles (C⁴⁰⁴ C⁵²³ T⁵⁴⁹), such as individual D10 in whom a gene coding for the KIR2DL1 with a P¹⁶ was detected. While 8C11 recognized KIR2DL1 molecules encoded by the KIR2DL1*001, *002 or *008 alleles, as illustrated in Fig. 7(c) for individual D10, 1F12 did not recognize the KIR2DL1 molecule with P¹⁶. None of the studied KIR2DL1*001, *002 or *008 positive individuals (n = 3) expressed a KIR2DL1 recognized by 1F12 upon flow cytometry analysis. Consequently, in contrast to the 8C11 mAb whose recognition is dependent on P¹⁵⁴, the P¹⁶ binding site is important, but not sufficient for 1F12 recognition. We can therefore hypothesize that the three-dimensional structure of the KIR2DL1 molecule around the P¹⁶ site observed for the KIR2DL1*001, *002 and *008 allele-encoded proteins prevents 1F12 recognition.

Figure 7.

1F12 monoclonal antibody (mAb) recognizes only the KIR2DS2 and KIR2DL3. (a) Codon at position 16 for all referenced KIR2DL1, KIR2DL2, KIR2DL3 and KIR2DS2 alleles. P¹⁶ is indicated in bold on grey background. (b) Alignment of nucleotide sequences for all KIR2DL1 alleles around the polymorphic positions 404, 523 and 549. The corresponding electrophoretogram obtained for donor D10 is presented below the nucleotide KIR2DL1 sequences and polymorphic positions are boxed. (c) Peripheral blood mononuclear cells were stained with CD3- and CD56-specific mAbs to target natural killer (NK) cells (CD3⁻ CD56⁺) and the combined use of GL183 with 8C11 or 1F12 mAb to target KIR2DL1⁺ KIR2DL2/2DL3/2DS1/2DS2⁻ and KIR2DL2⁺ KIR2DL3/2DS2⁻ NK cell subsets, respectively, for the D10 individual. D10 is representative of three individuals of 11 studied individuals.

Discussion

In this paper, we describe the precise characterization of several newly generated KIR-specific mAbs. Our results show that some of these mAbs allow for the specific assessment of KIR2DS1, KIR2DL1 and KIR2DL2 expression. Using KIR2DL1/2DL2/2DL3/2DS2-specific 8C11 in combination with KIR2DL1/2DS1-specific EB6, we targeted KIR2DS1 (8C11⁻ EB6⁺) by excluding the expression of the major HLA-Cw-specific KIR2DL1/2DL2/2DL3/2DS2. We demonstrated the cell surface expression of KIR2DS1 from all of the studied KIR2DS1 genotyped individuals and found that the frequency of KIR2DS1⁺ 2DL1⁻ NK cells is relatively constant (mean of approximately 6·25%). KIR2DS1 is barely expressed on CD56⁻ CD3⁺ and CD56⁺ CD3⁺ T lymphocytes and is mainly detected on the surface of NK cells and especially on CD56^dim NK cells. Conversely, using the KIR2DL1/2DL2/2DL3/2DS2-specific 8C11 with KIR2DL2/2DL3/2DS2-specific GL183, we targeted the KIR2DL1 (8C11⁺ GL183⁻) by excluding KIR2DL2/2DL3/2DS1/2DS2 expression. We found the KIR2DS1 and KIR2DL1 to be independently expressed on NK cells. Interestingly, we showed that the frequency of this KIR2DL1⁺ NK cell population is lower in C1C2 versus C2C2 individuals, and that the frequency of this KIR2DL1⁺ NK cell subset is lower in C1C2 individuals who express the corresponding activating KIR2DS1 gene than in KIR2DS1⁻ C1C2 individuals. Among all of the KIR2DS2-specific mAbs evaluated, only one mAb, 1F12, did not recognize the corresponding inhibitory KIR2DL2. The combined use of KIR2DL3/2DS2-specific 1F12 with KIR2DL2/2DL3/2DS2-specific GL183 led us to target KIR2DL2 (1F12⁻ GL183⁺) by excluding expression of the KIR2DL3 and KIR2DS2. We found the frequency of KIR2DL2⁺ KIR2DL3/2DS2⁻ NK cells to be significantly higher in KIR2DL3⁻ than in KIR2DL3⁺ genotyped individuals. This observation may be explained by a gene–dose effect because KIR2DL2 and KIR2DL3 segregate as alleles of one locus.⁶

In most studies to date, inhibitory KIR2DL and activating KIR2DS have been studied simultaneously using first developed antibodies EB6 or GL183. Studies using these antibodies provided new insight into the mechanisms regulating NK cell function via KIR.¹² While no activating KIR2DS1-specific mAbs have helped to identify their functional surface expression, several studies using KIR2DS1 transcript⁺ NK clones have shown that KIR2DS1 is an activating receptor that recognizes the C2 ligand.^12,21,22 Recently, using the combined association of 8C11/EB6 mAb to exclude KIR2DL1/2DL2/2DL3/2DS2 expression, we have shown that only KIR2DS1⁺ NK cells from C2^– individuals are C2 alloreactive.²³

The presence of cognate HLA class I ligand for a given KIR has been reported to increase the frequency of NK cells expressing that particular KIR, and to decrease the frequency of NK cells expressing other inhibitory KIR.²⁴ Yawata et al. studied KIR2DL1⁺ KIR2DS1⁻ genotyped Japanese individuals and showed that the presence of cognate C2 almost doubled the frequency of KIR2DL1⁺ NK cells. We observed a significant difference in KIR2DL1⁺ NK cell frequency only between C1C2 and C2C2 individuals. Interestingly, by distinguishing KIR2DS1⁻ and KIR2DS1⁺ genotyped individuals, we observed a significant increase in KIR2DL1⁺ NK cells in C1C2 individuals who do not carry the KIR2DS1 gene. Our results suggest that not only does the HLA KIR ligand have an impact on specific KIR NK cell frequency but so does activating homologous KIR. However, we did not note any significant differences in KIR2DL2⁺ NK cell frequency when comparing C1C1, C1C2 and C2C2 individuals. This may be explained by the fact that KIR2DL2 recognizes a large spectrum of HLA-Cw molecules including not only C1 molecules (Asp⁸⁰) but also C2 molecules (Lys⁸⁰) and HLA-B molecules, as shown recently.²⁵

Allelic KIR polymorphism can have an impact on KIR expression and function, as described for KIR3DL1.²⁴ The allelic KIR recognition of 8C11, revealed by the precise characterization of its specificity, underlines the caution required when studying KIR expression, because of the high allelic KIR polymorphism. It will be necessary to characterize the KIR specificity of mAbs at the KIR allele level to avoid misleading interpretation of KIR NK cell phenotype. The impact of allelic KIR polymorphism on NK function has not been investigated widely except in the case of KIR3DL1. Using these new mAbs, it will be conceivable to study the functional impact of KIR2DL1 and KIR2DL2 polymorphisms. The use of combined 1F12 and GL183 would make it possible to determine the impact of different C2 ligands, such as Cw*0501-encoded or Cw*0202-encoded molecules, on KIR2DL2⁺ NK cell education, even though this has not yet been determined.²⁵ We showed that 8C11 recognizes KIR2DL1*003 allele-encoded molecules, which are the KIR most frequently detected in different populations.^11,26

From a clinical point of view, these newly characterized mAbs should represent new and interesting tools in numerous applications. NK cells present a phenotypic and functional diversity in distinct tissues, notably uterine NK cells versus blood NK cells. An investigation of KIR2DS1 expression on uterine NK cells using a combination of 8C11 and EB6 should be interesting because this activating KIR gene has been implicated in miscarriage and pre-eclampsia.^27,28 KIR2DS2 expression has also been reported on CD4⁺ T cells in healthy individuals,²⁹ as well as in patients affected by rheumatoid arthritis³⁰ or acute coronary syndrome.³¹ In agreement with reports on the frequency of KIR⁺ T cells in healthy individuals,²⁹ we observed that the majority of T cells expressed KIR2DL2/2DL3 but that KIR2DL1⁺ and KIR2DS1⁺ T-cell frequencies were low. The combined use of 1F12 with GL183 would be useful to study the impact of KIR2DL2 and KIR2DS2 on T lymphocytes expressing only KIR2DL2 or KIR2DS2. Indeed, it has been recently shown that even though CD4⁺ T lymphocytes carry KIR2DL2, -2DL3 and -2DS2 transcripts, they selectively express inhibitory KIR2DL2/2DL3 or KIR2DS2 receptors.³² A number of associations have been identified between the risk of autoimmune diseases and the expression of activating KIR2DS1 and KIR2DS2.³³ This risk seems to be increased in the absence of ligands for the inhibitory counterparts, KIR2DL1 and KIR2DL2/2DL3, respectively. Studies of KIR genotypes in viral infections suggest a beneficial role of activating KIR genes but a deleterious impact of activating KIR genes in autoimmune diseases.^33,34 A better knowledge of activating KIR expression and interactions would be essential to understand their contribution to regulating NK cell function. 8C11 and 1F12 constitute useful tools to investigate the functional contribution not only of activating KIR2DS1 and KIR2DS2 but also of inhibitory KIR2DL1 and KIR2DL2. The combined use of these new mAbs with commercial EB6 or GL183 mAbs should help to improve our knowledge of individual KIR contributions to NK cell biology.

Glossary

Abbreviations:

ELISA

enzyme-linked immunosorbent assay

FACS

fluorescence-activated cell sorting

FITC

fluorescein isothiocyanate

HLA

human leucocyte antigen

ITAM

immunoreceptor tyrosine-based activation motif

ITIM

immunoreceptor tyrosine-based inhibition motif

KIR

killer cell immunoglobulin-like receptor

mAb

monoclonal antibody

NK

natural killer

OD

optical density

PBMC

peripheral blood mononuclear cells

PCR-SSP

polymerase chain reaction–sequence-specific primer

PE

phycoerythrin

Disclosures

The authors declare no financial or commercial conflict of interest.

Supporting Information

Additional Supporting Information may be found in the online version of this article:

Fig. S1: 1A6 recognize some KIR2DL1 alleles encoded molecules. (a) PBMC were stained with CD3 and CD56 specific mAbs to target NK cells (CD3- CD56 +) and the combined use of 1A6 with GL183 and 8C11 with GL183 to target KIR2DL1 + KIR2DL2/2DL3/2DS1/2DS2- NK cells and KIR2DL1 + KIR2DL2/2DL3/2DS2- NK cell subsets respectively from 4 different KIR2DS1- KIR2DL1 + genotyped individuals (D10, 11, D12 and D13). (b) Codon at position 114 for all referenced KIR2DS2, KIR2DL2, KIR2DL3, KIR2DS1 and KIR2DL1 alleles. P114 is indicated in bold on grey background. (c) Electrophoretograms obtained for D10, D11, D12 and D13 individuals are presented above the aligned nucleotide sequences of KIR2DL1 alleles around the position 404. Polymorphic position is boxed on the electrophoretograms and indicated in bold on grey background in all KIR2DL1 allele sequences. D10, D11, D12 and D13 are representative of 3, 4, 2 and 2 respectively of 11 studied individuals.

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