A human histocompatibility leukocyte antigen (HLA)-G-specific receptor expressed on all natural killer cells - PubMed (original) (raw)

A human histocompatibility leukocyte antigen (HLA)-G-specific receptor expressed on all natural killer cells

S Rajagopalan et al. J Exp Med. 1999.

Erratum in

Abstract

Human natural killer (NK) cells express several killer cell immunoglobulin (Ig)-like receptors (KIRs) that inhibit their cytotoxicity upon recognition of human histocompatibility leukocyte antigen (HLA) class I molecules on target cells. Additional members of the KIR family, including some that deliver activation signals, have unknown ligand specificity and function. One such KIR, denoted KIR2DL4, is structurally divergent from other KIRs in the configuration of its two extracellular Ig domains and of its transmembrane and cytoplasmic domains. Here we show that recombinant soluble KIR2DL4 binds to cells expressing HLA-G but not to cells expressing other HLA class I molecules. Unlike other HLA class I-specific KIRs, which are clonally distributed on NK cells, KIR2DL4 is expressed at the surface of all NK cells. Furthermore, functional transfer of KIR2DL4 into the cell line NK-92 resulted in inhibition of lysis of target cells that express HLA-G, but not target cells that express other class I molecules including HLA-E. Therefore, given that HLA-G expression is restricted to fetal trophoblast cells, KIR2DL4 may provide important signals to maternal NK decidual cells that interact with trophoblast cells at the maternal-fetal interface during pregnancy.

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Figures

Figure 1

Figure 1

Binding of soluble KIR2DL4–Ig to 221–G cells. (A) 721.221 (221) and 721.221 transfectants expressing HLA-Cw*0304 (221-Cw3) or HLA-G (221-G) were incubated with no fusion protein, 25 μg/ml of 2DL2–Ig, or 25 μg/ml of 2DL4–Ig. The bound fusion proteins were detected by flow cytometry after reaction with FITC-conjugated goat anti– human Fc specific antibodies. Data are expressed as median fluorescence intensity (MFI). Inset, histogram profile (filled) of ungated 221-G cells stained with 2DL4–Ig. Open histogram is control staining with secondary antibodies alone. (B) Binding of KIR2DL4–Ig to 721.221 transfectants was detected as described in A. Cell surface expression of HLA class I on the different cells, as detected by staining with the mAb DX17, was as follows (MFI in parenthesis): 221 (21); 221–B7 (1,731); 221–Cw3 (588); and 221–G (1,540).

Figure 1

Figure 1

Binding of soluble KIR2DL4–Ig to 221–G cells. (A) 721.221 (221) and 721.221 transfectants expressing HLA-Cw*0304 (221-Cw3) or HLA-G (221-G) were incubated with no fusion protein, 25 μg/ml of 2DL2–Ig, or 25 μg/ml of 2DL4–Ig. The bound fusion proteins were detected by flow cytometry after reaction with FITC-conjugated goat anti– human Fc specific antibodies. Data are expressed as median fluorescence intensity (MFI). Inset, histogram profile (filled) of ungated 221-G cells stained with 2DL4–Ig. Open histogram is control staining with secondary antibodies alone. (B) Binding of KIR2DL4–Ig to 721.221 transfectants was detected as described in A. Cell surface expression of HLA class I on the different cells, as detected by staining with the mAb DX17, was as follows (MFI in parenthesis): 221 (21); 221–B7 (1,731); 221–Cw3 (588); and 221–G (1,540).

Figure 2

Figure 2

Cell surface expression of KIR2DL4 on NK cells. (A) Flow cytometry profiles of the T leukemia cell line Jurkat, the B lymphoblastoid cell line 721.221, the monocytic cell line HL60 and the NK cell lines NK-92 and NK3.3 as well as a CD3−CD56+CD94− NK clone. Cells were incubated with anti-2DL4 antiserum followed by FITC anti–rabbit IgG. (B) Flow cytometry analysis of CD3−CD56+ NK cells isolated from the peripheral blood of a normal donor and double stained with PE-conjugated CD56 and rabbit anti-2DL4 antiserum followed by FITC-conjugated goat anti–rabbit IgG. The percentages of total cells in each quadrant are listed. (C) Flow cytometry analysis of a primary T cell culture from a normal donor gated on CD3+ cells and double stained for PE-conjugated CD3 and rabbit anti-2DL4 followed by FITC-conjugated goat anti–rabbit IgG. The percentages of total cells in each quadrant are listed.

Figure 3

Figure 3

Functional transfer of KIR2DL4 in NK-92 cells results in HLA-G–mediated inhibition. (A) Flow cytometry analysis of cell surface KIR2DL4 in NK-92 cells either uninfected, or infected with vac-2DL4 (15 PFU/cell). The cells were stained with either anti-2DL4 rabbit antiserum followed by FITC goat anti–rabbit IgG or with the mAb VV1-1G10 specific for a vaccinia cell surface protein. The histograms show log fluorescence of ungated cells. (B) Aliquots of uninfected or vac-2DL4–infected cells in A were tested for their ability to lyse 721.221 cells and 721.221 cells transfected with HLA-Cw*0401 and HLA-G, in a 4-h 51Cr-release assay. Lysis is shown for an E/T ratio of 5. Similar data was obtained at other E/T ratios and in five independent experiments. (C) Aliquots of NK-92 cells infected with vac-2DL4 were tested for their ability to lyse 721.221 (squares) or 221-G cells (triangles) in the presence of 5 μg/ml of the anti-class I mAb DX17 (open symbols) or control IgG1 (filled symbols). Similar results were obtained in two independent experiments.

Figure 3

Figure 3

Functional transfer of KIR2DL4 in NK-92 cells results in HLA-G–mediated inhibition. (A) Flow cytometry analysis of cell surface KIR2DL4 in NK-92 cells either uninfected, or infected with vac-2DL4 (15 PFU/cell). The cells were stained with either anti-2DL4 rabbit antiserum followed by FITC goat anti–rabbit IgG or with the mAb VV1-1G10 specific for a vaccinia cell surface protein. The histograms show log fluorescence of ungated cells. (B) Aliquots of uninfected or vac-2DL4–infected cells in A were tested for their ability to lyse 721.221 cells and 721.221 cells transfected with HLA-Cw*0401 and HLA-G, in a 4-h 51Cr-release assay. Lysis is shown for an E/T ratio of 5. Similar data was obtained at other E/T ratios and in five independent experiments. (C) Aliquots of NK-92 cells infected with vac-2DL4 were tested for their ability to lyse 721.221 (squares) or 221-G cells (triangles) in the presence of 5 μg/ml of the anti-class I mAb DX17 (open symbols) or control IgG1 (filled symbols). Similar results were obtained in two independent experiments.

Figure 3

Figure 3

Functional transfer of KIR2DL4 in NK-92 cells results in HLA-G–mediated inhibition. (A) Flow cytometry analysis of cell surface KIR2DL4 in NK-92 cells either uninfected, or infected with vac-2DL4 (15 PFU/cell). The cells were stained with either anti-2DL4 rabbit antiserum followed by FITC goat anti–rabbit IgG or with the mAb VV1-1G10 specific for a vaccinia cell surface protein. The histograms show log fluorescence of ungated cells. (B) Aliquots of uninfected or vac-2DL4–infected cells in A were tested for their ability to lyse 721.221 cells and 721.221 cells transfected with HLA-Cw*0401 and HLA-G, in a 4-h 51Cr-release assay. Lysis is shown for an E/T ratio of 5. Similar data was obtained at other E/T ratios and in five independent experiments. (C) Aliquots of NK-92 cells infected with vac-2DL4 were tested for their ability to lyse 721.221 (squares) or 221-G cells (triangles) in the presence of 5 μg/ml of the anti-class I mAb DX17 (open symbols) or control IgG1 (filled symbols). Similar results were obtained in two independent experiments.

Figure 4

Figure 4

KIR2DL4-mediated recognition of HLA-G is reversed using a HLA-G–specific mAb that does not recognize HLA-E. (A) mAb G233 does not recognize HLA-E. RMAS-E cells were loaded with 500 μM of the HLA-G signal sequence–derived peptide VMAPRTLFL at 26°C and stained with either the anti-HLA reactive mAb B9.12.1 or the HLA-G specific mAb G233. The staining of 221-G cells using both antibodies is shown in the bottom panel. (B) Flow cytometry analysis of cell surface KIR2DL1 and KIR2DL4 in NK-92 cells infected with either vac-2DL1 or vac-2DL4. Cells were stained with mAb EB6 (specific for KIR2DL1), mAb VV1-1G10 (anti-vac), or anti-2DL4 rabbit antiserum followed by FITC-labeled species-specific secondary reagents. (C) Aliquots of infected cells expressing KIR2DL1 or KIR2DL4 were tested for their ability to lyse 221-Cw3, 221-Cw4, or 221-G target cells. The interaction between vac-2DL4 expressing NK-92 cells and those expressing 221-G was tested in the presence of either 5 μg/ml of isotype-matched control IgG2a (cIg) or HLA-G–specific mAb G233. Similar results were obtained in three independent experiments.

Figure 4

Figure 4

KIR2DL4-mediated recognition of HLA-G is reversed using a HLA-G–specific mAb that does not recognize HLA-E. (A) mAb G233 does not recognize HLA-E. RMAS-E cells were loaded with 500 μM of the HLA-G signal sequence–derived peptide VMAPRTLFL at 26°C and stained with either the anti-HLA reactive mAb B9.12.1 or the HLA-G specific mAb G233. The staining of 221-G cells using both antibodies is shown in the bottom panel. (B) Flow cytometry analysis of cell surface KIR2DL1 and KIR2DL4 in NK-92 cells infected with either vac-2DL1 or vac-2DL4. Cells were stained with mAb EB6 (specific for KIR2DL1), mAb VV1-1G10 (anti-vac), or anti-2DL4 rabbit antiserum followed by FITC-labeled species-specific secondary reagents. (C) Aliquots of infected cells expressing KIR2DL1 or KIR2DL4 were tested for their ability to lyse 221-Cw3, 221-Cw4, or 221-G target cells. The interaction between vac-2DL4 expressing NK-92 cells and those expressing 221-G was tested in the presence of either 5 μg/ml of isotype-matched control IgG2a (cIg) or HLA-G–specific mAb G233. Similar results were obtained in three independent experiments.

Figure 4

Figure 4

KIR2DL4-mediated recognition of HLA-G is reversed using a HLA-G–specific mAb that does not recognize HLA-E. (A) mAb G233 does not recognize HLA-E. RMAS-E cells were loaded with 500 μM of the HLA-G signal sequence–derived peptide VMAPRTLFL at 26°C and stained with either the anti-HLA reactive mAb B9.12.1 or the HLA-G specific mAb G233. The staining of 221-G cells using both antibodies is shown in the bottom panel. (B) Flow cytometry analysis of cell surface KIR2DL1 and KIR2DL4 in NK-92 cells infected with either vac-2DL1 or vac-2DL4. Cells were stained with mAb EB6 (specific for KIR2DL1), mAb VV1-1G10 (anti-vac), or anti-2DL4 rabbit antiserum followed by FITC-labeled species-specific secondary reagents. (C) Aliquots of infected cells expressing KIR2DL1 or KIR2DL4 were tested for their ability to lyse 221-Cw3, 221-Cw4, or 221-G target cells. The interaction between vac-2DL4 expressing NK-92 cells and those expressing 221-G was tested in the presence of either 5 μg/ml of isotype-matched control IgG2a (cIg) or HLA-G–specific mAb G233. Similar results were obtained in three independent experiments.

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