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Comparative Study
. 1997 Dec 1;186(11):1809-18.
doi: 10.1084/jem.186.11.1809.

A common inhibitory receptor for major histocompatibility complex class I molecules on human lymphoid and myelomonocytic cells

M Colonna  1 , F Navarro, T Bellón, M Llano, P García, J Samaridis, L Angman, M Cella, M López-Botet
Affiliations
Comparative Study

A common inhibitory receptor for major histocompatibility complex class I molecules on human lymphoid and myelomonocytic cells

M Colonna et al. J Exp Med. .

Abstract

Natural killer (NK) cell-mediated lysis is negatively regulated by killer cell inhibitory receptors specific for major histocompatibility complex (MHC) class I molecules. In this study, we characterize a novel inhibitory MHC class I receptor of the immunoglobulin-superfamily, expressed not only by subsets of NK and T cells, but also by B cells, monocytes, macrophages, and dendritic cells. This receptor, called Ig-like transcript (ILT)2, binds MHC class I molecules and delivers a negative signal that inhibits killing by NK and T cells, as well as Ca2+ mobilization in B cells and myelomonocytic cells triggered through the B cell antigen receptor and human histocompatibility leukocyte antigens (HLA)-DR, respectively. In addition, myelomonocytic cells express receptors homologous to ILT2, which are characterized by extensive polymorphism and might recognize distinct HLA class I molecules. These results suggest that diverse leukocyte lineages have adopted recognition of self-MHC class I molecules as a common strategy to control cellular activation during an immune response.

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Figures

Figure 1
Figure 1
(a) The HP-F1 mAb reconstitutes NK cell–mediated lysis of HLA class I transfectants. Lysis of the class I–negative mutant cell line 721.221 by NKL is inhibited upon transfection of HLA-B*2702, -B*2705, -B*5101, -A*0301, -B*0702, -Cw*0301, and -G1 alleles in 721.221 (white bars), regardless of the presence of a control antibody (C218, anti-CD56, IgG1). F(ab′)2 fragments of the HP-F1 mAb (black bars) completely reconstitute lysis of HLA-B*2702, -B*2705, and -B*5101 transfectants. F(ab′)2 fragments of the anti-CD94 HP-3B1 mAb (gray bars) reconstitute lysis of the HLA-Cw*0301 transfectant almost completely. A combination of both HP-F1 and HP-3B1 mAbs (hatched bars) is necessary to reconstitute lysis of HLA-A*0301, -B*0702, and -G1 transfectants. The anti–class I HP-1F7 mAb (vertical bars) reconstitutes lysis of all of the class I transfectants. Cytotoxicity was determined in a standard 4 h 51Cr-release assay. NKL expresses the CD94–NKG2A heterodimer but no KIRs, as determined by cell surface staining with Z199 (anti–CD94– NKG2A heterodimer), HP-3E4, GL183, EB6 (anti-p58 KIRs), DX9, 5.133 (anti-p70 and -p70/140 KIRs) mAbs, and by RT-PCR. (b) HP-F1 mAb inhibits CD16-dependent redirected lysis of P815 cells by NKL. 51Cr-labeled P815 cells were preincubated for 15 min with anti-CD16 alone, with HP-F1 (5–10 μg/ml), or with HP-3B1 (anti-CD94, 5 μg/ml). In control samples, cells were incubated with medium, with anti-CD56 mAb (IgG1), or with HP-F1 (5–10 μg/ml). After incubation, NKL cells were added at different effector/target ratios. HP-F1 significantly reduced CD16-mediated lysis of P815 cells, mimicking the ligand of an inhibitory receptor. HP-F1 mAb also reduced the low basal lysis of P815 by NKL in the absence of anti-CD16. Similar results were obtained with the anti-CD94 mAb.
Figure 1
Figure 1
(a) The HP-F1 mAb reconstitutes NK cell–mediated lysis of HLA class I transfectants. Lysis of the class I–negative mutant cell line 721.221 by NKL is inhibited upon transfection of HLA-B*2702, -B*2705, -B*5101, -A*0301, -B*0702, -Cw*0301, and -G1 alleles in 721.221 (white bars), regardless of the presence of a control antibody (C218, anti-CD56, IgG1). F(ab′)2 fragments of the HP-F1 mAb (black bars) completely reconstitute lysis of HLA-B*2702, -B*2705, and -B*5101 transfectants. F(ab′)2 fragments of the anti-CD94 HP-3B1 mAb (gray bars) reconstitute lysis of the HLA-Cw*0301 transfectant almost completely. A combination of both HP-F1 and HP-3B1 mAbs (hatched bars) is necessary to reconstitute lysis of HLA-A*0301, -B*0702, and -G1 transfectants. The anti–class I HP-1F7 mAb (vertical bars) reconstitutes lysis of all of the class I transfectants. Cytotoxicity was determined in a standard 4 h 51Cr-release assay. NKL expresses the CD94–NKG2A heterodimer but no KIRs, as determined by cell surface staining with Z199 (anti–CD94– NKG2A heterodimer), HP-3E4, GL183, EB6 (anti-p58 KIRs), DX9, 5.133 (anti-p70 and -p70/140 KIRs) mAbs, and by RT-PCR. (b) HP-F1 mAb inhibits CD16-dependent redirected lysis of P815 cells by NKL. 51Cr-labeled P815 cells were preincubated for 15 min with anti-CD16 alone, with HP-F1 (5–10 μg/ml), or with HP-3B1 (anti-CD94, 5 μg/ml). In control samples, cells were incubated with medium, with anti-CD56 mAb (IgG1), or with HP-F1 (5–10 μg/ml). After incubation, NKL cells were added at different effector/target ratios. HP-F1 significantly reduced CD16-mediated lysis of P815 cells, mimicking the ligand of an inhibitory receptor. HP-F1 mAb also reduced the low basal lysis of P815 by NKL in the absence of anti-CD16. Similar results were obtained with the anti-CD94 mAb.
Figure 2
Figure 2
The HP-F1 antigen is expressed on CD56+ NK cells (23– 77% in six different donors), α/β T cells (3–28%), γ/δ T cells (16–50%), CD19+ B cells, CD14+ monocytes, HLA-DRhigh DCs derived from CD34+ precursors, and CD1a+ DCs derived from monocytes under appropriate culture conditions. Macrophages derived in vitro from purified monocytes also expressed the HP-F1 antigen, whereas neutrophils were HP-F1− (data not shown).
Figure 3
Figure 3
The HP-F1γ receptor is an ∼110-kD monomeric glycoprotein. (Left) Immunoprecipitation from 125I-labeled NKL cells with HP-F1 mAb yields a protein which runs as a 110-kD band in SDS-PAGE under reducing conditions. After treatment with N-glycosidase, the molecular mass is reduced to ∼90 kD. (Right) Identical electrophoretic patterns were obtained by SDS-PAGE analysis of immunoprecipitates from ILT2-transfected COS7 cells. No bands were detected using the culture supernatant from X63 murine myeloma in control experiments.
Figure 4
Figure 4
ILT2 soluble protein binds HLA-A, -B, and -G1 transfectants. HLA-B*2702, -B*2705, -A*0301, -G1, and -Cw*0301 transfectants in 721.221 and untransfected cells were incubated with a soluble ILT2– IgG1 fusion protein, followed by a PE-labeled goat anti–human IgG antibody. Binding was assessed by FACS® analysis. HLA class I expression of the transfectants was determined in the same experiment by FACS® analysis using the w6/32 mAb (IgG2a; American Type Culture Collection). MFI were as follows: 721.221, 121; HLA-B*2702, 2128; -B*2705, 3045; -A*0301, 3854; -G1, 1084; -Cw*0301, 1582. The binding pattern did not correlate with the level of class I expression on the transfectants.
Figure 5
Figure 5
Inhibition of IgE-induced serotonin release in ILT2-transfected RBL cells. Transfected and control cells were stimulated with purified mouse IgE (20 μg/ml) alone and in combination with either HP-F1 [20 μg/ml of whole antibody or F(ab′)2 fragments] or with the isotype-matched antibody HP-1F7 [20 μg/ml of whole antibody or F(ab′)2 fragments] immobilized on plastic. The percentage of serotonin release, as compared to total and spontaneous release, was determined after 1 h at 37°C. The expression of ILT2 on transfected RBL cells was assessed by indirect immunofluorescence with HP-F1 mAb (not shown).
Figure 6
Figure 6
ILT2 is associated with SHP-1. NKL cells and the EBV-transformed B cell line C1R were incubated for 10 min at 37°C either with medium alone or with pervanadate. ILT2 was then immunoprecipitated with the HP-F1 mAb and immunoprecipitates were separated by SDS-PAGE and immunoblotted with anti–SHP-1 antibody. Whole cell lysate is included as a positive control. SHP-1 was recruited to ILT2 after cell stimulation with pervanadate.
Figure 7
Figure 7
ILT2-class I interaction inhibits TSST-1–mediated T cell cytotoxicity. HP-F1+ T cell clones were generated from the peripheral blood of a healthy donor and selected for expression of TCR-Vβ2 and for lack of KIR expression. T cell clones were then tested in a 4-h 51Cr-release assay for cytotoxicity against the class I–negative B lymphoblastoid cell line 721.221 cells or 721.221 cells stably transfected with HLA-B*2705, in the presence of serial dilutions of TSST-1 at an effector/target ratio of 20:1. The T cell clone OKT8-24 killed 721.221 in the presence of TSST-1, but did not kill B*2705-transfected cells. F(ab′)2 fragments of the HP-F1 mAb partially restored the lysis, while F(ab′)2 fragments of the isotype-matched anti-CD56 mAb had no effect.
Figure 8
Figure 8
Intracellular Ca2+ mobilization induced by anti–human IgG antibodies in the EBV-B cell line C1R (a) is inhibited upon crosslinking with ILT2 (b). Similarly, the increased [Ca2+]i triggered through HLA-DR by the 3.8 B1 mAb in monocytes (c) and dendritic cells (not shown) is downregulated, although to a lesser extent, when ILT2 is coligated with HLA-DR (d). HP-1F7 is an isotype-matched control antibody.
Figure 8
Figure 8
Intracellular Ca2+ mobilization induced by anti–human IgG antibodies in the EBV-B cell line C1R (a) is inhibited upon crosslinking with ILT2 (b). Similarly, the increased [Ca2+]i triggered through HLA-DR by the 3.8 B1 mAb in monocytes (c) and dendritic cells (not shown) is downregulated, although to a lesser extent, when ILT2 is coligated with HLA-DR (d). HP-1F7 is an isotype-matched control antibody.
Figure 9
Figure 9
Alignment of ILTs with four Ig-SF extracellular domains. The alignment was generated by the Clustal method using Lasergene analysis software. (DNASTAR, Inc., Madison, WI). Amino acid sequences were aligned with ILT2. Amino acid variants are indicated. Gaps (dashes) were introduced to maximize homologies. Amino acids are numbered on the right side. SS, signal sequence; EC, extracellular domain; TM, transmembrane domain; CY, cytoplasmic domain.
Figure 10
Figure 10
Diversity of the extracellular region of ILT5. 15 variants of ILT5 were identified from a pool of bone marrow cells derived from 51 donors, while only 1 variant (clone DC.1) was detected from a single donor. Amino acid variants are clustered in variable regions (underlined). ILT5-cl41 is prematurely truncated as a consequence of an alternative splicing which generates a stop codon. D1–D4, extracellular domains.
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