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. 1997 Aug 4;186(3):473-8.
doi: 10.1084/jem.186.3.473.

Negative signaling pathways of the killer cell inhibitory receptor and Fc gamma RIIb1 require distinct phosphatases

N Gupta  1 , A M Scharenberg, D N Burshtyn, N Wagtmann, M N Lioubin, L R Rohrschneider, J P Kinet, E O Long
Affiliations

Negative signaling pathways of the killer cell inhibitory receptor and Fc gamma RIIb1 require distinct phosphatases

N Gupta et al. J Exp Med. .

Abstract

Inhibition of natural killer (NK) cells by the killer cell inhibitory receptor (KIR) involves recruitment of the tyrosine phosphatase SHP-1 by KIR and is prevented by expression of a dominant negative SHP-1 mutant. Another inhibitory receptor, the low affinity Fc receptor for immunoglobulin G (IgG) (Fc gamma RIIb1), has been shown to bind SHP-1 when cocross-linked with the antigen receptor on B cells (BCR). However, coligation of Fc gamma RIIb1 with BCR and with Fc epsilon RI on mast cells leads to recruitment of the inositol 5' phosphatase SHIP and to inhibition of mast cells from SHP-1-deficient mice. In this study, we evaluated the ability of these two inhibitory receptors to block target cell lysis by NK cells, and the contribution of SHP-1 and SHIP to inhibition. Recombinant vaccinia viruses encoding chimeric receptors and dominant negative mutants of SHP-1 and SHIP were used for expression in mouse and human NK cells. When the KIR cytoplasmic tail was replaced by that of Fc gamma RIIb1, recognition of HLA class I on target cells by the extracellular domain resulted in inhibition. A dominant negative mutant of SHP-1 reverted the inhibition mediated by the KIR cytoplasmic tail but not that mediated by Fc gamma RIIb1. In contrast, a dominant negative mutant of SHIP reverted only the inhibition mediated by the Fc gamma RIIb1 tail, providing functional evidence that SHIP plays a role in the Fc gamma RIIb1-mediated negative signal. These data demonstrate that inhibition of NK cells by KIR involves primarily the tyrosine phosphatase SHP-1, whereas inhibition mediated by Fc gamma RIIb1 requires the inositol phosphatase SHIP.

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Figures

Figure 1
Figure 1
Schematic representation of the chimeric receptors. Hybrid molecules bearing the cytoplasmic tail of human FcγRIIb1 and the extracellular domains of KIR-cl6 or KIR-cl42 were generated as described. Open, hatched, and shaded boxes represent sequences from KIR-6, KIR-42, and FcγRIIb1, respectively. Closed boxes represent transmembrane regions and open bars in the cytoplasmic tails indicate the position of ITIMs.
Figure 2
Figure 2
The cytoplasmic tail of human FcγRIIb1 can deliver an inhibitory signal in mouse NK cells. (A) Surface expression of KIR-6, KIR-6/RIIb1, and KIR-6tr on cells infected with 5, 5, and 10 PFU/cell of the indicated recombinant vaccinia viruses, respectively. (B) Specific lysis of the B cell line .221 (open circles), and its HLA-Cw3 transfectant (closed circles) by uninfected mouse NK cells or those infected with Vac-6, Vac-6/ RIIb1 or Vac-6tr. ADCC was induced by precoating the targets with 0.1 μg/ml of anti-HLA-DR mAb L243 for 30 min on ice. Effectors and targets were plated at the indicated ratios.
Figure 3
Figure 3
SHP-1 is not required for inhibition of NK cells by FcγRIIb1. The KIR-negative human NK92 cell line was infected with recombinant vaccinia viruses expressing KIR-6, KIR-42, or their chimeric derivatives bearing the cytoplasmic tail of human FcγRIIb1 (KIR-6/RIIb1 and KIR-42/RIIb1), alone or with dominant negative SHP-1 (dnSHP-1), as indicated. The mean fluorescence intensity of staining with mAbs GL183 (for KIR-6 and KIR-6/RIIb1) and EB6 (for KIR-42 and KIR-42/RIIb1) is indicated next to each bar in the .221 panel. Lysis of .221, .221-Cw3, and .221-Cw4 targets was determined in a 4-h 51Cr release assay at an E/T of 4. Similar results were observed at an E/T of 1 and in two independent experiments.
Figure 4
Figure 4
SHIP is required for inhibition of NK cells by FcγRIIb1 but not by KIR. The experiment was as described in Fig. 3 except that a dominant negative SHIP (dnSHIP) was used. The data shown were obtained at an E/T of 6. Similar results were obtained at an E/T of 1 and in two independent experiments.
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