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

Gupta N, Scharenberg AM, Burshtyn DN, Wagtmann N, Lioubin MN, Rohrschneider LR, Kinet JP, Long EO - J. Exp. Med. (1997)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, Rockville, Maryland 20852, USA.

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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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.
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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.

Mentions: Expression of a catalytically inactive mutant of SHP-1 in human NK cells reverts the KIR-mediated inhibition of target cell lysis (2, 6). KIR-6, KIR-42, and their chimeric counterparts KIR-6/RIIb1 and KIR-42/RIIb1 were expressed in the human NK cell line NK92 either individually or together with the dominant negative mutant of SHP-1. The surface staining of Vac-6 and Vac-6/RIIb1 infected cells by GL183 and that of Vac-42 and Vac-42/RIIb1 infected cells by EB6 was comparable as seen by the mean fluorescence intensities (Fig. 3). Coinfection of NK92 cells with the receptors along with dominant negative SHP-1 did not alter their surface expression (Fig. 3). NK92 cells infected with Vac-6 or with Vac-6/RIIb1 killed .221 and .221-Cw4 targets but were inhibited from lysing .221-Cw3 targets. Reciprocally, cells infected with Vac-42 and Vac-42/RIIb1 lysed .221 and .221-Cw3 but not .221-Cw4 targets (Fig. 3). Therefore, as shown in mouse NK cells, the cytoplasmic tail of FcγRIIb1 was able to deliver an inhibitory signal in human NK cells. Expression of dominant negative SHP-1 in NK92 reverted the KIR-mediated inhibition of target cell lysis but not that mediated by the cytoplasmic tail of FcγRIIb1 (Fig. 3). Thus, SHP-1 appears to play an important role in KIR-mediated inhibition of NK cells, as reported earlier (2), but not in FcγRIIb1-mediated inhibition of NK cells.


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

Gupta N, Scharenberg AM, Burshtyn DN, Wagtmann N, Lioubin MN, Rohrschneider LR, Kinet JP, Long EO - J. Exp. Med. (1997)

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.
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Related In: Results  -  Collection

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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.
Mentions: Expression of a catalytically inactive mutant of SHP-1 in human NK cells reverts the KIR-mediated inhibition of target cell lysis (2, 6). KIR-6, KIR-42, and their chimeric counterparts KIR-6/RIIb1 and KIR-42/RIIb1 were expressed in the human NK cell line NK92 either individually or together with the dominant negative mutant of SHP-1. The surface staining of Vac-6 and Vac-6/RIIb1 infected cells by GL183 and that of Vac-42 and Vac-42/RIIb1 infected cells by EB6 was comparable as seen by the mean fluorescence intensities (Fig. 3). Coinfection of NK92 cells with the receptors along with dominant negative SHP-1 did not alter their surface expression (Fig. 3). NK92 cells infected with Vac-6 or with Vac-6/RIIb1 killed .221 and .221-Cw4 targets but were inhibited from lysing .221-Cw3 targets. Reciprocally, cells infected with Vac-42 and Vac-42/RIIb1 lysed .221 and .221-Cw3 but not .221-Cw4 targets (Fig. 3). Therefore, as shown in mouse NK cells, the cytoplasmic tail of FcγRIIb1 was able to deliver an inhibitory signal in human NK cells. Expression of dominant negative SHP-1 in NK92 reverted the KIR-mediated inhibition of target cell lysis but not that mediated by the cytoplasmic tail of FcγRIIb1 (Fig. 3). Thus, SHP-1 appears to play an important role in KIR-mediated inhibition of NK cells, as reported earlier (2), but not in FcγRIIb1-mediated inhibition of NK cells.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, Rockville, Maryland 20852, USA.

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.

Show MeSH
Related in: MedlinePlus