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Mouse Ly-49A interrupts early signaling events in natural killer cell cytotoxicity and functionally associates with the SHP-1 tyrosine phosphatase.

Nakamura MC, Niemi EC, Fisher MJ, Shultz LD, Seaman WE, Ryan JC - J. Exp. Med. (1997)

Bottom Line: We show that ligation of Ly-49A by H-2Dd inhibits early signaling events during target cell stimulation, including polyphosphoinositide turnover and tyrosine phosphorylation.Finally, we demonstrate that mutational substitution of the tyrosine within the proposed SHP-1 binding motif in Ly-49A completely abrogates inhibition of NK cell cytotoxicity through this receptor.These results demonstrate that Ly-49A interrupts early activating signals in NK cells, and that SHP-1 is an important mediator of Ly-49A function.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of California, San Francisco 94143, USA.

ABSTRACT
The lytic activity of natural killer (NK) cells is inhibited by the expression of class I major histocompatibility complex (MHC) antigens on target cells. In murine NK cells, Ly-49A mediates inhibition of cytotoxicity in response to the class I MHC antigen H-2Dd. In this report, we studied the function of mouse Ly-49A in both the rat NK cell tumor line, RNK-16, transfected with Ly-49A cDNA, and in primary NK cells. We show that ligation of Ly-49A by H-2Dd inhibits early signaling events during target cell stimulation, including polyphosphoinositide turnover and tyrosine phosphorylation. We also show that Ly-49A directly associates with the cytoplasmic tyrosine phosphatase SHP-1, and that Ly-49A function is impaired in NK cells from SHP-1 mutant viable motheaten mice and from SHP-1-deficient motheaten mice. Finally, we demonstrate that mutational substitution of the tyrosine within the proposed SHP-1 binding motif in Ly-49A completely abrogates inhibition of NK cell cytotoxicity through this receptor. These results demonstrate that Ly-49A interrupts early activating signals in NK cells, and that SHP-1 is an important mediator of Ly-49A function.

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Cold-target competition of unlabeled P388D1 and B-16S in  the lysis of labeled B-16S targets. 4-h cytotoxicity assays were performed  with RNK-16 (left) or RNK-mLy-49A.9 cells (right) as effectors. B-16S  (H-2b) target cells were labeled with 51Cr and tested at an effector to labeled target ratio of 10:1. Cold targets were either unlabeled B-16S cells  (H-2b) or P388D1 cells (H-2Dd). Cold targets were added in 10-fold excess to labeled targets. 105 cold targets and 104 labeled targets were added  at the same time to 105 effectors in a total volume of 0.2 ml. Effectors  were preincubated with no antibody, F(ab′)2 anti–Ly-49A or F(ab′)2 antiNK1.1. Results are expressed as percent inhibition = (1 − [percent cytotoxicity with cold target/percent cytotoxicity without cold target]) × 100.
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Figure 9: Cold-target competition of unlabeled P388D1 and B-16S in the lysis of labeled B-16S targets. 4-h cytotoxicity assays were performed with RNK-16 (left) or RNK-mLy-49A.9 cells (right) as effectors. B-16S (H-2b) target cells were labeled with 51Cr and tested at an effector to labeled target ratio of 10:1. Cold targets were either unlabeled B-16S cells (H-2b) or P388D1 cells (H-2Dd). Cold targets were added in 10-fold excess to labeled targets. 105 cold targets and 104 labeled targets were added at the same time to 105 effectors in a total volume of 0.2 ml. Effectors were preincubated with no antibody, F(ab′)2 anti–Ly-49A or F(ab′)2 antiNK1.1. Results are expressed as percent inhibition = (1 − [percent cytotoxicity with cold target/percent cytotoxicity without cold target]) × 100.

Mentions: Initial reports demonstrated that ligation of H-2Dd targets by the Ly-49A receptor resulted in inhibition of natural killing, of antibody-dependent cellular cytoxicity (ADCC), and of lectin-induced cytotoxicity by NK cells (6, 38). These studies, performed on bulk populations of Ly-49A+ NK cells, suggested that Ly-49A might transduce signals that globally inhibit NK cell function. Using our RNK-Ly-49A transfectants, we were able to examine mouse Ly-49A function in a clonal cell population. To examine the effect of inhibitory H-2Dd targets on the lysis of labeled bystander non–H-2Dd targets, we performed cold-target competition experiments with wild-type RNK-16 or RNK-mLy-49A.9 as effectors, and 51Cr-labeled B-16S (H-2b) melanoma target cells. The effect of unlabeled H-2Dd or non–H-2Dd targets on lysis of B-16S was determined for each effector (Fig. 9). As expected, unlabeled B-16S (H-2b) were effective cold-target competitors for the lysis of 51Cr-labeled B-16S by both RNK-16 and by RNK-mLy-49A.9. Unlabeled P388D1 (H-2Dd) targets, which are sensitive to lysis by RNK-16, effectively inhibited B-16S lysis by RNK-16. In contrast, P388D1, which was not killed by RNK-mLy49A.9, was relatively ineffective as a cold-target inhibitor of B-16S lysis by RNK-mLy-49A.9. In the presence of F(ab′)2 anti–Ly-49A, P388D1 became sensitive to lysis by RNKmLy-49A.9. This allowed cold P388D1 cells to compete effectively with labeled B-16S for the lytic machinery of RNK-mLy-49A.9. Control F(ab′)2 anti-NK1.1 fragments had no effect on cold-target competition.


Mouse Ly-49A interrupts early signaling events in natural killer cell cytotoxicity and functionally associates with the SHP-1 tyrosine phosphatase.

Nakamura MC, Niemi EC, Fisher MJ, Shultz LD, Seaman WE, Ryan JC - J. Exp. Med. (1997)

Cold-target competition of unlabeled P388D1 and B-16S in  the lysis of labeled B-16S targets. 4-h cytotoxicity assays were performed  with RNK-16 (left) or RNK-mLy-49A.9 cells (right) as effectors. B-16S  (H-2b) target cells were labeled with 51Cr and tested at an effector to labeled target ratio of 10:1. Cold targets were either unlabeled B-16S cells  (H-2b) or P388D1 cells (H-2Dd). Cold targets were added in 10-fold excess to labeled targets. 105 cold targets and 104 labeled targets were added  at the same time to 105 effectors in a total volume of 0.2 ml. Effectors  were preincubated with no antibody, F(ab′)2 anti–Ly-49A or F(ab′)2 antiNK1.1. Results are expressed as percent inhibition = (1 − [percent cytotoxicity with cold target/percent cytotoxicity without cold target]) × 100.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2196152&req=5

Figure 9: Cold-target competition of unlabeled P388D1 and B-16S in the lysis of labeled B-16S targets. 4-h cytotoxicity assays were performed with RNK-16 (left) or RNK-mLy-49A.9 cells (right) as effectors. B-16S (H-2b) target cells were labeled with 51Cr and tested at an effector to labeled target ratio of 10:1. Cold targets were either unlabeled B-16S cells (H-2b) or P388D1 cells (H-2Dd). Cold targets were added in 10-fold excess to labeled targets. 105 cold targets and 104 labeled targets were added at the same time to 105 effectors in a total volume of 0.2 ml. Effectors were preincubated with no antibody, F(ab′)2 anti–Ly-49A or F(ab′)2 antiNK1.1. Results are expressed as percent inhibition = (1 − [percent cytotoxicity with cold target/percent cytotoxicity without cold target]) × 100.
Mentions: Initial reports demonstrated that ligation of H-2Dd targets by the Ly-49A receptor resulted in inhibition of natural killing, of antibody-dependent cellular cytoxicity (ADCC), and of lectin-induced cytotoxicity by NK cells (6, 38). These studies, performed on bulk populations of Ly-49A+ NK cells, suggested that Ly-49A might transduce signals that globally inhibit NK cell function. Using our RNK-Ly-49A transfectants, we were able to examine mouse Ly-49A function in a clonal cell population. To examine the effect of inhibitory H-2Dd targets on the lysis of labeled bystander non–H-2Dd targets, we performed cold-target competition experiments with wild-type RNK-16 or RNK-mLy-49A.9 as effectors, and 51Cr-labeled B-16S (H-2b) melanoma target cells. The effect of unlabeled H-2Dd or non–H-2Dd targets on lysis of B-16S was determined for each effector (Fig. 9). As expected, unlabeled B-16S (H-2b) were effective cold-target competitors for the lysis of 51Cr-labeled B-16S by both RNK-16 and by RNK-mLy-49A.9. Unlabeled P388D1 (H-2Dd) targets, which are sensitive to lysis by RNK-16, effectively inhibited B-16S lysis by RNK-16. In contrast, P388D1, which was not killed by RNK-mLy49A.9, was relatively ineffective as a cold-target inhibitor of B-16S lysis by RNK-mLy-49A.9. In the presence of F(ab′)2 anti–Ly-49A, P388D1 became sensitive to lysis by RNKmLy-49A.9. This allowed cold P388D1 cells to compete effectively with labeled B-16S for the lytic machinery of RNK-mLy-49A.9. Control F(ab′)2 anti-NK1.1 fragments had no effect on cold-target competition.

Bottom Line: We show that ligation of Ly-49A by H-2Dd inhibits early signaling events during target cell stimulation, including polyphosphoinositide turnover and tyrosine phosphorylation.Finally, we demonstrate that mutational substitution of the tyrosine within the proposed SHP-1 binding motif in Ly-49A completely abrogates inhibition of NK cell cytotoxicity through this receptor.These results demonstrate that Ly-49A interrupts early activating signals in NK cells, and that SHP-1 is an important mediator of Ly-49A function.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of California, San Francisco 94143, USA.

ABSTRACT
The lytic activity of natural killer (NK) cells is inhibited by the expression of class I major histocompatibility complex (MHC) antigens on target cells. In murine NK cells, Ly-49A mediates inhibition of cytotoxicity in response to the class I MHC antigen H-2Dd. In this report, we studied the function of mouse Ly-49A in both the rat NK cell tumor line, RNK-16, transfected with Ly-49A cDNA, and in primary NK cells. We show that ligation of Ly-49A by H-2Dd inhibits early signaling events during target cell stimulation, including polyphosphoinositide turnover and tyrosine phosphorylation. We also show that Ly-49A directly associates with the cytoplasmic tyrosine phosphatase SHP-1, and that Ly-49A function is impaired in NK cells from SHP-1 mutant viable motheaten mice and from SHP-1-deficient motheaten mice. Finally, we demonstrate that mutational substitution of the tyrosine within the proposed SHP-1 binding motif in Ly-49A completely abrogates inhibition of NK cell cytotoxicity through this receptor. These results demonstrate that Ly-49A interrupts early activating signals in NK cells, and that SHP-1 is an important mediator of Ly-49A function.

Show MeSH
Related in: MedlinePlus