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Killer cell inhibitory receptor recognition of human leukocyte antigen (HLA) class I blocks formation of a pp36/PLC-gamma signaling complex in human natural killer (NK) cells.

Valiante NM, Phillips JH, Lanier LL, Parham P - J. Exp. Med. (1996)

Bottom Line: The killer cell inhibitory receptors (KIR) of human natural killer (NK) cells recognize human leukocyte antigen class I molecules and inhibit NK cell cytotoxicity through their interaction with protein tyrosine phosphatases (PTP).Here, we report that KIR recognition of class I ligands inhibits distal signaling events and ultimately NK cell cytotoxicity by blocking the association of an adaptor protein (pp36) with phospholipase C-gamma in NK cells.In addition, we demonstrate that pp36 can serve as a substrate in vitro for the KIR-associated PTP, PTP-1C (also called SHP-1), and that recognition of class I partially disrupts tyrosine phosphorylation of NK cell proteins, providing evidence for KIR-induced phosphatase activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Structural Biology, Stanford University Medical School, California 94305, USA.

ABSTRACT
The killer cell inhibitory receptors (KIR) of human natural killer (NK) cells recognize human leukocyte antigen class I molecules and inhibit NK cell cytotoxicity through their interaction with protein tyrosine phosphatases (PTP). Here, we report that KIR recognition of class I ligands inhibits distal signaling events and ultimately NK cell cytotoxicity by blocking the association of an adaptor protein (pp36) with phospholipase C-gamma in NK cells. In addition, we demonstrate that pp36 can serve as a substrate in vitro for the KIR-associated PTP, PTP-1C (also called SHP-1), and that recognition of class I partially disrupts tyrosine phosphorylation of NK cell proteins, providing evidence for KIR-induced phosphatase activity.

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Related in: MedlinePlus

NKB1 recognition  of Bw4+ HLA-B class I allotypes  inhibits IP3 generation and calcium flux in NK cells. (A) The  NKB1+ NK cell clone EN1.6  was labeled with [3H]myoinositol, and IP3 levels were determined at various times after challenge with different target cells:  (▪) untransfected 221 cells, (•)  HLA-B*5501 (Bw4−) transfectant and (▴) −B*5801 (Bw4+)  transfectant. Assays were performed in triplicate and results  are presented as mean cpm ±  SD. In control cell killing assays  performed at the same time, the  sensitivities of the target cells to  lysis by clone EN1.6 were the  following: untransfected 221  cells (94.9%); Bw4− transfectant  (99.6%); and Bw4+ transfectant  (6.5%). (B) Clone EN1.6 was labeled with the fluorescent calcium indicator, fluo-3. After challenge with target cells (at 60 s)  changes in fluorescence were recorded over time using a flow cytometer. Target cells were the following: untransfected 221 cells  (left); HLA-B*5501 (Bw4−) transfectant (center), and −B*5101 (Bw4+) transfectant (right). Addition (at 330 s) of a calcium ionophore (10 nM A23187) to  the NK clone challenged with Bw4+ transfectants demonstrated that increased calcium levels in the NK cells could be detected under these conditions  (right). The sensitivities of the target cells to lysis were the same as in (A) except that the HLA-B*5101 (Bw4+) transfectant was lysed by clone EN1.6 at  4.7%. (C) The NK clone JG1.6 (NKB1+) was challenged for 2 min with the indicated target cells in the presence (striped bar) or absence (solid bar) of an  anti-NKB1 antibody (mAb DX9). IP3 levels were determined for triplicate samples and the results are presented as percent increase over untreated controls + SD. The basal IP3 level (cpm) in untreated NK cells was 250 ± 26 (mean ± SD). The sensitivities of the target cells to lysis by clone JG1.6 were  the following: untransfected 221 cells 92.4% without antibody and 89.7% with mAb DX9; HLA-B*5501 (Bw4−) transfectant 93.1% without antibody  and 86.3% with mAb DX9; and HLA-B*5101 (Bw4+) transfectant 2.4% without antibody and 76.5% with mAb DX9. Control cell killing and IP3 assays  were also performed in the presence of an anti-CD56 antibody (mAb L185), which had no effect on target cell lysis or IP3 accumulation (data not  shown). (D) Fluo-3-labeled NK cells (clone PP1.13) were analyzed by flow cytometry after interaction with Bw4+ target cells with or without addition of  mAb DX9. Changes in fluorescence were recorded over time after challenge (at 60 s) with HLA-B*2705 (Bw4+) transfectants. The sensitivities of the  target cells to lysis by clone PP1.13 were the following: 1.1% without antibody and 76.9% with mAb DX9. Calcium concentration was also determined  in clone PP1.13 after challenge with untransfected target cells and Bw4− transfectants and the presence of the DX9 mAb did not alter the ability of the  NK cells to flux calcium (data not shown). As for (C), the presence of an anti-CD56 antibody did not affect target cell lysis or the accumulation of calcium (data not shown).
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Figure 1: NKB1 recognition of Bw4+ HLA-B class I allotypes inhibits IP3 generation and calcium flux in NK cells. (A) The NKB1+ NK cell clone EN1.6 was labeled with [3H]myoinositol, and IP3 levels were determined at various times after challenge with different target cells: (▪) untransfected 221 cells, (•) HLA-B*5501 (Bw4−) transfectant and (▴) −B*5801 (Bw4+) transfectant. Assays were performed in triplicate and results are presented as mean cpm ± SD. In control cell killing assays performed at the same time, the sensitivities of the target cells to lysis by clone EN1.6 were the following: untransfected 221 cells (94.9%); Bw4− transfectant (99.6%); and Bw4+ transfectant (6.5%). (B) Clone EN1.6 was labeled with the fluorescent calcium indicator, fluo-3. After challenge with target cells (at 60 s) changes in fluorescence were recorded over time using a flow cytometer. Target cells were the following: untransfected 221 cells (left); HLA-B*5501 (Bw4−) transfectant (center), and −B*5101 (Bw4+) transfectant (right). Addition (at 330 s) of a calcium ionophore (10 nM A23187) to the NK clone challenged with Bw4+ transfectants demonstrated that increased calcium levels in the NK cells could be detected under these conditions (right). The sensitivities of the target cells to lysis were the same as in (A) except that the HLA-B*5101 (Bw4+) transfectant was lysed by clone EN1.6 at 4.7%. (C) The NK clone JG1.6 (NKB1+) was challenged for 2 min with the indicated target cells in the presence (striped bar) or absence (solid bar) of an anti-NKB1 antibody (mAb DX9). IP3 levels were determined for triplicate samples and the results are presented as percent increase over untreated controls + SD. The basal IP3 level (cpm) in untreated NK cells was 250 ± 26 (mean ± SD). The sensitivities of the target cells to lysis by clone JG1.6 were the following: untransfected 221 cells 92.4% without antibody and 89.7% with mAb DX9; HLA-B*5501 (Bw4−) transfectant 93.1% without antibody and 86.3% with mAb DX9; and HLA-B*5101 (Bw4+) transfectant 2.4% without antibody and 76.5% with mAb DX9. Control cell killing and IP3 assays were also performed in the presence of an anti-CD56 antibody (mAb L185), which had no effect on target cell lysis or IP3 accumulation (data not shown). (D) Fluo-3-labeled NK cells (clone PP1.13) were analyzed by flow cytometry after interaction with Bw4+ target cells with or without addition of mAb DX9. Changes in fluorescence were recorded over time after challenge (at 60 s) with HLA-B*2705 (Bw4+) transfectants. The sensitivities of the target cells to lysis by clone PP1.13 were the following: 1.1% without antibody and 76.9% with mAb DX9. Calcium concentration was also determined in clone PP1.13 after challenge with untransfected target cells and Bw4− transfectants and the presence of the DX9 mAb did not alter the ability of the NK cells to flux calcium (data not shown). As for (C), the presence of an anti-CD56 antibody did not affect target cell lysis or the accumulation of calcium (data not shown).

Mentions: Generation of IP3 and the dependent increase in intracellular Ca++ are critical steps in the activation of NK cell cytotoxicity (7, 9). To determine whether KIR recognition of HLA class I affects generation of these second messengers, NK cell clones expressing the KIR NKB1 were challenged with untransfected target cells and transfectants expressing inhibitory (Bw4+) or permissive (Bw4−) HLA-B allotypes. The NK cells were prelabeled with [3H]myoinositol, so that after addition of target cells the kinetics of IP3 generation could be measured (Fig. 1 A). Only those target cells that were lysed by the NK cells induced accumulation of IP3, and no difference was observed between untransfected target cells and those transfected with a Bw4− HLA-B allele. In contrast, target cells transfected with a Bw4+ HLA-B allele induced no changes in intracellular IP3 levels. Analogous experiments were performed to measure increases in intracellular Ca++ concentration in NK cells labeled with the fluorescent calcium indicator, fluo-3. The results paralleled those of the experiments to measure IP3: a Ca++ flux was induced by untransfected target cells and those transfected with a Bw4− HLA-B allele, but not by a transfectant expressing a Bw4+ HLA-B molecule (Fig. 1 B). That the inhibition of IP3 and Ca++ mobilization induced by a Bw4+ HLA-B molecule was eliminated by inclusion of a mAb specific for the KIR NKB1 during stimulation (Fig. 1 C and D, respectively) demonstrates that the changes in second messengers are dependent upon interaction of KIR and HLA class I.


Killer cell inhibitory receptor recognition of human leukocyte antigen (HLA) class I blocks formation of a pp36/PLC-gamma signaling complex in human natural killer (NK) cells.

Valiante NM, Phillips JH, Lanier LL, Parham P - J. Exp. Med. (1996)

NKB1 recognition  of Bw4+ HLA-B class I allotypes  inhibits IP3 generation and calcium flux in NK cells. (A) The  NKB1+ NK cell clone EN1.6  was labeled with [3H]myoinositol, and IP3 levels were determined at various times after challenge with different target cells:  (▪) untransfected 221 cells, (•)  HLA-B*5501 (Bw4−) transfectant and (▴) −B*5801 (Bw4+)  transfectant. Assays were performed in triplicate and results  are presented as mean cpm ±  SD. In control cell killing assays  performed at the same time, the  sensitivities of the target cells to  lysis by clone EN1.6 were the  following: untransfected 221  cells (94.9%); Bw4− transfectant  (99.6%); and Bw4+ transfectant  (6.5%). (B) Clone EN1.6 was labeled with the fluorescent calcium indicator, fluo-3. After challenge with target cells (at 60 s)  changes in fluorescence were recorded over time using a flow cytometer. Target cells were the following: untransfected 221 cells  (left); HLA-B*5501 (Bw4−) transfectant (center), and −B*5101 (Bw4+) transfectant (right). Addition (at 330 s) of a calcium ionophore (10 nM A23187) to  the NK clone challenged with Bw4+ transfectants demonstrated that increased calcium levels in the NK cells could be detected under these conditions  (right). The sensitivities of the target cells to lysis were the same as in (A) except that the HLA-B*5101 (Bw4+) transfectant was lysed by clone EN1.6 at  4.7%. (C) The NK clone JG1.6 (NKB1+) was challenged for 2 min with the indicated target cells in the presence (striped bar) or absence (solid bar) of an  anti-NKB1 antibody (mAb DX9). IP3 levels were determined for triplicate samples and the results are presented as percent increase over untreated controls + SD. The basal IP3 level (cpm) in untreated NK cells was 250 ± 26 (mean ± SD). The sensitivities of the target cells to lysis by clone JG1.6 were  the following: untransfected 221 cells 92.4% without antibody and 89.7% with mAb DX9; HLA-B*5501 (Bw4−) transfectant 93.1% without antibody  and 86.3% with mAb DX9; and HLA-B*5101 (Bw4+) transfectant 2.4% without antibody and 76.5% with mAb DX9. Control cell killing and IP3 assays  were also performed in the presence of an anti-CD56 antibody (mAb L185), which had no effect on target cell lysis or IP3 accumulation (data not  shown). (D) Fluo-3-labeled NK cells (clone PP1.13) were analyzed by flow cytometry after interaction with Bw4+ target cells with or without addition of  mAb DX9. Changes in fluorescence were recorded over time after challenge (at 60 s) with HLA-B*2705 (Bw4+) transfectants. The sensitivities of the  target cells to lysis by clone PP1.13 were the following: 1.1% without antibody and 76.9% with mAb DX9. Calcium concentration was also determined  in clone PP1.13 after challenge with untransfected target cells and Bw4− transfectants and the presence of the DX9 mAb did not alter the ability of the  NK cells to flux calcium (data not shown). As for (C), the presence of an anti-CD56 antibody did not affect target cell lysis or the accumulation of calcium (data not shown).
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Figure 1: NKB1 recognition of Bw4+ HLA-B class I allotypes inhibits IP3 generation and calcium flux in NK cells. (A) The NKB1+ NK cell clone EN1.6 was labeled with [3H]myoinositol, and IP3 levels were determined at various times after challenge with different target cells: (▪) untransfected 221 cells, (•) HLA-B*5501 (Bw4−) transfectant and (▴) −B*5801 (Bw4+) transfectant. Assays were performed in triplicate and results are presented as mean cpm ± SD. In control cell killing assays performed at the same time, the sensitivities of the target cells to lysis by clone EN1.6 were the following: untransfected 221 cells (94.9%); Bw4− transfectant (99.6%); and Bw4+ transfectant (6.5%). (B) Clone EN1.6 was labeled with the fluorescent calcium indicator, fluo-3. After challenge with target cells (at 60 s) changes in fluorescence were recorded over time using a flow cytometer. Target cells were the following: untransfected 221 cells (left); HLA-B*5501 (Bw4−) transfectant (center), and −B*5101 (Bw4+) transfectant (right). Addition (at 330 s) of a calcium ionophore (10 nM A23187) to the NK clone challenged with Bw4+ transfectants demonstrated that increased calcium levels in the NK cells could be detected under these conditions (right). The sensitivities of the target cells to lysis were the same as in (A) except that the HLA-B*5101 (Bw4+) transfectant was lysed by clone EN1.6 at 4.7%. (C) The NK clone JG1.6 (NKB1+) was challenged for 2 min with the indicated target cells in the presence (striped bar) or absence (solid bar) of an anti-NKB1 antibody (mAb DX9). IP3 levels were determined for triplicate samples and the results are presented as percent increase over untreated controls + SD. The basal IP3 level (cpm) in untreated NK cells was 250 ± 26 (mean ± SD). The sensitivities of the target cells to lysis by clone JG1.6 were the following: untransfected 221 cells 92.4% without antibody and 89.7% with mAb DX9; HLA-B*5501 (Bw4−) transfectant 93.1% without antibody and 86.3% with mAb DX9; and HLA-B*5101 (Bw4+) transfectant 2.4% without antibody and 76.5% with mAb DX9. Control cell killing and IP3 assays were also performed in the presence of an anti-CD56 antibody (mAb L185), which had no effect on target cell lysis or IP3 accumulation (data not shown). (D) Fluo-3-labeled NK cells (clone PP1.13) were analyzed by flow cytometry after interaction with Bw4+ target cells with or without addition of mAb DX9. Changes in fluorescence were recorded over time after challenge (at 60 s) with HLA-B*2705 (Bw4+) transfectants. The sensitivities of the target cells to lysis by clone PP1.13 were the following: 1.1% without antibody and 76.9% with mAb DX9. Calcium concentration was also determined in clone PP1.13 after challenge with untransfected target cells and Bw4− transfectants and the presence of the DX9 mAb did not alter the ability of the NK cells to flux calcium (data not shown). As for (C), the presence of an anti-CD56 antibody did not affect target cell lysis or the accumulation of calcium (data not shown).
Mentions: Generation of IP3 and the dependent increase in intracellular Ca++ are critical steps in the activation of NK cell cytotoxicity (7, 9). To determine whether KIR recognition of HLA class I affects generation of these second messengers, NK cell clones expressing the KIR NKB1 were challenged with untransfected target cells and transfectants expressing inhibitory (Bw4+) or permissive (Bw4−) HLA-B allotypes. The NK cells were prelabeled with [3H]myoinositol, so that after addition of target cells the kinetics of IP3 generation could be measured (Fig. 1 A). Only those target cells that were lysed by the NK cells induced accumulation of IP3, and no difference was observed between untransfected target cells and those transfected with a Bw4− HLA-B allele. In contrast, target cells transfected with a Bw4+ HLA-B allele induced no changes in intracellular IP3 levels. Analogous experiments were performed to measure increases in intracellular Ca++ concentration in NK cells labeled with the fluorescent calcium indicator, fluo-3. The results paralleled those of the experiments to measure IP3: a Ca++ flux was induced by untransfected target cells and those transfected with a Bw4− HLA-B allele, but not by a transfectant expressing a Bw4+ HLA-B molecule (Fig. 1 B). That the inhibition of IP3 and Ca++ mobilization induced by a Bw4+ HLA-B molecule was eliminated by inclusion of a mAb specific for the KIR NKB1 during stimulation (Fig. 1 C and D, respectively) demonstrates that the changes in second messengers are dependent upon interaction of KIR and HLA class I.

Bottom Line: The killer cell inhibitory receptors (KIR) of human natural killer (NK) cells recognize human leukocyte antigen class I molecules and inhibit NK cell cytotoxicity through their interaction with protein tyrosine phosphatases (PTP).Here, we report that KIR recognition of class I ligands inhibits distal signaling events and ultimately NK cell cytotoxicity by blocking the association of an adaptor protein (pp36) with phospholipase C-gamma in NK cells.In addition, we demonstrate that pp36 can serve as a substrate in vitro for the KIR-associated PTP, PTP-1C (also called SHP-1), and that recognition of class I partially disrupts tyrosine phosphorylation of NK cell proteins, providing evidence for KIR-induced phosphatase activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Structural Biology, Stanford University Medical School, California 94305, USA.

ABSTRACT
The killer cell inhibitory receptors (KIR) of human natural killer (NK) cells recognize human leukocyte antigen class I molecules and inhibit NK cell cytotoxicity through their interaction with protein tyrosine phosphatases (PTP). Here, we report that KIR recognition of class I ligands inhibits distal signaling events and ultimately NK cell cytotoxicity by blocking the association of an adaptor protein (pp36) with phospholipase C-gamma in NK cells. In addition, we demonstrate that pp36 can serve as a substrate in vitro for the KIR-associated PTP, PTP-1C (also called SHP-1), and that recognition of class I partially disrupts tyrosine phosphorylation of NK cell proteins, providing evidence for KIR-induced phosphatase activity.

Show MeSH
Related in: MedlinePlus