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The Ly-49D receptor activates murine natural killer cells.

Mason LH, Anderson SK, Yokoyama WM, Smith HR, Winkler-Pickett R, Ortaldo JR - J. Exp. Med. (1996)

Bottom Line: An Ly-49D+ subset of NK cells that did not express Ly-49A, C, and G2 was isolated and examined for their functional capabilities.Furthermore, the cytoplasmic domain of Ly-49D does not contain the V/Ix-YxxL immunoreceptor tyrosine-based inhibitory motif found in Ly-49A, C, or G2 that has been characterized in the human p58 killer inhibitory receptors.Therefore, Ly-49D is the first member of the Ly-49 family characterized as transmitting positive signals to NK cells, rather than inhibiting NK cell function.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Experimental Immunology, SAIC Frederick, National Cancer Institute-FCRDC, Maryland 21702-1201, USA.

ABSTRACT
Proteins encoded by members of the Ly-49 gene family are predominantly expressed on murine natural killer (NK) cells. Several members of this gene family have been demonstrated to inhibit NK cell lysis upon recognizing their class I ligands on target cells. In this report, we present data supporting that not all Ly-49 proteins inhibit NK cell function. Our laboratory has generated and characterized a monoclonal antibody (mAb) (12A8) that can be used to recognize the Ly-49D subset of murine NK cells. Transfection of Cos-7 cells with known members of the Ly-49 gene family revealed that 12A8 recognizes Ly-49D, but also cross-reacts with the Ly-49A protein on B6 NK cells. In addition, 12A8 demonstrates reactivity by both immuno-precipitation and two-color flow cytometry analysis with an NK cell subset that is distinct from those expressing Ly-49A, C, or G2. An Ly-49D+ subset of NK cells that did not express Ly-49A, C, and G2 was isolated and examined for their functional capabilities. Tumor targets and concanovalin A (ConA) lymphoblasts from a variety of H2 haplotypes were examined for their susceptibility to lysis by Ly-49D+ NK cells. None of the major histocompatibility complex class I-bearing targets inhibited lysis of Ly-49D+ NK cells. More importantly, we demonstrate that the addition of mAb 12A8 to Ly-49D+ NK cells can augment lysis of Fc gamma R+ target cells in a reverse antibody-dependent cellular cytotoxicity-type assay and induces apoptosis in Ly-49D+ NK cells. Furthermore, the cytoplasmic domain of Ly-49D does not contain the V/Ix-YxxL immunoreceptor tyrosine-based inhibitory motif found in Ly-49A, C, or G2 that has been characterized in the human p58 killer inhibitory receptors. Therefore, Ly-49D is the first member of the Ly-49 family characterized as transmitting positive signals to NK cells, rather than inhibiting NK cell function.

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mAb 12A8 mediates RADCC of FcγR+ target cells by Ly-49D+, but not Ly-49D−, NK cells. Splenic NK cells were depleted of cells expressing Ly-49A, C, and G2. Ly-49D+ and Ly-49D− cells were sorted (using biotinylated 12A8 followed by streptavidin PE) into populations that were  >98% 12A8+ (Ly-49D+) or 12A8− (Ly-49D−). These subsets were cultured in high dose IL-2 for 9 d, washed, and tested against the indicated targets in  a 4-h 51Cr-release assay at E/T ratios starting at 10:1. mAbs were added at a final concentration of 2 μg/well. Data are presented as lytic units per 107 cells  at 30% lysis. This experiment is one of at least three representative assays (NT, not tested). Both the Ly-49D+ and Ly-49D− populations were <5% Ly49A, C, and G2+ at the time of testing.
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Figure 5: mAb 12A8 mediates RADCC of FcγR+ target cells by Ly-49D+, but not Ly-49D−, NK cells. Splenic NK cells were depleted of cells expressing Ly-49A, C, and G2. Ly-49D+ and Ly-49D− cells were sorted (using biotinylated 12A8 followed by streptavidin PE) into populations that were >98% 12A8+ (Ly-49D+) or 12A8− (Ly-49D−). These subsets were cultured in high dose IL-2 for 9 d, washed, and tested against the indicated targets in a 4-h 51Cr-release assay at E/T ratios starting at 10:1. mAbs were added at a final concentration of 2 μg/well. Data are presented as lytic units per 107 cells at 30% lysis. This experiment is one of at least three representative assays (NT, not tested). Both the Ly-49D+ and Ly-49D− populations were <5% Ly49A, C, and G2+ at the time of testing.

Mentions: Since Ly-49D+ NK cells failed to exhibit any pattern of class I restriction, we decided to examine mAb 12A8 for its ability to activate the lytic potential of Ly-49D+ NK cells. A panel of different tumor target cells, including both FcγR+ and FcγR− targets, were examined for susceptibility to lysis by Ly-49D+ and Ly-49D− NK cells in the presence of various antibodies. Fig. 5 presents the data obtained from a representative redirected lysis (RADCC) assay. The addition of mAb 12A8 to Ly-49D+, but not Ly-49D−, NK cells, results in augmented lysis of FcγR+ target cells. Both P815 (Fig. 5 A) and WEHI-3 (Fig. 5 B) targets (FcγR+) are lysed much more efficiently in the presence of mAb 12A8 when compared to either media alone, a negative control mAb (Rγ2A), or mAbs reactive with NK cells (e.g., RM21 and 2.4G2). This RADCC effect has been reported for mAb PK136, which detects the NK-1.1 molecule (17), and can be blocked by the addition of mAb 2.4G2, which is specific for FcγRII/III (18). As a further control in these experiments, it can be seen that mAb PK136 is capable of augmenting the lysis of both the Ly49D+ and D− subsets (>95% NK-1.1+) against the FcγR+ targets P815 and WEHI-3. The Ly-49D− subset, however, is not augmented in the presence of mAb 12A8 to lyse these same targets. These results also reveal that the augmented lysis observed in the presence of mAb 12A8 can be abrogated upon addition of mAb 2.4G2 (identical to that seen using PK136 + 2.4G2). Similar results have been obtained upon pretreatment and washing of Ly-49D+ NK cells with mAb 12A8 before performing cytotoxicity assays (data not shown). FcγR− target cells such as SST (Fig. 5 C) and WEHI 164 (Fig. 5 D) do not demonstrate any significant augmentation of lysis by Ly-49D+ NK cells in the presence of mAb 12A8. The data shown in Fig. 5 (C) shows that mAb 12A8 weakly augments killing by Ly-49D+ NK cells against the SST target. This finding has not been reproducible in other experiments using this target and was not considered significant. Based on the above experiments, we conclude that mAb 12A8 engagement of the Ly-49D molecule augments the lytic function of NK cells. These results establish for the first time that a member of the Ly-49 gene family may upregulate NK lytic function upon binding the appropriate ligand.


The Ly-49D receptor activates murine natural killer cells.

Mason LH, Anderson SK, Yokoyama WM, Smith HR, Winkler-Pickett R, Ortaldo JR - J. Exp. Med. (1996)

mAb 12A8 mediates RADCC of FcγR+ target cells by Ly-49D+, but not Ly-49D−, NK cells. Splenic NK cells were depleted of cells expressing Ly-49A, C, and G2. Ly-49D+ and Ly-49D− cells were sorted (using biotinylated 12A8 followed by streptavidin PE) into populations that were  >98% 12A8+ (Ly-49D+) or 12A8− (Ly-49D−). These subsets were cultured in high dose IL-2 for 9 d, washed, and tested against the indicated targets in  a 4-h 51Cr-release assay at E/T ratios starting at 10:1. mAbs were added at a final concentration of 2 μg/well. Data are presented as lytic units per 107 cells  at 30% lysis. This experiment is one of at least three representative assays (NT, not tested). Both the Ly-49D+ and Ly-49D− populations were <5% Ly49A, C, and G2+ at the time of testing.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2196364&req=5

Figure 5: mAb 12A8 mediates RADCC of FcγR+ target cells by Ly-49D+, but not Ly-49D−, NK cells. Splenic NK cells were depleted of cells expressing Ly-49A, C, and G2. Ly-49D+ and Ly-49D− cells were sorted (using biotinylated 12A8 followed by streptavidin PE) into populations that were >98% 12A8+ (Ly-49D+) or 12A8− (Ly-49D−). These subsets were cultured in high dose IL-2 for 9 d, washed, and tested against the indicated targets in a 4-h 51Cr-release assay at E/T ratios starting at 10:1. mAbs were added at a final concentration of 2 μg/well. Data are presented as lytic units per 107 cells at 30% lysis. This experiment is one of at least three representative assays (NT, not tested). Both the Ly-49D+ and Ly-49D− populations were <5% Ly49A, C, and G2+ at the time of testing.
Mentions: Since Ly-49D+ NK cells failed to exhibit any pattern of class I restriction, we decided to examine mAb 12A8 for its ability to activate the lytic potential of Ly-49D+ NK cells. A panel of different tumor target cells, including both FcγR+ and FcγR− targets, were examined for susceptibility to lysis by Ly-49D+ and Ly-49D− NK cells in the presence of various antibodies. Fig. 5 presents the data obtained from a representative redirected lysis (RADCC) assay. The addition of mAb 12A8 to Ly-49D+, but not Ly-49D−, NK cells, results in augmented lysis of FcγR+ target cells. Both P815 (Fig. 5 A) and WEHI-3 (Fig. 5 B) targets (FcγR+) are lysed much more efficiently in the presence of mAb 12A8 when compared to either media alone, a negative control mAb (Rγ2A), or mAbs reactive with NK cells (e.g., RM21 and 2.4G2). This RADCC effect has been reported for mAb PK136, which detects the NK-1.1 molecule (17), and can be blocked by the addition of mAb 2.4G2, which is specific for FcγRII/III (18). As a further control in these experiments, it can be seen that mAb PK136 is capable of augmenting the lysis of both the Ly49D+ and D− subsets (>95% NK-1.1+) against the FcγR+ targets P815 and WEHI-3. The Ly-49D− subset, however, is not augmented in the presence of mAb 12A8 to lyse these same targets. These results also reveal that the augmented lysis observed in the presence of mAb 12A8 can be abrogated upon addition of mAb 2.4G2 (identical to that seen using PK136 + 2.4G2). Similar results have been obtained upon pretreatment and washing of Ly-49D+ NK cells with mAb 12A8 before performing cytotoxicity assays (data not shown). FcγR− target cells such as SST (Fig. 5 C) and WEHI 164 (Fig. 5 D) do not demonstrate any significant augmentation of lysis by Ly-49D+ NK cells in the presence of mAb 12A8. The data shown in Fig. 5 (C) shows that mAb 12A8 weakly augments killing by Ly-49D+ NK cells against the SST target. This finding has not been reproducible in other experiments using this target and was not considered significant. Based on the above experiments, we conclude that mAb 12A8 engagement of the Ly-49D molecule augments the lytic function of NK cells. These results establish for the first time that a member of the Ly-49 gene family may upregulate NK lytic function upon binding the appropriate ligand.

Bottom Line: An Ly-49D+ subset of NK cells that did not express Ly-49A, C, and G2 was isolated and examined for their functional capabilities.Furthermore, the cytoplasmic domain of Ly-49D does not contain the V/Ix-YxxL immunoreceptor tyrosine-based inhibitory motif found in Ly-49A, C, or G2 that has been characterized in the human p58 killer inhibitory receptors.Therefore, Ly-49D is the first member of the Ly-49 family characterized as transmitting positive signals to NK cells, rather than inhibiting NK cell function.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Experimental Immunology, SAIC Frederick, National Cancer Institute-FCRDC, Maryland 21702-1201, USA.

ABSTRACT
Proteins encoded by members of the Ly-49 gene family are predominantly expressed on murine natural killer (NK) cells. Several members of this gene family have been demonstrated to inhibit NK cell lysis upon recognizing their class I ligands on target cells. In this report, we present data supporting that not all Ly-49 proteins inhibit NK cell function. Our laboratory has generated and characterized a monoclonal antibody (mAb) (12A8) that can be used to recognize the Ly-49D subset of murine NK cells. Transfection of Cos-7 cells with known members of the Ly-49 gene family revealed that 12A8 recognizes Ly-49D, but also cross-reacts with the Ly-49A protein on B6 NK cells. In addition, 12A8 demonstrates reactivity by both immuno-precipitation and two-color flow cytometry analysis with an NK cell subset that is distinct from those expressing Ly-49A, C, or G2. An Ly-49D+ subset of NK cells that did not express Ly-49A, C, and G2 was isolated and examined for their functional capabilities. Tumor targets and concanovalin A (ConA) lymphoblasts from a variety of H2 haplotypes were examined for their susceptibility to lysis by Ly-49D+ NK cells. None of the major histocompatibility complex class I-bearing targets inhibited lysis of Ly-49D+ NK cells. More importantly, we demonstrate that the addition of mAb 12A8 to Ly-49D+ NK cells can augment lysis of Fc gamma R+ target cells in a reverse antibody-dependent cellular cytotoxicity-type assay and induces apoptosis in Ly-49D+ NK cells. Furthermore, the cytoplasmic domain of Ly-49D does not contain the V/Ix-YxxL immunoreceptor tyrosine-based inhibitory motif found in Ly-49A, C, or G2 that has been characterized in the human p58 killer inhibitory receptors. Therefore, Ly-49D is the first member of the Ly-49 family characterized as transmitting positive signals to NK cells, rather than inhibiting NK cell function.

Show MeSH
Related in: MedlinePlus