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Mouse Ly-49D recognizes H-2Dd and activates natural killer cell cytotoxicity.

Nakamura MC, Linnemeyer PA, Niemi EC, Mason LH, Ortaldo JR, Ryan JC, Seaman WE - J. Exp. Med. (1999)

Bottom Line: As expected, anti- Ly-49D monoclonal antibody 12A8 specifically stimulated redirected lysis of the Fc receptor- bearing rat target YB2/0 by RNK.mLy-49D transfectants.RNK.mLy-49D cells lysed YB2/0.Dd targets more efficiently than untransfected YB2/0 or YB2/0 transfected with Db, Kk, or Kb.These experiments show that the activating receptor Ly-49D specifically interacts with the MHC I antigen, H-2Dd, demonstrating the existence of alloactivating receptors on murine NK cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of California San Francisco, San Francisco, California 94143, USA. marynak@itsa.ucsf.edu

ABSTRACT
Although activation of natural killer (NK) cytotoxicity is generally inhibited by target major histocompatibility complex (MHC) class I expression, subtle features of NK allorecognition suggest that NK cells possess receptors that are activated by target MHC I. The mouse Ly-49D receptor has been shown to activate NK cytotoxicity, although recognition of MHC class I has not been demonstrated previously. To define Ly-49D-ligand interactions, we transfected the mouse Ly-49D receptor into the rat NK line, RNK-16 (RNK.mLy-49D). As expected, anti- Ly-49D monoclonal antibody 12A8 specifically stimulated redirected lysis of the Fc receptor- bearing rat target YB2/0 by RNK.mLy-49D transfectants. RNK.mLy-49D effectors were tested against YB2/0 targets transfected with the mouse MHC I alleles H-2Dd, Db, Kk, or Kb. RNK.mLy-49D cells lysed YB2/0.Dd targets more efficiently than untransfected YB2/0 or YB2/0 transfected with Db, Kk, or Kb. This augmented lysis of H-2Dd targets was specifically inhibited by F(ab')2 anti-Ly-49D (12A8) and F(ab')2 anti-H-2Dd (34-5-8S). RNK.mLy-49D effectors were also able to specifically lyse Concanavalin A blasts isolated from H-2(d) mice (BALB/c, B10.D2, and DBA/2) but not from H-2(b) or H-2(k) mice. These experiments show that the activating receptor Ly-49D specifically interacts with the MHC I antigen, H-2Dd, demonstrating the existence of alloactivating receptors on murine NK cells.

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The α1/α2 region of  H-2Dd is required to activate mLy-49D. RNK-16 cells (A and D),  RNK.mLy-49A transfectants (B and  E), and RNK.mLy-49D transfectants (C and F) were used as effectors  against YB2/0 cells expressing chimeric class I molecules Dbα1,2Dd  (A–C) or Ddα1,2Db (D–F) as target  cells in standard 4-h cytotoxicity  assays. Effector cells were preincubated with media alone (□), anti– Ly-49D (12A8) F(ab′)2 fragments  (♦), or control (2C7) F(ab′)2 fragments (○).
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Figure 4: The α1/α2 region of H-2Dd is required to activate mLy-49D. RNK-16 cells (A and D), RNK.mLy-49A transfectants (B and E), and RNK.mLy-49D transfectants (C and F) were used as effectors against YB2/0 cells expressing chimeric class I molecules Dbα1,2Dd (A–C) or Ddα1,2Db (D–F) as target cells in standard 4-h cytotoxicity assays. Effector cells were preincubated with media alone (□), anti– Ly-49D (12A8) F(ab′)2 fragments (♦), or control (2C7) F(ab′)2 fragments (○).

Mentions: We next examined the domains of H-2Dd that are recognized by Ly-49D. To this end, we examined the lysis by RNK.mLy-49D effectors of YB2/0 cells transfected with genes in which exons for the α1, α2, and α3 domains of H-2Dd and H-2Db were recombined to create chimeric class I molecules. As shown in Fig. 4 C, the chimeric molecule Dbα1,2Dd, containing only the α3 domain of Dd, failed to activate cytotoxicity by RNK.mLy-49D cells, whereas the chimeric H-2 molecule Ddα1,2Db, which contains the α1,2 domains of Dd and the α3 domain of Db, activated cytotoxicity by these cells (panel F). Augmentation of RNK.mLy-49D lysis of Ddα1,2Db was inhibited by F(ab′)2 anti–Ly-49D, but not by control F(ab′)2 (panel F). As controls for the specific effect of the mAb on the Ly-49D receptor, lysis of targets expressing Dbα1,2Dd by either RNK-16 or RNK.mLy-49A cells was unchanged by F(ab′)2 12A8 or control F(ab′)2 (panels A and B). As we have previously shown, RNK.mLy-49A effectors were unable to lyse YB2/0 cells expressing Ddα1,2Db (29). However, lysis of this target by RNK.mLy-49A could be restored by the addition of blocking F(ab′)2 12A8 (panel E). Thus, both Ly-49D and Ly-49A bind to the α1 and α2 domains, but not the α3 domain, of H-2Dd.


Mouse Ly-49D recognizes H-2Dd and activates natural killer cell cytotoxicity.

Nakamura MC, Linnemeyer PA, Niemi EC, Mason LH, Ortaldo JR, Ryan JC, Seaman WE - J. Exp. Med. (1999)

The α1/α2 region of  H-2Dd is required to activate mLy-49D. RNK-16 cells (A and D),  RNK.mLy-49A transfectants (B and  E), and RNK.mLy-49D transfectants (C and F) were used as effectors  against YB2/0 cells expressing chimeric class I molecules Dbα1,2Dd  (A–C) or Ddα1,2Db (D–F) as target  cells in standard 4-h cytotoxicity  assays. Effector cells were preincubated with media alone (□), anti– Ly-49D (12A8) F(ab′)2 fragments  (♦), or control (2C7) F(ab′)2 fragments (○).
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Figure 4: The α1/α2 region of H-2Dd is required to activate mLy-49D. RNK-16 cells (A and D), RNK.mLy-49A transfectants (B and E), and RNK.mLy-49D transfectants (C and F) were used as effectors against YB2/0 cells expressing chimeric class I molecules Dbα1,2Dd (A–C) or Ddα1,2Db (D–F) as target cells in standard 4-h cytotoxicity assays. Effector cells were preincubated with media alone (□), anti– Ly-49D (12A8) F(ab′)2 fragments (♦), or control (2C7) F(ab′)2 fragments (○).
Mentions: We next examined the domains of H-2Dd that are recognized by Ly-49D. To this end, we examined the lysis by RNK.mLy-49D effectors of YB2/0 cells transfected with genes in which exons for the α1, α2, and α3 domains of H-2Dd and H-2Db were recombined to create chimeric class I molecules. As shown in Fig. 4 C, the chimeric molecule Dbα1,2Dd, containing only the α3 domain of Dd, failed to activate cytotoxicity by RNK.mLy-49D cells, whereas the chimeric H-2 molecule Ddα1,2Db, which contains the α1,2 domains of Dd and the α3 domain of Db, activated cytotoxicity by these cells (panel F). Augmentation of RNK.mLy-49D lysis of Ddα1,2Db was inhibited by F(ab′)2 anti–Ly-49D, but not by control F(ab′)2 (panel F). As controls for the specific effect of the mAb on the Ly-49D receptor, lysis of targets expressing Dbα1,2Dd by either RNK-16 or RNK.mLy-49A cells was unchanged by F(ab′)2 12A8 or control F(ab′)2 (panels A and B). As we have previously shown, RNK.mLy-49A effectors were unable to lyse YB2/0 cells expressing Ddα1,2Db (29). However, lysis of this target by RNK.mLy-49A could be restored by the addition of blocking F(ab′)2 12A8 (panel E). Thus, both Ly-49D and Ly-49A bind to the α1 and α2 domains, but not the α3 domain, of H-2Dd.

Bottom Line: As expected, anti- Ly-49D monoclonal antibody 12A8 specifically stimulated redirected lysis of the Fc receptor- bearing rat target YB2/0 by RNK.mLy-49D transfectants.RNK.mLy-49D cells lysed YB2/0.Dd targets more efficiently than untransfected YB2/0 or YB2/0 transfected with Db, Kk, or Kb.These experiments show that the activating receptor Ly-49D specifically interacts with the MHC I antigen, H-2Dd, demonstrating the existence of alloactivating receptors on murine NK cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of California San Francisco, San Francisco, California 94143, USA. marynak@itsa.ucsf.edu

ABSTRACT
Although activation of natural killer (NK) cytotoxicity is generally inhibited by target major histocompatibility complex (MHC) class I expression, subtle features of NK allorecognition suggest that NK cells possess receptors that are activated by target MHC I. The mouse Ly-49D receptor has been shown to activate NK cytotoxicity, although recognition of MHC class I has not been demonstrated previously. To define Ly-49D-ligand interactions, we transfected the mouse Ly-49D receptor into the rat NK line, RNK-16 (RNK.mLy-49D). As expected, anti- Ly-49D monoclonal antibody 12A8 specifically stimulated redirected lysis of the Fc receptor- bearing rat target YB2/0 by RNK.mLy-49D transfectants. RNK.mLy-49D effectors were tested against YB2/0 targets transfected with the mouse MHC I alleles H-2Dd, Db, Kk, or Kb. RNK.mLy-49D cells lysed YB2/0.Dd targets more efficiently than untransfected YB2/0 or YB2/0 transfected with Db, Kk, or Kb. This augmented lysis of H-2Dd targets was specifically inhibited by F(ab')2 anti-Ly-49D (12A8) and F(ab')2 anti-H-2Dd (34-5-8S). RNK.mLy-49D effectors were also able to specifically lyse Concanavalin A blasts isolated from H-2(d) mice (BALB/c, B10.D2, and DBA/2) but not from H-2(b) or H-2(k) mice. These experiments show that the activating receptor Ly-49D specifically interacts with the MHC I antigen, H-2Dd, demonstrating the existence of alloactivating receptors on murine NK cells.

Show MeSH
Related in: MedlinePlus