Natural Cell Mediated Immunity Mechanism of Lysis

Ortaldo, J R.

Abstract

Members of the Ly-49 gene family encode type-II integral transmembrane proteins and are primarily expressed on the surface of murine NK cells. Our monoclonal antibody (4D11) reacts with Ly-49G2 represents a type II transmembrane protein of 268 amino acids with its carboxyl end exposed extracellularly. Ly-49G2+ cells failed to lyse several H-2Dd expressing tumor targets and Con A lymphoblasts from BALB/c, but not C57BL/6 mice. This inhibition of lysis by Ly-49G2+ NK cells was negated by addition of monoclonal antibody (mAb) 4D11 or anti-class I antibodies to Dd or Ld. Our recent studies have generated and characterized a mAb (12A8) that can recognize the Ly-49D subset of murine NK cells. Ly-49D+ subset of NK cells that did not express Ly-49A, C, and G2 failed to demonstrate an inhibitory pattern when tested on targets [tumor and lymphoblast] expressing a variety of H-2 haplotypes. More importantly we demonstrate that the addition of mAb 12A8 to Ly-49D+ NK cells can augment lysis of Fc-gammaR+ target cells in a RADCC-type assay and induces apoptosis in Ly-49D+ NK cells. Examination of the cytoplasmic domain of Ly-49D indicates that it does not contain the V/IxYxxL ITIM motif found in Ly-49A, C or G2 that has been characterized in the human p58 KIRs. 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. These studies are critical to the study of the structure/function of the various Ly-49 receptors. We have cloned and analyzed a 7 kB mRNA in NK cells that has been termed the NK-TR. Antibodies to the NK-TR or its peptides have indicated a role for this molecule in the cytolytic process of NK cells. Direct evidence for the importance of NK-TR in NK killing was obtained by generating antisense NK-TR transfectants of the rat NK-cell line, RNK-16. Introduction of a cDNA vector producing antisense NK-TR mRNA abolished the ability of these cells to kill several tumor targets as well as virus-infected [NK-sensitive] cell lines. Lectin-mediated killing ADCC and reverse-ADCC were unaffected by antisense expression. We are currently determining if there are alterations in kinase activity in the antisense transfectants in order to identify potential lesions in the normal signal transduction pathway.

Funding Agency

• Agency: National Institute of Health (NIH)
• Institute: National Cancer Institute (NCI)
• Type: Intramural Research (Z01)
• Project #: 1Z01BC009247-16
• Application #: 2463756
• Study Section: Special Emphasis Panel (LEI)
• Support Year: 16
• Fiscal Year: 1996

Institution

• Name: National Cancer Institute Division of Basic Sciences
• Country: United States

Publications

Ortaldo, John R; Winkler-Pickett, Robin T; Bere Jr, Earl W et al. (2005) In vivo hydrodynamic delivery of cDNA encoding IL-2: rapid, sustained redistribution, activation of mouse NK cells, and therapeutic potential in the absence of NKT cells. J Immunol 175:693-9

Ortaldo, John R; Young, Howard A (2005) Mouse Ly49 NK receptors: balancing activation and inhibition. Mol Immunol 42:445-50

Ortaldo, John R; Young, Howard A; Winkler-Pickett, Robin T et al. (2004) Dissociation of NKT stimulation, cytokine induction, and NK activation in vivo by the use of distinct TCR-binding ceramides. J Immunol 172:943-53

Mason, Llewellyn H; Willette-Brown, Jami; Anderson, Stephen K et al. (2003) Receptor glycosylation regulates Ly-49 binding to MHC class I. J Immunol 171:4235-42

Ortaldo, John R; Young, Howard A (2003) Expression of IFN-gamma upon triggering of activating Ly49D NK receptors in vitro and in vivo: costimulation with IL-12 or IL-18 overrides inhibitory receptors. J Immunol 170:1763-9

Raziuddin, A; Bennett, M; Winkler-Pickett, R et al. (2000) Synergistic effects of in vivo depletion of Ly-49A and Ly-49G2 natural killer cell subsets in the rejection of H2(b) bone marrow cell allografts. Blood 95:3840-4

Peacock, C D; Lin, M Y; Ortaldo, J R et al. (2000) The virus-specific and allospecific cytotoxic T-lymphocyte response to lymphocytic choriomeningitis virus is modified in a subpopulation of CD8(+) T cells coexpressing the inhibitory major histocompatibility complex class I receptor Ly49G2. J Virol 74:7032-8

Mason, L H; Willette-Brown, J; Mason, A T et al. (2000) Interaction of Ly-49D+ NK cells with H-2Dd target cells leads to Dap-12 phosphorylation and IFN-gamma secretion. J Immunol 164:603-11

Bosco, M C; Curiel, R E; Zea, A H et al. (2000) IL-2 signaling in human monocytes involves the phosphorylation and activation of p59hck. J Immunol 164:4575-85

Ortaldo, J R; Winkler-Pickett, R; Wiegand, G (2000) Activating Ly-49D NK receptors: expression and function in relation to ontogeny and Ly-49 inhibitor receptors. J Leukoc Biol 68:748-56