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. 2002 Jun 25;99(13):8826-31.
doi: 10.1073/pnas.092258599. Epub 2002 Jun 11.

Recognition of a virus-encoded ligand by a natural killer cell activation receptor

Hamish R C Smith  1 , Jonathan W Heusel, Indira K Mehta, Sungjin Kim, Brigitte G Dorner, Olga V Naidenko, Koho Iizuka, Hiroshi Furukawa, Diana L Beckman, Jeanette T Pingel, Anthony A Scalzo, Daved H Fremont, Wayne M Yokoyama
Affiliations

Recognition of a virus-encoded ligand by a natural killer cell activation receptor

Hamish R C Smith et al. Proc Natl Acad Sci U S A. .

Abstract

Natural killer (NK) cells express inhibitory and activation receptors that recognize MHC class I-like molecules on target cells. These receptors may be involved in the critical role of NK cells in controlling initial phases of certain viral infections. Indeed, the Ly49H NK cell activation receptor confers in vivo genetic resistance to murine cytomegalovirus (MCMV) infections, but its ligand was previously unknown. Herein, we use heterologous reporter cells to demonstrate that Ly49H recognizes MCMV-infected cells and a ligand encoded by MCMV itself. Exploiting a bioinformatics approach to the MCMV genome, we find at least 11 ORFs for molecules with previously unrecognized features of predicted MHC-like folds and limited MHC sequence homology. We identify one of these, m157, as the ligand for Ly49H. m157 triggers Ly49H-mediated cytotoxicity, and cytokine and chemokine production by freshly isolated NK cells. We hypothesize that the other ORFs with predicted MHC-like folds may be involved in immune evasion or interactions with other NK cell receptors.

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Figures

Figure 1
Figure 1
Specific activation of Ly49H-expressing reporter cells by MCMV-infected cells. (A) Schematic illustration of retroviral vector constructs and the HD12 reporter assay. cDNA cassettes for murine DAP12, Ly49H, or Ly49D were cloned into the multiple cloning site (MCS) upstream from the internal ribosomal entry site (IRES) of pMX-IRES-GFP to generate the control vectors DAP12-GFP, H-GFP, and D-GFP. The GFP cassette in the downstream position was replaced with either a wild-type or ITAM-deficient mutant murine DAP12 cassette to generate the pMX-HD12, pMX-HD12.Y2F, pMX-DD12, and pMX-DD12.Y2F expression vectors. Individual constructs were transfected into BWZ cells. For example, the pMX-HD12 construct was used to produce HD12 indicator cells in which Ly49H is associated with DAP12 (KARAP) that contains ITAMs (boxes). Activation through Ly49H and DAP12 leads to nuclear factor of activated T cells (NFAT)-induced production of β-gal. (B) Chlorophenol red β-d-galactoside analysis of BWZ.36 reporter lines after coculture with mock- or MCMV-infected primary bone marrow macrophages, as indicated. Ly49D-expressing lines (DD12 and DD12.Y2F) were additionally tested with CHO cells as indicated. For HD12 and DD12 lines, the effect of addition of mAbs 3D10 (anti-Ly49H) or 4E4 (anti-Ly49D) are indicated (arrows). Data are expressed as a percentage of maximal β−gal induction by phorbol 12-myristate 13-acetate (PMA) + ionomycin for each reporter-stimulator combination. (C) X-Gal staining of the indicated BWZ reporter lines after coculture with mock- or MCMV-infected IC-21 cells. (Insets) Maximal β-gal induction after stimulation with PMA + ionomycin. The Ly49D-expressing DD12 and DD12.Y2F lines were additionally stimulated with CHO cells, known to express a cognate ligand for Ly49D (24).
Figure 2
Figure 2
Recognition of m157 by Ly49H. (A) Specific activation of HD12 cells by m157 transfectants. HD12 or DD12 cells were incubated with C1498 cells transfected with m157, or control m158, as indicated. (B) Specific blockade of m157 activation of HD12 by anti-Ly49H mAb. HD12 cells were incubated with J774 cells transfected with GFP or m157, in the presence of anti-Ly49H or isotype control mAb, as indicated. (Inset) Maximal β-gal induction with PMA + ionomycin. (C) Ly49H+ NK cells kill MCMV-infected C57BL/6 BM macrophages and BaF3-m157 transfectants. A standard 4-h 51Cr-release assay was performed with Ly49H sorted LAK cells from C57BL/6 mice. Ly49H+ (filled symbols) or Ly49H− (open symbols) LAKs were incubated with indicated targets at various effector-to-target (E:T) ratios in media alone (squares) or with the anti-Ly49H mAb 3D10 (diamonds) or isotype control (circles). In the BM macrophage experiments, F(ab′)2 fragments were used to avoid redirected lysis whereas whole antibodies were used for other panels. The isotype control for BM macrophage experiments was mAb B8-24-3, and mAb 9E10 was used as isotype control in the remaining experiments. The lysis of uninfected BM macrophages is represented by a triangle.
Figure 3
Figure 3
Interaction of Ly49H with m157 induces production of IFN-γ and ATAC by fresh NK cells. Freshly isolated splenocytes from B6.RAG-1-deficient mice were cocultured with (A) uninfected or MCMV-infected IC-21 cells, (B) parental BaF3 cells or BaF3 cells transfected with either m157 or GFP, (C) BaF3 cells transfected with m157, and (D) BaF3 cells transfected with m157 or MCMV-infected IC-21 cells. All cocultures were performed at effector-to-target ratio of 1:1. Where indicated, F(ab′)2 fragments of anti-Ly49H or anti-Ly49D were added during incubation. Six to 8 h later, cells were stained for intracellular IFN-γ (A, B, and C) or ATAC (D) after cell surface marker staining for NK1.1 and Ly49H or Ly49D. As controls, profiles of cytokine production by NK cells in the absence of target cells are shown (w/o target). Gated NK1.1+ cells are shown.
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