Vital involvement of a natural killer cell activation receptor in resistance to viral infection.

Natural killer (NK) cells are lymphocytes that can be distinguished from T and B cells through their involvement in innate immunity and their lack of rearranged antigen receptors. Although NK cells and their receptors were initially characterized in terms of tumor killing in vitro, we have determined that the NK cell activation receptor, Ly-49H, is critically involved in resistance to murine cytomegalovirus in vivo. Ly-49H requires an immunoreceptor tyrosine-based activation motif (ITAM)-containing transmembrane molecule for expression and signal transduction. Thus, NK cells use receptors functionally resembling ITAM-coupled T and B cell antigen receptors to provide vital innate host defense.

Inducible expression of the gp49B inhibitory receptor on NK cells.

Murine NK cells express inhibitory receptors belonging to the C-type lectin-like (Ly-49, CD94/NKG2) and Ig superfamily-related (gp49) receptors. The murine gp49B receptor displays structural homology with human killer inhibitory receptors, and was previously identified to be a receptor on mast cells and activated NK cells. The gp49B receptor is highly related to gp49A, a receptor with unknown function. In this study, using a novel mAb produced against soluble gp49B molecules that cross-reacts with gp49A, we examined the cellular distribution and function of these receptors. gp49 is constitutively expressed on cells of the myeloid lineage throughout development, as well as on mature cells. Importantly, gp49 is not expressed on spleen- and liver-derived lymphocytes, including NK cells, but its expression is induced in vitro on NK cells following IL-2 stimulation, or in vivo by infection with murine CMV. Molecular studies revealed that both the immunoreceptor tyrosine-based inhibitory motif-containing gp49B as well as immunoreceptor tyrosine-based inhibitory motif-less gp49A receptors are up-regulated on NK cells following murine CMV infection. When co-cross-linked with NK1.1, gp49B can inhibit NK1.1-mediated cytokine release by NK cells. Taken together, these studies demonstrate that the expression of gp49B on NK cells is regulated, providing the first example of an in vivo activation-induced NK cell inhibitory receptor, in contrast to the constitutively expressed Ly49 family.

Analysis of in situ NK cell responses during viral infection.

NK cells are required for early control of murine CMV (MCMV) infection, but the distribution of murine NK cells in situ has not been clearly defined. We tested the reactivity of all available NK cell receptor-specific mAbs by immunohistochemistry. Only one mAb, 4D11 (anti-Ly-49G2), was reactive with C57BL/6 tissue sections. mAb 4D11-reactive cells expressed the nuclear morphology and flow cytometric profile of NK cells. In lymphoid organs, NK cells were distributed primarily in the splenic red pulp, between adjacent lobes in lymph node and randomly in the cortex and medulla of the thymus. No NK cells were detected in normal liver sections. Two days following MCMV infection, most splenic NK cells were associated with the lymphoid follicles and marginal zone. By day 3 following infection, the number of liver NK cells had increased significantly and the cells were detected within inflammatory foci. These changes were independent of IL-12, IFN-gamma, and TNF-alpha, as assessed in mice with targeted mutations. Concurrent immunostaining for NK cells and viral Ags revealed close association of NK cells and MCMV-infected cells in the spleen and liver. Similar results were obtained in CD1(-/-) and recombination activation gene-1(-/-) mice lacking NK T or T and B cells, respectively, indicating specificity of staining for NK cells. Thus, following MCMV infection, NK cells accumulate at sites of viral replication in an IL-12-, IFN-gamma-, and TNF-alpha-independent manner.

Specific and nonspecific NK cell activation during virus infection.

The natural killer (NK) cell activation receptor Ly49H is required for resistance to murine cytomegalovirus (MCMV). We show here that NK cell proliferation and production of interferon-gamma (IFN-gamma) was not dependent on Ly49H expression during early MCMV infection. During a later phase of infection, however, Ly49H+ NK cells selectively proliferated and this expansion was blocked by anti-Ly49H administration. With vaccinia virus infection, neither the early nor late phase of NK cell proliferation was selective for Ly49H+ NK cells. These findings indicated that Ly49H+ NK cells were specifically activated by MCMV and that MCMV infection was characterized by nonspecific and specific phases of NK cell activation in vivo.