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.

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.

Ly49H(+)NK1.1(+) natural killer (NK) cells are essential for the control of murine cytomegalovirus (MCMV) during the acute stage of infection. This cell subset expands at the later stages of infection in an MCMV-specific fashion. Here we demonstrate a critical interaction between Ly49H(+) NK cells and CD8alpha(+) dendritic cells (DCs) whereby the presence of Ly49H(+) NK cells results in maintenance of CD8alpha(+) DCs in the spleen during acute MCMV infection. Reciprocally, CD8alpha(+) DCs are essential for the expansion of Ly49H(+) NK cells by a mechanism involving interleukin 18 (IL-18) and IL-12. This study provides evidence for a functional interrelationship between DCs and NK cells during viral infection and defines some of the critical cytokines.

Effective immunity requires the coordinated activation of innate and adaptive immune responses. Natural killer (NK) cells are central innate immune effectors, but can also affect the generation of acquired immune responses to viruses and malignancies. How NK cells influence the efficacy of adaptive immunity, however, is poorly understood. Here, we show that NK cells negatively regulate the duration and effectiveness of virus-specific CD4+ and CD8+ T cell responses by limiting exposure of T cells to infected antigen-presenting cells. This impacts the quality of T cell responses and the ability to limit viral persistence. Our studies provide unexpected insights into novel interplays between innate and adaptive immune effectors, and define the critical requirements for efficient control of viral persistence.

The genetic basis of the control of acute splenic MCMV infection was studied after intraperitoneal inoculation of the virus. Classical Mendelian analyses using C57BL/6 (resistant) and BALB/c (susceptible) parental strains disclosed an autosomal dominant non-H-2 gene that regulates splenic virus replication. The probable location of this gene, to which we have assigned the symbol Cmv-1, is on chromosome 6 as defined by the strain distribution pattern of splenic MCMV replication in CXB recombinant inbred mice. Although there is a similar hierarchy of resistance to MCMV and HSV-1 with respect to the C57BL and BALB genetic backgrounds, the strain distribution pattern of HSV-1 replication in recombinant inbred mice suggests that Cmv-1 is not involved in restricting the spread of this virus. This is the first clear identification of a non-H-2 gene regulating the magnitude of MCMV infection. Elucidation of the function of this gene may be a fundamental step towards understanding the control of systemic CMV infection.

CD1d-restricted Vα14-Jα281 invariant αβTCR+ (NKT) cells are well defined in the C57BL/6 mouse strain, but they remain poorly characterized in non-NK1.1-expressing strains. Surrogate markers for NKT cells such as αβTCR+CD4−CD8− and DX5+CD3+ have been used in many studies, although their effectiveness in defining this lineage remains to be verified. Here, we compare NKT cells among C57BL/6, NK1.1-congenic BALB/c, and NK1.1-congenic nonobese diabetic mice. NKT cells were identified and compared using a range of approaches: NK1.1 expression, surrogate phenotypes used in previous studies, labeling with CD1d/α-galactosylceramide tetramers, and cytokine production. Our results demonstrate that NKT cells and their CD4/CD8-defined subsets are present in all three strains, and confirm that nonobese diabetic mice have a numerical and functional deficiency in these cells. We also highlight the hazards of using surrogate phenotypes, none of which accurately identify NKT cells, and one in particular (DX5+CD3+) actually excludes these cells. Finally, our results support the concept that NK1.1 expression may not be an ideal marker for CD1d-restricted NKT cells, many of which are NK1.1-negative, especially within the CD4+ subset and particularly in NK1.1-congenic BALB/c mice.

Effective natural killer (NK) cell recognition of murine cytomegalovirus (MCMV)-infected cells depends on binding of the Ly49H NK cell activation receptor to the m157 viral glycoprotein. Here we addressed the immunological consequences of variation in m157 sequence and function. We found that most strains of MCMV possess forms of m157 that evade Ly49H-dependent NK cell activation. Importantly, repeated passage of MCMV through resistant Ly49H ؉ mice resulted in the rapid emergence of m157 mutants that elude Ly49H-dependent NK cell responses. These data provide the first molecular evidence that NK cells can exert sufficient immunological pressure on a DNA virus, such that it undergoes rapid and specific mutation in an NK cell ligand enabling it to evade efficient NK cell surveillance.

The susceptibility of certain inbred mouse strains to murine cytomegalovirus (MCMV) is related to their inability to generate a strong natural killer (NK) cell response. We addressed here whether the MCMV susceptibility of the BALB/c strain is due to viral functions that control NK cell activation in a strain-specific manner. MCMV expresses two proteins, gp48 and gp40, that are encoded by the genes m06 and m152, respectively; they down-regulate major histocompatibility complex (MHC) class I expression at the plasma membrane. Using MCMV deletion mutants and revertants, we found that gp40 but not gp48 controls NK cell activation. Absence of gp40 improved antiviral NK cell control in BALB/c, but not C57BL/6, mice. Down-regulation of H-60, the high-affinity ligand for the NKG2D receptor, was the mechanism by which gp40 modulates NK cell activation. Thus, a single herpesvirus protein has a dual function in inhibiting both the adaptive as well as the innate immune response.