MyD88 is an essential regulator of NK cell-mediated clearance of MCMV infection

Abstract Natural killer (NK) cells are innate immune cells that express members of the leukocyte β2 integrin family in humans and mice. These CD11/CD18 heterodimers play critical roles in leukocyte trafficking, immune synapse formation, and costimulation. The cell-surface expression of one of these integrins, CD11b/CD18, is also recognized as a major marker of mouse NK-cell maturation, but its function on NK cells has been largely ignored. Using N-ethyl-N-nitrosourea (ENU) mutagenesis, we generated a mouse carrying an A → T transverse mutation in the Itgb2 gene, resulting in a mutation that prevented the cell-surface expression of CD18 and its associated CD11a, CD11b, and CD11c proteins. We show that β2 integrin–deficient NK cells have a hyporesponsive phenotype in vitro, and present an alteration of their in vivo developmental program characterized by a selective accumulation of c-kit+ cells. NK-cell missing-self recognition was partially altered in vivo, whereas the early immune response to mouse cytomegalovirus (MCMV) infection occurred normally in CD18-deficient mice. Therefore, β2 integrins are required for optimal NK-cell maturation, but this deficiency is partial and can be bypassed during MCMV infection, highlighting the robustness of antiviral protective responses.

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NK cell activation through the NKG2D ligand MULT-1 is selectively prevented by the glycoprotein encoded by mouse cytomegalovirus gene m145

Journal of Experimental Medicine ◽

10.1084/jem.20041617 ◽

2005 ◽

Vol 201 (2) ◽

pp. 211-220 ◽

Cited By ~ 115

Author(s):

Astrid Krmpotic ◽

Milena Hasan ◽

Andrea Loewendorf ◽

Tanja Saulig ◽

Anne Halenius ◽

...

Keyword(s):

Cell Activation ◽

Nk Cell ◽

Surface Expression ◽

Viral Gene ◽

Nkg2d Ligand ◽

Mouse Cytomegalovirus ◽

Mcmv Infection ◽

Viral Survival ◽

Necessary And Sufficient

The NK cell–activating receptor NKG2D interacts with three different cellular ligands, all of which are regulated by mouse cytomegalovirus (MCMV). We set out to define the viral gene product regulating murine UL16-binding protein-like transcript (MULT)-1, a newly described NKG2D ligand. We show that MCMV infection strongly induces MULT-1 gene expression, but surface expression of this glycoprotein is nevertheless completely abolished by the virus. Screening a panel of MCMV deletion mutants defined the gene m145 as the viral regulator of MULT-1. The MCMV m145-encoded glycoprotein turned out to be necessary and sufficient to regulate MULT-1 by preventing plasma membrane residence of MULT-1. The importance of MULT-1 in NK cell regulation in vivo was confirmed by the attenuating effect of the m145 deletion that was lifted after NK cell depletion. Our findings underline the significance of escaping MULT-1/NKG2D signaling for viral survival and maintenance.

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Ly49P recognition of cytomegalovirus-infected cells expressing H2-Dk and CMV-encoded m04 correlates with the NK cell antiviral response

Journal of Experimental Medicine ◽

10.1084/jem.20080954 ◽

2009 ◽

Vol 206 (3) ◽

pp. 515-523 ◽

Cited By ~ 94

Author(s):

Agnieszka Kielczewska ◽

Michal Pyzik ◽

Tianhe Sun ◽

Astrid Krmpotic ◽

Melissa B. Lodoen ◽

...

Keyword(s):

Nk Cells ◽

Deletion Mutant ◽

Viral Infections ◽

Nk Cell ◽

Cell Receptor ◽

Major Histocompatability Complex ◽

Wild Type ◽

Mcmv Infection ◽

Host Protection ◽

Infected Cells

Natural killer (NK) cells are crucial in resistance to certain viral infections, but the mechanisms used to recognize infected cells remain largely unknown. Here, we show that the activating Ly49P receptor recognizes cells infected with mouse cytomegalovirus (MCMV) by a process that requires the presence of H2-Dk and the MCMV m04 protein. Using H2 chimeras between H2-Db and -Dk, we demonstrate that the H2-Dk peptide-binding platform is required for Ly49P recognition. We identified m04 as a viral component necessary for recognition using a panel of MCMV-deletion mutant viruses and complementation of m04-deletion mutant (Δm04) virus infection. MA/My mice, which express Ly49P and H2-Dk, are resistant to MCMV; however, infection with Δm04 MCMV abrogates resistance. Depletion of NK cells in MA/My mice abrogates their resistance to wild-type MCMV infection, but does not significantly affect viral titers in mice infected with Δm04 virus, implicating NK cells in host protection through m04-dependent recognition. These findings reveal a novel mechanism of major histocompatability complex class I–restricted recognition of virally infected cells by an activating NK cell receptor.

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Natural killer (NK) cell response to virus infections in mice with severe combined immunodeficiency. The stimulation of NK cells and the NK cell-dependent control of virus infections occur independently of T and B cell function.

Journal of Experimental Medicine ◽

10.1084/jem.173.5.1053 ◽

1991 ◽

Vol 173 (5) ◽

pp. 1053-1063 ◽

Cited By ~ 118

Author(s):

R M Welsh ◽

J O Brubaker ◽

M Vargas-Cortes ◽

C L O'Donnell

Keyword(s):

Nk Cells ◽

Natural Killer ◽

Cell Response ◽

Nk Cell ◽

Severe Combined Immunodeficiency ◽

Scid Mice ◽

Combined Immunodeficiency ◽

Virus Infections ◽

Mcmv Infection

The activation, proliferation, and antiviral properties of natural killer (NK) cells were examined in severe combined immunodeficiency (SCID) mice to determine the influence of mature T or B cells on virus-induced NK cell functions and to more conclusively determine the antiviral properties of prototypical CD3- NK cells. NK cells were activated to high levels of cytotoxicity 3 d after infection of mice with lymphocytic choriomeningitis virus (LCMV) or murine cytomegalovirus (MCMV). Analyses of spleen leukocytes from LCMV-infected mice by a variety of techniques indicated that the NK cells proliferated and increased in number during infection. Propidium iodide staining of the DNA of cycling cells revealed that the great majority of proliferating spleen leukocytes 3 d after LCMV infection was of the NK cell phenotype (CD3-, Ig-, Mac-1+, CZ1+, 50% Thy-1+), in contrast to uninfected mice, whose proliferating cells were predominantly of other lineages. Analyses of the NK cell responses over a 2 wk period in control CB17 mice infected with MCMV indicated a sharp rise in serum interferon (IFN) and spleen NK cell activity early (days 3-5) in infection, followed by sharp declines at later stages. In SCID mice the IFN levels continued to rise over a 10-d period, whereas the NK cell response peaked on day 3-5 and gradually tapered. In contrast to the immunocompetent CB17 mice, SCID mice did not clear the MCMV infection and eventually succumbed. SCID mice, again in contrast to immunocompetent CB17 mice, also failed to clear infections with LCMV and Pichinde virus (PV); these mice, infected as adults, did not die but instead developed long-term persistent infections. Depletion of the NK cells in vivo with antiserum to asialo GM1 rendered both SCID and CB17 control mice much more sensitive to MCMV infection, as shown by titers of virus in organs and by survival curves. In contrast, similar depletions of NK cells did not enhance the titers of the NK cell-resistant virus, LCMV. Two variants of PV, one sensitive to NK cells and the other selected for resistance to NK cells by in vivo passage, were also tested in NK cell-depleted SCID mice. The NK-sensitive PV replicated to higher titers in NK cell-depleted SCID mice, whereas the titers of the NK cell-resistant PV were the same, whether or not the mice had NK cells. These experiments support the concept that CD3- prototypical NK cells mediate resistance to NK cell-sensitive viruses via a mechanism independent of antiviral or "natural" antibody.(ABSTRACT TRUNCATED AT 400 WORDS)

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Continuous engagement of a self-specific activation receptor induces NK cell tolerance

Journal of Experimental Medicine ◽

10.1084/jem.20072446 ◽

2008 ◽

Vol 205 (8) ◽

pp. 1829-1841 ◽

Cited By ~ 119

Author(s):

Sandeep K. Tripathy ◽

Peter A. Keyel ◽

Liping Yang ◽

Jeanette T. Pingel ◽

Tammy P. Cheng ◽

...

Keyword(s):

Nk Cells ◽

Cell Activation ◽

Nk Cell ◽

Murine Cytomegalovirus ◽

Cell Tolerance ◽

Major Histocompatability Complex ◽

Inhibitory Receptors ◽

Mcmv Infection ◽

Functional Defects

Natural killer (NK) cell tolerance mechanisms are incompletely understood. One possibility is that they possess self-specific activation receptors that result in hyporesponsiveness unless modulated by self–major histocompatability complex (MHC)–specific inhibitory receptors. As putative self-specific activation receptors have not been well characterized, we studied a transgenic C57BL/6 mouse that ubiquitously expresses m157 (m157-Tg), which is the murine cytomegalovirus (MCMV)–encoded ligand for the Ly49H NK cell activation receptor. The transgenic mice were more susceptible to MCMV infection and were unable to reject m157-Tg bone marrow, suggesting defects in Ly49H+ NK cells. There was a reversible hyporesponsiveness of Ly49H+ NK cells that extended to Ly49H-independent stimuli. Continuous Ly49H–m157 interaction was necessary for the functional defects. Interestingly, functional defects occurred when mature wild-type NK cells were adoptively transferred to m157-Tg mice, suggesting that mature NK cells may acquire hyporesponsiveness. Importantly, NK cell tolerance caused by Ly49H–m157 interaction was similar in NK cells regardless of expression of Ly49C, an inhibitory receptor specific for a self-MHC allele in C57BL/6 mice. Thus, engagement of self-specific activation receptors in vivo induces an NK cell tolerance effect that is not affected by self-MHC–specific inhibitory receptors.

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Tracking the fate of antigen-specific versus cytokine-activated natural killer cells after cytomegalovirus infection

Journal of Experimental Medicine ◽

10.1084/jem.20160726 ◽

2016 ◽

Vol 213 (12) ◽

pp. 2745-2758 ◽

Cited By ~ 38

Author(s):

Tsukasa Nabekura ◽

Lewis L. Lanier

Keyword(s):

Nk Cells ◽

Natural Killer ◽

Nk Cell ◽

Interleukin 12 ◽

Mcmv Infection ◽

Specific Memory ◽

Important Host ◽

Free Environment ◽

Memory Nk Cells

Natural killer (NK) cells provide important host defense and can generate long-lived memory NK cells. Here, by using novel transgenic mice carrying inducible Cre expressed under the control of Ncr1 gene, we demonstrated that two distinct long-lived NK cell subsets differentiate in a mouse model of cytomegalovirus (MCMV) infection. NK cells expressing the MCMV-specific Ly49H receptor differentiated into memory NK cells by an activating signaling through Ly49H and Ly49H− NK cells differentiated into cytokine-activated NK cells by exposure to inflammatory cytokines during infection. Interleukin-12 is indispensable for optimal generation of both antigen-specific memory NK cells and cytokine-activated NK cells. MCMV-specific memory NK cells show enhanced effector function and augmented antitumor activity in vivo as compared with cytokine-activated NK cells, whereas cytokine-activated NK cells exhibited a more robust response to IL-15 and persisted better in an MCMV-free environment. These findings reveal that NK cells are capable of differentiation into distinct long-lived subsets with different functional properties.

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Proinflammatory cytokine signaling required for the generation of natural killer cell memory

Journal of Experimental Medicine ◽

10.1084/jem.20111760 ◽

2012 ◽

Vol 209 (5) ◽

pp. 947-954 ◽

Cited By ~ 176

Author(s):

Joseph C. Sun ◽

Sharline Madera ◽

Natalie A. Bezman ◽

Joshua N. Beilke ◽

Mark H. Kaplan ◽

...

Keyword(s):

Nk Cells ◽

Natural Killer ◽

Nk Cell ◽

Killer Cell ◽

Interleukin 12 ◽

Cytokine Signaling ◽

Mcmv Infection ◽

Ifn Γ ◽

Deficient Mice ◽

Memory Nk Cells

Although natural killer (NK) cells are classified as innate immune cells, recent studies demonstrate that NK cells can become long-lived memory cells and contribute to secondary immune responses. The precise signals that promote generation of long-lived memory NK cells are unknown. Using cytokine receptor-deficient mice, we show that interleukin-12 (IL-12) is indispensible for mouse cytomegalovirus (MCMV)-specific NK cell expansion and generation of memory NK cells. In contrast to wild-type NK cells that proliferated robustly and resided in lymphoid and nonlymphoid tissues for months after MCMV infection, IL-12 receptor–deficient NK cells failed to expand and were unable to mediate protection after MCMV challenge. We further demonstrate that a STAT4-dependent IFN-γ–independent mechanism contributes toward the generation of memory NK cells during MCMV infection. Understanding the full contribution of inflammatory cytokine signaling to the NK cell response against viral infection will be of interest for the development of vaccines and therapeutics.

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Selective Down-Regulation of the NKG2D Ligand H60 by Mouse Cytomegalovirus m155 Glycoprotein

Journal of Virology ◽

10.1128/jvi.79.5.2920-2930.2005 ◽

2005 ◽

Vol 79 (5) ◽

pp. 2920-2930 ◽

Cited By ~ 77

Author(s):

Milena Hasan ◽

Astrid Krmpotic ◽

Zsolt Ruzsics ◽

Ivan Bubic ◽

Tihana Lenac ◽

...

Keyword(s):

Gene Family ◽

Nk Cell ◽

Cell Receptor ◽

Mouse Strains ◽

Mcmv Infection ◽

Partial Restoration ◽

Golgi Compartment ◽

Infected Cells ◽

Nkg2d Receptor

ABSTRACT Both human and mouse cytomegaloviruses (CMVs) encode proteins that inhibit the activation of NK cells by down-regulating cellular ligands for the activating NK cell receptor NKG2D. Up to now, three ligands for the NKG2D receptor, named RAE-1, H60, and MULT-1, have been identified in mice. The resistance of mouse strains to murine CMV (MCMV) infection is determined by their ability to generate an effective NK cell response. The MCMV gene m152, a member of the m145 gene family, down-regulates the expression of RAE-1 in order to avoid NK cell control in vivo. Here we report that the m155 gene, another member of the m145 gene family, encodes a protein that interferes with the expression of H60 on the surfaces of infected cells. Deletion of the m155 gene leads to an only partial restoration of H60 expression on the cell surface, suggesting the involvement of another, so far unknown, viral inhibitor. In spite of this, an m155 deletion mutant virus shows NK cell-dependent attenuation in vivo. The acquisition of endo-β-N-acetylglucosaminidase H resistance and the preserved half-life of H60 in MCMV-infected cells indicate that the m155-mediated effect must take place in a compartment after H60 exits from the ERGIC-cis-Golgi compartment.

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Natural Killer Cells Utilize both Perforin and Gamma Interferon To Regulate Murine Cytomegalovirus Infection in the Spleen and Liver

Journal of Virology ◽

10.1128/jvi.79.1.661-667.2005 ◽

2005 ◽

Vol 79 (1) ◽

pp. 661-667 ◽

Cited By ~ 118

Author(s):

Joy Loh ◽

Dortha T. Chu ◽

Andrew K. O'Guin ◽

Wayne M. Yokoyama ◽

Herbert W. Virgin

Keyword(s):

Nk Cells ◽

Natural Killer ◽

Acute Phase ◽

Nk Cell ◽

Gamma Interferon ◽

Murine Cytomegalovirus ◽

Mcmv Infection ◽

Independent Manner ◽

Organ Specific ◽

Ifn Γ

ABSTRACT Natural killer (NK) cells are critical for innate regulation of the acute phase of murine cytomegalovirus (MCMV) infection and have been reported to utilize perforin (Pfp)- and gamma interferon (IFN-γ)-dependent effector mechanisms in an organ-specific manner to regulate MCMV infection in the spleen and liver. In this study, we further examined the roles of NK cells, Pfp, and IFN-γ in innate immunity to MCMV infection. With the recently described NK cell-deficient (NKD) mouse, we confirmed previous findings that NK cells, but not NKT cells, are required for control of the acute phase of MCMV infection in spleen and liver cells. Interestingly, we found that Pfp and IFN-γ are each important for regulating MCMV replication in both the spleen and the liver. Moreover, NK cells can regulate MCMV infection in the spleens and livers of Pfp−/− mice in a Pfp-independent manner and can use an IFN-γ-independent mechanism to control MCMV infection in IFN-γ−/− mice. Thus, contrary to previous reports, NK cells utilize both Pfp and IFN-γ to control MCMV infection in the spleen and liver.

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Role for TLR2 in NK Cell-Mediated Control of Murine Cytomegalovirus In Vivo

Journal of Virology ◽

10.1128/jvi.80.9.4286-4291.2006 ◽

2006 ◽

Vol 80 (9) ◽

pp. 4286-4291 ◽

Cited By ~ 86

Author(s):

Eva Szomolanyi-Tsuda ◽

Xueya Liang ◽

Raymond M. Welsh ◽

Evelyn A. Kurt-Jones ◽

Robert W. Finberg

Keyword(s):

Nk Cells ◽

Nk Cell ◽

Innate Immune ◽

Murine Cytomegalovirus ◽

Innate Immune Responses ◽

Toll Like Receptor ◽

Mcmv Infection ◽

Toll Like Receptor 2 ◽

Antiviral Control

ABSTRACT Natural killer (NK) cells are essential for the early control of murine cytomegalovirus (MCMV) infection. Here, we demonstrate that toll-like receptor 2 (TLR2) plays a role in the NK cell-mediated control of MCMV. TLR2 knockout (KO) mice had elevated levels of MCMV in the spleen and liver on day 4 postinfection compared to C57BL/6 mice. In vivo depletion of NK cells with anti-NK1.1 antibodies, however, eliminated the differences in viral titers between the two groups, suggesting that the effect of TLR2 on MCMV clearance on day 4 was NK cell mediated. The defect in early antiviral control was associated with a decreased NK cell population in the spleen and liver and reduced amounts of interleukin-18 and α/β interferon secreted in the TLR2 KO mice. Our studies suggest that in addition to the reported involvement of TLR9 and TLR3, TLR2 is also involved in innate immune responses to MCMV infection.

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