Ly49R activation receptor drives self-MHC–educated NK cell immunity against cytomegalovirus infection

Natural killer (NK) cells mediate vital control of cancer and viral infection. They rely on MHC class I (MHC I)-specific self-receptors to identify and lyse diseased cells without harming self-MHC I-bearing host cells. NK cells bearing inhibitory self-receptors for host MHC I also undergo education, referred to as licensing, which causes them to become more responsive to stimulation via activation receptor signaling. Previous work has shown that licensed NK cells selectively expand during virus infections and they are associated with improved clinical response in human patients experiencing certain chronic virus infections, including HIV and hepatitis C virus. However, the importance of inhibitory self-receptors in NK-mediated virus immunity is debated as they also limit signals in NK cells emanating from virus-specific activation receptors. Using a mouse model of MHC I-dependent (H-2Dk) virus immunity, we discovered that NK cells depend on the Ly49G2 inhibitory self-receptor to mediate virus control, which coincided with host survival during murine cytomegalovirus infection. This antiviral effect further requires active signaling in NK cells via the Ly49R activation receptor that also binds H-2Dk. In tandem, these functionally discordant Ly49 self-receptors increase NK cell proliferation and effector activity during infection, resulting in selective up-regulation of CD25 and KLRG1 in virus-specific Ly49R+Ly49G2+NK cells. Our findings establish that paired self-receptors act as major determinants of NK cell-mediated virus sensing and immunity.

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Soluble and Exosome-Bound α-Galactosylceramide Mediate Preferential Proliferation of Educated NK Cells with Increased Anti-Tumor Capacity

Cancers ◽

10.3390/cancers13020298 ◽

2021 ◽

Vol 13 (2) ◽

pp. 298

Author(s):

Arnika K. Wagner ◽

Ulf Gehrmann ◽

Stefanie Hiltbrunner ◽

Valentina Carannante ◽

Thuy T. Luu ◽

...

Keyword(s):

Nk Cells ◽

Cancer Immunotherapy ◽

Nk Cell ◽

Mouse Strains ◽

Target Cells ◽

Class I Mhc ◽

Mhc I ◽

Cell Responses ◽

Missing Self

Natural killer (NK) cells can kill target cells via the recognition of stress molecules and down-regulation of major histocompatibility complex class I (MHC-I). Some NK cells are educated to recognize and kill cells that have lost their MHC-I expression, e.g., tumor or virus-infected cells. A desired property of cancer immunotherapy is, therefore, to activate educated NK cells during anti-tumor responses in vivo. We here analyze NK cell responses to α-galactosylceramide (αGC), a potent activator of invariant NKT (iNKT) cells, or to exosomes loaded with αGC. In mouse strains which express different MHC-I alleles using an extended NK cell flow cytometry panel, we show that αGC induces a biased NK cell proliferation of educated NK cells. Importantly, iNKT cell-induced activation of NK cells selectively increased in vivo missing self-responses, leading to more effective rejection of tumor cells. Exosomes from antigen-presenting cells are attractive anti-cancer therapy tools as they may induce both innate and adaptive immune responses, thereby addressing the hurdle of tumor heterogeneity. Adding αGC to antigen-loaded dendritic-cell-derived exosomes also led to an increase in missing self-responses in addition to boosted T and B cell responses. This study manifests αGC as an attractive adjuvant in cancer immunotherapy, as it increases the functional capacity of educated NK cells and enhances the innate, missing self-based antitumor response.

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IL-15 trans-presentation promotes human NK cell development and differentiation in vivo

Journal of Experimental Medicine ◽

10.1084/jem.20082013 ◽

2008 ◽

Vol 206 (1) ◽

pp. 25-34 ◽

Cited By ~ 331

Author(s):

Nicholas D. Huntington ◽

Nicolas Legrand ◽

Nuno L. Alves ◽

Barbara Jaron ◽

Kees Weijer ◽

...

Keyword(s):

Nk Cells ◽

Nk Cell ◽

Cell Development ◽

Class I Mhc ◽

Mhc I ◽

Hematopoietic Stem ◽

Receptor Agonists ◽

Development And Differentiation ◽

Therapeutic Tools

The in vivo requirements for human natural killer (NK) cell development and differentiation into cytotoxic effectors expressing inhibitory receptors for self–major histocompatability complex class I (MHC-I; killer Ig-like receptors [KIRs]) remain undefined. Here, we dissect the role of interleukin (IL)-15 in human NK cell development using Rag2−/−γc−/− mice transplanted with human hematopoietic stem cells. Human NK cell reconstitution was intrinsically low in this model because of the poor reactivity to mouse IL-15. Although exogenous human IL-15 (hIL-15) alone made little improvement, IL-15 coupled to IL-15 receptor α (IL-15Rα) significantly augmented human NK cells. IL-15–IL-15Rα complexes induced extensive NK cell proliferation and differentiation, resulting in accumulation of CD16+KIR+ NK cells, which was not uniquely dependent on enhanced survival or preferential responsiveness of this subset to IL-15. Human NK cell differentiation in vivo required hIL-15 and progressed in a linear fashion from CD56hiCD16−KIR− to CD56loCD16+KIR−, and finally to CD56loCD16+KIR+. These data provide the first evidence that IL-15 trans-presentation regulates human NK cell homeostasis. Use of hIL-15 receptor agonists generates a robust humanized immune system model to study human NK cells in vivo. IL-15 receptor agonists may provide therapeutic tools to improve NK cell reconstitution after bone marrow transplants, enhance graft versus leukemia effects, and increase the pool of IL-15–responsive cells during immunotherapy strategies.

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Immunoreceptor tyrosine-based inhibitory motif–dependent functions of an MHC class I-specific NK cell receptor

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1713064114 ◽

2017 ◽

Vol 114 (40) ◽

pp. E8440-E8447 ◽

Cited By ~ 10

Author(s):

Michael D. Bern ◽

Diana L. Beckman ◽

Takashi Ebihara ◽

Samantha M. Taffner ◽

Jennifer Poursine-Laurent ◽

...

Keyword(s):

Nk Cells ◽

Mhc Class I ◽

Nk Cell ◽

Single Gene ◽

Cell Receptor ◽

Class I ◽

Class I Mhc ◽

Mhc I ◽

Receptor Repertoire

Natural killer (NK) cells express MHC class I (MHC-I)-specific receptors, such as Ly49A, that inhibit killing of cells expressing self–MHC-I. Self–MHC-I also “licenses” NK cells to become responsive to activating stimuli and regulates the surface level of NK-cell inhibitory receptors. However, the mechanisms of action resulting from these interactions of the Ly49s with their MHC-I ligands, particularly in vivo, have been controversial. Definitive studies could be derived from mice with targeted mutations in inhibitory Ly49s, but there are inherent challenges in specifically altering a single gene within a multigene family. Herein, we generated a knock-in mouse with a targeted mutation in the immunoreceptor tyrosine-based inhibitory motif (ITIM) of Ly49A that abolished the inhibitory function of Ly49A in cytotoxicity assays. This mutant Ly49A caused a licensing defect in NK cells, but the surface expression of Ly49A was unaltered. Moreover, NK cells that expressed this mutant Ly49A exhibited an altered inhibitory receptor repertoire. These results demonstrate that Ly49A ITIM signaling is critical for NK-cell effector inhibition, licensing, and receptor repertoire development.

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Murine NK-cell licensing is reflective of donor MHC-I following allogeneic hematopoietic stem cell transplantation in murine cytomegalovirus responses

Blood ◽

10.1182/blood-2013-02-483503 ◽

2013 ◽

Vol 122 (8) ◽

pp. 1518-1521 ◽

Cited By ~ 16

Author(s):

Can M. Sungur ◽

Yajarayma J. Tang-Feldman ◽

Anthony E. Zamora ◽

Maite Alvarez ◽

Claire Pomeroy ◽

...

Keyword(s):

Stem Cell ◽

Nk Cells ◽

Cell Expansion ◽

Nk Cell ◽

Murine Cytomegalovirus ◽

Mcmv Infection ◽

Mhc I ◽

Hematopoietic Stem ◽

Allogeneic Hsct ◽

Key Points

Key Points Licensed NK cells based on the donor MHC-I haplotype show greater anti-MCMV resistance than unlicensed cells in allogeneic HSCT. Ly49H+ licensed NK-cell expansion based on donor MHC-I with greater IFNγ production than unlicensed NK cells is seen after MCMV infection.

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Expression of m157, a Murine Cytomegalovirus-Encoded Putative Major Histocompatibility Class I (MHC-I)-Like Protein, Is Independent of Viral Regulation of Host MHC-I

Journal of Virology ◽

10.1128/jvi.80.1.545-550.2006 ◽

2006 ◽

Vol 80 (1) ◽

pp. 545-550 ◽

Cited By ~ 28

Author(s):

Sandeep K. Tripathy ◽

Hamish R. C. Smith ◽

Erika A. Holroyd ◽

Jeanette T. Pingel ◽

Wayne M. Yokoyama

Keyword(s):

Antigen Processing ◽

Cell Activation ◽

Nk Cell ◽

Class I ◽

Murine Cytomegalovirus ◽

Viral Gene ◽

Major Histocompatibility ◽

Class I Mhc ◽

Mhc I ◽

Beta 2 Microglobulin

ABSTRACT A murine cytomegalovirus (MCMV)-encoded protein, m157, has a putative major histocompatibility complex class I (MHC-I) structure and is recognized by the Ly49H NK cell activation receptor. Using a monoclonal antibody against m157, in this study we directly demonstrated that m157 is a cell surface-expressed glycophosphatidylinositol-anchored protein with early viral gene kinetics. Beta-2 microglobulin and TAP1 (transporter associated with antigen processing 1) were not required for its expression. MCMV-encoded proteins that down-regulate MHC-I did not affect the expression of m157. Thus, m157 is expressed on infected cells in a manner independent of viral regulation of host MHC-I.

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NK cells force cytomegalovirus to use hematopoietic cells and immune evasion for dissemination after mucosal infection

10.1101/618132 ◽

2019 ◽

Author(s):

Shunchuan Zhang ◽

Finn Grey ◽

Christopher M. Snyder

Keyword(s):

T Cells ◽

Salivary Gland ◽

Nk Cells ◽

Immune Evasion ◽

Nk Cell ◽

Hematopoietic Cells ◽

Murine Cytomegalovirus ◽

Viral Gene ◽

Mhc I ◽

Intranasal Infection

AbstractCytomegalovirus (CMV) infects most people in the world and causes clinically important disease in immune compromised and immune immature individuals. How the virus disseminates from the initial site of infection is poorly understood. We used an innovative approach, involving insertion of target sites for the haematopoietic specific miRNA miR-142-3p into an essential viral gene in murine cytomegalovirus. This virus was unable to disseminate to the salivary gland following intranasal infection, demonstrating a strict need for hematopoietic cells for dissemination from the natural site of infection. Viral immune evasion genes that modulate MHC-I expression and NKG2D activation were also required in this setting, as MCMV lacking these genes exhibited impaired dissemination of the viral genome to the salivary gland, and there was no detectable viral replication in the salivary gland. Depletion of T cells rescued the replication of this evasion-deficient virus in the salivary gland. Surprisingly however, the early dissemination to the salivary gland of this evasion-deficient virus, could be rescued by depletion of NK cells, but not T cells. These data are the first to show a profound loss of MCMV fitness in the absence of its MHC-I evasion genes and suggest that they protect the virus from NK cells during hematopoietic dissemination to the salivary gland, where they continued to need the three evasion genes to avoid T cell responses. Remarkably, we found that depletion of NK cells also freed the virus from the need to infect hematopoietic cells in order to reach the salivary gland. Thus, our data show that MCMV adapts to NK cell pressure after intranasal infection by using hematopoietic cells for dissemination while immune evasion genes protect the virus from NK cells during dissemination and from T cells within mucosal tissues.

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Regulation of MHC I Molecules in Glioblastoma Cells and the Sensitizing of NK Cells

Pharmaceuticals ◽

10.3390/ph14030236 ◽

2021 ◽

Vol 14 (3) ◽

pp. 236

Author(s):

Timo Burster ◽

Fabian Gärtner ◽

Christiane Bulach ◽

Anuar Zhanapiya ◽

Adrian Gihring ◽

...

Keyword(s):

Nk Cells ◽

Nk Cell ◽

Immune Recognition ◽

Complex Class ◽

Glioblastoma Cells ◽

Independent Manner ◽

Class I Mhc ◽

Mhc I ◽

Histocompatibility Complex ◽

Subsequent Elimination

Immunotherapy has been established as an important area in the therapy of malignant diseases. Immunogenicity sufficient for immune recognition and subsequent elimination can be bypassed by tumors through altered and/or reduced expression levels of major histocompatibility complex class I (MHC I) molecules. Natural killer (NK) cells can eliminate tumor cells in a MHC I antigen presentation-independent manner by an array of activating and inhibitory receptors, which are promising candidates for immunotherapy. Here we summarize the latest findings in recognizing and regulating MHC I molecules that affect NK cell surveillance of glioblastoma cells.

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Impaired natural killer cell self-education and “missing-self” responses in Ly49-deficient mice

Blood ◽

10.1182/blood-2012-02-408732 ◽

2012 ◽

Vol 120 (3) ◽

pp. 592-602 ◽

Cited By ~ 46

Author(s):

Simon Bélanger ◽

Megan M. Tu ◽

Mir Munir Ahmed Rahim ◽

Ahmad B. Mahmoud ◽

Rajen Patel ◽

...

Keyword(s):

Tumor Cell ◽

Nk Cells ◽

Natural Killer ◽

Nk Cell ◽

Killer Cell ◽

Host Cells ◽

Target Cells ◽

Mhc I ◽

Missing Self ◽

Deficient Mice

Abstract Ly49-mediated recognition of MHC-I molecules on host cells is considered vital for natural killer (NK)–cell regulation and education; however, gene-deficient animal models are lacking because of the difficulty in deleting this large multigene family. Here, we describe NK gene complex knockdown (NKCKD) mice that lack expression of Ly49 and related MHC-I receptors on most NK cells. NKCKD NK cells exhibit defective killing of MHC-I–deficient, but otherwise normal, target cells, resulting in defective rejection by NKCKD mice of transplants from various types of MHC-I–deficient mice. Self–MHC-I immunosurveillance by NK cells in NKCKD mice can be rescued by self–MHC-I–specific Ly49 transgenes. Although NKCKD mice display defective recognition of MHC-I–deficient tumor cells, resulting in decreased in vivo tumor cell clearance, NKG2D- or antibody-dependent cell-mediated cytotoxicity–induced tumor cell cytotoxicity and cytokine production induced by activation receptors was efficient in Ly49-deficient NK cells, suggesting MHC-I education of NK cells is a single facet regulating their total potential. These results provide direct genetic evidence that Ly49 expression is necessary for NK-cell education to self–MHC-I molecules and that the absence of these receptors leads to loss of MHC-I–dependent “missing-self” immunosurveillance by NK cells.

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Inhibitory receptors specific for MHC class I educate murine NK cells but not CD8αα intestinal intraepithelial T lymphocytes

Blood ◽

10.1182/blood-2011-01-331124 ◽

2011 ◽

Vol 118 (2) ◽

pp. 339-347 ◽

Cited By ~ 11

Author(s):

Sylvie Taveirne ◽

Jessica Filtjens ◽

Els Van Ammel ◽

Veerle De Colvenaer ◽

Tessa Kerre ◽

...

Keyword(s):

Nk Cells ◽

Nk Cell ◽

Cell Types ◽

Cell Receptor ◽

Intraepithelial Lymphocytes ◽

Class I ◽

Inhibitory Receptors ◽

Class I Mhc ◽

Mhc I ◽

Inhibitory Function

Abstract The engagement of inhibitory receptors specific for major histocompatibility complex class I (MHC-I) molecules educates natural killer (NK) cells, meaning the improvement of the response of activation receptors to subsequent stimulation. It is not known whether inhibitory MHC-I receptors educate only NK cells or whether they improve the responsiveness of all cell types, which express them. To address this issue, we analyzed the expression of inhibitory MHC-I receptors on intestinal intraepithelial lymphocytes (iIELs) and show that T-cell receptor (TCR)-αβ CD8αα iIELs express multiple inhibitory receptors specific for MHC-I molecules, including CD94/NKG2A, Ly49A, and Ly49G2. However, the presence of MHC-I ligand for these receptors did not improve the response of iIELs to activation via the TCR. The absence of iIEL education by MHC-I receptors was not related to a lack of inhibitory function of these receptors in iIELs and a failure of these receptors to couple to the TCR. Thus, unlike NK cells, iIELs do not undergo an MHC-I–guided education process. These data suggest that education is an NK cell–specific function of inhibitory MHC-I receptors.

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The m15 Locus of Murine Cytomegalovirus Modulates Natural Killer Cell Responses to Promote Dissemination to the Salivary Glands and Viral Shedding

Pathogens ◽

10.3390/pathogens10070866 ◽

2021 ◽

Vol 10 (7) ◽

pp. 866

Author(s):

Baca Chan ◽

Maja Arapović ◽

Laura Masters ◽

Francois Rwandamuiye ◽

Stipan Jonjić ◽

...

Keyword(s):

Nk Cells ◽

Salivary Glands ◽

Natural Killer ◽

Nk Cell ◽

Acute Infection ◽

Killer Cell ◽

Viral Escape ◽

Murine Cytomegalovirus ◽

Viral Shedding ◽

Cell Responses

As the largest herpesviruses, the 230 kb genomes of cytomegaloviruses (CMVs) have increased our understanding of host immunity and viral escape mechanisms, although many of the annotated genes remain as yet uncharacterised. Here we identify the m15 locus of murine CMV (MCMV) as a viral modulator of natural killer (NK) cell immunity. We show that, rather than discrete transcripts from the m14, m15 and m16 genes as annotated, there are five 3′-coterminal transcripts expressed over this region, all utilising a consensus polyA tail at the end of the m16 gene. Functional inactivation of any one of these genes had no measurable impact on viral replication. However, disruption of all five transcripts led to significantly attenuated dissemination to, and replication in, the salivary glands of multiple strains of mice, but normal growth during acute infection. Disruption of the m15 locus was associated with heightened NK cell responses, including enhanced proliferation and IFNγ production. Depletion of NK cells, but not T cells, rescued salivary gland replication and viral shedding. These data demonstrate the identification of multiple transcripts expressed by a single locus which modulate, perhaps in a concerted fashion, the function of anti-viral NK cells.

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