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

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.

scholarly journals Hematopoietic cell-mediated dissemination of murine cytomegalovirus is regulated by NK cells and immune evasion

2021 ◽  
Vol 17 (1) ◽  
pp. e1009255
Author(s):  
Shunchuan Zhang ◽  
Lauren E. Springer ◽  
Han-Zhi Rao ◽  
Renee G. Espinosa Trethewy ◽  
Lindsey M. Bishop ◽  
...  
Keyword(s):  
T Cells ◽  
Salivary Gland ◽  
Nk Cells ◽  
Immune Evasion ◽  
Hematopoietic Cells ◽  
Hematopoietic Cell ◽  
Murine Cytomegalovirus ◽  
Cell Mediated Immunity ◽  
Class I Mhc ◽  
Intranasal Infection

Cytomegalovirus (CMV) causes clinically important diseases in immune compromised and immune immature individuals. Based largely on work in the mouse model of murine (M)CMV, there is a consensus that myeloid cells are important for disseminating CMV from the site of infection. In theory, such dissemination should expose CMV to cell-mediated immunity and thus necessitate evasion of T cells and NK cells. However, this hypothesis remains untested. We constructed a recombinant MCMV encoding target sites for the hematopoietic specific miRNA miR-142-3p in the essential viral gene IE3. This virus disseminated poorly to the salivary gland following intranasal or footpad infections but not following intraperitoneal infection in C57BL/6 mice, demonstrating that dissemination by hematopoietic cells is essential for specific routes of infection. Remarkably, depletion of NK cells or T cells restored dissemination of this virus in C57BL/6 mice after intranasal infection, while dissemination occurred normally in BALB/c mice, which lack strong NK cell control of MCMV. These data show that cell-mediated immunity is responsible for restricting MCMV to hematopoietic cell-mediated dissemination. Infected hematopoietic cells avoided cell-mediated immunity via three immune evasion genes that modulate class I MHC and NKG2D ligands (m04, m06 and m152). MCMV lacking these 3 genes spread poorly to the salivary gland unless NK cells were depleted, but also failed to replicate persistently in either the nasal mucosa or salivary gland unless CD8+ T cells were depleted. Surprisingly, CD8+ T cells primed after intranasal infection required CD4+ T cell help to expand and become functional. Together, our data suggest that MCMV can use both hematopoietic cell-dependent and -independent means of dissemination after intranasal infection and that cell mediated immune responses restrict dissemination to infected hematopoietic cells, which are protected from NK cells during dissemination by viral immune evasion. In contrast, viral replication within mucosal tissues depends on evasion of T cells.

scholarly journals The Putative Natural Killer Decoy Early Genem04 (gp34) of Murine Cytomegalovirus Encodes an Antigenic Peptide Recognized by Protective Antiviral CD8 T Cells

2000 ◽  
Vol 74 (4) ◽  
pp. 1871-1884 ◽  
Author(s):  
Rafaela Holtappels ◽  
Doris Thomas ◽  
Jürgen Podlech ◽  
Gernot Geginat ◽  
Hans-Peter Steffens ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Surface ◽  
Nk Cells ◽  
Natural Killer ◽  
Gene Product ◽  
Immune Evasion ◽  
Cd8 T Cells ◽  
Antigenic Peptide ◽  
Murine Cytomegalovirus ◽  
Mhc I

ABSTRACT Several early genes of murine cytomegalovirus (MCMV) encode proteins that mediate immune evasion by interference with the major histocompatibility complex class I (MHC-I) pathway of antigen presentation to cytolytic T lymphocytes (CTL). Specifically, the m152 gene product gp37/40 causes retention of MHC-I molecules in the endoplasmic reticulum (ER)-Golgi intermediate compartment. Lack of MHC-I on the cell surface should activate natural killer (NK) cells recognizing the “missing self.” The retention, however, is counteracted by the m04early gene product gp34, which binds to folded MHC-I molecules in the ER and directs the complex to the cell surface. It was thus speculated that gp34 might serve to silence NK cells and thereby complete the immune evasion of MCMV. In light of these current views, we provide here results demonstrating an in vivo role for gp34 in protective antiviral immunity. We have identified an antigenic nonapeptide derived from gp34 and presented by the MHC-I molecule Dd. Besides the immunodominant immediate-early nonapeptide consisting of IE1 amino acids 168-176 (IE1168-176), the early nonapeptide m04243-251 is the second antigenic peptide described for MCMV. The primary immune response to MCMV generates significant m04-specific CD8 T-cell memory. Upon adoptive transfer into immunodeficient recipients, an m04-specific CTL line controls MCMV infection with an efficacy comparable to that of an IE1-specific CTL line. Thus, gp34 is the first noted early protein of MCMV that escapes viral immune evasion mechanisms. These data document that MCMV is held in check by a redundance of protective CD8 T cells recognizing antigenic peptides in different phases of viral gene expression.

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

2019 ◽  
Vol 116 (52) ◽  
pp. 26768-26778 ◽  
Author(s):  
Awndre Gamache ◽  
John M. Cronk ◽  
William T. Nash ◽  
Patryk Puchalski ◽  
Alyssa Gillespie ◽  
...  
Keyword(s):  
Nk Cells ◽  
Cytomegalovirus Infection ◽  
Nk Cell ◽  
Host Cells ◽  
Murine Cytomegalovirus ◽  
Virus Infections ◽  
Class I Mhc ◽  
Mhc I ◽  
Cell Immunity ◽  
Virus Immunity

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.

scholarly journals Efficient genome editing of human natural killer cells by CRISPR RNP

2018 ◽  
Author(s):  
Jai Rautela ◽  
Elliot Surgenor ◽  
Nicholas D. Huntington
Keyword(s):  
T Cells ◽  
Gene Delivery ◽  
Nk Cells ◽  
Natural Killer ◽  
Nk Cell ◽  
Genetically Modify ◽  
Viral Gene ◽  
Human Natural Killer Cells ◽  
Viral Gene Delivery ◽  
Cancer Immunotherapies

AbstractThe ability to genetically modify CD8 T cells using viral gene delivery has facilitated the development of next generation of cancer immunotherapies such as chimeric antigen receptor (CAR) T cells engineered to specifically kill tumor cells. Development of immunotherapies targeting natural killer (NK) cells have stalled in part by their resistance to viral gene delivery. Here, we describe an efficient approach to genetically edit human NK cells by electroporation and CRISPR-Cas9 ribonucleoprotein (RNP) complexes. We detail electroporation pulse codes and buffer optimization for protein uptake by human NK cells and viability, and the efficiency of this approach over other methods. To highlight the transformative step this technique will have for NK cell immunotherapy drug discovery, we deleted NKp46 and CIS in primary human NK cells and validated murine findings on their key roles in regulating NK cell anti-tumor function.

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

Blood ◽  
2013 ◽  
Vol 122 (8) ◽  
pp. 1518-1521 ◽  
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.

scholarly journals 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

2006 ◽  
Vol 80 (1) ◽  
pp. 545-550 ◽  
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.

scholarly journals Induction of Natural Killer Cell Responses by Ectromelia Virus Controls Infection

2007 ◽  
Vol 81 (8) ◽  
pp. 4070-4079 ◽  
Author(s):  
April Keim Parker ◽  
Scott Parker ◽  
Wayne M. Yokoyama ◽  
John A. Corbett ◽  
R. Mark L. Buller
Keyword(s):  
T Cells ◽  
Nk Cells ◽  
Natural Killer ◽  
Nk Cell ◽  
Intracellular Cytokine Staining ◽  
Killer Cell ◽  
Murine Cytomegalovirus ◽  
Virus Infectivity ◽  
Ectromelia Virus ◽  
Ifn Γ

ABSTRACT Natural killer (NK) cells play a pivotal role in the innate immune response to viral infections, particularly murine cytomegalovirus (MCMV) and human herpesviruses. In poxvirus infections, the role of NK cells is less clear. We examined disease progression in C57BL/6 mice after the removal of NK cells by both antibody depletion and genetic means. We found that NK cells were crucial for survival and the early control of virus replication in spleen and to a lesser extent in liver in C57BL/6 mice. Studies of various knockout mice suggested that γδ T cells and NKT cells are not important in the C57BL/6 mousepox model and CD4+ and CD8+ T cells do not exhibit antiviral activity at 6 days postinfection, when the absence of NK cells has a profound effect on virus titers in spleen and liver. NK cell cytotoxicity and/or gamma interferon (IFN-γ) secretion likely mediated the antiviral effect needed to control virus infectivity in target organs. Studies of the effects of ectromelia virus (ECTV) infection on NK cells demonstrated that NK cells proliferate within target tissues (spleen and liver) and become activated following a low-dose footpad infection, although the mechanism of activation appears distinct from the ligand-dependent activation observed with MCMV. NK cell IFN-γ secretion was detected by intracellular cytokine staining transiently at 32 to 72 h postinfection in the lymph node, suggesting a role in establishing a Th1 response. These results confirm a crucial role for NK cells in controlling an ECTV infection.

scholarly journals The complex of MCMV proteins and MHC class I evades NK cell control and drives the evolution of virus-specific activating Ly49 receptors

2019 ◽  
Vol 216 (8) ◽  
pp. 1809-1827 ◽  
Author(s):  
Jelena Železnjak ◽  
Vanda Juranić Lisnić ◽  
Branka Popović ◽  
Berislav Lisnić ◽  
Marina Babić ◽  
...  
Keyword(s):  
Nk Cells ◽  
Immune Evasion ◽  
Nk Cell ◽  
Cytotoxic T Cells ◽  
Mhc I ◽  
Immune Evasion Mechanism ◽  
Viral Immune Evasion ◽  
Nk Cell Receptors ◽  
Self Recognition ◽  
Missing Self

CMVs efficiently target MHC I molecules to avoid recognition by cytotoxic T cells. However, the lack of MHC I on the cell surface renders the infected cell susceptible to NK cell killing upon missing self recognition. To counter this, mouse CMV (MCMV) rescues some MHC I molecules to engage inhibitory Ly49 receptors. Here we identify a new viral protein, MATp1, that is essential for MHC I surface rescue. Rescued altered-self MHC I molecules show increased affinity to inhibitory Ly49 receptors, resulting in inhibition of NK cells despite substantially reduced MHC I surface levels. This enables the virus to evade recognition by licensed NK cells. During evolution, this novel viral immune evasion mechanism could have prompted the development of activating NK cell receptors that are specific for MATp1-modified altered-self MHC I molecules. Our study solves a long-standing conundrum of how MCMV avoids recognition by NK cells, unravels a fundamental new viral immune evasion mechanism, and demonstrates how this forced the evolution of virus-specific activating MHC I–restricted Ly49 receptors.

Pediatric T-ALL but Not cALL Blasts Express Ligands for Siglec-7 Inhibiting NK Cell Mediated Lysis.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1844-1844
Author(s):  
Susanne Viebahn ◽  
Friederike Gieseke ◽  
Matthias Pfeiffer ◽  
Rupert Handgretinger ◽  
Ingo Müller
Keyword(s):  
T Cells ◽  
Sialic Acid ◽  
Nk Cells ◽  
Immune Evasion ◽  
Nk Cell ◽  
Leukemic Cells ◽  
Malignant Cells ◽  
Inhibitory Receptor ◽  
Specific Lysis ◽  
Effector Cells

Abstract Changes in the glycosylation pattern of cell surface proteins occur during early stages of malignant transformation. These alterations in the glycan repertoire may contribute to numerous processes of tumor cell survival such as adhesion, migration and immune evasion. Sialic acid is the most common terminal carbohydrate moiety of glycan structures in humans binding to a family of eleven receptors termed sialic acid binding Ig-like lectins (siglecs). Siglecs are expressed on virtually all effector cells of the immune system and several members function as inhibitory receptors. Siglec-7 is known as an inhibitory receptor expressed by NK cells and T cells. Therefore, siglec-7 ligands may contribute to immune evasion of malignant cells. In order to analyze the expression of siglec-7 ligands by malignant cells, we cloned the extracellular domain of human siglec-7. The recombinant protein was expressed in 293 cells to allow glycosylation, which is supposed to be important for specificity. As the interaction of a single glycan and lectin is of low affinity, we tetramerized siglec-7 in analogy to MHC-tetramers to increase avidity. This strategy allowed for reliable use of siglec-7 fusion protein in flow cytometry. Analysis of PBMC from healthy donors revealed that siglec-7 ligands are expressed on all subpopulations of PBMC: expression was detected on CD4+ and CD8+ T-cells as well as on monocytes and NK cells. B cells could be subdivided into a positive and negative population after staining with siglec-7 tetramers. Subsequently, we analyzed primary lymphatic blasts from childhood leukemias. Interestingly, T-ALL blasts expressed ligands for siglec-7, whereas these ligands were absent from cALL blasts. As cALL blasts are known to be better targets for NK cell-mediated cytotoxicity than T-ALL blasts, we hypothesized that siglec-7 ligands on the surface of leukemic blasts inhibit NK cells. To test the functional relevance of siglec-7 ligand expression for NK cell-mediated cytotoxicity, we used soluble monomeric siglec-7 as competitive inhibitor in cytotoxicity assays. In these competition assays, NK cell-mediated specific lysis of leukemic cells increased roughly twofold in the presence of soluble siglec-7. These results show that engagement of inhibitory siglecs on the surface of effector cells might contribute to immune escape mechanisms of malignant cells. It underlines the function of siglec-7 as a non-MHC-specific inhibitory receptor on NK cells and the important role of altered glycans expression on malignant cells in tumor immunology.

Siglec-7 Tetramers Characterize B Cell Subpopulations and Identify Immunomodulatory Ligands on Malignant Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1728-1728
Author(s):  
Philippa Mang ◽  
Friederike Gieseke ◽  
Susanne Viebahn ◽  
Inga Gondesen ◽  
Anne Kruchen ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Surface ◽  
B Cell ◽  
Nk Cells ◽  
Immune Evasion ◽  
Nk Cell ◽  
Sialic Acids ◽  
Target Cells ◽  
Malignant Cells ◽  
Neuraminidase Treatment

Abstract Abstract 1728 Cell surface glycoconjugates have important functions in the modulation of immune cell maturation, activation and homeostasis. Glycans on cell surfaces are generally sialylated. Sialic acids are predominantly found on the non-reducing end of oligosaccharide chains of glycoconjugates. They are recognized by sialic acid-binding immunoglobulin-like lectins (siglecs). CD33-related siglecs are receptors predominantly expressed on immune cells. Most of the CD33-related siglecs display a tyrosine-based inhibitory motif (ITIM) known to downregulate effector cell functions. So far, identification of ligands for siglecs was hampered by low affinity between proteins and glycans, but by glycan array analysis α2,8-, α2,3- as well as α2,6-linked sialic acids were identified as the carbohydrate part of the ligand. In order to analyse the expression of physiological siglec ligands on different lymphocyte subpopulations, the extracellular three domains of siglec-7 were cloned. To allow for mammalian glycosylation, the protein was expressed in 293T cells. To overcome the low affinity of lectins, siglec-7 was enzymatically biotinylated and oligomerized using streptavidin in analogy to HLA tetramers. Flow cytometric analysis of lymphocytes revealed reproducible binding patterns of siglec-7 tetramers. Specificity was ensured by treatment of PBMC with neuraminidase, specifically cleaving terminal sialic acids in α2,3, α2,6 and α2,8 linkages. On T cells, the density of siglec-7 ligands increased 3.5-fold during activation. This is contradictory to results using plant lectins, like Maackia amurensis lectin and Sambucus nigra lectin, which indicated an 1.8- and 1.5-fold decrease, respectively. Activation of NK cells did not affect the siglec-7 ligand expression. Interestingly, B cells could be divided in a siglec-7 ligand positive and a siglec-7 ligand negative population. This may reflect different differentiation or activation stages, which were not found with any antibody combination directed against typical protein B cell markers. Malignant transformation is often associated with aberrant cell surface glycosylation. In acute lymphoblastic leukemia (ALL), blasts of the more aggressive T-ALL express 17 fold higher amounts of siglec-7 ligand than blasts of the B cell lineage (cALL), where the siglec-7 ligand was nearly not detectable. These findings may contribute to the poor prognosis of T-ALL by a possible immune escape achieved by siglec-ligand binding induced inhibition. Since immune evasion mechanisms are also described for other malignancies, we additionally analysed rhabdomyosarcoma (RMS) cells. Siglec-7 ligand could be found on all tested RMS cell lines in different expression levels, independent of the classification. Siglec-7 is a known inhibitory receptor expressed by NK cells and T cells. The effect of siglec-7 ligand expressed by malignant cells on NK cell cytotoxicity was analysed. By the removal of all terminal sialic acids on the surface of RMS cells by neuraminidase treatment, the NK cell-induced cytotoxicity could be increased by 20%. Coating of siglec-7 ligands on target cells by preincubation with recombinant siglec-7 protein also resulted in an increased cytotoxicity. These results show that sialic acids play an important role in the immune system as physiologic ligands. However, the expression of siglec-7 ligands on malignant cells may contribute to immune evasion mechanisms. Disclosures: No relevant conflicts of interest to declare.

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