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

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.

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

Journal of Virology ◽

10.1128/jvi.74.4.1871-1884.2000 ◽

2000 ◽

Vol 74 (4) ◽

pp. 1871-1884 ◽

Cited By ~ 59

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.

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CD8+ and CD4+ T Cells Contribute to the Exacerbation of Class I MHC Disparate Graft-vs-Host Reaction by Concurrent Murine Cytomegalovirus Infection

Clinical Immunology and Immunopathology ◽

10.1006/clin.1993.1048 ◽

1993 ◽

Vol 67 (1) ◽

pp. 84-90 ◽

Cited By ~ 12

Author(s):

Carolyn Cray ◽

Robert B. Levy

Keyword(s):

T Cells ◽

Cd4 T Cells ◽

Cytomegalovirus Infection ◽

Class I ◽

Murine Cytomegalovirus ◽

Host Reaction ◽

Class I Mhc

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

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1913064117 ◽

2019 ◽

Vol 116 (52) ◽

pp. 26768-26778 ◽

Cited By ~ 2

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.

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Absence of Cross-Presenting Cells in the Salivary Gland and Viral Immune Evasion Confine Cytomegalovirus Immune Control to Effector CD4 T Cells

PLoS Pathogens ◽

10.1371/journal.ppat.1002214 ◽

2011 ◽

Vol 7 (8) ◽

pp. e1002214 ◽

Cited By ~ 52

Author(s):

Senta M. Walton ◽

Sanja Mandaric ◽

Nicole Torti ◽

Albert Zimmermann ◽

Hartmut Hengel ◽

...

Keyword(s):

T Cells ◽

Salivary Gland ◽

Immune Evasion ◽

Cd4 T Cells ◽

Immune Control ◽

Viral Immune Evasion

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Reconstitution of T and NK cells after haploidentical hematopoietic cell transplantation using αβ T cell-depleted grafts and the clinical implication of γδ T cells

Clinical Transplantation ◽

10.1111/ctr.13147 ◽

2017 ◽

Vol 32 (1) ◽

pp. e13147 ◽

Cited By ~ 7

Author(s):

Meerim Park ◽

Ho Joon Im ◽

Yu-Jin Lee ◽

Nuree Park ◽

Seongsoo Jang ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Nk Cells ◽

Cell Transplantation ◽

Clinical Implication ◽

Hematopoietic Cell Transplantation ◽

Γδ T Cells ◽

Hematopoietic Cell ◽

Haploidentical Hematopoietic Cell Transplantation

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Identification of a Kd-restricted antigenic peptide encoded by murine cytomegalovirus early gene M84

Journal of General Virology ◽

10.1099/0022-1317-81-12-3037 ◽

2000 ◽

Vol 81 (12) ◽

pp. 3037-3042 ◽

Cited By ~ 23

Author(s):

Rafaela Holtappels ◽

Doris Thomas ◽

Matthias J. Reddehase

Keyword(s):

Immune Evasion ◽

Antigen Processing ◽

Matrix Protein ◽

Early Gene ◽

Antigenic Peptide ◽

Murine Cytomegalovirus ◽

Class I Mhc ◽

Mhc I ◽

Cmv Matrix ◽

Phase Protein

The two sister cytomegaloviruses (CMVs), human and murine CMV, have both evolved immune evasion functions that interfere with the major histocompatibility complex class I (MHC-I) pathway of antigen processing and presentation and are effectual in the early (E) phase of virus gene expression. However, studies on murine CMV have shown that E-phase immune evasion is leaky. An E-phase protein involved in immune evasion, namely m04-gp34, was found to simultaneously account for an antigenic peptide presented by the MHC-I molecule Dd. Recent work has demonstrated the induction of protective immunity specific for the E-phase protein M84-p65, one of two murine CMV homologues of the human CMV matrix protein UL83-pp65. In this study, the identification of the MHC-I Kd-restricted M84 peptide 297AYAGLFTPL305 is documented. This peptide is the third antigenic peptide described for murine CMV and the second that escapes immunosubversive mechanisms.

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Comparing the Kinetics of NK Cells, CD4, and CD8 T Cells in Murine Cytomegalovirus Infection

The Journal of Immunology ◽

10.4049/jimmunol.1100416 ◽

2011 ◽

Vol 187 (3) ◽

pp. 1385-1392 ◽

Cited By ~ 20

Author(s):

Timothy E. Schlub ◽

Joseph C. Sun ◽

Senta M. Walton ◽

Scott H. Robbins ◽

Amelia K. Pinto ◽

...

Keyword(s):

T Cells ◽

Nk Cells ◽

Cd8 T Cells ◽

Cytomegalovirus Infection ◽

Murine Cytomegalovirus ◽

Kinetics Of

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NK Cell KIR Reconstitution after Allogeneic Hematopoietic Cell Transplant (HCT) Is Affected by T Cell Number and Function.

Blood ◽

10.1182/blood.v106.11.1406.1406 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1406-1406

Author(s):

Sarah Cooley ◽

Valarie McCullar ◽

Rosanna Wangen ◽

Tracy L. Bergemann ◽

John E. Wagner ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Growth Factor ◽

Nk Cells ◽

Clinical Outcomes ◽

Nk Cell ◽

Cell Number ◽

Hematopoietic Cell ◽

Naive T Cells ◽

Naïve T Cells

Abstract NK cell KIR interactions are among the variables known to affect clinical outcomes including relapse, graft versus host disease (GVHD) and survival after HCT. We hypothesized that T cells in graft sources available for HCT may affect KIR recovery and the therapeutic potential of KIR alloreactivity. We studied KIR reconstitution (the percentage of KIR+ NK cells measured by flow cytometry) in blood collected from recipients at day +100 after T cell deplete (TCD-BMT) and unmanipulated (U-BMT) unrelated BM transplants. We found that KIR reconstitution was suppressed compared to the healthy donors, significantly more so after U-BMT transplants (donor: 48.42 ± 2.35% KIR+ NK cells versus recipient: 26.74 ± 1.94, n = 36; P < .001) than after TCD-BMT transplants (donor: 53.34 ± 3.25% versus recipient: 42.68 ± 3.32%, n = 38; P = .017), with P = .001 between the recipient groups. Additionally, multivariate Cox proportional hazards models showed that improved KIR recovery independently correlated with improved survival and that higher NK cell IFN-γ production independently correlated with more frequent acute GVHD in that patient cohort. These data suggested that T cell number in the graft affects KIR reconstitution and transplant outcome. We next examined other sources of hematopoietic cells in which T cell function may be suppressed either by growth factor mobilization (sibling donor unmanipulated peripheral blood: SibU-PB) or the innate naivety of the T cells (umbilical cord blood: UCB). KIR+ NK reconstitution on recovering cells at day +100 after all HCT graft types was significantly less than that on normal donor cells (normals 55.33 ± 1.73%, n = 124; all P < .0006). U-BMT recipients had significantly lower KIR+ NK recovery (27.31 ± 2.06%, n = 36 vs. SibU-PB: 37.58 ± 3.29%, n = 29; TCD-BMT: 42.68 ± 3.32%, n = 38; or UCB, 37.99 ± 2.54%, n = 49) when compared to all other transplant types. The highest absolute T cell inoculum, found in SibU-PB, showed KIR reconstitution similar to that of TCD-BMT, which had the lowest T cell content (p=0.29), perhaps due to the lower alloreactivity of the Sib grafts and to the G-CSF-priming which preferentially mobilizes T cells with a suppressive phenotype. Similarly, KIR reconstitution was better after UCB compared to U-BMT (P = .0027), possibly due to the more permissive interactions with naive T cells. These results suggest that reduced T cell number after T cell depletion, suppressed T cells found after growth factor mobilization, or naive T cells present in UCB grafts enhance in vivo KIR reconstitution after allogeneic HCT when compared to unmanipulated marrow grafts. Such enhanced KIR reconstitution may have clinical consequences. Graft T cells may directly compete for cytokines and growth factors, or may be a surrogate marker for other transplant factors such as the development of GVHD and the requirement for intensive post-transplant immunosuppression. Understanding these interactions will allow judicious selection of hematopoietic cell source to select for enhanced KIR recovery. For example, among unrelated unmanipulated donor grafts, KIR+ NK recovery was significantly better using UCB than adult donors and further investigation may show that this is advantageous to improve clinical outcomes.

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