Download Two Novel Human Cytomegalovirus nk cell evasion

IMMUNE EVASION
“Methods used by pathogenic organisms to evade a host’s immune system.”
VIRAL IMMUNE EVASION STRATEGIES
•
Reducing Class 1 expression to evade the cytotoxic T lymphocytes (CTL).
•
Suppression of Class 1 proteins from ever reaching the surface of the cell.
•
Producing “decoy” Class 1 homologs to evade Natural Killer cells (NK).
•
Preventing apoptosis (programmed cell death).
TWO NOVEL HUMAN CYTOMEGALOVIRUS NK
CELL EVASION FUNCTIONS TARGET MICA FOR
LYSOSOMAL DEGRADATION
By: Ceri A. Fielding, Rebecca Aicheler, Richard J. Stanton, Eddie C. Y. Wang, Song
Han, Sepehr Seirafian, James Davies, Brian P. McSharry, Michael P. Weekes, P.
Robin Antrobus, Virginie Prod'homme, Fabien P. Blanchet, Daniel Sugrue, Simone
Cuff, Dawn Roberts, Andrew J. Davison, Paul J. Lehner, Gavin W. G.
Wilkinson, and Peter Tomasec
Funding: Medical Research Council and the Wellcome Trust
Published: online, May 1, 2014
HUMAN CYTOMEGALOVIRUS (HCMV)
• Herpes virus (life long infection).
• High morbidity and mortality in
those with weakened immune
systems.
HUMAN CYTOMEGALOVIRUS
•
Can cause graft rejection, pneumonia, enteritis, hepatitis, retinitis in immune
compromised individuals.
•
Major cause of birth defects (mental retardation and hearing loss).
•
Correlation between infection and two brain tumors (medulloblastoma and
glioblastoma multiforme).
•
Can cause heart disease and arthritis.
•
However, most infections do not get this severe.
HCMV AND IMMUNE EVASION
HCMV evades natural killer (NK) cells by suppressing the stress proteins from ever
reaching the infected cells surface. These stress proteins would normally signal
the NK cells of a cell’s abnormality. However, they never reach the surface,
allowing HCMV to “hide” in the host cell.
A vast majority of HCMV’s large (263 kb) genome is dedicated to evading the immune
system.
NKG2D AND NKG2DL’S
NKG2D is an activating receptor on NK cells that recognize 8 ligands (NKG2DL’s)
present in HCMV cells:
Ligands that are recognized by NKG2D
MICA
MICB
ULBP1
ULBP2
ULBP3
ULBP4
ULBP5
ULBP6
NKG2DL’S
Become present on the cell surface when the cell undergoes a form of stress such as:
•
Genotoxic damage
•
Growth stimulation
•
Viral infection
OVERVIEW
Genes
UL122
&
UL123
Immediate Early
proteins
IE1/2
NK Cell Evasion
Genes
US18 & US20
Ligands
• MICA/B
• ULBP(1-6)
HYPOTHESIS
“We hypothesized that HCMV infected cells may be vulnerable to NK cell surveillance
during the early phase if there were a temporal window between activation of
NKG2DL’s by IE1 and IE2 and the expression of effective HCMV counter measure.”
The results and findings of this study will hopefully help to fight HCMV lifelong
infections.
METHODS
•
Cell lines
•
MICA typing
•
Viruses
•
Infect cells with mutated virus
•
Analysis:
• Flow cytometry- cell surface marker expression
• Triton X-114 membrane protein extraction
• Immunoblotting of target cells
• Immunofluorescence staining
• Intracellular flow cytometry
• NK cytotoxicity assay
• NK degradation assay
Cell lines usedimmortalized human fetal foreskin fibroblasts with human telomerase- HF-TERTS
transfected with adenovirus receptor and cells expressing U373 MICA-YFP
MICA typing
DNA extracted
Viruses
mutated HCMV with deleted genes, Rad-1E1 and 1E2 from HCV strain
Infected cells with mutated virus
FLOW CYTOMETRY
Analyze cell surface marker expression
of viable and infected cells
Picture source: http://www.abcam.com/index.html?pageconfig=resource&rid=11446
IMMUNOBLOTTING
Identify target protein
Using antibodies
http://www.virology.ws/2010/07/07/virology-toolbox-the-western-blot/
IMMUNOFLUORESCENCE STAINING
http://www.wieslab.se/index.php?langId=1&headId=72&subId=92&pageId=123
http://fanosmcb.blogspot.com/2010/06/immunofluorescence.html
NK (NATURAL KILLER CELLS) ANALYSIS
Standard 51Cr chromium release assay
Target: labelled RAd-infected
fibroblast target cells
Effector: NK cells
Analyze cytotoxicity and
Degranulation of NK cells
http://www.perkinelmer.com/resources/technicalresources/applicationsupportknowledgebase/radiometric/chromium51.xhtml
FIGURE 8A-1
“Before designating a virus gene as being an NK cell evasion function, it is
important to monitor its biological activity during infection. The effects of
US18 and US20 on NK cell recognition were therefore analyzed using the
HCMV deletion mutants.”
FIGURE 8A-2
“Relative to uninfected cells, infection with strain Merlin elicited robust
protection against NK cells in all donors tested. A significant increase in
NK cell degranulation was associated with loss of US18 or US20 or the
US18–22 ‘block’ deletion, and an additive effect was observed when both
genes were absent.”
FIGURE 8B
“The use of a MICA blocking antibody led to a small decrease in NK
degranulation in response to targets infected with the US18 and US20
double deletion virus, whilst the same antibody had no effect on NK
activation in response to Mock or HCMV-infected targets.”
WHAT DOES IT MEAN?
‘It was concluded that US18 and US20 were effective in suppressing NK cell
activation in the context of a productive HCMV infection.’
‘These data suggest that either the blockade of MICA was incomplete, or that US18 &
US20 target other cellular molecules capable of regulating NK cell activation.’
WHY IS IT IMPORTANT?
In layman’s terms, the two genes – US18 and US20 – successfully prevent Natural
Killer cells from being activated.
It also points out the possibility that the Major Histocompatibility Complex A was
altered or incompletely formed, otherwise genes US18 and US20 could be
targeting other molecular components that activate Natural Killer cells.
RESULTS
When expressed using ad vectors, IE1 induced relatively modest increase in MICA
and MICB, but provided for a major upregulation in ULBP2 both as the level of
total protein expression and specifically on the cell surface.
In contrast, IE2 induced strong activation of MICA and MICB, yet only a small increase
of ULBP2 levels.
IE1 and IE2 were thus found to differentially activate individual ligands recognized by
the NKG2D activator receptor.
RESULTS
Expression of US18 or US20 led to reductions in MICA/B levels
Deletion of US18 alone had no visible effect on cell surface
Deletion of US20 induced a modest but significant increase in surface levels of MICA
compared to parental virus
Deletion of both US18 and US20 caused a much more dramatic increase in MICA
levels
IN CONCLUSION:
The infected cell responded to differential sensing of the:
a. Expression of IE1 to promote the upregulation of ULBP2
b. Expression of IE2 to promote the upregulation of MICA/B
Expression of the US18–US22 genes individually was each associated with a minor
reduction in MICA cell surface expression
Deletion of both US18 and US20 caused a much more dramatic increase in MICA
levels
REFERENCES
Fielding, C. A., & et al. (2014). Two Novel Human Cytomegalovirus NK Cell Evasion Functions Target MICA for Lysosomal
Degradation. PLoS Pathogens,10(5). Retrieved May 26, 2014, from
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4006889/
Immune Evasion (definition). (n.d.).Immune Evasion (definition). Retrieved May 28, 2014, from
http://www.reference.md/files/D057/mD057131.html
Ploegh, H. L. (1998). Viral Strategies of Immune Evasion. Science Magazine,280(5361), 248-253. Retrieved May 28, 2014,
from http://www.sciencemag.org/content/280/5
Trasnfection Protocols & Applications (definition). Sample & Assay Technologies. Retrieved May 28, 2014. from
http://www.qiagen.com/resources/molecular-biology-methods/transfection/
Copeland, N., Jenkins, N. & Court, D. Et al. (2001). Recombineering: A Power New Tool for Mouse Functional Genomics.
Nature Reviews Genetics 2 (769-779). Retrieved May 28, 2014. from
http://www.nature.com/nrg/journal/v2/n10/full/nrg1001-769a.html
Magi, B., Liberatori, S. et al. (2005). Immunoblotting Techniques. Methods in Molecular Biology. Retreived May 28, 2014,
from http://www.ncbi.nlm.nih.gov/pubmed/15596900
TEAM ANALYSIS
1. The researchers started out exploring if there was a temporal window of
activation pre-expression of suppression genes.
2. The methods were appropriate for their alternate exploration, however not for
their initial hypothesis.
3. The conclusions were not overstated—if anything they were understated.
4. This research could potentially have an effect on any individual who is infected
with a herpes virus.
5. It is super complicated but it HCMV is a perfect virus to study immunize evasion
because of how much of its viral genome is dedicated to immunize evasion
6. The experimental step would be to take the procedure into animal testing