PowerPointPrsentation

1
Immune Sensing of Latent Cytomegalovirus
Reactivation Impact on Immune Senescence?
Molecular Virology Lab
Immunology Lab
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Cytomegalic Inclusion Disease (CID) a disease of
the immunocompromised
Congenital infection (in the US) Cases per
annum 40,000 Asymptomatic at birth 36,000
5,400 Neurol. sequelae Symptomatic at birth
4,000 3,600 Neurol. sequelae 400 Fatal
cases
Transplantation-associated (in the US) Cases per
annum 2,800 Moderate disease 1,200 Severe
disease 1,600 160
Fatal cases
IOM study Vaccines for the 21st century
CMV vaccine is 1 out of 7 ranked in level 1 out
of 26 candidate vaccines included in the
study Annual gain of 18,000 - 70,000
QALYs Annual saving to the health system US
1.1 billion - 4.0 billion
Courtesy of G.Jahn Institute of Medical
Virology Tuebingen
Jahn et al., Dtsch. Med. Wochenschrift, 1988
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Immune senescence A new indication for a CMV
vaccine?
People aged 65 and older in the US 40
million Carriers of latent CMV 30 million
4
Immune surveillance of CMV in the murine BMT model
6 Gy
mCMV
BMC
BALB/c
BALB/c
Recipient
Donor
Lungs
Latency
Acute phase
Virus
CD8 T cells
1
5
10
months
Holtappels et al., JVI, 1998 Podlech et al., JVI,
2000
5
Control of acute CMV infection in the lungs
6 Gy
mCMV
Inflammatory focus
BMC
BALB/c
BALB/c
Recipient
Donor
Lungs
Acute phase
Latency
Virus
CD8 T cells
1
10
5
months
Red staining of intranuclear IE1 protein Black
staining of CD8 T cells
Podlech et al., JVl, 2000
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Control of acute CMV infection in the lungs
Effector CD8 T cells

• Activated phenotype CD62Llow
• Secrete IFN-g
• Lyse infected target cells

Protection upon adoptive transfer
mCMV
6 Gy
BALB/c
Recipient
Podlech et al., JVI, 2000 Holtappels et al., JVI,
1998 2000
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Persistence of protective CD8 T cell infiltrates
during latency
Effector-memory CD8 T cells
6 Gy
mCMV

• Activated phenotype CD62Llow
• Secrete IFN-g
• Protect upon adoptive transfer

BMC
BALB/c
BALB/c
Recipient
Donor
Lungs
Latency
Acute phase
Episome
Virus
CD8 T cells
1
10
5
months
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The immunodominant IE1 peptide of murine CMV
ORF Phase Sequence
Restriction Reference
m123 (ie1) IE
168YPHFMPTNL176 Ld Reddehase,
Rothbard, and Koszinowski, Nature, 1989

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IE1
IFN-g
IFN-g
Processing and presentation of the IE1 peptide
Processing
Immuno- proteasome
Constitutive proteasome
ERAAP
ER
For a review, see Reddehase, Nature Rev.
Immunol., 2002
Golgi
Presentation
TCR
CD8 T cell
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Enrichment of IE1 peptide-specific CD8 T cells
during latency
Lungs
Latency
Acute phase
Holtappels et al., J.Virol., 2000
Virus
CD8 T cells
mo
1
10
5
60.000
5
5
4
4
3
3
ELISPOT-reactive CD8 T cells
ELISPOT-reactive CD8 T cells
2
2
30.000
1
1


3.000
12.000
M84
m18
M83
M45
M84
m18
M83
M45
m04
m04
m123/IE1
m123/IE1
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What is the motor that drives the selective
expansion of the IE1-specific CD8 T cells?
Major Immediate-Early (MIE) region of mCMV
Selective and stochastic IE1 gene expression
during latency
Signal
Receptor
ie1/3 transcription unit
TF
TFBS
1
2
3
4
5
M122
m123
P1/3
enhancer
Activation
Differential splicing
IE1 specific RT-PCR
2
3
4
1
IE1 mRNA
IE3 mRNA
12
What is the motor that drives the selective
expansion of the IE1 specific CD8 T cells?
Major Immediate-Early (MIE) region of mCMV
Selective and stochastic IE1 gene expression
during latency
Signal
Receptor
ie1/3 transcription unit
TF
TFBS
1
2
3
4
5
M122
m123
P1/3
enhancer
Activation
Differential splicing
IE1 specific RT-PCR
2
3
4
1
IE1 mRNA
IE3 mRNA
13
What is the motor that drives the selective
expansion of the IE1 specific CD8 T cells?
Major Immediate-Early (MIE) region of mCMV
Selective and stochastic IE1 gene expression
during latency
Signal
Receptor
ie1/3 transcription unit
TF
TFBS
1
2
3
4
5
M122
m123
P1/3
enhancer
Activation
Differential splicing
IE1 specific RT-PCR
2
3
4
1
IE1 mRNA
IE3 mRNA
14
What is the motor that drives the selective
expansion of the IE1 specific CD8 T cells?
Major Immediate-Early (MIE) region of mCMV
Selective and stochastic IE1 gene expression
during latency
Signal
Receptor
ie1/3 transcription unit
TF
TFBS
1
2
3
4
5
M122
m123
P1/3
enhancer
Activation
Differential splicing
IE1 specific RT-PCR
2
3
4
1
IE1 mRNA
IE3 mRNA
15
What is the motor that drives the selective
expansion of the IE1 specific CD8 T cells?
Major Immediate-Early (MIE) region of mCMV
Selective and stochastic IE1 gene expression
during latency
Signal
Receptor
ie1/3 transcription unit
TF
TFBS
1
2
3
4
5
M122
m123
P1/3
enhancer
Activation
Differential splicing
Poisson distribution analysis of variegated gene
expression
2
3
4
1
IE1 mRNA
Kurz et al., JVI, 1999 Grzimek et al., JVI,
2001 Simon et al., JVI, 2005
IE3 mRNA
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Generation of recombinant CMV with a point
mutation in the codon of the C-terminal MHC
anchor residue of the IE1 peptide
2
3
4
IE1
N
A
P
T
M
M
P
F
F
T
H
H
GCA
N
Y
Y
P
P
L
L176A
A176L
CTA
WT and revertant
MHC class-I anchor mutant
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Elimination of IE1 antigenicity enhances the
frequency of latency-associated IE1 transcription
150
131
105
100
Incidence of IE1 transcription
84
50
36
27
21
10
Revertant
Mutant
5 lungs
Simon et al., in preparation
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Desilencing and immune sensing hypothesis
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Desilencing and immune sensing hypothesis
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Desilencing and immune sensing hypothesis
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Desilencing and immune sensing hypothesis
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Desilencing and immune sensing hypothesis
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Desilencing and immune sensing hypothesis
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Multiple checkpoints for immune sensing
MIE locus latency
Stages of transcriptional reactivation
Recurrence
IE1
MIE Locus
Immunosuppression
anti-IE1
anti-X1
anti-Xn

• Open viral chromatin structure at all essential
  loci
• Abrogation of immune sensing by
  immunosuppression

Conditions for virus recurrence
25
How could latency-associated CMV gene expression
contribute to immune senescence?

• Cellular aging of clonotypic memory cells by a
  high number
• of cell divisions
• 2. Misallocation of immune system resources by
  clonal expansion
• of memory CD8 T cells specific for a
  single pathogen