Immune Sensing of Latent Cytomegalovirus Reactivation: Impact on Immune Senescence? Molecular Virology LabImmunology Lab Institute for Virology

Cytomegalic Inclusion Disease (CID): a disease of the immunocompromised Courtesy of G.Jahn Institute of Medical Virology Tuebingen Jahn et al., Dtsch. Med. Wochenschrift, 1988 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 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 QALYs Annual saving to the health system US $ 1.1 billion billion Transplantation-associated (in the US) Cases per annum: 2,800 Moderate disease: 1,200 Severe disease: 1, Fatal cases IOM study: Vaccines for the 21st century

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

Holtappels et al., JVI, 1998 Podlech et al., JVI, 2000 Recipient mCMV 6 Gy BMC Donor BALB/c ~ Immune surveillance of CMV in the murine BMT model months Lungs Acute phase Latency 1 Virus CD8 T cells 10 5

months Lungs Acute phase Latency 1 Virus CD8 T cells 10 5 Recipient mCMV 6 Gy BMC Donor BALB/c ~ Podlech et al., JVl, 2000 Inflammatory focus Red staining of intranuclear IE1 protein Black staining of CD8 T cells Control of acute CMV infection in the lungs

Podlech et al., JVI, 2000 Holtappels et al., JVI, 1998; 2000 Recipient mCMV 6 Gy BALB/c ~ Effector CD8 T cells Activated phenotype CD62L low Secrete IFN-  Lyse infected target cells Protection upon adoptive transfer Control of acute CMV infection in the lungs

CD8 T cell months Lungs Acute phase Latency 1 Virus CD8 T cells 10 5 Recipient mCMV 6 Gy BMC Donor BALB/c ~ Effector-memory CD8 T cells Activated phenotype CD62L low Secrete IFN-  Protect upon adoptive transfer Persistence of protective CD8 T cell infiltrates during latency ~ Episome

The immunodominant IE1 peptide of murine CMV ORF Phase Sequence Restriction Reference m123 (ie1) IE 168 YPHFMPTNL 176 L d Reddehase, Rothbard, and Koszinowski, Nature, 1989

IFN-  + IFN-  ERAAP Presentation ER Golgi CD8 + T cell TAP TCR IE1 Processing and presentation of the IE1 peptide Processing For a review, see: Reddehase, Nature Rev. Immunol., 2002 Constitutive proteasome Immuno- proteasome

Lungs Acute phase Latency 1 Virus CD8 T cells 10 5 Enrichment of IE1 peptide-specific CD8 T cells during latency 1 3 M84m18 m04 M83 m123/IE1 4 5 M45  % ELISPOT-reactive CD8 T cells M84m18 m04 M83 m123/IE1 4 5 M45  % ELISPOT-reactive CD8 T cells mo Holtappels et al., J.Virol., 2000

Major Immediate-Early (MIE) region of mCMV IE1 specific RT-PCR P 1/3 enhancer 1 ie1/3 transcription unit M122 m123 Differential splicing IE1 mRNA IE3 mRNA TFBS Activation What is the motor that drives the selective expansion of the IE1-specific CD8 T cells? Selective and stochastic IE1 gene expression during latency Signal Receptor TF

IE1 specific RT-PCR What is the motor that drives the selective expansion of the IE1 specific CD8 T cells? Selective and stochastic IE1 gene expression during latency P 1/3 enhancer 1 ie1/3 transcription unit M122 m123 Differential splicing IE1 mRNA IE3 mRNA TFBS Activation Signal Receptor TF Major Immediate-Early (MIE) region of mCMV

IE1 specific RT-PCR What is the motor that drives the selective expansion of the IE1 specific CD8 T cells? Selective and stochastic IE1 gene expression during latency P 1/3 enhancer 1 ie1/3 transcription unit M122 m123 Differential splicing IE1 mRNA IE3 mRNA TFBS Activation Signal Receptor TF Major Immediate-Early (MIE) region of mCMV

IE1 specific RT-PCR What is the motor that drives the selective expansion of the IE1 specific CD8 T cells? Selective and stochastic IE1 gene expression during latency P 1/3 enhancer 1 ie1/3 transcription unit M122 m123 Differential splicing IE1 mRNA IE3 mRNA TFBS Activation Signal Receptor TF Major Immediate-Early (MIE) region of mCMV

Poisson distribution analysis of variegated gene expression What is the motor that drives the selective expansion of the IE1 specific CD8 T cells? Selective and stochastic IE1 gene expression during latency P 1/3 enhancer 1 ie1/3 transcription unit M122 m123 Differential splicing IE1 mRNA IE3 mRNA TFBS Activation Signal Receptor TF Major Immediate-Early (MIE) region of mCMV Kurz et al., JVI, 1999 Grzimek et al., JVI, 2001 Simon et al., JVI, 2005

Generation of recombinant CMV with a point mutation in the codon of the C-terminal MHC anchor residue of the IE1 peptide P Y H F M P T N A GCA L P Y H F M P T N CTA 2 34 IE1 WT and revertant MHC class-I anchor mutant L176A A176L

Elimination of IE1 antigenicity enhances the frequency of latency-associated IE1 transcription RevertantMutant Incidence of IE1 transcription* ~ ~ * 5 lungs Simon et al., in preparation

IE1 Desilencing and immune sensing hypothesis

IE1-specific CD8 T cell Desilencing and immune sensing hypothesis

IE1 Desilencing and immune sensing hypothesis IE1-specific CD8 T cell

IE1-specific CD8 T cell Desilencing and immune sensing hypothesis

IE1 Desilencing and immune sensing hypothesis IE1-specific CD8 T cell

IE1-specific CD8 T cell Effector function Desilencing and immune sensing hypothesis

Multiple checkpoints for immune sensing Open viral chromatin structure at all essential loci Abrogation of immune sensing by immunosuppression MIE locus latency Stages of transcriptional reactivation Recurrence IE1 anti-IE1 anti-X 1 anti-X n Immunosuppression MIE Locus ~ Conditions for virus recurrence:

How could latency-associated CMV gene expression contribute to immune senescence? 1.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