Immune Sensing of Latent Cytomegalovirus Reactivation: Impact on Immune Senescence?

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

2. 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

3. 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 Latency Acute phase Virus CD8 T cells 1 5 10 months Red staining of intranuclear IE1 protein Black staining of CD8 T cells Podlech et al., JVl, 2000

6. ~ 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

7. CD8 T cell ~ ~ 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 5 10 months

8. The immunodominant IE1 peptide of murine CMV ORF Phase Sequence Restriction Reference m123 (ie1)IE 168YPHFMPTNL176 Ld Reddehase, Rothbard, and Koszinowski, Nature, 1989

9. TAP 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

10. 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 5 10 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 m18 M45 M83 M84 m18 M45 M83 M84 m04 m04 m123/IE1 m123/IE1

11. 1 5 3 2 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 5 4 3 2 1 M122 m123 P1/3 enhancer Activation Differential splicing IE1 specific RT-PCR 4 3 2 1 IE1 mRNA IE3 mRNA

12. 1 5 3 2 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 5 4 3 2 1 M122 m123 P1/3 enhancer Activation Differential splicing IE1 specific RT-PCR 4 3 2 1 IE1 mRNA IE3 mRNA

13. 1 5 3 2 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 5 4 3 2 1 M122 m123 P1/3 enhancer Activation Differential splicing IE1 specific RT-PCR 4 3 2 1 IE1 mRNA IE3 mRNA

14. 1 5 3 2 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 5 4 3 2 1 M122 m123 P1/3 enhancer Activation Differential splicing IE1 specific RT-PCR 4 3 2 1 IE1 mRNA IE3 mRNA

15. 1 5 3 2 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 5 4 3 2 1 M122 m123 P1/3 enhancer Activation Differential splicing Poisson distribution analysis of variegated gene expression 4 3 2 1 IE1 mRNA Kurz et al., JVI, 1999 Grzimek et al., JVI, 2001 Simon et al., JVI, 2005 IE3 mRNA

16. Generation of recombinant CMV with a point mutation in the codon of the C-terminal MHC anchor residue of the IE1 peptide 2 4 3 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

17. ~ ~ 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

18. IE1 Desilencing and immune sensing hypothesis

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

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

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

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

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

24. ~ Multiple checkpoints for immune sensing MIE locus latency Recurrence Stages of transcriptional reactivation 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