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Lymphocyte kinetics in HTLV-1 infection
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Lymphocyte kinetics in HTLV-1 infection

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Bangham, charles

Bangham, charles

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  • 1. Lymphocyte kinetics inHTLV-1 infectionCharles BanghamDepartment of ImmunologyWright-Fleming Institute
  • 2. Human T-lymphotropic virus type 1 (HTLV-1)• infects 10-20 million people worldwide.• endemic (1-20% of adults) in South America, Caribbean, Central Africa, southern Japan.• 2-3% develop an aggressive T-cell leukaemia/lymphoma• 2-3% develop a chronic inflammatory disease either of CNS, eyes, muscles, joints, lungs or skin.• 95% remain healthy carriers of HTLV-1.
  • 3. HTLV-1 persistence and inflammatory diseaseThree main questions:1. How does HTLV-1 persist?2. How does it spread?3. Why do some develop HAM/TSP, whereas most remain healthy carriers?
  • 4. The proviral load of HTLV-1 is high and correlates with the risk of HAM/TSP median proviral load (copies/100 PBMCs) HAM/TSP: 5.4 asymptomatic: 0.34 Nagai et al 1998 J. Neurovirol. 4, 586
  • 5. How is the high proviral load maintained? Retroviruses replicate by two routes: host cell - provirus latentmitotic polymerase - little sequence variation in virus reverse - provirusinfectious expressed transcriptase - sequence variation
  • 6. Evidence for latency of HTLV-1 variability (Kabat-Wu) Env1. HTLV-1 varies little in HIV-1 sequence: HTLV-1 amino acid position Daenke et al. 1990: J. Virol. 64, 12782. HTLV-1 mRNA and proteins are usually undetectable in PBMCs.3. Virions are absent and plasma is non-infectious.
  • 7. ‘Standard model’ of HTLV-1 persistenceHTLV-1 is maintained by passive proliferation of provirus- containing lymphocytes.A fraction of cells express HTLV-1, but too few to allow the immune response to make an impact on proviral load.Supported by observation oflarge clones of HTLV-1+lymphocytes in vivo: Wattel et al. 1995: J. Virol.69, 2863
  • 8. What is wrong with the ‘standard model’?There is a strong T-cell and antibody response to HTLV-1.Anti-HTLV-1 cytotoxic T lymphocytes (CTLs) are chronically activated; virus-specific IgM is produced.Passively proliferating HTLV-1+ cells would be outgrown if any start to express HTLV-1: 1%/day 40-fold drop in load over 1 year. - does the CTL response make any impact?
  • 9. Anti-HTLV-1 CTLs are - • abundant • chronically activated • chiefly directed against the HTLV-1 Tax proteinbut CTL frequency, specificity and activation do not differ between HAM/TSP and asymptomatics Bangham 2000 Curr Opin Immunol 12, 397
  • 10. HTLV-1-specific CTL frequency is positively correlated with proviral load Kubota et al. 2000 Wodarz et al. 2001- so do CTLs determine load, or passively reflect the load?
  • 11. CTLs exert selection on HTLV-1 in vivo1. The tax gene is under positive selection in asymptomatic carriers but not in HAM/TSP patients: AC (N=4) HAM (N=4) DN / DS = 1.15 0.42 % specific lysis2. Naturally occurring Tax variants escape CTL recognition: Niewiesk et al 1994: J. Virol. 68, 6778 Niewiesk et al 1995: J. Virol. 69, 2649
  • 12. Positive selection at individual Tax residues HLA-A2- restricted CTL epitopes Positive selection at individual residues
  • 13. 9 genes were overexpressed in CD8+ cells from individuals with a low HTLV-1 proviral load AAC1 CD8L AC2 CD8L 1 (AJ001687): NKG2Dcluster: cluster: 2 (M57888): Granzyme B 3 (U20350): CX3CR133 genes 16 genes 4 (M30894): TCR-gamma 5 (M12824): CD8 A 20 3 2 6 (AF031824): Leukocystatin 7 (M18737): Granzyme A 8 (M85276): Granulysin (NKG5) 9 (S69115): NKG7 9 B 1 (X57352): IFITM3 (I-8U) 1 2 C 1 (U26174): Granzyme K 2 (M28393): Perforin 5 HAM CD8L D cluster: 1 (M17016): Granzyme B precursor 2 (M1121): RANTES (1) 17 genes 3 (M1121): RANTES (2) Vine, Heaps et al., 2004: J Immunol 173, 5121
  • 14. Protective role of HLA class 1 indicates that CTLs limit HTLV-1 expression in vivo1. Possession of either HLA-A*02 or HLA-Cw*08 : • reduced proviral load by 3-fold HLA-A2 and HLA-Cw8 prevent 36% of potential HAM/TSP • halved the odds of HAM/TSP cases.2. HLA class 1 heterozygosity was associated with a lower proviral load. Jeffery et al: PNAS (1999) 96, 3848; J Immunol (2000) 165, 7278 Vine et al: J Infect Dis (2002) 186, 932
  • 15. Alternative scheme of HTLV-1 persistence CTLSpontaneously expressed Tax protein drives proliferation of provirus+ cells.CTLs kill virus-expressing cells.A fraction of daughter cells survive by shutting down expression (how?)Proviral load is determined by an equilibrium between virus and CTLs : thechief determinant of load is the rate – the ‘efficiency’ – of CTL killing.Nowak and Bangham 1996: Science 272, 74-79; Bangham and Osame 2005: Oncogene 24, 6035–6046.
  • 16. Predictions of model 21. Proviral load is determined by a) CTL efficiency & b) rate of Tax expression.2. Mean lymphocyte turnover rate a) is abnormally high in HTLV-1 infection, especially in HAM/TSP patients, and b) correlates with [Tax].3. Proviral load correlates with Tax protein expression.4. The advantage to the virus conferred by Tax expression diminishes as the CTL lysis rate increases.
  • 17. Quantification of anti-viral CTL efficiency dy = c − εyz HAM dt patient y = freq. of infected cells z = freq. of CTLs ε = lysis rate constant - ‘CTL efficiency’ carrier c = constant Asquith, Mosley et al., 2005: J Gen Virol 86, 1515
  • 18. Quantification of anti-viral CTL efficiency 6 % T a x + C D 4 + c e lls repeat 1: observed data 5 repeat 1: best theoretical fit a fte r 1 8 h rs 4 3 repeat 2: observed data 2 repeat 2: best theoretical fit 1 0 0 10 20 30 40 50 60 70 % CD8+ cellsε is reduced: 80% by block of perforin 29% by partial HLA mismatch Asquith, Mosley et al., 2005 100% by full HLA mismatch J Gen Virol 86, 1515
  • 19. Prediction 1: CTL lytic efficiency determines HTLV-1 proviral load in vivo proviral load (copies/100 PBMC) Asquith, Mosley et al. 2005: J Gen Virol 86, 1515 lytic efficiency ε (%Tax+ cells killed/CD8+ cell/day)Conclusion: 30% to 50% of observed variation in HTLV-1 proviral load is accounted for by variation in ε.
  • 20. Impact of CTL activity in HTLV-1 infection1. The rate of CD8+ cell-mediated lysis is an importantdeterminant - perhaps the largest single determinant - ofvariation in HTLV-1 load between individuals.2. In a typical infected individual, each CD8+ cell kills ~5 HTLV-1-infected cells/day. turnover rate of Tax+ cells of ~7% per day. i.e. total of ~2 x 109 infected CD4+ cells killed/day.
  • 21. Measurement of lymphocyte turnover rates in vivo• infuse 2H-labelled glucose (5% ) i.v. overnight• carbon ring is incorporated into newly synthesizednucleosides genomic DNA of newly divided cells• decay of 2H/1H in DNA direct estimate of t½ ofspecific (sorted) lymphocyte subsets Macallan et al (1998) PNAS 95, 708 Asquith et al (2002) Trends Immunol. 23, 596
  • 22. 2H-glucose kinetics in vivo: CD45RO+ cells turn over faster than CD45RA+ CD45RO+0.15 0.15 L01-HS L07-TBI AC 1 HAM 1 0.1 0.100.05 0.05 0 0.00 0 2 4 6 8 10 0 2 4 6 8 10 CD45RA+0.15 2H incorporation L10-HBO AC 20.100.050.00 0 2 4 6 8 10 Asquith et al. 2006: submitted time (d)
  • 23. Prediction 2a: mean proliferation rate of CD4+ T cells in HAM/TSP > asymptomatic carriers C D 4 + C D 4 5 R O + T ly m p h o cy te 5 p ro life ra tio n ra te (d a y-1 ) 4 p = 0.01 (Mann-Whitney, 3 2-tailed) 2 1 0 Asymptomatic HAM/TSP carriers patients Asquith et al. 2007 PNAS 104, 8035-8040
  • 24. In vivo turnover rate of CD4+CD45RO+ cells correlates with Tax expression ex vivo CD4 CD45RO proliferation rate in 5 4 vivo (% per day) 3 2 P = 0.016 + (Spearman rank 1 correlation) + 0 0 2 4 6 8 10 Tax expression ex vivo (Tax CD4+/CD4+ after 18h culture) + Asquith et al. 2007 PNAS 104, 8035-8040
  • 25. Prediction 2b:Tax-expressing cells turn over faster than Tax – cells in vivo 0.07 L02-HAY 0.06 Tax+ turnover rate: 0.05 0.04 0.03 7% per day 0.02 Tax – 0.01 0 0 2 4 6 8 10 0.14 L07-TBI 0.12 Tax+ 0.1 0.08 13% per day 0.06 0.04 Tax – 0.02 Asquith et al. 2007 0 0 2 4 6 8 10 PNAS 104, 8035-8040
  • 26. Prediction 3: proviral load correlates with Tax expression 25 20(Tax+CD4+/CD4+)% Tax expression y = 0.86x + 3.08 2 R = 0.71 15 HAM/TSP AC 10 5 y = 0.61x + 0.56 2 R = 0.82 0 0 5 10 15 20 Proviral load (% of PBMC infected) Asquith et al. 2005: Retrovirology 2, 75
  • 27. Prediction 4: advantage conferred by Taxexpression falls as CTL lysis rate increases Increase in proviral load caused by model prediction increase in % Tax expression experimental results Rate of CTL lysis Asquith et al. 2005: Retrovirology 2, 75 (Tax+ cells killed/day/CD8+ cell)
  • 28. Quantification of HTLV-1 infection dynamics in vivo cytokines (Il-2, IL-15) 1d (70d) antigen(30d) 0.05 – 5% per day 1d 1d X CTLrestingHTLV-1+ HTLV-1- 1dcell expressing ? cell Asquith and Bangham 2008: Trends Immunol. 29, 4-11
  • 29. Tax expression in HAM/TSP > carriers at a given proviral load Asquith et al. 2005: Retrovirology 2, 75-83- and a givenFoxP3+ Toulza et al.frequency 2008: Blood 111, 5047-5053
  • 30. What determines the rate of HTLV-1 proviral expression in vivo?- strain (sequence) of virus? No.- proportion of defective proviruses? Unknown.- T cell activation (ag, cytokines)? Unlikely to explain observed between-individual variation.- HTLV-1 regulatory products (p30II, Rex, HBZ) Limit existing proviral expression, but do not control onset.- epigenetic changes?- genomic integration site?
  • 31. Is HTLV-1 integration random?Linker-mediated PCRPBMCs taken from 10 HAM patients + 10 ACs311 genomic integration sites mappedObserved integration sites compared with random NlaIII sites in genomeKiran Meekings, 2008: PLoS Pathogens 4(3): e1000027 .Statistical analysis carried out in collaboration with Rick Bushman,Jeremy Leipzig, Chuck Berry
  • 32. HTLV-1 integration frequency in vivo correlates with gene density 10Observed/Expected 8 6 in vitro 4 in vivo 2 0 1 2 3 4 5 6 Gene density (number genes within 25kb) Meekings et al. 2008: p = 1.8 x 10-5 (logistic regression) PLoS Pathogens 4(3): e1000027
  • 33. HTLV-1 proviral integration in vivo predominates: Observed / Expected 3 Observed / Expected 2.5 4 2 3 In Vitro 1.5 2 1 In Vivo 1 0.5 0 0 1 2 5 10 25 1 2 5 10 25 Distance from integration site (kb) Distance from Integration site (kb) - near CpG islands - near transcription start sites- and is associated with 50% 40% % in g e n e 0.4 30% Proportion of sites 0.35 0.3 20% 0.25 10% Tax Positive 0.2 0.15 Tax Negative 0% 0.1 Meekings et al. AC H A M /T S P 0.05 0 2008: PLoS 1 2 5 10 25 Integration Site Window (kb) Pathogens 4(3): e1000027 - Tax expression - and with HAM-TSP
  • 34. Rate of proviral expression & rate of CTL lysis determine HTLV-1 load and risk of HAM/TSPproviral expression HAM HAM fast moderate load high load HTLV-1 AC AC slow low load moderate load fast slow CTL lysis
  • 35. Conclusion: persistence of HTLV-1 in vivo p12I CD4+FoxP3+ cells histone deacetylation CTLsintegration into proviral infectious spreadtranscriptionally expression (viral synapse)active units T-cell proliferation p30, Rex, HBZ