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Luis J. Montaner, D.V.M., M.Sc., D.Phil. Monday July 20, 2009 IAS 2009, Cape Town

Differential regulation of plasma TRAIL and IFN- a levels following therapy interruption during chronic HIV-1 infection Understanding the role of IFN-  …. Luis J. Montaner, D.V.M., M.Sc., D.Phil. Monday July 20, 2009 IAS 2009, Cape Town. Innate Compartment & HIV Pathogenesis. NK-T Gd- T.

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Luis J. Montaner, D.V.M., M.Sc., D.Phil. Monday July 20, 2009 IAS 2009, Cape Town

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  1. Differential regulation of plasma TRAIL and IFN-a levels following therapy interruption during chronic HIV-1 infectionUnderstanding the role of IFN-…. Luis J. Montaner, D.V.M., M.Sc., D.Phil. Monday July 20, 2009 IAS 2009, Cape Town

  2. Innate Compartment & HIV Pathogenesis NK-T Gd-T

  3. PDC, NK response & HIV-infected Subjects Steady-state HIV-1 replication decreases the functionality of the innate effector compartment and PDC-mediated responses (cross-sectional studies to uninfected subjects). Immune reconstitution of innate effector cell function is possible following antiretroviral therapy yet degree of recovery varies relative to pre-therapy immune status. Journal of Immunology 168:4796, 2002 Journal of Immunology 168:5764, 2002 Journal of Infectious Diseases 188: 873, 2003 Journal of Infectious Diseases 191:1451, 2005 AIDS. 2007 Jan 30;21(3):293-305. Journal of Immunology 179:2642, 2007.

  4. PDC, TRAIL, Interferon-alpha & HIV Viremia • Working Questions (#1) • Does plasma Interferon- levels increase in parallel to increases in sTRAIL levels after therapy interruption in chronic infection? • Are circulating PDC activated by viremia and is their state associated with enhanced or decreased potential for Interferon- secretion?

  5. Higher plasma TRAIL but not IFN- after viral rebound during ART interruption p=0.0016 ns p=0.0003 p=0.002 p=0.0005 100 8000 7000 75 6000 5000 IFN- (pg/ml) 50 4000 3000 25 2000 1000 0 0 Baseline <50) Week 2 (50-19,700) Week 4 (50-517,000) Week 6 (824-493,000) Baseline (<50) Week 2 (50-19,700) Week 4 (50-517,000) Week 6 (824-493,000) sTRAIL (pg/ml) ns p=0.011 p=0.0024 400 p=0.0023 p<0.0001 6000 300 4800 IFN- (pg/ml) 3600 200 2400 100 1200 0 0 Aviremic (N=32) Controls (N=31) Viremic (N=37) All HIV+ (N=69) Controls (N=31) Viremic (N=37) Aviremic (N=32) All HIV+ (N=69)

  6. Evidence for IFN amplification loop? IFN- IFNAR-1/2 CYTOPLASM STAT1 IRF9 STAT2 IRF-7 IRF-3 IRF-5 JAK/STAT pathway p-IRF-7 p-IRF-3 p-IRF-5 p-STAT1 p-STAT1 IRF-9 ISGF3 Dimer Dimer p-IRF-7 p-IRF-3 p-IRF-3 p-IRF-3 p-IRF-5 P-IRF-7 p-IRF-7 p-IRF-3 p-IRF-5 p-IRF-5 NUCLEUS IFN-stimulated genes: Type 1 IFNs Promoters Promoters PKR, OAS, ISG, MX, IRF-7 Autocrine IFN amplification loop

  7. 1 PDC Protein Expression of mTRAIL, IRF-7 Control HIV+ (VL=10,807)) Cell Count IRF-7-PE 0.002 0.0035 70 20.0 60 17.5 50 15.0 % PDC expressing mTRAIL GMF/IRF-7 40 12.5 10.0 30 7.5 20 5.0 2.5 10 0.0 0 Controls HIV+ (N=10 (N=11) Controls (N=8) HIV+ (N=11)

  8. Section Conclusions • Working Questions #1 • Do Interferon- plasma levels increase in parallel to increases in sTRAIL levels after therapy interruption in chronic infection? ANSWER: Evidence for sTRAIL increase but not soluble IFN- in asymptomatic subjects upon ART interruption. • Are circulating PDC as target cells activated by viremia in association with Interferon- secretion? ANSWER: PDC upregulates mTRAIL upon viremia in cojunction with decreased IRF-7 levels consistent with decreased IFN- secretion potential.

  9. PDC, Interferon-alpha, DR5 & CD4 T Cell Loss • Working Question (#2) • Do HIV-induced TRAIL expressing PDCs (or Interferon- mediate cytotoxic responses against autologous HIV-infected CD4 T-cell targets? • Does HIV-1 infection of CD4 T-cells (in vivo or in vitro) result in increased DR5 as a ligand for TRAIL? Herbeuval et al. PNAS 103:7000, 2006; 104:17453, 2007 Boasso, et al. Blood 109:3357, 2006.

  10. Day 1 Day 3 Day 4 PHA/IL-2 IL-2 IL-2 Spinfection PBMCs CD4+ T Cells Day 7 Day 7 51Cr + 4 hr. Effector: PBMCs NKs Targets Ficoll Purify CD107a Cytotoxicity against Autologous HIV-1-Infected CD4+ T Cells

  11. PBMC 100:1 PBMC 100:1 p=0.0039 Percentage Cytotoxicity Percentage Cytotoxicity PBMC + HIV CD4 PBMC + HIV CD4 PBMC + uCD4 PBMC + uCD4 Wilcoxon Matched Pairs Test Non-parametric, n=9 aCD4-HIV stimulation can Augment NK-mediated Lysis of Autologous Infected CD4 T-cells

  12. Uninfected primary aCD4 HIV-infected primary aCD4 HIV-infected SupT1 HIV-infected SupT1 60 50 40 % specific lysis 30 20 10 0 Ig anti-TRAIL Activated PDC do not Lyse aHIV-infected CD4+ T cells 60 40 % specific lysis 20 0 Activated NK Activated PDC

  13. 4 3 2 1 0 0.058% 0.22% CD3+CD4+ T cells 53% 1500 1000 500 0 No evidence for DR5 expression on HIV-infected CD4+ T cells In Vivo-derived In Vitro-derived Control HIV+ Uninfected NL4-3-infected 1.7% 0.58% 0.076% 0.02% DR5 DR5 0.013% 0 1 2 3 4 0 1 2 3 4 10 10 10 10 10 10 10 10 10 10 p24 Ag Uninfected HIV-IIIB HIV-SHIP Sup-T1 % DR5+ CD4+T Cell Count Controls HIV+ (N=10) (N=16) 0 1 2 3 0 1 2 3 0 1 2 3 0 1 2 3 4 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 DR5-PE

  14. PDC, Interferon-alpha & CD4 Cell Loss • Working Question (#2) • Do HIV-activated PDCs (via TLR interactions) or Interferon- mediate PDC-mediated TRAIL-dependent cytotoxic responses against autologous CD4 T-cell HIV-infected targets? ANSWER: PDC show no TRAIL-mediated activity against autologous infected CD4 cells but instead show activity to mediate NK cytotoxic responses upon HIV-infected cell activation. • Do infected CD4 T-cells express DR5 as a ligand for TRAIL? ANSWER: Evidence for lack of surface DR5 expression in infected CD4 T-cells (in vivo or following in vitro PDC activation) in contrast to HIV-infected transformed T cell lines.

  15. CD4+ T cells pDC, IFN- NK cytotoxicity NK cells T-cell activation Serum HIV RNA Suppressive Anti-retroviral Therapy delayed benefit Viremia early benefit Using Innate Activation as Antiviral Strategy after ART? Discussion question (#3) Can we directly test (proof-of-concept) the effect of innate activation and/or viral control via IFN-  following immune reconstitution? No study to date has investigated the effects of Interferon-alpha against HIV-1 if the immune system is fully recovered following ART.

  16. Test of IFN-alpha monotherapy after ART

  17. Conclusions Activation of PDC in chronic HIV Infection unlikely to be a major factor in progressive CD4 T cell depletion via direct cytotoxic activity. Functional PDC/NK axis may represent a major innate antiviral response mechanism acutely lost upon acute infection in progressive disease. Remains to be determined how suppressive is IFN- in the presence of a fully functional immune reconstituted subject.

  18. Acknowledgments Livio Azzoni Craig Carty Jihed Chehimi Jennifer Dubin Betsy Gekonge Agnes Mackiewicz Emmanouil Papasavvas Maria Picone Max Pistilli Griffin Reynolds Andrea Raymond Brian Ross Costin Tomescu Kavita Vinekar

  19. Acknowledgments The Wistar Institute L. Showe & Lab H. Ertl Jeffrey Faust Phlebotomy Unit University of Pennsylvania Ronald Collman Jay Kostman Robert Doms Robert Gross Lan Zou CFAR Cores BD BioSciences Skip Maino Maria Suni Jonathan Lax Immune Disorder Clinic Cele Gallo Karam Mounzer Jane Shull & Clinical Research Staff DAIDS, NIH Larry Fox Daniella Livnat Carolyn Williams Multicenter AIDS Cohort Study Women’s Interagency HIV Study U. Mass Andrea Foulkes Gladstone Inst. Robert Grant Michael McCune Douglas Nixon Gabriel Ortiz Children’s Hospital Of Philadelphia Alma Nowmos Rick M Rutstein Carol Vincent NIAID, NIH The Philadelphia Foundation Commonwealth of Pennsylvania

  20. THANK YOU!

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