PD-1, SOCS-1, Tim-3 in HCV infection -WHY WE CARE?

1. PD-1, SOCS-1, Tim-3 in HCV infection -WHY WE CARE? Yao, Z. Q. M.D. Ph.D. Director, Hepatitis (HCV/HIV) Program, JHQ-VAMC Associate Professor, Division of Infectious Diseases Department of Internal Medicine, Quillen COM ETSU

2. Disclosures • Grant funding from NIH NIAID, NIDDK, and ETSU/WFU • No other financial interests involved in this presentation

3. In this Presentation We’ll talk: • 1. Clinical features and immunodysregulations of HCV infection • Negative signaling molecules such as PD-1, SOCS-1, and Tim-3 • in control of human innate to adaptive immune responses • We expect to know: • A) how HCV employ negative signaling molecules to establish • chronic infection; • B) why we care about this – its application in the HCV • pathogenesis, treatment, and vaccine development

4. 200 M WW 4 M U.S. Clinical features of HCV infection 15% PD-1, SOCS-1, Tim-3 HIV Why the majority of infected individuals become chronic?

5. B cell hyperactivation mixed cryoglobulinemia T cell dysfunction and exhaustion non-Hodgkins lymphoma Immunodysregulation in chronic HCV infection Impaired Monocyte maturation into DC Decreased IL-12 Viral Persistence B cell clonal expansion Th17 cell and Foxp3+ Treg cell expansions What is the underlying mechanism leading to these immunodysregulations?

6. Mechanism leading to these immunodysregulations The primary site of HCV infection is within the liver, where hepatic sinusoids lack basal membrane with a very low velocity of blood flow PD-1 SOCS-1 HCV+ Huh-7 HCV- Huh-7 PD-1 Tim-3 So HCV-infected hepatocytes has ample opportunity to contact circulating or infiltrating immune cells Tim-3+ CD14+ M/MØ 6 h 12 h 24 h 48 h

7. Programmed Death-1, first identified on apoptotic cells • Inducible expressed receptor on immune cells upon activation • Provides a negative signaling to TCR positive signaling pathway • A powerful negative feedback mechanism to balance the +/- signal • Blocking PD-1 signaling will reverse T cell dysfunction What is PD-1

8. PD-1 and T cell function / exhaustion

9. Tim-3 : a molecule different from PD-1 A new negative molecule first identified on Th1, but not Th2, and now also found on other cell types: M/MФ, NK cells

10. Suppressor of cytokine signaling (SOCS) – a family of negative inhibitors of cell signaling Cytokine

11. Why we care about this? -Negative signaling molecules in HCV pathogenesis PD-1 & Tim-3 in Monocyte IL-12 regulation

12. Immunodysregulation in chronic HCV infection Impaired Monocyte maturation into DC Decreased IL-12 Viral Persistence

13. Monocyte IL-12 expression is significantly suppressed in chronic HCV infection Healthy HCV 15.2% Gating strategy CD14 ▲ ▲ ▲ ▲ ▲ IL-12 □ 60% ** __________________________ □ 50% □ □ * ____________ □ ■ ■ ** ▲ ____________________________ 40% PD-1+ CD14+ cells 60% * _______________ ▲ ■ 30% ▲ □ 50% □ ▲ □ ▲ ▲ ▲ 20% ■ □ □ ▲ ■ 40% □ ▲ □ ▲ □ ■ ▲ ▲ ▲ ▲ □ □ ▲ □ ▲ ▲ ■ □ ■ ▲ ■ 10% ▲ ■ □ ■ □ ▲ □ ■ ■ ■ 30% ▲ □ ■ ■ ■ ▲ IL-12+ CD14+ cells ▲ ▲ ■ ■ ▲ ▲ ■ □ □ ■ ■ □ □ 0% ▲ ■ ■ ▲ ▲ 20% ■ ■ □ ▲ HCV-InfectedHCV-Resolved Healthy ■ ▲ ▲ □ ▲ ▲ ■ ▲ ▲ ▲ ▲ □ ▲ 10% □ ▲ ▲ ▲ □ ▲ ▲ ▲ 0% HCV-InfectedHCV-Resolved Healthy Ma et al. Immunology 2010; Zhang et al. J Immunol 2011

14. PD-1 is inversely associated with IL-12 production by monocytes B) Pearson Correlation = -0.464* A) LPS/R848 PD-1 PD-1+ CD14+ cells IL-12 IL-12 production C) D) 50% 50% P = 0.003 P = 0.034 40% 40% PD-1+ CD14+ Cells IL-12+ CD14+ Cells 30% 30% 20% 20% 10% 10% 0 0 before after IFN/RBV IFN/RBV before after IFN/RBV IFN/RBV

15. Tim-3 is a negative molecule expressed on resting monocytes to control IL-12 expression Positive cells (%) isoun-stimulatedTLR-stimulated isoun-stimulatedTLR-stimulated 2.7% 0.2% 1.8% Tim-3 55.3% 0.4% 2.2% Tim-3 1.2% 3.0% 45.8% 0.1% 0.1% 38.9% Time of TLR stimulation IL-12 CD14 CD14 0.3% 0.2% 27.2% IL-12 Zhang et al. JLB 2011 Tim-3 functions as a break, and TLR as the driving force for IL-12 expression

16. Tim-3/IL-12 expression in resting and activated monocytes in HCV patients A Healthy Subject HCV Patient Isotype control Naïve Activated Naïve Activated 2.8 4.1 4.4 54.6 3.4 8.7 7.4 38.3 Tim-3 47.6 18.8 74.4 45.5 22.3 13.5 27.3 27.0 CD14 40.9 33.9 54.0 15.3 71.3 0.2 67.7 0.4 CD14 2.5 1.0 0.1 22.7 0.2 29.6 28.5 31.8 IL-12 ** *** B C NS ** *** *** *** *** % of IL12+ CD14+ M/MØ % of Tim-3+ CD14+ M/MØ Healthy Subjects Chronic HCV Patients Naïve Activated Naïve Activated Naïve Activated Naïve Activated Zhang et al. PLoS One 2011

17. It’s not because of TLR expression, but due to defect of intracellular signaling A) Healthy Subjects HCV patients B) Healthy Subjects HCV patients 99.5% 10.3% 40.7% 99.7% * TLR7+ CD14+ Cells TLR4+ CD14+ Cells Healthy Subjects HCV patients Healthy Subjects HCV patients C) Healthy subject HCV-infected HCV-resolved STAT-1+ CD14+ Cells 21.6% 9.6% 23.7% * Healthy HCV-infected HCV-resolved D) IgG anti-Tim-3 + + Core Phospho Stat1 Total Stat1

18. gC1qR PD-1 Isotype 1.2% LPS R848 11.7% LPS R848+core+IgG 3.2% LPS R848+core+a-PDL-1 8.9% Control IgG a-PDL-1 + + Core SOCS-1 β-actin HCV core TLR Count * * M/MФ IL-12 production SOCS-1 % IL-12+CD14+ cells * * IL-12

19. Silencing SOCS-1 inhibits PD-1 expression and improve IL-12 production A) + + + + Core Isotype 0.73% LPS R848 Core control siRNA 39.1% Isotype 2.88% 48 h after transfection72 h after transfection Control siRNASOCS-1 siRNAControl siRNASOCS-1 siRNA LPS R848 Core SOCS-1 siRNA 2.9% LPS R848 Core control siRNA 10.1% LPS R848 Core SOCS-1 siRNA 19.4% B) SOCS-1 Count β-Actin PD-1 C) Count IL-12

20. Crosstalk between PD-1 and SOCS-1 to inhibit STAT-1/5 phosphorylations A) B) Control IgG a-PDL-1 Control siRNA SOCS-1 siRNA + + Core - + + + Core pSTAT-1 pSTAT-1 Total STAT-1 Total STAT-1 D) Control IgG Anti-PDL-1 Control IgG Anti-PDL-1 STAT-1 STAT-5 CD14 CD14 ** * %STAT-5+CD14+ cells %STAT-1+CD14+ cells

21. gC1qR Our Model LPS/R848 Gal-9 HCV core PD-L1 Tim-3 PD-1 TLRs Signaling pathways for IL-12 expression (Jak/STAT) SOCS-1 Th1/Tc1 dysregulation Viral persistence Viral clearance

22. B cell hyperactivation mixed cryoglobulinemia T cell dysfunction / exhaustion non-Hodgkins lymphoma Viral Persistence Immunodysregulation in chronic HCV infection Impaired Monocyte maturation into DC Decreased IL-12 B cell clonal expansion What is the underlying mechanism leading to these immunodysregulations?

23. Why we care about this? -Negative signaling molecules in HCV pathogenesis HCV infection lead to a differential effect on T/B lymphocytes - what is the underlying mechanism ?

24. CD20 CD20 CD20 CD4 CD8 19.4% 45.3% Cell Immunology & Biology 2011 5.8% 10.4% 96.2% 83.3% IgM IgG TALL-1 HCV-NHL CD69 HCV-NHL HCV-Tetramer 19.1% 4.2% 72.3% 48.8% HS HS 70.0% 6.9% HCV-NHL HCV-NHL PD-1 PD-1 PD-1 47.3% 37.2% 53.5% 13.7% HCV HS CD4 Tetramer CD8 CD20

25. T cells B cells + - + - Core SOCS-1 -Actin Differential regulation of T/B lymphocyte activation in patients with HCV-NHL Differential regulation of T / B lymphocyte signaling by HCV core protein T cells B cells T cells B cells T cells B cells ____________ ____________ ____________ ____________ + - + - Core HCV-NHL HS HCV-NHL HS HCV-NHL HS HCV-NHL HS SOCS-1 SOCS-1 pSTAT1 SOCS-1 -Actin hβ2M Conclusion: HCV induces a differential regulation of PD-1/SOCS-1 expression, which translate into a differential regulation of T/B lymphocyte functions through Jak/STAT pathway

26. Blocking PD-1 signaling restores T cell activation and proliferation A) B) Anti-PD-L1 Control Ab Anti-PD-L1 Control Ab 29.8% 17.1% 80% 10% 9% 1% 46% 13% 36% 5% CD69 HS T cell Counts CD4 22% 36% 38% 4% 1% 9% 77% 13% 13.7% 21.3% HCV-NHL HCV-Tetramer CFSE CD8

27. Why we care about this? -Negative signaling molecules in HCV pathogenesis Differential regulation of IL-12/IL-23 expressions by M/MФ leads to TH17 cell and Foxp3+ Treg development

28. Differential regulation of IL-12/IL-23 expressions by M/MФ leads to TH17 cell development during HCV infection HCV HS ** IL-17A HCV HS CD4 IL-12 p35 IL-23 p19 ** * Pearson r=0.465 p＜0.05 IL-23/IL-12 by CD14+ cells

29. Hepatocyte Hepatocyte Hepatocyte TLR TLR monocyte monocyte monocyte monocyte  Tim-3 STAT-3 STAT-1  IL-23  IL-12 HCV  Foxp3+ Tregs Tim-3  TH17 Gal-9

30. Differential regulation of IL-12/IL-23 expressions by M/MФ leads to TH17 cell and Foxp3+ Treg development during HCV infection HS HCV A) ** 11.94 20.85 8.91 13.54 11.93 74.75 53.68 4.40 B) HS HCV *** * 5.68 9.41 1.54 2.44 39.95 25.45 52.82 62.70

31. A) B) HS HCV HS HCV 15.78 7.42 4.89 25.86 20.06 15.94 7.26 14.53 29.85 27.44 16.7 28.50 45.08 62.47 19.03 59.18 NS NS * ** Pearson Correlation = 0.75 Sig. (1-tailed)=0.0002; (2-tailed)=0.0004 CD4+ CD4+ CD25+ CD4+ CD25+ Foxp3+ CD4+ CD25+ Foxp3-

32. Differential regulation of IL-12/IL-23 expressions by monocytes/macrophages leads to TH17 cell and CD4+CD25+Foxp3+development during HCV infection HCV-infected hepatocytes produce Gal-9 and TGF-β CD4+ T cell  IL-23/IL-12 CD25+ FoxP3- T eff  IL-17 CD25+ FoxP3+ T reg Tim-3 is up-regulated more on Foxp3+ Tregs than on Teffs TGF-β/IL-10  monocyte IL-2   proliferation  apoptosis  apoptosis  proliferation Tim-3/Gal-9 interactions shift the balance of Tregs/Teffs by regulating T cell proliferation and apoptosis Teff Treg Treg Treg α-Tim-3 α-Tim-3 may correct the imbalance of Tregs/Teffs ratio induced by HCV Tim-3 /Gal-9 Moorman JP et al J Immunol 2012

33. Immunodysregulation during chronic HCV infection HCV-infected Hepatocytes Increased PD-1, SOCS-1, Tim-3 Decreased IL-12 Impaired CD14+ M/MΦ maturation into DC Increased IL-23 Increased IL-17 HCV chronic infection Accumulated TH17 & Foxp3+ Treg cells Increased IL-10 Increased TGF-β Autoimmune disorders Decreased IL-2 Diminished CD4+/ CD8+ T cells Decreased TNF-α Decreased IFN-γ Increased IgG Aberrant CD19+ B cell activation Increased IgM

34. Why we care about this? -Negative signaling molecules in Vaccine response HBV vaccine response and HCV vaccine development

35. Tim-3 on HBV vaccine failure in HCV-infected individuals HBV Vaccine response: 90% in Healthy Subjects; 50% in HCV-infected patients A) *** HBV-R ** HBV-NR Isotype * B) % Tim-3/CD14+ cells % IL-23p19/CD14+ cells % IL-12p35/CD14+ cells

36. PD-1/SOCS-1 on HBV vaccine failure in HCV-infected individuals 9.4% vs 4.9%, P=0.007 12.1% vs 7.0%, P=0.002 5.6% vs 4.5%, P>0.05 Pearson Corr. = - 0.374** Sig.(2-tailed) = 0.001 PD-1 expression on CD4+ T cells CD69+ CD4+ T cells PD-1+ CD4+ T cells HCV patients HBV-NR (n=29) HCV patients HBV-R (n=32) HCV resolved individuals(n=6) Healthy Subjects(n=10) HBsAg stimulation a-CD3/28 stimulation * HBV-R HBV-NR HBV-R HBV-NR * SOCS-1/actin SOCS-1 β-actin HBV-R HBV-NR HBV-R HBV-NR HBsAg stimulation a-CD3/28 stimulation

37. Control Ab a-PD-L1 Ab A) IgG * 18.1% 29.9% a-PDL-1 HBsAg stimulation * % CD69+ in CD4+ T cells 31.6% 57.8% HBsAg stimulation a-CD3/28 stimulation a-CD3/28 stimulation IgG Control Ab a-PD-L1 Ab B) a-PDL-1 0% 0.3% 7% 92% 1.5% 8% 18% 73% CFSE / HBsAg * HBsAg stimulation * M1 M2 M3 M4 IgG 1% 21% 58% 20% 0% 1.5% 19% 79% * a-PDL-1 a-CD3/28 stimulation * * CFSE / a-CD3/28 M1 M2 M3 M4

38. Why do we care - Listeriamonocytogenes (Lm)-based DC-targeting HCV vaccine Development HCV vaccine development: HCV-quasispecies; HCV-delivery; HCV-models; HCV-exhaustion Listeria monocytogenes HCV antigens Virulence determinants Lm-NS5B ∆actA/∆inlB Lm vector NS5B Lm-infected DC

39. Why do we care? Improve Lm-based DC-targeting HCV vaccine by blocking Tim-3 signaling M/MФ BSA-FITC Uptake (ΔMFI) iDC %Tim-3+ cells %IL-12 + cells mDC Un-infected HCV HCV+IgG HCV+a-Tim-3

40. Why do we care? Improve Lm-based DC-targeting HCV vaccine by blocking Tim-3 signaling %HCV-Tet+/CD3+CD8+ HCV-Tetramer %Tim-3+/HCV-Tet+CD3+CD8+ Lm-control Lm-NS5B SSC HCV-Tetramer α-Tim-3 IgG IgG α-Tim-3 A) Gating strategy CD3+CD8+ Tim-3 CD3+CD8+ B) Iso HCV-resolved HCV-infected

41. Why do we care? Listeria monocytogenes (Lm)-based DC-targeting HCV vaccine A) %IFN-r+/HCV-Tet+CD3+CD8+ IFN-γ B) %GranzymeB+/HCV-Tet+CD3+CD8+ Granzyme-B

42. Why do we care?- novel therapeutics HCV Core MHC/peptide/B7 PD-L1 LPS Gal-9 gC1qR TLR PD-1 CD3 CD28 Tim-3 STAT Monocyte IL-12 α-gC1qR α-HCV core SOCS T cell activation α-PD-1 α-Tim-3 Negative T cell regulators Improve HBV vaccine response in HCV/HIV-infected individuals T cell dysfunction viral clearance Improve HCV - DC therapeutic Vaccine HCV persistence

43. Acknowledgements Dr. T. Niki: President of GalPharm, Japan; Dr. T.J. Liang, Chief Liver Dis, NIH NIDDK Dr. T Wakita, Director Virology Lab, NIH, Japan; Dr. D. Brockstedt, VP of Aduro BioTech, CA