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 1. Clinical features and immunodysregulations of HCV infection 2.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 We’ll talk:

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

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

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 So HCV-infected hepatocytes has ample opportunity to contact circulating or infiltrating immune cells PD-1 SOCS-1 Tim-3 PD-1 6 h 12 h 24 h 48 h Tim-3 + CD14 + M/M Ø HCV + Huh-7 HCV - Huh-7

7. What is PD-1 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

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

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

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

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

16. Naïve Activated Naïve Activated *** ** % of Tim-3 + CD14 + M/M Ø Naïve Activated Naïve Activated Healthy Subjects Chronic HCV Patients *** NS % of IL12 + CD14 + M/M Ø BC A 7.438.3 27.0 27.3 2.84.1 45.5 47.6 4.454.6 22.3 18.8 3.48.7 13.5 74.4 71.30.2 28.5 0.1 40.933.9 22.7 2.5 67.70.4 31.8 0.2 54.015.3 29.6 1.0 IL-12 Tim-3 CD14 Isotype control Naïve Activated Naïve Activated Healthy Subject HCV Patient Tim-3/IL-12 expression in resting and activated monocytes in HCV patients Zhang et al. PLoS One 2011

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

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

19. 48 h after transfection 72 h after transfection Control siRNA SOCS-1 siRNA SOCS-1 β-Actin + + + + Core A) B) C) Isotype 0.73% LPS R848 Core control siRNA 39.1% LPS R848 Core SOCS-1 siRNA 2.9% Isotype 2.88% LPS R848 Core control siRNA 10.1% LPS R848 Core SOCS-1 siRNA 19.4% PD-1 Count IL-12 Count Silencing SOCS-1 inhibits PD-1 expression and improve IL-12 production

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

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

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

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

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

25. 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 + - + - Core T cells B cells SOCS-1  -Actin + - + - Core T cells B cells SOCS-1  -Actin Differential regulation of T / B lymphocyte signaling by HCV core protein HCV-NHL HS ____________ T cells B cells SOCS-1 hβ2M T cells B cells ____________ HCV-NHL HS SOCS-1 Differential regulation of T/B lymphocyte activation in patients with HCV-NHL pSTAT1

26. B) Anti-PD-L1 Control Ab HS HCV-NHL 80% 10% 9% 1% 46% 13% 36% 5% 22% 36% 38% 4% 1% 9% 77% 13% CFSE T cell Counts 21.3% 13.7% Anti-PD-L1Control Ab CD4 CD8 HCV-Tetramer A) CD69 29.8%17.1% Blocking PD-1 signaling restores T cell activation and proliferation

27. Why we care about this? -Negative signaling molecules in HCV pathogenesis Differential regulation of IL-12/IL-23 expressions by M/M Ф leads to T H 17 cell and Foxp3 + Treg development

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

29. STAT-1 STAT-3  T H 17  IL-12  IL-23  Tim-3 TLR monocyte Gal-9 HCV Tim-3 monocyte Hepatocyte  Foxp3+ Tregs

30. A) B) ** **** 8.91 11.94 74.75 4.40 13.54 20.85 53.68 11.93 1.54 5.68 52.82 39.95 2.44 9.41 62.70 25.45 HSHCV HSHCV Differential regulation of IL-12/IL-23 expressions by M/M Ф leads to T H 17 cell and Foxp3 + Treg development during HCV infection

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

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

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

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

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

36. PD-1 + CD4 + T cells CD69 + CD4 + T cells Pearson Corr. = - 0.374** Sig.(2-tailed) = 0.001 HBV-R HBV-NR HBV-R HBV-NR SOCS-1 β-actin HBsAg stimulation a-CD3/28 stimulation SOCS-1/actin HBsAg stimulation a-CD3/28 stimulation HBV-R HBV-NR HBV-R HBV-NR * * PD-1/SOCS-1 on HBV vaccine failure in HCV-infected individuals HCV patients HBV-NR (n=29) HCV patients HBV-R (n=32) HCV resolved individuals(n=6) Healthy Subjects(n=10) PD-1 expression on CD4 + T cells 12.1% vs 7.0%, P=0.0025.6% vs 4.5%, P>0.05 9.4% vs 4.9%, P=0.007

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

38. Monocyte iDC induced by GM-CSF + IL-4 mDC induced by TNF-α + Poly I:C mDC infected by Lm-HCV vaccine CD14HLA-ABCHLA-DRCD209CD1aCD80CD83CD86 Lm-NS5B Lm-infected DC Why do we care - Listeria monocytogenes (Lm)-based DC-targeting HCV vaccine Development HCV vaccine development: HCV-quasispecies; HCV-delivery; HCV-models; HCV-exhaustion Lm vectorNS5B ∆actA/∆inlB Listeria monocytogenes HCV antigens Virulence determinants

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

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

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

42. HCV persistence PD-1 CD28CD3 gC1qR HCV Core PD-L1 MHC/peptide/B7 T cell dysfunction T cell activation α-PD-1 α-Tim-3 α-gC1qR α-HCV core viral clearance SOCS Negative T cell regulators LPS TLR STAT Monocyte IL-12 Tim-3 Gal-9 Why do we care?- novel therapeutics Improve HBV vaccine response in HCV/HIV-infected individuals Improve HCV - DC therapeutic Vaccine

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