GSIG Journal Club March 15, 2012

1. GSIG Journal Club March 15, 2012

3. Hepatocellular carcinoma (HCC) • Malignant adult tumor with multiple suspected origins- hepatocyte, biliary, or adult progenitor cells, occurs at age 50+, incidence of ~5/100,000 (US) and rising (HCV) • 90% of primary liver cancers in US, 7th most common cancer globally, 3rd leading cause of cancer mortality globally • 10% survival at 5 yrs Chemical carcinogens Farazi & DePinho, Nat Rev Can, 2006

4. Effector loop mutant

5. Presumably, all Nras G12V+ cells are senescent and express pERK, p21 and p16 (double stains or serial sections would have been helpful).

6. Whole liver lysates Purified hepatocytes

7. Increased infiltration of immune cells in Nras G12V livers

8. Suppl Figure 6 | Immunohistochemical staining of immune cell infiltrates surrounding senescent hepatocytes in C57BL/6 mouse livers stably transduced with NrasG12V. Murine livers were harvested on day 12 after intrahepatic delivery of oncogenicNrasand paraffin sections were stained and analyzed for the indicated markers to identify NASDCL-positive cells (granulocytes and monocytes); CD3+ T lymphocytes and CD68+ histiocytes (macrophages) (400X).

9. Disappearance of Nras G12V+ cells in liver Disappearance not caused by apoptosis

10. Clearance of Nras G12V+ senescent cells requires an adaptive immune response Figure 3 | Impaired senescence surveillance results in liver cancer development. a, Experimental setup and description of immune status of C.B-17 WT, C.B-17 SCID and C.B-17 SCID/beige mice. b–e, Quantification of Nras- (b), p21- (c), p-Erk- (d) and p16-positive cells (e) on liver sections from indicated mice after stable intrahepatic delivery of NrasG12V. Values represent mean +/-s.d. (n=4).

11. Nras G12V+ cells may escape senescence by day 30 in CB 17 SCID mice livers

12. HCC arises when senescence surveillance is impaired Suppl Figure 9 | Histopathological analyses detect early hepatocellular carcinomas (HCC) in mice. a, H&E staining of liver sections from C.B-17 (left), C.B-17 SCID (middle) and C.B-17 SCID/beige mice (right) 3 months after stable intrahepatic delivery of NrasG12V via transposable elements. Shown liver section of a C.B-17 mouse reveals normal acinar architecture (left, 100X). Livers of C.B-17 SCID (middle) and C.B-17 SCID/beige (right) mice show dysplastic nodules (DN), a bona fide precursor lesion of HCC. Fig 3f. Impaired immune surveillance of pre-malignant senescent hepatocytes results in the development of HCC in C.B-17 SCID and C.B-17 SCID/beige mice 7 months following NrasG12V transduction. Shown are representative photographs of explanted livers (n=10). Arrows, intrahepatictumour nodules. Values represent mean +/-s.d. of macroscopically detectable tumour nodules bigger than 3mm in diameter.

14. Ag specific T cell activation by peptides expressed in Nras G12V hepatocytes How do they know the IFN production is T cell mediated? What cells are producing IFN? Are T-cells recognizing G12V peptide as an Ag presented by senescent hepatocytes?

15. T cell activation requires a functional senescence program. p19 Arf is required to induce senescence. In its absence, Ag specific T cell response not induced. Intrahepatic delivery of NrasG12V into p19Arf-/- mice caused rapid onset of invasive HCC, which was independent of the CD4+T-cell status Figure 4

16. Senescence surveillance occurs in Tak -/- (inflammation) and DEN (toxicity) models of HCC Suppl Figure 21 Suppl Figure 22 Rag -/- mice are deficient in CD4 and CD8 cells

17. Professional APCs sufficient for T cell activation but senescence surveillance requires both professional and hepatocyte Ag presentation Figure 4 Suppl Figure 23 To distinguish whether senescent hepatocytes can directly present antigens and prime CD4+ T cells or whether antigen presentation occurs via professional APCs, we stably co-delivered oncogenic NrasG12V and ovalbumin (Ova) as a model antigen (NrasG12V-Ova) into mice with liver specific MHCII deficiency (transplantation of wt C57BL/6 BM into sublethally irradiated MHCII-/- mice) or into mice with normal hepatic MHCII expression and MHCII-/- BM (syngeneic transplantation of MHCII-/- BM into sublethally irradiated C57BL/6 wt mice). Subsequently, CFSE-labeled transgenic CD4+ T lymphocytes (OT-II, isolated from transgenic mice, which T cell receptor (TCR) is able to recognize the Ova323-339 peptide presented in the context of I-Ab) were administered and proliferation of these cells was quantified after five days

18. Senescence surveillance requires infiltration of nonresident macrophages and monocytes Gadolinium depletes macrophages and affects monocytes Ly-6G antibody depletes neutrophils Anti-asialo antibody depletes natural killer cells (Ly-6G/Ly-6C) antibody depletes neutrophils and monocytes, but not tissue-resident macrophages (Kupffer cells of the liver)

19. Impaired senescence surveillance in HCV-infected individuals that are immunosuppressed or HIV+ Quantified senescent cells in patients infected with hepatitis C virus (HCV) with or without concomitant human immunodeficiency virus (HIV) infection (impaired CD41 T-cell function) and in a unique cohort of patients who underwent two subsequent liver transplantations due to HCV-induced liver cirrhosis with a comparable grade of liver damage.

20. Concluding remarks and questions • Removal of senescent cells in the liver is required to prevent or attenuate HCC. • Process requires activated CD4+ T cells and monocytes/macrophages • Do the senescent cells escape senescence and give rise to HCC or cause neighbors to become carcinogenic? Possibly “escaping cells initiate the senescence program (and thus display senescence markers) but never achieve the final state… or “factors secreted from pre-malignant senescent hepatocytes also contribute to the oncogenic transformation of neighbouring cells. • How do T cells recognize senescent cells- are there particular antigens presented or cytokines secreted? • How specific is T-cell mediated killing- are any neighboring normal cells eliminated as well?