Hepatocellular carcinoma (HCC) can have viral or non-viral causes1–5. Non-alcoholic steatohepatitis (NASH) is an important driver of HCC. Immunotherapy has been approved for treating HCC, but biomarker-based stratification of patients for optimal response to therapy is an unmet need6,7. Here we report the progressive accumulation of exhausted, unconventionally activated CD8+PD1+ T cells in NASH-affected livers. In preclinical models of NASH-induced HCC, therapeutic immunotherapy targeted at programmed death-1 (PD1) expanded activated CD8+PD1+ T cells within tumours but did not lead to tumour regression, which indicates that tumour immune surveillance was impaired. When given prophylactically, anti-PD1 treatment led to an increase in the incidence of NASH–HCC and in the number and size of tumour nodules, which correlated with increased hepatic CD8+PD1+CXCR6+, TOX+, and TNF+ T cells. The increase in HCC triggered by anti-PD1 treatment was prevented by depletion of CD8+ T cells or TNF neutralization, suggesting that CD8+ T cells help to induce NASH–HCC, rather than invigorating or executing immune surveillance. We found similar phenotypic and functional profiles in hepatic CD8+PD1+ T cells from humans with NAFLD or NASH. A meta-analysis of three randomized phase III clinical trials that tested inhibitors of PDL1 (programmed death-ligand 1) or PD1 in more than 1,600 patients with advanced HCC revealed that immune therapy did not improve survival in patients with non-viral HCC. In two additional cohorts, patients with NASH-driven HCC who received anti-PD1 or anti-PDL1 treatment showed reduced overall survival compared to patients with other aetiologies. Collectively, these data show that non-viral HCC, and particularly NASH–HCC, might be less responsive to immunotherapy, probably owing to NASH-related aberrant T cell activation causing tissue damage that leads to impaired immune surveillance. Our data provide a rationale for stratification of patients with HCC according to underlying aetiology in studies of immunotherapy as a primary or adjuvant treatment.

In contrast to most other malignancies, hepatocellular carcinoma (HCC), which accounts for approximately 90% of primary liver cancers, arises almost exclusively in the setting of chronic inflammation. Irrespective of etiology, a typical sequence of chronic necroinflammation, compensatory liver regeneration, induction of liver fibrosis and subsequent cirrhosis often precedes hepatocarcinogenesis. The liver is a central immunomodulator that ensures organ and systemic protection while maintaining immunotolerance. Deregulation of this tightly controlled liver immunological network is a hallmark of chronic liver disease and HCC. Notably, immunotherapies have raised hope for the successful treatment of advanced HCC. Here we summarize the roles of specific immune cell subsets in chronic liver disease, with a focus on non-alcoholic steatohepatitis and HCC. We review new advances in immunotherapeutic approaches for the treatment of HCC and discuss the challenges posed by the immunotolerant hepatic environment and the dual roles of adaptive and innate immune cells in HCC.

Hepatocellular carcinoma (HCC), the fastest rising cancer in the United States and increasing in Europe, often occurs with nonalcoholic steatohepatitis (NASH). Mechanisms underlying NASH and NASH-induced HCC are largely unknown. We developed a mouse model recapitulating key features of human metabolic syndrome, NASH, and HCC by long-term feeding of a choline-deficient high-fat diet. This induced activated intrahepatic CD8(+) T cells, NKT cells, and inflammatory cytokines, similar to NASH patients. CD8(+) T cells and NKT cells but not myeloid cells promote NASH and HCC through interactions with hepatocytes. NKT cells primarily cause steatosis via secreted LIGHT, while CD8(+) and NKT cells cooperatively induce liver damage. Hepatocellular LTβR and canonical NF-κB signaling facilitate NASH-to-HCC transition, demonstrating that distinct molecular mechanisms determine NASH and HCC development.

X-linked inhibitor of apoptosis protein (XIAP) has been identified as a potent regulator of innate immune responses, and loss-of-function mutations in XIAP cause the development of the X-linked lymphoproliferative syndrome type 2 (XLP-2) in humans. Using gene-targeted mice, we show that loss of XIAP or deletion of its RING domain lead to excessive cell death and IL-1β secretion from dendritic cells triggered by diverse Toll-like receptor stimuli. Aberrant IL-1β secretion is TNF dependent and requires RIP3 but is independent of cIAP1/cIAP2. The observed cell death also requires TNF and RIP3 but proceeds independently of caspase-1/caspase-11 or caspase-8 function. Loss of XIAP results in aberrantly elevated ubiquitylation of RIP1 outside of TNFR complex I. Virally infected Xiap(-/-) mice present with symptoms reminiscent of XLP-2. Our data show that XIAP controls RIP3-dependent cell death and IL-1β secretion in response to TNF, which might contribute to hyperinflammation in patients with XLP-2.

Intrahepatic cholangiocarcinoma (ICC) is a highly malignant, heterogeneous cancer with poor treatment options. We found that mitochondrial dysfunction and oxidative stress trigger a niche favoring cholangiocellular overgrowth and tumorigenesis. Liver damage, reactive oxygen species (ROS) and paracrine tumor necrosis factor (Tnf) from Kupffer cells caused JNK-mediated cholangiocellular proliferation and oncogenic transformation. Anti-oxidant treatment, Kupffer cell depletion, Tnfr1 deletion, or JNK inhibition reduced cholangiocellular pre-neoplastic lesions. Liver-specific JNK1/2 deletion led to tumor reduction and enhanced survival in Akt/Notch- or p53/Kras-induced ICC models. In human ICC, high Tnf expression near ICC lesions, cholangiocellular JNK-phosphorylation, and ROS accumulation in surrounding hepatocytes are present. Thus, Kupffer cell-derived Tnf favors cholangiocellular proliferation/differentiation and carcinogenesis. Targeting the ROS/Tnf/JNK axis may provide opportunities for ICC therapy.

Alternatively polarized macrophages (MU) shape the microenvironment of hepatocellular carcinoma (HCC) and temper anticancer immune responses. We investigated if sorafenib alters the HCC microenvironment by restoring classical macrophage polarization and triggering tumor-directed natural killer (NK) cell responses. In vivo experiments were conducted with sorafenib (25 mg/kg)-treated C57BL/6 wildtype as well as hepatitis B virus (HBV) and lymphotoxin transgenic mice with and without HCC. Monocyte-derived MU or tumor-associated macrophages (TAM) isolated from HCC tissue were treated with sorafenib (0.07-5.0 lg/mL) and cocultured with autologous NK cells. MU and NK cell activation was analyzed by flow cytometry and killing assays, respectively. Cytokine and growth factor release was measured by enzyme-linked immunosorbent assay. Short-term administration of sorafenib triggered activation of hepatic NK cells in wildtype and tumor-bearing mice. In vitro, sorafenib sensitized MU to lipopolysaccharide, reverted alternative MU polarization and enhanced IL12 secretion (P 5 0.0133). NK cells activated by sorafenib-treated MU showed increased degranulation (15.3 6 0.2% versus 32.0 6 0.9%, P < 0.0001) and interferon-gamma (IFN-c) secretion (2.1 6 0.2% versus 8.0 6 0.2%, P < 0.0001) upon target cell contact. Sorafenib-triggered NK cell activation was verified by coculture experiments using TAM. Sorafenib-treated MU increased cytolytic NK cell function against K562, Raji, and HepG2 target cells in a dose-dependent manner. Neutralization of interleukin (IL)12 or IL18 as well as inhibition of the nuclear factor kappa B (NF-jB) pathway reversed NK cell activation in MU/NK cocultures. Conclusion: Sorafenib triggers proinflammatory activity of TAM and subsequently induces antitumor NK cell responses in a cytokine-and NF-jB-dependent fashion. This observation is relevant for HCC therapy, as sorafenib is a compound in clinical use that reverts alternative polarization of TAM in HCC. (HEPATOLOGY 2013;57:2358-2368 T umor-associated macrophages (TAM) located in the hepatocellular carcinoma (HCC) environment increase HCC recurrence after resection and reduce patient survival. 1,2 TAM thereby fosters tumor cell proliferation and tumor spread. 3 Natural killer (NK) cell numbers and activity, on the other hand, are associated with lower HCC stages and improved patient survival. 4,5 An outstanding feature of Abbreviations: AFP, alpha-fetoprotein; C57BL/6 wt , C57BL/6

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Mouse transgenesis has provided fundamental insights into pancreatic cancer, but is limited by the long duration of allele/model generation. Here we show transfection-based multiplexed delivery of CRISPR/Cas9 to the pancreas of adult mice, allowing simultaneous editing of multiple gene sets in individual cells. We use the method to induce pancreatic cancer and exploit CRISPR/Cas9 mutational signatures for phylogenetic tracking of metastatic disease. Our results demonstrate that CRISPR/Cas9-multiplexing enables key applications, such as combinatorial gene-network analysis, in vivo synthetic lethality screening and chromosome engineering. Negative-selection screening in the pancreas using multiplexed-CRISPR/Cas9 confirms the vulnerability of pancreatic cells to Brca2-inactivation in a Kras-mutant context. We also demonstrate modelling of chromosomal deletions and targeted somatic engineering of inter-chromosomal translocations, offering multifaceted opportunities to study complex structural variation, a hallmark of pancreatic cancer. The low-frequency mosaic pattern of transfection-based CRISPR/Cas9 delivery faithfully recapitulates the stochastic nature of human tumorigenesis, supporting wide applicability for biological/preclinical research.