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.

Genome-wide association study Histologically confirmed NAFLD Replication cohort of 559 NAFLD cases and 945 controls genotyped for top SNPs Highlights Genome-wide association study involved 1,483 biopsied NAFLD cases and 17,781 controls. Main analysis shows genome-wide significance for PNPLA3, TM6SF2, HSD17B13 and GCKR. Sub-analyses show significance near LEPR for NASH and near PYGO1 for steatosis. Except for GCKR, the genome-wide significant signals were replicated.

The mechanisms that drive nonalcoholic fatty liver disease (NAFLD) remain incompletely understood. This large multicenter study characterized the transcriptional changes that occur in liver tissue across the NAFLD spectrum as disease progresses to cirrhosis to identify potential circulating markers. We performed high-throughput RNA sequencing on a discovery cohort comprising histologically characterized NAFLD samples from 206 patients. Unsupervised clustering stratified NAFLD on the basis of disease activity and fibrosis stage with differences in age, aspartate aminotransferase (AST), type 2 diabetes mellitus, and carriage of PNPLA3 rs738409, a genetic variant associated with NAFLD. Relative to early disease, we consistently identified 25 differentially expressed genes as fibrosing steatohepatitis progressed through stages F2 to F4. This 25-gene signature was independently validated by logistic modeling in a separate replication cohort (n = 175), and an integrative analysis with publicly available single-cell RNA sequencing data elucidated the likely relative contribution of specific intrahepatic cell populations. Translating these findings to the protein level, SomaScan analysis in more than 300 NAFLD serum samples confirmed that circulating concentrations of proteins AKR1B10 and GDF15 were strongly associated with disease activity and fibrosis stage. Supporting the biological plausibility of these data, in vitro functional studies determined that endoplasmic reticulum stress up-regulated expression of AKR1B10, GDF15, and PDGFA, whereas GDF15 supplementation tempered the inflammatory response in macrophages upon lipid loading and lipopolysaccharide stimulation. This study provides insights into the pathophysiology of progressive fibrosing steatohepatitis, and proof of principle that transcriptomic changes represent potentially tractable and clinically relevant markers of disease progression.

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The rapid improvements in single cell sequencing technologies and analyses afford greater scope for dissecting organoid cultures composed of multiple cell types and create an opportunity to interrogate these models to understand tissue biology, cellular behavior and interactions. To this end, retinal organoids generated from human embryonic stem cells (hESCs) were analyzed by single cell RNA‐sequencing (scRNA‐Seq) at three time points of differentiation. Combinatorial data from all time points revealed the presence of nine clusters, five of which corresponded to key retinal cell types: retinal pigment epithelium (RPE), retinal ganglion cells (RGCs), cone and rod photoreceptors, and Müller glia. The remaining four clusters expressed genes typical of mitotic cells, extracellular matrix components and those involved in homeostasis. The cell clustering analysis revealed the decreasing presence of mitotic cells and RGCs, formation of a distinct RPE cluster, the emergence of cone and rod photoreceptors from photoreceptor precursors, and an increasing number of Müller glia cells over time. Pseudo‐time analysis resembled the order of cell birth during retinal development, with the mitotic cluster commencing the trajectory and the large majority of Müller glia completing the time line. Together, these data demonstrate the feasibility and potential of scRNA‐Seq to dissect the inherent complexity of retinal organoids and the orderly birth of key retinal cell types. Stem Cells 2019;37:593–598