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.

Tertiary lymphoid structures (TLS) are associated with favorable outcome in non-metastatic colorectal carcinoma (nmCRC), but the dynamics of TLS maturation and its association with effective anti-tumor immune surveillance in nmCRC are unclear. Here, we hypothesized that not only the number of TLS but also their composition harbors information on recurrence risk in nmCRC. In a comprehensive molecular, tissue, laboratory, and clinical analysis of 109 patients with stage II/III nmCRC, we assessed TLS numbers and degree of maturation in surgical specimens by multi-parameter immunofluorescence of follicular dendritic cell (FDC) and germinal center (GC) markers. TLS formed in most tumors and were significantly more prevalent in highly-microsatellite-instable (MSI-H) and/or BRAF-mutant nmCRC. We could distinguish three sequential TLS maturation stages which were characterized by increasing prevalence of FDCs and mature B-cells: [1] Early TLS, composed of dense lymphocytic aggregates without FDCs, [2] Primary follicle-like TLS, having FDCs but no GC reaction, and [3] Secondary follicle-like TLS, having an active GC reaction. A simple integrated TLS immunoscore reflecting these parameters identified a large subgroup of nmCRC patients with a very low risk of recurrence independently of clinical co-variables such as ECOG performance status, age, stage, and adjuvant chemotherapy. We conclude that (1) mismatch repair and BRAF mutation status are associated with the formation of TLS in nmCRC, (2) TLS formation in nmCRC follows sequential maturation steps, culminating in germinal center formation, and (3) this maturation process harbors important prognostic information on the risk of disease recurrence.

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