We performed bulk RNA sequencing of HCC tissue as well as flow cytometry and single-cell RNA sequencing of enriched ILCs from non-tumour liver, margin and tumour core derived from 48 patients with HCC. Simultaneous measurement of protein and RNA expression at the single-cell level (AbSeq) identified precise signatures of ILC subgroups. In vitro culturing of ILCs was used to validate findings from in silico analysis. Analysis of RNA-sequencing data from large HCC cohorts allowed stratification and survival analysis based on transcriptomic signatures.
RNA sequencing of tumour, non-tumour and margin identified tumour-dependent gradients, which were associated with poor survival and control of ILC plasticity. Single-cell RNA sequencing and flow cytometry of ILCs from HCC livers identified natural killer (NK)-like cells in the non-tumour tissue, losing their cytotoxic profile as they transitioned into tumour ILC1 and NK-like-ILC3 cells. Tumour ILC composition was mediated by cytokine gradients that directed ILC plasticity towards activated tumour ILC2s. This was liver-specific and not seen in ILCs from peripheral blood mononuclear cells. Patients with high ILC2/ILC1 ratio expressed interleukin-33 in the tumour that promoted ILC2 generation, which was associated with better survival.
Our results suggest that the tumour cytokine milieu controls ILC composition and HCC outcome. Specific changes of cytokines modify ILC composition in the tumour by inducing plasticity and alter ILC function.
A highly multiplexed imaging mass cytometry (IMC) panel was designed to simultaneously quantify 36 biomarkers of tissues from 134 patients with HCC and 7 healthy donors to generate 562 highly multiplexed histology images at single-cell resolution. Different function units were defined by topological analysis of TME. CN relevant to the patients’ prognosis was identified as specific target for HCC therapy. Transgenic mouse models were used to validate the novel immunotherapy target for HCC.
Three major types of intratumour areas with distinct distribution patterns of tumorous, stromal and immune cells were identified. 22 cellular metaclusters and 16 CN were defined. CN composed of various types of cells formed regional function units and the regional immunity was regulated reversely by resident Kupffer cells and infiltrating macrophages with protumour and antitumour function, respectively. Depletion of Kupffer cells in mouse liver largely enhances the T cell response, reduces liver tumour growth and sensitises the tumour response to antiprogrammed cell death protein-1 treatment.
Our findings reveal for the first time the various topological function units of HCC TME, which also presents the largest depository of pathological landscape for HCC. This work highlights the potential of Kupffer cell-specific targeting rather than overall myeloid cell blocking as a novel immunotherapy for HCC treatment.
目的
识别免疫eparesis(慢性肝衰竭的标志之一)的组成部分对我们了解肝硬化并发症至关重要。多种抑制性T细胞CD4+已被证实为系统免疫激活的有效抑制剂。在这里，我们建立了表达人类白细胞抗原G (HLA-G)的CD4+ T细胞亚群在急性肝硬化失代偿(AD)患者中的存在、调节和作用机制。< / p > < /秒> Flow cytometry was used to determine the proportion and immunophenotype of CD4+HLA-G+ T cells from peripheral blood of 20 healthy controls (HCs) and 98 patients with cirrhosis (28 with stable cirrhosis (SC), 20 with chronic decompensated cirrhosis (CD) and 50 with AD). Transcriptional and functional signatures of cell-sorted CD4+HLA-G+ cells were delineated by NanoString technology and suppression assays, respectively. The role of immunosuppressive cytokine interleukin (IL)-35 in inducing this population was investigated through in vitro blockade experiments. Immunohistochemistry (IHC) and cultures of primary human Kupffer cells (KCs) were performed to assess cellular sources of IL-35. HLA-G-mediated T cell suppression was explored using neutralising antibodies targeting co-inhibitory pathways. Patients with AD were distinguished by an expansion of a CD4+HLA-G+CTLA-4+IL-35+ immunosuppressive population associated with disease severity, clinical course of AD, infectious complications and poor outcome. Transcriptomic analyses excluded the possibility that these were thymic-derived regulatory T cells. IHC analyses and in vitro cultures demonstrate that KCs represent a potent source of IL-35 which can induce the observed HLA-G+ phenotype. These exert cytotoxic T lymphocyte antigen-4-mediated impaired responses in T cells paralleled by an HLA-G-driven downregulation of T helper 17-related cytokines. We have identified a cytokine-driven peripherally derived suppressive population that may contribute to immuneparesis in AD.
目的
改变的代谢物对肝细胞癌(HCC)的成瘤性很重要。我们对肝细胞癌患者和健康肝供体的门静脉血液中的代谢物变化进行了综合代谢组学分析，并与肝组织和粪便样品中的代谢物变化进行了比较。< / p > < /秒> Serum (portal and central vein), liver tissue (HCC tumour and adjacent non-tumour, normal liver) and stool samples were collected from 102 subjects (52 HCC patients and 50 healthy controls) in the discovery cohort; and 100 subjects (50 HCC patients and 50 healthy controls) in an independent validation cohort. Untargeted metabolomic profiling was performed using high-performance liquid chromatography-mass spectrometry. The function of candidate metabolites was validated in hepatocyte cell lines. Detailed metabolomic evaluation showed distinct clusters of metabolites in serum, liver tissue and stool samples from patients with HCC and control individuals (p<0.001). HCC patients had significantly higher levels of portal vein serum and HCC tissue metabolites of DL-3-phenyllactic acid, L-tryptophan, glycocholic acid and 1-methylnicotinamide than healthy controls, which were associated with impaired liver function and poor survival. On the other hand, HCC patients had lower levels of linoleic acid and phenol in portal vein and stool samples than healthy controls. Linoleic acid and phenol significantly inhibited HCC proliferation, inferring their anti-HCC function as protective metabolites. The integrative metabolome analysis of serum, tissue and stool metabolites revealed unreported metabolic alterations in HCC patients. In portal vein, we identified elevated and depleted metabolites signifying that they might play a role in HCC development.