Interleukin-21 receptor signaling promotes metabolic dysfunction-associated steatohepatitis-driven hepatocellular carcinoma by inducing immunosuppressive IgA+ B cells

Background Dysregulation of immune surveillance is tightly linked to the development of metabolic dysfunction-associated steatohepatitis (MASH)-driven hepatocellular carcinoma (HCC); however, its underlying mechanisms remain unclear. Herein, we aimed to determine the role of interleukin-21 receptor (IL-21R) in MASH-driven HCC. Methods The clinical significance of IL-21R was assessed in human HCC specimens using immunohistochemistry staining. Furthermore, the expression of IL-21R in mice was assessed in the STAM model. Thereafter, two different MASH-driven HCC mouse models were applied between IL-21R-deficient mice and wild type controls to explore the role of IL-21R in MASH-driven HCC. To further elucidate the potential mechanisms by which IL-21R affected MASH-driven HCC, whole transcriptome sequencing, flow cytometry and adoptive lymphocyte transfer were performed. Finally, flow cytometry, enzyme-linked immunosorbent assay, immunofluorescent staining, chromatin immunoprecipitation assay and western blotting were conducted to explore the mechanism by which IL-21R induced IgA+ B cells. Results HCC patients with high IL-21R expression exhibited poor relapse-free survival, advanced TNM stage and severe steatosis. Additionally, IL-21R was demonstrated to be upregulated in mouse liver tumors. Particularly, ablation of IL-21R impeded MASH-driven hepatocarcinogenesis with dramatically reduction of lipid accumulation. Moreover, cytotoxic CD8+ T lymphocyte activation was enhanced in the absence of IL-21R due to the reduction of immunosuppressive IgA+ B cells. Mechanistically, the IL-21R-STAT1-c-Jun/c-Fos regulatory axis was activated in MASH-driven HCC and thus promoted the transcription of Igha, resulting in the induction of IgA+ B cells. Conclusions IL-21R plays a cancer-promoting role by inducing IgA+ B cells in MASH-driven hepatocarcinogenesis. Targeting IL-21R signaling represents a potential therapeutic strategy for cancer therapy. Supplementary Information The online version contains supplementary material available at 10.1186/s12943-024-02001-2.

Either the PCR products for lncRNA library or the purified PCR products for small RNA library were denatured into single-stranded DNA for circularization, while the uncyclized linear DNA molecules were digested.Single-stranded cyclized products were further replicated via rolling cycle amplification, resulting in a DNA nanoball (DNB) containing multiple copies of DNA.Sufficient quality DNBs were subsequently loaded into patterned nanoarrays by using high-intensity DNA nanochip technique and sequenced through combinatorial Probe-Anchor Synthesis (cPAS).
The sequencing data from lncRNA library was filtered with SOAPnuke [2] by several steps: (1) Removing reads containing sequencing adapter; (2) Removing reads whose low-quality base ratio (base quality less than or equal to 15) is more than 20%; (3) Removing reads whose unknown base ('N' base) ratio is more than 5%.Thereafter, the clean reads were mapped to the mouse genome (GRCm38/mm10) using HISAT2 [3] for messenger RNA (mRNA) and lncRNA, or applied to CircBase to annotate circular RNA (circRNA).After that, the expression levels of mRNA and lncRNA were calculated by RSEM (v1.3.1) [4], while the expression level of circRNA was calculated using reads spanning the junction site (the head-to-tail junction of the circRNA sequence) with at least 10 bp coverage at both ends.
The data analysis was further performed on Dr. Tom Multi-omics Data Mining System (https://biosys.bgi.com).
Particularly, the CD107a staining antibody was added to the culture during the stimulation for CD107a measurement.After four hours stimulation, cells were fixed and permeabilized either with BD Cytofix/Cytoperm reagent (#51-2091KZ, BD Biosciences, San Diego, CA, USA) for cytokine staining, or Foxp3/Transcription Factor staining buffer (#00-5523-00, Invitrogen, Carlsbad, CA, USA) for combined staining of cytokines and transcription factors.After fixation/permeabilization, cells were stained with labelled antibodies of interest.Cells were analyzed on BD FACS Verse Flow Cytometer (BD Biosciences, San Jose, CA, USA) and the data were analyzed using FlowJo X 10.0.7 software (Tree Star, Ashland, OR, USA).Absolute numbers of particular immune cells in spleen or livers/tumor tissues were calculated as described previously [5] .The antibodies used for flow cytometry are provided in Table S2.

Real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) analysis
Total RNA was extracted using the TRI reagent (#T9424, Sigma-Aldrich, MO, USA).The levels of target genes were normalized against the level of reference gene (Actb) by 2 -ΔCt , where ΔCt=Ct target -Ct reference .The primer sequences used for qRT-PCR are listed in Supplementary Table S3.

Western blotting
Mouse liver/tumor extracts or cell lysates were separated in a SDS-polyacrylamide gel, electrophoretically transferred to a PVDF membrane (Roche Diagnostics, Mannheim, Germany), and incubated sequentially with primary and secondary antibodies.The signal was developed with a commercial ECL kit (Millipore, Billerica, MA, USA).The antibodies used for western blotting are listed in Supplementary Table S2.

Immunohistochemistry staining
Paraffin-embedded tissue sections were processed for antigen retrieval by microwave heating in 10 mM citrate buffer (pH 6.0), and immunostained with primary antibody against human or mouse IL-21R, mouse F4/80, mouse Ki67 at 4 °C overnight.Next, the sections were incubated with corresponding HRP conjugates and developed with DAB substrate solution.