Essential roles of B-cell subsets in the progression of MASLD and HCC

Highlights•CD19+B220+CD5+CD1d+/CD19-B220+CD5+CD1d- Bregs are strongly increased locally in the livers of mice with MASLD and HCC•IgM+IgD+ Bregs demonstrate an immunosuppressive phenotype shown by PD-L1 and IL-10 expression in murine MASLD and HCC•PD-L1+-, IL-10+-, and IgM+-expressing nonswitched memory B-cells are strongly increased in the livers of mice with HCC•PD-L1+-, IL-10+-, and IgM+IgD+-expressing plasmablasts are strongly increased in the livers of mice with MASLD and HCC•Reduction of IL-10+-expressing CD19-B220+CD5+CD1d- Bregs in vaccinated mice correlates with protection against HCCAbstractBackground & AimsHepatocellular carcinoma (HCC) is the third leading cause of cancer-related death. Metabolic dysfunction-associated steatotic liver disease (MASLD) is a significant cause of HCC. Current treatment options for HCC are very limited. Recent evidence highlights B-cells as key drivers in MASLD progression toward HCC. However, it remains unclear whether multiple B-cell populations or a distinct B-cell subset regulates inflammatory responses during liver disease progression. The scope of this study was to define protumorigenic B-cell subsets in MASLD and HCC.MethodsMulticolor flow cytometry, immunohistochemistry and immunofluorescence analyses were performed to investigate B-cell populations locally (in liver tissue) and systemically (in the blood) in mice with MASLD (n=6) and HCC (n=5-6). The results obtained in mice were also verified in patients with MASLD (n=19) and HCC (n=16).ResultsOur study revealed an increase of two regulatory B-cell (Breg) subsets, CD19+B220+CD5+CD1d+ (****p<0.0001) and CD19-B220+CD5+CD1d- (****p<0.0001), both of which highly overexpress IgM/IgD, PD-L1, IL-10, in the livers of mice with MASLD and HCC. Furthermore, we showed that B-cell depletion therapy in combination with a Listeria-based vaccine decreased CD19-B220+CD5+CD1d- Bregs (*p=0.0103), and improved survival of mice with HCC. We also found CD19+CD5+IL-10+ (*p=0.0167), CD19+CD5+PD-L1+ (*p=0.0333) and CD19+CD5+IgM+IgD+ (*p=0.0317) B-cells in human HCCs. In addition, strong overexpression of IgM/IgD, PD-L1, IL-10, were detected on nonswitched memory B-cells (NSw MBCs, **p=0.0049) and plasmablasts (**p=0.0020). The examination of blood samples obtained from MASLD patients showed an increase of total B-cells expressing IL-10 (****p<0.0001) and IgM/IgD (p=0.3361), CD19+CD20+CD5+CD1d+ Bregs (p=0.6424) and CD19+CD20+CD27+ NSw MBCs (***p=0.0003).ConclusionsOur results provide novel insights into the protumorigenic roles of several B-cell subsets, the specific targeting of which could abrogate the progression of liver disease.Graphical abstractGraphical abstract. Essential roles of IgM+IgD+ Bregs, NSw MBCs and PBs in the progression of MASLD and HCC. Murine models were used to clarify the phenotypic characteristics and investigate the role of B lymphocytes in precancerous (MASLD) and cancerous (HCC) liver diseases. The results obtained in mice have also been verified in patients with MASLD and HCC. Blood and liver tissues obtained from mice and patients were analyzed using multicolor FACS, ELISA, IHC and IF. Our study revealed an increase in numbers of several specific B-cell subsets: 1) CD19+B220+CD5+CD1d+ B10 Bregs; 2) CD19-B220+CD5+CD1d- Bregs; 3) CD19+B220+CD5-CD27+ NSw MBCs; and 4) CD19+B220+CD138+ PBs, all of which highly expressed IgM, IgD receptors, and the inhibitory molecules PD-L1 and IL-10. Two pie chart diagrams, demonstrating the proportions of IL-10+- and PD-L1+-expressing B-cell subsets in the livers of HCC/CaMIN mice and depicting the most immunosuppressive B-cell subset, CD19-B220+CD5+CD1d- Bregs, in murine HCC. All four identified B-cell subsets might play a protumorigenic role in precancerous (MASLD) and cancerous (HCC) liver disease progression.