Background & 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.