Lack of the E3-ubiquitin Ligase TRIM21 Promotes Higher Emergence of Hepatocellular Carcinoma Nodules in Diabetic Mice with Non-Alcoholic Steatohepatitis

Background and aims: The gut microbiota and their metabolites play a key role in the response to checkpoint receptor (CR) inhibitors.Interestingly, antibiotics can also limit cancer progression by modulating the gut microbiota and their metabolites.The role of bacteria and their metabolites in modulating anti-tumour immunity in hepatocellular carcinoma (HCC) is not understood and is the focus of this study.Method: Peripheral blood mononuclear cells (PBMCs) from HCC patients were stimulated in vitro with a peptide pool spanning seven HCC tumour-associated antigens with/without priming with formaldehyde-fixed Escherichia coli DH5a (EC, 50 bacteria-per-cell) or EC supernatants (20% v/v, representing bacterial metabolites).After 3 days, the expression of inhibitory immune checkpoints and functional markers (including PD-1, PD-L1, Granzyme-B, Perforin, IL-10 and IFNg) were assessed on CD4, CD8 and MAIT T cells by flow cytometry.Thirty-four secreted cytokines (including IL-6, IL-23, IL-2, IL-1a, IL-10, IL-18 and IFNg) were measured in PBMC culture supernatants by Luminex.For the analysis, peptide-only PBMC cultures were compared with EC-cell-primed or EC-supernatantprimed peptide-stimulated PBMC cultures.Results: Immunosuppressive PD-1, PD-L1 and IL-10 expression was significantly higher in bacteria-primed HCC-specific CD4+ T-cells ( p = 0.024, 0.0061 and 0.037 respectively) compared to HCC peptide-stimulated only PBMCs.A significant increase in pro-and anti-inflammatory cytokines (including IL-6, IL-23, IL-2, IL-1a, IL-18, IL-10 and IFN gamma) was observed in bacteria-primed HCC peptidestimulated PBMC cultures compared to HCC peptide-only stimulated PBMCs (p = 0.037, 0.037, 0.0098, 0.0061, 0.0061, 0.0061, respectively).No significant differences were seen in MAIT cells, or granzyme-B/ perforin expression between HCC peptide-stimulated only and bacteria-primed HCC peptide-stimulated PBMC cultures.Conclusion: These results reveal that bacterial priming induces inhibitory checkpoint receptor expression on anti-tumour T-cells and promotes an immunosuppressive inflammatory landscape in HCC.Further studies investigating bacterial modulation of anti-tumour immunity in HCC are warranted.