Fig 2: CD36 reversed the phenotype induced by BCL6 overexpression.A A Schedule of manipulating the overexpression of CD36 and BCL6. Six-week-old mice were injected with AAV-Control, AVV-BCL6, AAV-Control + AAV-CD36, or AAV-BCL6 + AAV-CD36 via the tail vein and fed an HFD. B Protein level of CD36 and BCL6 in livers of the mice from the indicated groups after being injected with AAV. C Bodyweight, (D) food intake, and (E) LW/BW of the mice from the indicated groups (n = 6/group). F Representative images of H&E and Oil red O staining of liver tissues from the mice from the indicated groups. Scale bar, 100 µm. G Total cholesterol, H triglyceride, I NEFA, J ALT, and (K) AST levels in the livers from the mice of indicated groups fed an HFD for 16 weeks. (n = 6/group). L Representative images of immunohistochemistry staining of F4/80 of liver tissues from four group mice. Scale bar, 100 µm. M Protein level of CD36 and BCL6 in LO2 cells after transfected adv-Control, adv-BCL6, adv-Control + adv-CD36, or adv-BCL6 + adv-CD36. N Representative images of Oil red O staining of LO2 cells of indicated groups stimulated with PA (0.3 mM) for 24 h. Data represent the mean ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001 and n.s. indicates no significance between the two indicated groups.

Fig 3: Treg recruitment as potential mechanism of decreased CD8+ T cell infiltration in tertiary lymphoid structures(A) Comparison of immune suppressive cell infiltration estimated from XCELL between MIA tumor and adjacent normal tissues. p values are calculated using Mann-Whitney U test; ***p < 0.001; **p < 0.01; *p < 0.05; ns p > 0.05.(B) Plot of Tfr/Tfh marker ratio vs CD8+ T cell infiltration. FoxP3, PDCD1, and IL2RA are markers of Tfr cells, while BCL6, ICOS, and IL7R are markers of Tfh cells. See text. p values are calculated using Spearman correlation test; ***p < 0.001; **p < 0.01; *p < 0.05; ns p > 0.05.(C) Typical observation field of MIA 7-plex panel staining for Tfr and Tfh. Merged multispectral image was captured after 6-cycle TSA staining and DAPI counterstain and reveals a mature TLS architecture. Single channels are shown for the area bounded by the white rectangle. Fake colors are assigned as DAPI: blue, CD8: yellow, CXCR5: pink, CD4: green, FOXP3: red, BCL6: cyan and CD3: orange. Sub-populations of CD4+ T cells are highlighted by arrows (Tfr: red, Treg: purple, Th: green and Tfh: cyan).(D) Paired comparison of CD8+ T cell density between non-follicle (Non-Fol) and TLS areas.(E) Paired comparison of ratio of regulatory T cells to helper T cells between TLS and non-follicle areas.(F) Comparison of CD8+ T cell density between groups with high and low Tfr/Tfh ratio

Fig 4: Insulin resistance induced by the HFD can be mitigated by hepatocyte-specific BCL6 overexpression and exacerbated by hepatocyte-specific BCL6 knockout.A Intraperitoneal glucose tolerance tests (GTTs; 1 g/kg) and (B) intraperitoneal insulin tolerance tests (ITTs; 0.75 U/kg) were performed on the AAV-Control mice and the AAV-BCL6 mice at the 16th week of food administration. The corresponding area under the curve (AUC) of the blood glucose level was calculated (n = 6/group). C Representative western blot analysis (n = 3 western blots for each band) of phosphorylated (p-) and total IRS1, AKT, and GSK3ß expression in the livers of the AAV-Control mice and the AAV-BCL6 mice fed an HFD for 16 weeks that received insulin treatment (n = 2 mice in each group without insulin injection; n = 4 mice in each group with insulin injection). D Intraperitoneal glucose tolerance tests (GTTs; 1 g/kg) and (E) intraperitoneal insulin tolerance tests (ITTs; 0.75 U/kg) were performed on the BCL6-CKO and BCL6-flox mice at the 16th week of food administration. The corresponding area under the curve (AUC) of the blood glucose level was calculated (n = 6/group). F Representative western blot analysis (n = 3 western blots for each band) of phosphorylated (p-) and total IRS1, AKT, and GSK3ß expression in the livers of the BCL6-CKO and BCL6-flox mice fed an HFD for 16 weeks that received insulin treatment (n = 2 mice in each group without insulin injection; n = 4 mice in each group with insulin injection). Data represent the mean ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001 and n.s. indicates no significance between the two indicated groups.

Fig 5: Overexpression of BCL6 alleviates HFD-induced hepatic steatosis.A Schedule of BCL6 overexpression. Six-week-old mice were injected with AAV-Control (as a control) or AAV-BCL6 Flag (overexpressing BCL6) via the tail vein. B The protein expression of BCL6 was tested by western blot analysis. C Bodyweight, D liver weight, E LW/BW, and F food intake of the AAV-Control mice and the AAV-BCL6 mice (n = 6/group). G Triglyceride, H NEFA, I total cholesterol in the livers of the AAV-Control mice and the AAV-BCL6 mice fed an HFD for 16 weeks (n = 6/group). J Representative images of H&E and Oil red O staining of liver tissues from the AAV-Control mice and the AAV-BCL6 mice fed an HFD for 16 weeks. Scale bar, 100 µm. K AST and L ALT levels in liver tissues from the AAV-Control mice and the AAV-BCL6 mice fed an HFD for 16 weeks (n = 6/group). M Representative images of immunohistochemistry staining of F4/80 and sirian red staining of liver tissues from two group mice. Scale bar, 100 µm. Hepatic mRNA levels of (N)IL-6 and TNFa, (O) COL1a1and aSMA from two group mice fed an HFD for 16 weeks (n = 6/group). Data represent the mean ± SEM, *P < 0.05, **P < 0.01,***P < 0.001 and N.S. indicates no significance between the two indicated groups.