Fig 2: METTL3, YTHDF1, and TLR9 are highly correlated with immune cell infiltration in B lymphomas samples.A and B Timer 2.0 was used to analyze the positive correlation between METTL3, YTHDF1, and TLR9 and immune infiltration in diffuse large B lymphomas, including CD8+ T cells, CD4+ T cells, and B cells. C, Kaplan–Meier plots of the relapse-free survival of diffuse large B cell lymphoma (DLBCL) patients. Patients were stratified with high (red) or low (blue) expression of TLR9. TLR9, toll-like receptor 9.

Fig 3: A working model depicting the main molecular mechanisms about the current study. EBV EBNA1 increases METTL3 protein degradation via K48-linked ubiquitin-proteasome pathway, which mediated by the E3 ligase PRKN, thus inhibits cellular m6A modification. Downregulation of METTL3 inhibits TLR9 m6A modification. YTHDF1 is an m6A “reader” for cellular TLR9 mRNA with m6A modification, and YTHDF1 binding increases TLR9 mRNA translation efficiency. EBV infection decreases the m6A levels of TLR9 mRNA, thus reduces TLR9 protein levels, as well as TLR9-induced cytokine secretions. EBV, Epstein-Barr virus; m6A, N6-methyladenosine; TLR9, toll-like receptor 9.

Fig 4: STM2457 inhibits CpG-ODN-induced B cell proliferation, Ig secretion and cytokine expressions.A, BJAB cells were treated with STM2457 for 96 h. Cellular m6A modification levels were assayed by dot blot analysis. TLR9 signaling molecules were assayed by Western blotting. B, gene set difference between the TLR9 high and low human immune cells was revealed by GSEA. The top gene set enriched in TLR9 high group comparing to low group is listed. C–E, PBMCs from healthy donors were stained with CFSE dyes and cultured with anti-IgM (10 mM), CD40L (1 mM), and IL-4 (10 nM), costimulated with STM2457(10 μM) or ODN-2006(5 μM) for 0 and 6 days. DMSO was used as a control. Only CD19+ B cells stained CFSE dyes were gated by flow cytometry analysis (C and D). E, histogram shows the percentage of B cells dividing under different conditions. F and G, fresh PBMCs cultured with anti-IgM, CD40L, IL-4, and costimulated with STM2457(10 μM) or ODN-2006(5 μM) for 8 days were collected to detect Ig-producing cells (F) and cytokine level in supernatants by ELISA analysis (G). H and I, BJAB-shRNA cells were transfected with HA-TLR9 plasmids for 48 h. Then, the levels of Ig (H) and cytokine (I) in supernatants were detected by ELISA analysis. J, model diagram demonstrates the role of STM2457 in the regulation of B-cell proliferation and cytokines secretion. Experiments were independently repeated three times, and results are presented as mean ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ns, not significant, compared with the control group. CFSE, carboxyfluorescein succinimidyl ester; DMSO, dimethylsulfoxide; GSEA, gene set enrichment analysis; Ig, immunoglobulin; IL, interleukin; m6A, N6-methyladenosine; ODN, oligodeoxynucleotide; PBMC, peripheral blood mononuclear cell; TLR9, toll-like receptor 9.

Fig 5: EBV infection suppresses TLR9 m6A modification and expression.A, schematic diagram of the MeRIP-seq protocol used to identify RNA m6A profile changes in BJAB cells upon EBV infection. B, GO and KEGG pathway enrichment results for downregulated m6A-modified genes upon EBV infection (p ≤ 0.05, date from our previous study: #GSE133936). C, BJAB cells were infected with 50 MOI EBV for 48 h. TLR9, EBNA1, and GAPDH protein expressions were examined. D, m6A -RIP-qPCR analysis of TLR9 mRNA expression in BJAB cells upon EBV infection. Control group was used for negative control. E and F, Flag-TLR9 was pretransfected into HEK293 cells for 6 h, then, Flag-NC, Flag-EBNA1, or Flag-EBNA1△NLS plasmids were respectively transfected for 24 h. The mRNA and protein expressions were measured by RT-qPCR and Western blotting (E). The m6A abundance of TLR9 mRNA was analyzed by m6A -RIP-qPCR (F). Three independent experiments were performed, and data are shown as the mean ± SD. ∗∗p < 0.01, ∗∗∗p < 0.001, ns, not significant. EBNA1, Epstein-Barr nuclear antigen 1; EBV, Epstein-Barr virus; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; m6A, N6-methyladenosine; MeRIP, methylated RNA immunoprecipitation; MOI, multiplicity of infection; TLR, toll-like receptor; RIP, RNA immunoprecipitation.