Fig. 6

Tumor-derived exosomal HMGB1 induced TIM-1⁺Breg cell expansion through the TLR2/4-MAPK pathway. a B cells were cultured with HMGB1 and TDEs in the presence or absence of an anti-HMGB1 antibody, and the phosphorylation of P38, JNK and ERK in B cells was determined by western blotting. b-c CD19⁺ B cells (2 × 10⁵ cells/well) purified from healthy donor PBMCs were treated with exosomes (derived from LO2, HepG2 or Huh7 cells) in the presence or absence of TLR2/4, P38, JNK and ERK inhibitors in 96-well plates. After 3 days, the B cells were collected and analyzed by flow cytometry to assess the frequency of TIM-1⁺ B cells. b One representative experiment of three is shown. c The data are represented as the mean ± s.e.m. of three independent experiments. d-i CD19⁺ B cells were cultured with TDEs as described previously. TDE-triggered B cells were collected and cocultured with autologous CD8⁺ T cells labeled with CFSE in the presence of anti-CD3/CD28 mAb bead stimulation. After 3 days, CD8⁺ T cell proliferation (d-e) and TNF-α (f-g) and IFN-γ (h-i) production were analyzed by flow cytometry. d, f and h One representative experiment of three is shown. e, g and i The data are represented as the mean ± s.e.m. of three independent experiments. *P < 0.05, **P < 0.01, *** P < 0.001, ^#P < 0.05, ^##P < 0.01, ^### P < 0.001