Fig. 4
Tumor-derived exosomes promoted TIM-1+B cell expansion via HMGB1. Isolated exosomes were characterized by electron microscopy (a), western blotting (b), flow cytometry (c) and NanoTracker analysis (d). (e) After 3 days, CD19+ B cells (2 × 105 cells/well) from healthy donor PBMCs were cultured in the presence or absence of exosomes (derived from cultures of LO2, HepG2, Huh7, Hep3B or LM3 cells) plus 2 μg/ml CpG ODN and then analyzed by flow cytometry to assess the frequency of TIM-1+B cells. f The dot plot represents the average percentages of TIM-1+B cells after culturing with or without exosomes (n = 3). g-h HMGB1 protein levels were measured using western blot analysis (g) and qPCR (h) in paired human HCC samples and their nontumor counterparts (N, nontumor tissue; T, tumor tissue; n = 20). i The HMGB1 level in exosomes (derived from LO2, HepG2, Huh7, Hep3B or LM3 cells) was measured by Wes automated simple western assay. j On day 3, CD19+ B cells (2 × 105 cells/well) purified from healthy donor PBMCs were treated with HMGB1 (10 μg/ml) or exosomes (derived from LO2, HepG2 or Huh7 cells) in the presence or absence of an anti-HMGB1 antibody (10 μg/ml) and were analyzed by flow cytometry to assess the frequency of TIM-1+ B cells. k Dot plots represent the average percentages of TIM-1+ B cells after culturing with HMGB1 or exosomes in the presence or absence of the anti-HMGB1 antibody (n = 3). *P < 0.05, **P < 0.01, *** P < 0.001