Fig 2: Exosomes from LSD1-abrogated GC cells promoted T-cell mediated tumor immunity through exosomal PD-L1 in vivo. a-c Tumor sizes (a), growth curve (b), tumor weight (c) of MFC cells in 615 mice treated with CON EXO or KO EXO in the presence of PD-1-recombinant or not (The data are presented as the mean ± SD, n = 6). d Tumor-infiltrating CD8 + T cell ratio in CD3 + T cells of MFC cells in 615 mice treated with CON EXO or KO EXO in the presence of PD-1-recombinant or not (The data are presented as the mean ± SD, n = 3). e and f Expression of CD3 and CD8 of MFC cells in 615 mice treated with CON EXO or KO EXO in the presence of PD-1-recombinant or not (The data are presented as the mean ± SD, n = 3). g and h IL-2 (g) and IFN-γ (h) levels in 615 mice tissues in different groups. Scale bar = 600 μm (The data are presented as the mean ± SD, n = 3). i Expression of PD-L1 in plasma exosomes of 615 mice treated with MFC cells in the presence or absence of LSD1. The data are presented as the mean ± SD; n.s, no significance, * P < 0.05; ** P < 0.01; *** P < 0.001, two-tailed unpaired Student’s t-test

Fig 3: Competitive binding experiments and pharmacokinetic study. (A–C) BiTP competitively inhibited the binding of TGFBR2 to TGF-β. TGFBR2-HIS and BiTP were incubated with precoated TGF-β. The competitive inhibition capability was evaluated with anti-HIS ELISA. (D) BiTP competitively inhibited the binding of PD-1 to PD-L1. PD-L1-HIS and BiTP were incubated with precoated PD-L1. The competitive inhibition capability was evaluated with anti-HIS ELISA. (E) CD-1 mice received 3 mg/kg, 9 mg/kg, 27 mg/kg BiTP treatment by intravenous injection. Then, 5 min, 2 hours, 8 hours, 24 hours, 3 days, 7 days, 14 days, 21 days, and 28 days later, 0.6 mL peripheral blood was collected to measure the concentration of BiTP in plasma. (F–H) The influence of BiTP on the concentration of TGF-β in plasma. B-cell-depleted (μMT) mice received 9 mg/kg BiTP and 6.6 mg/kg anti-PD-L1 treatment. Before treatment, as well as 2 hours, 6 hours, 24 hours, 24 hours, 72 hours, 120 hours, and 168 hours after treatment, peripheral blood was collected to measure the concentration of TGF-β. TGF-β, transforming growth factor-beta.

Fig 4: LSD1 deletion reverses the inhibitory effect of GC cell-derived exosomes in T-cell response by influencing the expression of PD-L1 in target cells. a Confocal image of 20 μg/ml B-EXO or B KO-EXO that were co-incubated with MGC-803 cells for 24 h. Exosomes were stained with PKH26, followed by membrane dye DIO and nucleus dye DAPI staining. Representative confocal images are shown on the left, whereas quantification is shown on the right. Scale bar = 20 µm. b-d Membrane (b) and total expression (c and d) of PD-L1 in BGC-823 cell lines when incubated with B-EXO or B KO-EXO. e Confocal images of recombinant PD-1-Fc binding to BGC-823 cell-PD-L1 when cells were incubated with B-EXO or B KO-EXO. Cells were incubated with anti-rabbit Alexa Fluor 488 dye conjugated antibody. Scale bar = 50 μm. f Mean fluorescence intensity (MFI) of PD-1-Fc binding when cells were incubated with B-EXO or B KO-EXO. g Expression of CD69 in CD8+ T-cell when cells were incubated with B-EXO or B KO-EXO. h Cell survival of BGC-823 cells in anti-CD3/CD28-stimulated PBMC when incubated with B-EXO or B KO-EXO. The data are presented as the mean ± SD; n = 3; n.s, no significance, * P < 0.05 *** P < 0.001, two-tailed unpaired Student’s t-test

Fig 5: The effect of XQ-P3 on the PD-1/PD-L1 interaction. (A) XQ-P3 gradually lost its binding to MDA-MB-231 PD-L1 OE cells when more PD-1 protein existed. MDA-MB-231 PD-L1 OE cells were preincubated with the indicated concentration of PD-1 protein before being incubated with Cy5-labeled XQ-P3 (250 nM). Competition binding was analyzed by flow cytometry. (B) T cells secreted elevated IL-2 after XQ-P3 treatment (n=3). RS, a random sequence served as a negative control of XQ-P3 aptamer.