Fig 2: AM-dLNPs mediated multiple ICs blockade and enhanced T cell cytotoxicity. (A) Schematic illustration of blockade and internalization of PD-L1 and GAL-3 on cancer cell by AM-dLNPs. (B–D) Representative flow cytometric histograms (left) and bar graph (right) of the relative levels of membrane PD-L1 (B), GAL-3 (C), CD155 (D) on IFN-γ-pretreated (20 ng/ml, 24h) CT-26 cells after incubation for 4h with dLNPs, M-dLNPs, AM-LNPs and AM-dLNPs or PBS (control) (n = 3 independent experiments). (E) Representative flow cytometric histograms (left) and bar graph (right) of the relative levels of membrane GAL-3 on SKBR3 tumor cells after incubation for 4h with dLNPs, M-dLNPs, AM-LNPs and AM-dLNPs or PBS (control) (n = 3 independent experiments). (F) The dynamic colocalization of PD-L1 (green) with early endosomes (Rab5a, purple), late endosomes (Rab7, purple), circulating endosome (Rab11, purple) and the lysosomal marker Lamp1 in 293-T cells incubated with AM-dLNPs (red), as shown by confocal images, scale bar, 10 μm. The white box indicates the magnified region. (G) IFN-γ-pretreated (20 ng/ml, 24h) 4T1-OVA/Luci cells were treated with diABZI, dLNPs, M-dLNPs, AM-LNPs or AM-dLNPs for 4h (diABZI = 5 μg/ml), fowolled by co-culture with OT-1 T cells for 24 h at an effector: target ratio of 5:1 (n = 3 independent experiments). (H–J) Representative flow cytometric percentage of IFN-γ+ (H), Perforin+ (I) and Gzm B+ (J) in the aforementioned T cells, gated on CD3+ CD8+T cells (n = 3 independent experiments). All the data are presented as the mean ± S.D. The p values were determined by one-way ANOVA with Tukey's post-test for (B–E) and (G–J). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)