Fig. 1

LAMTOR1 inhibits exosomal PD-L1 secretion. (A, B) Transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA) were utilized to detect the release of exosomes from NSCLC cells. (C) Western blot analysis was performed to assess the expression of PD-L1 in whole-cell lysates (WCL). (D) ELISA was employed to detect the expression of PD-L1 on exosomes derived from NSCLC cells. (E) The density gradient centrifugation confirmed that NSCLC cells (H1975 and H358) secreted exosomal PD-L1, identified by the presence of HRS, CD63, TSG101, and ALIX. (F) The correlation between LAMTOR1 and PD-L1 expression levels in human NSCLC specimens was assessed using H-score (Histochemistry score). (G) The correlation between LAMTOR1 and PD-L1 expression levels in human NSCLC specimens was assessed using immunohistochemistry. Scale bars indicate 50 μm. (H, I) Construction of the GST-LAMTOR1 plasmid enabled the analysis of pathways and functions regulated by LAMTOR1 in NSCLC cells (H1975 and H358) using mass spectrometry. BP: biological process.; CC: cellular component; MF: molecular function; KEGG: Kyoto Encyclopedia of Genes and Genomes. (J) Western blot analysis was performed to investigate the regulation of exosomal PD-L1 by LAMTOR1 in NSCLC cells (H1975 and H358) treated with OE-LAMTOR1 or sh-LAMTOR1. (K) Exosomes collected from NSCLC cells (H1975 and H358) with OE-LAMTOR1 or sh-LAMTOR1 were co-cultured with CD8⁺ T cells, and the functionality of CD8⁺ T cells was assessed using RT-PCR, ELISA, and flow cytometry. (L, M) In addition, flow cytometry was utilized to evaluate the proliferation (%CFSE⁻) and cytotoxicity (%GzmB⁺) of CD8⁺ T cells upon co-culturing with exosomes released from NSCLC cells (H1975 and H358) expressing different levels of LAMTOR1. When PD-L1 antibody blocking (10 μg/ml) was added to the co-culture system of CD8⁺ T cells with exosomes isolated from sh-LAMTOR1 NSCLC cells (H1975 and H358), the proliferation and cytotoxicity functions of CD8⁺ T cells were also evaluated. (N, O) The expression levels of IL-2, IFN-γ, and TNF-α in CD8⁺ T cells were determined through RT-PCR upon co-culturing with exosomes from NSCLC cells (H1975 and H358) OE-LAMTOR1 or sh-LAMTOR1. When PD-L1 antibody blocking (10 μg/ml) was added to the co-culture system of CD8⁺ T cells with exosomes isolated from sh-LAMTOR1 NSCLC cells (H1975 and H358), the expression levels of IL-2, IFN-γ, and TNF-α of CD8⁺ T cells were also evaluated. (P, Q) Additionally, ELISA was used to measure the expression levels of IL-2, IFN-γ, and TNF-α in CD8⁺ T cells treated with exosomes from NSCLC cells (H1975 and H358) overexpressing or underexpressing LAMTOR1. When PD-L1 antibody blocking (10 μg/ml) was added to the co-culture system of CD8⁺ T cells with exosomes isolated from sh-LAMTOR1 NSCLC cells (H1975 and H358), the expression levels of IL-2, IFN-γ, and TNF-α of CD8⁺ T cells were also evaluated. The results are presented as mean ± SEM from six assays, with statistical significance denoted as *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 based on Student’s t test