Exosomal PD-L1 promotes tumor growth through immune escape in non-small cell lung cancer

Abstract

Programmed cell death protein-1/programmed cell death ligand-1 (PD-1/PD-L1) pathway blockade is a promising new cancer therapy. Although PD-1/PD-L1 treatment has yielded clinical benefits in several types of cancer, further studies are required to clarify predictive biomarkers for drug efficacy and to understand the fundamental mechanism of PD-1/PD-L1 interaction between host and tumor cells. Here, we show that exosomes derived from lung cancer cells express PD-L1 and play a role in immune escape by reducing T-cell activity and promoting tumor growth. The abundance of PD-L1 on exosomes represented the quantity of PD-L1 expression on cell surfaces. Exosomes containing PD-L1 inhibited interferon-gamma (IFN-γ) secretion by Jurkat T cells. IFN-γ secretion was restored by PD-L1 knockout or masking on the exosomes. Both forced expression of PD-L1 on cells without PD-L1 and treatment with exosomes containing PD-L1 enhanced tumor growth in vivo. PD-L1 was present on exosomes isolated from the plasma of patients with non-small cell lung cancer, and its abundance in exosomes was correlated with PD-L1 positivity in tumor tissues. Exosomes can impair immune functions by reducing cytokine production and inducing apoptosis in CD8⁺ T cells. Our findings indicate that tumor-derived exosomes expressing PD-L1 may be an important mediator of tumor immune escape.

Fig. 1. The expression of PD-L1 in NSCLC cell lines and cells with acquired resistance to various anticancer drugs.

a The basal expression of PD-L1 in NSCLC cells was determined by western blot analysis. b, c PD-L1 expression in parental and resistant cells was determined by western blot analysis and flow cytometry. MFI, median fluorescence intensity; CR-1, cisplatin resistance; GR, gefitinib resistance; ER, erlotinib resistance; WR, WZ4002 resistance; CLR, CL-387785 resistance; CR, crizotinib resistance

Fig. 2. Identification of PD-L1 in exosomes isolated from lung cancer cell lines.

Exosomes were isolated by using ultracentrifugation as described in the methods. a Nanoparticle tracking analysis (NTA) of exosomes derived from A549, H460, and H1975 cells. b Representative electron micrograph of exosomes. Scale bars: 100 nm. c Western blot analysis was performed with 10 μg of exosomal protein to validate the expression of PD-L1 and exosome biomarker proteins. d Representative immunofluorescence images of exosomes. Exosomes were immunostained with anti-PD-L1 (green fluorescence) and DiI (red fluorescence). Scale bars: 100 nm. e Representative immuno-electron micrograph of exosomes. The red arrows indicate PD-L1 expression. Scale bars: 100 nm

Fig. 3. PD-L1-containing exosomes inhibit INF-γ production in Jurkat cells.

a Jurkat cells were treated with the indicated exosomes for 4 h in the presence of PMA (50 ng mL^–1) and ionomycin (500 ng mL^–1) after PD-L1 masking within the exosomes through treatment with control or anti-PD-L1 antibodies for 30 min. IFN-γ production was measured by ELISA. b H460/PD-L1^KO cells were generated as described in the methods. PD-L1 expression was confirmed by western blot analysis. c Nanoparticle tracking analysis (NTA) of H460 cell-derived and H460/PD-L1^KO cell-derived exosomes. d Jurkat cells were treated with H460-derived or H460/PD-L1^KO cell-derived exosomes for 4 h in the presence of PMA (50 ng mL^–1) and ionomycin (500 ng mL^–1), and IFN-γ production was then measured by ELISA. The results are from three experiments. All data are presented as the means ± standard deviations. *P < 0.05, **P < 0.005, and ***P < 0.0005 by a paired two-tailed Student’s t-test

Fig. 4. The effect of PD-L1-containing exosomes on tumor growth in vivo.

a Production of mPD-L1-containing exosomes by LLC-1 cells. Stable LLC-1/PD-L1 cells were generated by infection with pGIPZ-shmPD-L1/Flag-mPD-L1. PD-L1 expression levels in cell lysates (10 μg) and exosomes (10 μg) were determined by western blot analysis. b The cell proliferation rate was measured using a cell counter. c A xenograft tumor model was established by injecting LLC-1 or LLC-1/PD-L1 cells into the flank region of mice (n = 10). d Exosomes (50 μg 100 μL^–1) were injected into the tail vein of mice (n = 10) three times before and after the injection of LLC-1 cells. Tumor volumes were measured on the indicated day. e, f The numbers of tumor-infiltrating T cells (CD3+ cells) and proliferating tumor cells (Ki-67+ cells) were counted by using multiplex immunofluorescence in tumors harvested from the tumor-bearing mice described in (c) and (d). All data are presented as the means ± standard deviations. *P < 0.05 and **P < 0.005 by a paired two-tailed Student’s t-test

Fig. 5. Clinical implication of exosomes derived from the serum of patients with NSCLC.

a Representative images of immunohistochemistry for PD-L1. Scale bars: 50 μm. b Relationship between the number of PD-L1-positive exosomes and PD-L1 expression in tumor tissue. Each dot represents one individual; the horizontal lines represent the medians. c–j Inhibitory effect of exosomes on IL-2 and IFN-γ production in CD8⁺ T cells isolated from 24 patients with NSCLC. CD8⁺ T cells were stimulated with PMA (50 ng mL^–1), ionomycin (500 ng mL^–1), and brefeldin A (3 μg mL^–1) for 4 h after treatment with autologous exosomes from the same patient. Cells were stained with anti-CD8 antibody, fixed, and permeabilized, followed by intracellular staining with anti-human IL-2 and IFN-γ antibodies and analysis using a FACScan flow cytometer. c, d Inhibition of IL-2 and IFN-γ production in individual samples according to treatment with exosomes. e, h Summary of the analysis of IL-2 and IFN-γ production inhibition. Summary of the analysis of IL-2 and IFN-γ inhibition in patients with a high proportion of PD-L1-positive exosomes (f, i) and patients with a low proportion of PD-L1-positive exosomes (g, j). P = 0.0367 by an unpaired two-tailed Student’s t-test (B); P < 0.0001, P = 0.024, P = 0.012, and P = 0.074 by a paired two-tailed Student’s t-test (e–j)

Fig. 6. Induction of apoptosis by PD-L1-positive exosomes.

a CD8⁺ T cells (from patients #5 and #9) were labeled with 2.5 μM CFSE and cultured with H460 cell-derived exosomes or H460/PD-L1^KO cell-derived exosomes in the presence of 10 μg mL^–1 anti-CD3, 2 μg mL^–1 anti-CD28, and 2 μg mL^–1 anti-IL-2 for 72 h. b CD8⁺ T cells were incubated with H460 cell-derived exosomes or H460/PD-L1^KO cell-derived exosomes for 24 h and then double stained with Annexin V-FITC (1 μg mL^–1) and propidium iodide (50 μg mL^–1). Analyses were performed using a FACScan flow cytometer. All data are presented as the means ± standard deviations. *P < 0.05 and **P < 0.005 by a paired two-tailed Student’s t-test