Figure 1.
Expression of NKG2D ligands in prostate cancer cells.
Human prostate tumor cell lines LNCaP, PC3, and 22Rv1 were stained with monoclonal antibodies (mAbs) specific for NKG2D ligands (ULBP-1, ULBP-2, and MICA/B) and analyzed by flow cytometry. Histograms show the mean fluorescence intensity (MFI) and the shaded histogram represents negative controls in which isotype-matched antibodies were used.
Figure 2.
Characterization of prostate exosomes.
(A) Western blot analyses of CD63, TSG101, GRP78 and PSMA in whole cell lysates of the 22Rv1 cell line or isolated exosomes from supernatants from this cell line. (B) Representative electron microscopic image of 22Rv1 exosomes, showing the typical cup-shaped morphology. Scale bar represents 100 nm. (C) Flow cytometry of 22Rv1 exosomes captured by CD63-coated latex microbeads and stained with mAbs to NKG2D ligands (ULBP-1, ULBP-2, and MICA/B). The histogram shows the mean fluorescence intensity (MFI) and the shaded histogram represents negative controls in which isotype-matched antibodies were used. (D) Representative western blot analysis of CD63, GRP78, and PSMA in exosomes isolated from serum of PC patients. (E) The total amount of exosomal protein per 10 ml of human fresh plasma or serum from healthy donors (n = 6–8 in each group) and CRPC patients (n = 17–18 in each group) was determined by BCA protein assay. ** P<0.01.
Figure 3.
22Rv1 tumor exosomes downregulate cell-surface NKG2D expression on NK cells and CD8+ T cells.
Purified 22Rv1 tumor exosomes or control fibroblast (WPMY1) exosomes were incubated for 24 h with healthy PMBCs, and flow cytometry was performed to determine the surface expression of NKG2D. (A) Representative dot-plots of flow cytometry gating scheme used to analyze NKG2D expression. (B) The bar graphs show the proportion of NKG2D-positive CD8+ T cells and NK cells relative to untreated cells (normalized to a value of 100). (C) The line graphs show the proportion of NKG2D-positive cells relative to untreated cells (normalized to a value of 100) when 22Rv1 exosomes were added at doses of 2–20 µg. (D) The percentage inhibition of NKG2D expression on CD8+ T cells and NK cells when 22Rv1 exosomes were treated with a mixture of anti-ULBP1-5 and MIC mAbs or anti-CD63 mAb. The graphs show mean ± SE (n = 8) from 2 independent experiments. * P<0.05; ** P<0.01.
Figure 4.
Effector lymphocyte cytotoxicity is impaired by 22Rv1 prostate tumor-derived exosomes.
K562 target cells were incubated with PBMCs (effector cells), that were untreated, or treated with 22Rv1 exosomes, anti-NKG2D receptor-blocking mAb, or NKG2D-ligand blocked 22Rv1 exosomes. Additionally, target cells were blocked with a mixture of anti-ULBP1–5 and anti-MIC mAbs and the effect of 22Rv1 supernatant depleted from exosomes was also tested. The data represent mean values of 3 independent experiments ± SD. *P<0.05.
Figure 5.
Surface expression of NKG2D is downregulated on circulating CD8+ T cells and NK cells from patients with CRPC.
(A) and (B) NKG2D surface expression on circulating CD8+ T cells from healthy controls and CRPC patients was analyzed by flow cytometry. (C) and (D) NKG2D surface expression on circulating NK cells from healthy controls and CRPC patients. The graphs show the proportion of positive cells and the mean fluorescence intensity (MFI) of NKG2D as indicated. Each dot represents one individual and horizontal bars indicate mean values. *** P<0.001.
Figure 6.
Exosomes isolated from patients with CRPC downregulate NKG2D expression.
Serum-derived exosomes (2 µg) from healthy individuals or CRPC patients were incubated with healthy PMBCs, and flow cytometry was performed to determine NKG2D expression. (A) and (B) NKG2D surface expression on CD8+ T cells. (C) and (D) NKG2D surface expression on NK cells. The graphs show the proportion of positive cells and the mean fluorescence intensity (MFI) of NKG2D as indicated, relative to untreated PMBCs (normalized to a value of 100 and 1, respectively). Horizontal bars indicate mean values. *** P<0.001.