Differential mechanisms of shedding of the glycosylphosphatidylinositol (GPI)-anchored NKG2D ligands

Abstract

Tumor cells release NKG2D ligands to evade NKG2D-mediated immune surveillance. The purpose of our investigation was to explore the cellular mechanisms of release used by various members of the ULBP family. Using biochemical and cellular approaches in both transfectant systems and tumor cell lines, this paper shows that ULBP1, ULBP2, and ULBP3 are released from cells with different kinetics and by distinct mechanisms. Whereas ULBP2 is mainly shed by metalloproteases, ULBP3 is abundantly released as part of membrane vesicles known as exosomes. Interestingly, exosomal ULBP3 protein is much more potent for down-modulation of the NKG2D receptor than soluble ULBP2 protein. This is the first report showing functionally relevant differences in the biochemistry of the three members of the ULBP family and confirms that in depth study of the biochemical features of individual NKG2D ligands will be necessary to understand and manipulate the biology of these proteins for therapy.

FIGURE 1.

Different kinetics of release of ULBP1, -2, and -3. A, CHO cells were transiently transfected with ULBP1, ULBP2, and ULBP3. 24 h later, supernatants were collected and analyzed using a sandwich ELISA. B, cellular ULBP expression was analyzed by flow cytometry. C, kinetics of release of ULBP2 and ULBP3 in stably transfected CHO cells. ULBP release was assessed by ELISA after the cells had been in culture for 2, 4, and 8 h. Data shown here are representative from three experiments. Analysis of ULBP transfected CV1 cells gave similar results (not shown). D, flow cytometry analysis of ULBP2 and ULBP3 expression by stable transfectants.

FIGURE 2.

ULBP2 and -3 are released by the action of metalloproteases, but other mechanisms are also involved. A, CHO transfectants were treated with 1 μm leupeptin (LEU), 1 μm pepstatin (PEP) and a broad metalloproteinase inhibitor, 10 μm BB94. Data are expressed as percentage shedding of the untreated control at 2 h. The inhibition of the release of ULBP2 by the metalloproteinase inhibitor BB94 was the only statistically significant change (t test, p < 0.001), whereas ULBP3 release was only weakly, and not significantly, inhibited by BB94. B, CHO cells were treated with 100 ng/ml of PMA and 5 μm of ionomycin (IONO). The increase of ULBP2 shedding mediated by PMA is statistically significant (p < 0.005). Cells were incubated in the presence of the indicated compounds for 2, 4, 8, and 12 h. Detection of soluble ULBPs in tissue culture supernatant was performed by ELISA.

FIGURE 3.

ULBP3 is released in exosomes both in transfectants and the tumor cell line 293T. A, the exosome fraction and soluble proteins from CHO-ULBP2 and -3 transfectants were purified after 24 h in culture as described under “Experimental Procedures” and compared with total lysate from the same cells in Western blot analysis. Similar results were obtained on analysis of CV1-ULBP3 cells (supplemental Fig. 1). ULBP2 was shed mainly as a soluble protein, whereas ULBP3 was released both as a soluble protein and in exosomes. B, fractionation of exosomes in a sucrose gradient shows co-migration with CD63. C, exosomes from CHO-ULBP3 cells were negatively stained with 2% phosphotungstic acid and analyzed by electron microscopy. As expected, nano-sized vesicles from 30 to 120 nm were observed. Bar, 100 nm. D, analysis of tissue culture supernatant from the 293T cell line, which expresses ULBP2 and ULBP3 endogenously, confirmed the results obtained in the transfectant system. Soluble and exosome fractions were analyzed by Western blot. ULBP2 is mainly shed as a soluble protein, whereas ULBP3 is mainly released in exosomes.

FIGURE 4.

ULBP2 and ULBP3 can be recruited to exosomes and have different susceptibility to metalloproteases. A, the exosome fraction and soluble proteins from CHO-ULBP2 and -3 transfectants, either untreated or treated with the metalloprotease inhibitor BB94, were purified as in Fig. 3A. Western blot analysis of ULBP molecules and CD63 is shown. B, CV1-ULBP2 and CV1-ULBP3 transfectants were analyzed by confocal microscopy. Images show a single focal plane across the cell (depth of the plane 0.2035 μm) using the 63× objective. The series of images corresponding to the same cell are shown in supplemental Fig. 2. Scale bar, 25 μm.

FIGURE 5.

Both ULBP2 and ULBP3 containing supernatants provoke down-regulation of NK cell NKG2D. A, supernatants collected from CHO cells (control) and ULBP-transfected CHO cells were incubated with primary NK cells, prepared from healthy donors, for 48 h. Cell surface NKG2D was then quantitated by flow cytometry. B, t test of five experiments performed with two different donors. Data are expressed as percentage of NKG2D expression observed on NK cells incubated in medium alone. Down-modulation of NKG2D is significant after treatment with both ULBP2 and ULBP3 proteins (p < 0.05 and p < 0.01, respectively). C, ELISA assay to quantitate the amounts of ULBP2 and ULBP3 supernatants used in the down-regulation experiments.

FIGURE 6.

Purified exosomes containing ULBP3 down-modulate NKG2D. A, exosomes were incubated with primary NK cells for 24 h. Cell surface NKG2D was then quantitated by flow cytometry. B, t test of four experiments performed with two different donors. MFI, mean fluorescence intensity.

FIGURE 7.

Purified exosomes containing ULBP3 are potent modulators of NK cell NKG2D-mediated cytotoxicity. A, primary NK cells recognize CHO-MICA transfectants in an NKG2D-dependent fashion. B, effect of purified exosomes on NK cell cytotoxicity against CHO-MICA and untransfected CHO. Purified exosomes from either untransfected or ULBP3-transfected CHO cells (40–100 ng total protein) were incubated with primary NK cells for 24 h. After preincubation with exosomes, NK cells were used as effectors in a cytotoxicity assay (measured using a fluorimetric assay). E:T, effector:target ratio.