Obstructing shedding of the immunostimulatory MHC class I chain-related gene B prevents tumor formation

Abstract

Purpose: Clinical observations have suggested that shedding of the MHC class I chain-related molecule (MIC) may be one of the mechanisms by which tumors evade host immunosurveillance and progress. However, this hypothesis has never been proven. In this study, we tested this hypothesis using a prostate tumor model and investigated the effect of shedding of MIC on tumor development.

Experimental design: We generated a shedding-resistant noncleavable form of MICB (MICB.A2). We overexpressed MICB.A2, the wild-type MICB, and the recombinant soluble MICB (rsMICB) in mouse prostate tumor TRAMP-C2 (TC2) cells and implanted these cells into severe combined immunodeficient mice.

Results: No tumors were developed in animals that were implanted with TC2-MICB.A2 cells, whereas all the animals that were implanted with TC2, TC2-MICB, or TC2-rsMICB cells developed tumors. When a NKG2D-specific antibody CX5 or purified rsMICB was administered to animals before tumor implantation, all animals that were implanted with TC2-MICB.A2 cells developed tumors. In vitro cytotoxicity assay revealed the loss of NKG2D-mediated natural killer cell function in these prechallenged animals, suggesting that persistent levels of soluble MICB in the serum can impair natural killer cell function and thus allow tumor growth.

Conclusions: These data suggest that MIC shedding may contribute significantly to tumor formation by transformed cells and that inhibition of MIC shedding to sustain the NKG2D receptor-MIC ligand recognition may have potential clinical implication in targeted cancer treatment.

Figure 1

Putative MICB cleaved site(s) in TC2 cells. a. Western-blot showing the predicted size of cleaved soluble MICB in TC2 cells. Supernatant and lysates of TC2-MICB cells were immunoprecipitated with anti-MIC mAb 6D4.6. The immune complexes were treated with PNGase F and resolved on SDS-PAGE. Proteins were transferred to nitrocellulose membrane and blotted with goat anti-MICB polyclonal Ab. Lane 1 and 2, detection of sMICB from TC2-MICB supernatant. The molecular mass of the deglycosylated cleaved sMICB is estimated to be 31-33 kDa. Lane 3 and 4, detection of full-length MICB from TC2-MICB cell lysates. The full-length deglycosylated MICB is estimated to be 41 kDa on 4-15% SDS-PAGE. b. A diagram depicted the putative MICB cleavage site(s).

Figure 1

Putative MICB cleaved site(s) in TC2 cells. a. Western-blot showing the predicted size of cleaved soluble MICB in TC2 cells. Supernatant and lysates of TC2-MICB cells were immunoprecipitated with anti-MIC mAb 6D4.6. The immune complexes were treated with PNGase F and resolved on SDS-PAGE. Proteins were transferred to nitrocellulose membrane and blotted with goat anti-MICB polyclonal Ab. Lane 1 and 2, detection of sMICB from TC2-MICB supernatant. The molecular mass of the deglycosylated cleaved sMICB is estimated to be 31-33 kDa. Lane 3 and 4, detection of full-length MICB from TC2-MICB cell lysates. The full-length deglycosylated MICB is estimated to be 41 kDa on 4-15% SDS-PAGE. b. A diagram depicted the putative MICB cleavage site(s).

Figure 2

Construction and expression of the shedding-resistant non-cleavable and soluble recombinant forms of MICB (rsMICB) in TC2 cell lines. a. Diagram showing generation of the non-cleavable form MICB.A2 by replacing aa 215-274 of the MICB α3 domain with the corresponding sequence of HLA-A2. rsMICB was generated by deletion of the entire transmembrane and cytoplasmic region of MICB. b. Flow cytometry showing expression levels of MICB, MICB.A2, and rsMICB in TC2 cell lines. cDNAs of MICB, MICB.A2, or rsMICB were inserted into a IRES-GFP retroviral vector pBMNZ. TC2 cells were transduced with respective retrovirus. GFP-positive cells were sorted by flow cytometry. For detection of MICB and MICB.A2 expression, cells were directly incubated with anti-MIC 6D4.6 antibody followed a PE-conjugated secondary reagent. For detection of the secretable rsMICB expression, TC2-rsMICB cells were cultured in the presence of BD GolgiPlug for 3 hr to prevent the secretion of rsMICB before harvesting. Cells were resuspended in BD Fixation/permeablization solution for 20 min at 4°C and incubated with 6D4.6 followed with a PE-conjugated secondary reagent. c. MICB.A2 is shedding-resistant. Top graph showing amount of shed soluble MICB in the culture supernatant and MICB in the cell lysates. 4x10⁵ cells/well were plated on a 6-well plate o/n. Media were removed and replaced with 1ml serum-free media. 6hrs later, Media were collected and filtered. Cells were lysed with 1 ml lysis buffer. 50ul of culture spernatant and cell lysates were used for (s) MICB ELISA assay. Bar, SE. Bottom graph represents the degree of shedding as calculated by molars of soluble MICB in the supernatant vs. total molars of soluble MICB and MICB (a sum of supernatant and cell lysates). The final results were normalized by cell numbers at the time of the assay. Results represent three independent experiments. *, P < 0.001.

Figure 3

Overexpression of MICB and MICB.A2 increases the sensitivity of TC2 cells to NK cell killing. a. Binding of MICB and MICB.A2 by mouse NKG2D. Cells were incubated with the chimeric soluble mouse NKG2D-humanFc (smNKG2D-Fc) followed by PE-conjugated anti-human IgG. Cells were analyzed by flow cytometry. Data shown are mean fluorescence intensity (MFI). When smNKG2D-Fc was pre-absorbed with rsMICB, no binding was seen in any of the cell lines. No significant difference was shown in the binding ability between MICB and MICB.A2 (p = 0.24). b. Sensitivity of various MICB-expressing TC2 cells to mouse NK cells. NK cells were isolated from SCID mice and cultured in complete media with 1000 U/ml of IL-2 for 4 days before used as effectors in standard 4-h ⁵¹Cr release assay. For blocking NKG2D receptor, effector cells were pre-incubated with 30 μg/ml of CX5 antibody. For blocking RAE-1, target cells were pre-incubated with 100 ug/ml of anti-pan RAE-1 pAb for 1hr prior to the assay. RMA-Rae-1 cells were used positive controls for blocking antibodies. E:T, effector:target ratio. Bar, SE. *, P < 0.01 compared to TC2 cells as targets. c. Flow cytometry histograms showing surface expression of RAE-1 (left panel) and H-2K^b/D^b (right panel) in TC2, TC2-MICB, and TC2-MICB.A2 cells. Filled histograms, cells were stained with control isotype antibodies. Open histograms, cells were stained with specific antibodies. Results represent three independent experiments.

Figure 4

Expression of MICB.A2 but not the cleavable MICB prevents tumor formation in vivo. Six animals were used in each group. Tumor growth was monitored twice weekly. Tumor volume was estimated by the formula Volume = L²xW/2. a. Tumor growth of various MICB expressing TC2 cells in SCID mice. b. Rate of tumor formation of various MICB expressing TC2 cells in SCID mice. 1x10⁶ cells were injected s.c. into each animal in (a) and (b). c. Tumor growth of TC2-MICB cells when injected at lower doses (1x10⁵ and 1x10⁴ cells/animal). d. Representative flow cytometry histoplots showing MICB expression in tumor cells extracted from animals inoculated with TC2-MICB cells compared to prior inoculation. The MICB-specific antibody MAB1599 (R & D systems) was used as primary antibody. Filled histogram, MICB expression in TC2 cells. Open histogram, MICB expression in TC2-MICB cells or tumor cells. Results represent three independent experiments.

Figure 4

Expression of MICB.A2 but not the cleavable MICB prevents tumor formation in vivo. Six animals were used in each group. Tumor growth was monitored twice weekly. Tumor volume was estimated by the formula Volume = L²xW/2. a. Tumor growth of various MICB expressing TC2 cells in SCID mice. b. Rate of tumor formation of various MICB expressing TC2 cells in SCID mice. 1x10⁶ cells were injected s.c. into each animal in (a) and (b). c. Tumor growth of TC2-MICB cells when injected at lower doses (1x10⁵ and 1x10⁴ cells/animal). d. Representative flow cytometry histoplots showing MICB expression in tumor cells extracted from animals inoculated with TC2-MICB cells compared to prior inoculation. The MICB-specific antibody MAB1599 (R & D systems) was used as primary antibody. Filled histogram, MICB expression in TC2 cells. Open histogram, MICB expression in TC2-MICB cells or tumor cells. Results represent three independent experiments.

Figure 5

Shedding of MICB by TC2-MICB cells compromises NK cell activity in vivo. a. Serum levels of sMICB in all the tumor-bearing animals. b. Reduced NKG2D-dpenedent NK cells cytotoxicity of splenic NK cells from animals bearing TC2-sMICB and TC2-MICB tumors. Freshly isolated NK cells were used as effectors; TC2-MICB.A2 cells were used as target cells. **, P < 0.01 compared to which of TC2 or TC2-MICB.A2. Results represent three independent experiments.

Figure 6

Soluble MICB induced NK cell dysfunction. a. In vivo blocking NKG2D with CX5 antibody or neutralization the function of NKG2D with rsMICB enables TC2-MICB.A2 cells to form tumors. To block NKG2D receptor in vivo, 100 μg of NKG2D-specific antibody CX5 was injected i.p. on the day before and the day after tumor implantation and thereafter every three days. To modify NKG2D function, animals were i.p. injected with 50 ng of purified rsMICB prior to implantation of TC2-MICB.A2 cells and there after twice a week for four weeks. b. Measurements of NKG2D expression shown as mean fluorescence intensity (MFI) on splenic NK cells freshly isolated from SCID animals (n=6) injected with various tumor cells. bar, mean value of MFI. c, Compatitive binding assay indicating saturation of NKG2D receptor by sMICB in animals bearing TC2-MICB and TC2-rsMICB tumors. Freshly isolated splenocytes were incubated with 10 ng/μl of rsMICB-FLAG followed by FITC-conjugated anti-FLAG mAb M2 and PE-conjugated mAb DX5. Data shown are measurements of mean fluorescence intensity (MFI) of M2 staining from six animals of each experimental group. Bar, standard error.*, p < 0.01 when compared to animals injected with TC2 or TC2-MICB.A2 tumor cells.