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Comparative Study
, 114 (4), 560-8

Prevalent Expression of the Immunostimulatory MHC Class I Chain-Related Molecule Is Counteracted by Shedding in Prostate Cancer

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Comparative Study

Prevalent Expression of the Immunostimulatory MHC Class I Chain-Related Molecule Is Counteracted by Shedding in Prostate Cancer

Jennifer D Wu et al. J Clin Invest.

Abstract

The MHC class I chain-related molecules (MICs) have previously been shown to be induced on most epithelial tumor cells. Engagement of MIC by the activating immune receptor NKG2D triggers NK cells and augments antigen-specific CTL anti-tumor immunity. The MIC-NKG2D system was proposed to participate in epithelial tumor immune surveillance. Paradoxically, studies suggest that tumors may evade MIC-NKG2D-mediated immunity by MIC shedding-induced impairment of effector cell function. Here we demonstrate the first evidence to our knowledge of a significant correlation of MIC shedding and deficiency in NK cell function with the grade of disease in prostate cancer. MIC is widely expressed in prostate carcinoma. The presence of surface target MIC, however, is counteracted by shedding. A significant increase in serum levels of soluble MIC (sMIC) and deficiency in NK cell function was shown in patients with advanced cancer. Finally, the deficiency in NK cell function can be overcome by treatment with IL-2 or IL-15 in vitro. Our results suggest that (a) deficiency in MIC-NKG2D immune surveillance may contribute to prostate cancer progression, (b) sMIC may be a novel biomarker for prostate cancer, and (c) using cytokines to restore MIC-NKG2D-mediated immunity may have clinical significance for prostate cancer in cell-based adaptive immunotherapy.

Figures

Figure 1
Figure 1
MIC expression in prostate cancer cell lines and susceptibility of prostate cancer cells to NK cell activation in an NKG2D-dependent fashion. (A) Flow cytometry analysis of MIC expression in PrECs and the prostate cancer cell lines M12, PC-3, and LnCaP. White profiles represent staining with control mouse IgG (mIgG); black profiles represent staining with the BAMO1 mAb against MIC (MIC); and gray profiles represent staining with the W6/32 mAb against MHC I (MHC I). (B) Cytotoxicity of NK cells isolated from a healthy individual against M12 target cells at the indicated effector/target (E/T) ratios. NK cell cytotoxicity was inhibited by masking of NKG2D on NK cells or of MIC on M12 cells with 10 μg/ml of antibody M585 (+ anti-NKG2D) (31) or BAMO1 (+ anti-MIC) (30), respectively, but was not affected by control mouse IgG (+ IgG). Data shown are mean ± SD of triplicates. Results shown are representative of three independent experiments.
Figure 2
Figure 2
Immunohistochemical staining of MIC on human prostate biopsies. (A) No MIC immune reactivity in a benign prostate. (B and C) Predominant surface MIC immune reactivity in prostate secretory epithelia of HGPIN (B) and GS 5–6 prostate carcinoma (C). (DF) MIC staining was no longer abundantly present on carcinoma cell surface and diffuse stromal MIC immuoreactivity was shown in high-grade cancers (GS 8–10). Original magnification, ×40.
Figure 3
Figure 3
Serum levels of sMIC and PSA. (A) Serum levels of sMIC in healthy subjects (Healthy; n = 10) and prostate cancer patients with primary carcinomas with a GS of 6–7 (n = 13) or a GS of 8–10 (n = 10). Horizontal lines indicate mean value of respective groups. *P < 0.05; ***P < 0.001. Data shown are mean values of three independent ELISA measurements. (B) Lack of correlation of serum levels of sMIC with PSA in prostate cancer patients (r = 0.02, P = 0.48).
Figure 4
Figure 4
Surface NKG2D expression by NK cells from normal male donors and from prostate cancer patients. Cells were isolated and stained as described in Methods. (A) Plots show surface NKG2D expression of CD3CD56+ NK cells from a representative healthy subject and three representative prostate cancer patients (GS 7, GS 8, and GS 9). Note that NKG2Dlow and NKG2Dnormal populations were present in the patients with cancer with GS of 7 and 8. (B) Geometric mean fluorescence intensity (geo MFI) of surface NKG2D on CD3CD56+NK cells from 10 healthy subjects, 13 prostate cancer patients with primary carcinoma with a GS of 6–7, and 10 patients with prostate cancer with a GS of 8–10. Data shown are from three independent flow cytometry measurements. Horizontal lines indicate mean value of respective groups. *P < 0.05; **P < 0.01. Note that surface NKG2D expression on CD56+ cells was measured as geo MFI, due to the heterogeneous expression of surface NKG2D. (C) Inverse correlation of surface NKG2D expression on CD3CD56+ NK cells with serum levels of sMIC in prostate cancer patients (r = 0.57, P = 0.0049).
Figure 5
Figure 5
Deficiency in NK cell anti-tumor cytotoxicity in prostate cancer patients. Freshly isolated NK cells from peripheral blood of healthy subjects and representative prostate cancer patients (GS 6–7 and GS 8–10) were used as effector cells against M12 target cells in a 4-hour 51Cr-release assay. Data showed a significant reduction in cancer patient NK cell cytotoxicity against M12 cells. Effector/target: 10/1. *P < 0.05; **P < 0.01.
Figure 6
Figure 6
Effect of prostate cancer serum on normal NK cell surface NKG2D expression. Normal NK cells were cultured for 48 hours in media containing 20% serum from representative healthy donors (h1 and h2) and patients with prostate cancer with a GS of 9 (p14 and p23) or a GS of 10 (p21 and p22). (A) A reduction in NK cell surface NKG2D expression after cultured in serum from cancer patients was seen. (B) The effect on NKG2D expression was inhibited by pretreatment of serum from cancer patients with the BAMO1 mAb against MIC. Results shown are representative of three independent experiments.
Figure 7
Figure 7
Recovery of the NKG2D-mediated anti-tumor function of NK cells from prostate cancer patients by in vitro stimulation. NK cells from representative patients with cancer with a GS of 7 (p4) or a GS of 8 (p11) were cultured for 36 hours with 100 U/ml of IL-2 or 50 ng/ml of IL-15. (A) Increased NK cell surface NKG2D expression after IL-2 or IL-15 stimulation. (B) Increased NK cell cytotoxicity against M12 cells. Effector/target: 10/1. Data shown are representative of three independent experiments. (C) NK cell cytotoxicity against M12 cells were blocked by preincubation with the M585 mAb against NKG2D. Effector/target: 10/1. Fresh, freshly isolated NK cells.

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