Prevalent expression of the immunostimulatory MHC class I chain–related molecule is counteracted by shedding in prostate cancer (original) (raw)
NKG2D-dependent NK cell cytotoxicity against prostate cancer cell lines. Transformation-associated MIC expression renders tumor cells more susceptible to NK cell cytotoxicity (1–8). Therefore, we first examined the expression of MIC molecules in prostate cancer cell lines. As shown by flow cytometry analysis with mAb BAMO1, which recognizes both MICA and MICB (together called MIC), MIC was abundantly expressed in the prostate cancer cell lines M12, PC-3, and LnCaP but was absent from normal prostate primary epithelial cells (PrECs) (Figure 1A).
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.
Studies have shown that NK cell cytotoxicity against MIC-positive tumor cells may be NKG2D dependent or may be a result of combined activation of NKG2D and natural cytotoxicity receptors, depending upon the source of target cells (30). We thus tested whether the susceptibility of these MIC-expressing prostate cancer cells to NK cell cytotoxicity was NKG2D dependent. As shown in Figure 1B, M12 cells elicited NK cell target lytic activity and this response was substantially inhibited by either masking of MIC with mAb BAMO1 (31) or masking of NKG2D with mAb M585 (32). A similar NK cell lytic response was triggered by PC-3 or LnCaP cells but not by normal prostate primary epithelial cells (data not shown). Moreover, stimulating NK cells with these MIC-expressing prostate cancer cells resulted in a significant level of IFN-γ release (data not shown). These data indicate a MIC-specific, NKG2D-dependent activation of NK cells against prostate cancer cells.
MIC expression in primary prostate carcinomas. We investigated the expression of MIC by prostate secretory epithelial cells using a set of tissue microarray slides. Each slide was composed of the following: prostate biopsies from normal individuals and from individuals with prostatic neoplasia, carcinomas of varying Gleason scores (GS), and high-grade prostatic intraepithelial neoplasia (HGPIN). MIC immunoreactivity was seen only in neoplastic prostates but not in benign prostate glands (Figure 2A). Positive MIC immunoreactivity with varying patterns was exhibited in 95% of neoplastic prostate (Table 1). In HGPIN, MIC immunoreactivity was predominantly localized at the luminal surface of secretory epithelial cells (Figure 2B). In carcinomas with GS of 6–7, MIC immunoreactivity was no longer restricted to the epithelial cell surface but was diffusely distributed in the infiltrated glands (Figure 2C). In carcinomas with GS of 8–10, most tumor cells showed negative cell surface MIC immunoreactivity and diffuse MIC staining was shown in the stroma (Figure 2, D–F). These observations indicate that MIC is induced in the early stage of prostate luminal epithelial cell transformation (as early as HGPIN) and is expressed widely in prostate carcinoma. The data also suggest that loss of predominant surface localization of MIC may be associated with progression to invasive tumor or to progressively higher grades.
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). (D–F) 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.
MIC immunoreactivity in primary prostate carcinoma
Serum levels of sMIC in prostate cancer patients. Our observations in prostate biopsy studies suggested an association of increased loss of cell surface MIC with higher grades of prostate cancer. We thus seek to investigate serum levels of sMIC in patients with prostate cancer of various grades. Sera from 23 characterized prostate cancer patients (Table 2) and from 10 age-matched healthy male donors were assayed for sMICA by noncompeting solid-phase ELISA. AMO1 (specific for the α1α2 domain of MICA) and BAMO3 (specific for the α3 domain of MICA and MICB) were used as capture and detection antibodies, respectively (30). Significant amounts of sMICA, as high as 21 ng/ml, were detected in sera from nearly all patients (Student’s t test, P < 0.05, Figure 3A). No significant levels of sMICA were detected in sera from healthy donors. Furthermore, significantly higher levels of sMICA were seen in sera from patients with more advanced disease, that is, with primary carcinoma at a GS greater than 7 at the time of diagnosis and/or with recurrent androgen-independent disease (Student’s t test, P < 0.05; Figure 3A). Interestingly, although in some cases patients with high serum levels of prostate-specific antigen (PSA) tended to have higher levels of sMICA (p14, p17, p18, p20, p21, and p23; Table 3), serum levels of sMICA did not seem to correlate significantly with PSA overall (Figure 3B; ANOVA, r = 0.2, P = 0.38).
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).
Patient characteristics in this study
Serum levels of sMIC and PSA and relative surface NKG2D expression on CD56+ NK cells in patients with prostate cancer
Deficiency in NKG2D-mediated NK cell function in advanced prostate cancer. Our observation that MIC is induced frequently in transformed prostate epithelial cells suggests a potential role for NKG2D-mediated anti-tumor immunity in prostate cancer. However, the MIC-NKG2D immune surveillance may not be effective in prostate cancers, probably due to at least two factors. One is loss of the tumor cell surface target molecule MIC, as shown by our immunohistochemistry study of prostate tissue microarrays (Figure 2 and Table 1). The other is sMIC-induced downregulation of surface NKG2D expression on effector cells. The sMIC-induced impairment in NKG2D-mediated effector cell function has been demonstrated in other cancers (28, 29). To investigate whether deficiency in NKG2D-mediated effector cell function is associated with prostate cancer, we analyzed surface NKG2D expression on circulating NK cells freshly isolated from the 23 prostate cancer patients (Table 2) and from 10 healthy donors, using the M585 mAb against NKG2D in combination with anti-CD3 and anti-CD56. As demonstrated in Figure 4, A and B, significantly reduced surface NKG2D expression was seen in NK cells from prostate cancer patients compared with those of healthy donors (Student’s t test, P < 0.05). A more significant reduction in NK cell surface NKG2D expression was shown in patients with advanced disease (GS 8–10; P < 0.001). Interestingly, in nearly all patients with cancers with GS of 6–8 and nonmetastatic cancer with a GS of 9, two populations of CD3–CD56+ NK cells, NKG2Dlow and NKG2Dnormal, were seen and an increase in NKG2Dlow population was likely to be associated with cancer of a higher GS or with higher levels of serum sMIC. In all patients with cancer with a GS of 10 and metastatic cancer with a GS of 9, only the NKG2Dlow population was seen. In all cases, no difference in CD56 expression was noted between NKG2Dlow and NKG2Dnormal NK cells (data not shown). Nevertheless, a significant inverse correlation between serum levels of sMIC and levels of NK cell surface NKG2D expression was found in all patients by ANOVA (Figure 4C; r = 0.56, P = 0.0049).
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 CD3–CD56+ 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 CD3–CD56+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 CD3–CD56+ NK cells with serum levels of sMIC in prostate cancer patients (r = 0.57, P = 0.0049).
We next examined the NKG2D-dependent tumor lytic ability of freshly isolated polyclonal NK cells from representative prostate cancer patients and healthy donors. As presented in Figure 5, NK cell cytotoxicity against MIC-positive prostate cancer M12 cells was substantially decreased in all representative prostate cancer patients. A more pronounced decrease was seen in patients with high-grade cancers (GS 8–10). These cytotoxic responses were inhibited by pre-masking of NKG2D on NK cells with the antibody M585 or preincubation of M12 cells with the anti-MIC BAMO1 (data not shown). These results not only demonstrated deficiency of NKG2D-mediated NK cell anti-tumor function in prostate cancer patients but also demonstrated the correlation of severity of NK cell functional impairment with degree of disease.
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.
Downmodulation of normal NK cell surface NKG2D expression by sMIC in prostate cancer serum. Studies have shown that several soluble factors in prostate cancer serum can inhibit NK cell function (33–36). To ascertain the association of cancer serum sMIC with the deficiency in NKG2D-dependent NK cell cytotoxicity, we investigated the effect of serum from patients with prostate cancer with a GS of 9 (p14 and p23) or a GS of 10 (p21 and p22) on normal NK cell surface NKG2D expression. A decrease in surface NKG2D expression was observed after 12 hours (data not shown) and a maximum reduction of 5- to 7-fold was observed after 48 hours (Figure 6A). When the sera were preincubated with mouse IgG, the same reduction in surface NKG2D expression as in Figure 6 was observed (data not shown). In contrast, when the sera were pretreated with the BAMO1 mAb against MIC, no significant reduction in surface NKG2D expression was observed, even at 48 hours (Figure 6B), suggesting that sMIC in the sera of prostate cancer patients was the main factor that downregulated NK cell surface NKG2D expression. Subsequently, after 48 hours of incubation with cancer serum of NK cells from a healthy donor, we observed a significant reduction in IFN-γ secretion and NKG2D-dependent cytotoxicity of these NK cells against M12 cells (data not shown).
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.
Restoring NKG2D-mediated NK cell function by in vitro cytokine stimulation. Studies have shown that IL-15 can upregulate NKG2D expression in vitro in both CD8+ T cells (37) and NK cells (32). Very recent studies have demonstrated a similar effect of IL-2 on CD8+ T cells in vitro (38). We thus addressed whether in vitro cytokine stimulation could overcome the NKG2D-mediated functional deficiency of NK cells from prostate cancer patients. We cultured NK cells from patients with cancers with a GS of 7 (p6) or a GS of 8 (p11) in normal media containing 100 U/ml of IL-2 or 20 ng/ml of IL-15. After 24 hours, an increase in surface NKG2D expression on these NK cell was observed (data not shown). A maximum of increase was shown after 36 hours of culture (Figure 7A). In the presence of IL-15, a more pronounced increase in NKG2D expression was seen (Figure 7A). Moreover, these NK cells exhibited substantially enhanced cytotoxicity against M12 cells (Figure 7B). These lytic responses were inhibited when surface NKG2D was masked with mAb M585 (Figure 7C), indicating an NKG2D-dependent enhancement in NK cell cytotoxicity.
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.