Low NKp30, NKp46 and NKG2D expression and reduced cytotoxic activity on NK cells in cervical cancer and precursor lesions

Background Persistent high risk HPV infection can lead to cervical cancer, the second most common malignant tumor in women worldwide. NK cells play a crucial role against tumors and virus-infected cells through a fine balance between activating and inhibitory receptors. Expression of triggering receptors NKp30, NKp44, NKp46 and NKG2D on NK cells correlates with cytolytic activity against tumor cells, but these receptors have not been studied in cervical cancer and precursor lesions. The aim of the present work was to study NKp30, NKp46, NKG2D, NKp80 and 2B4 expression in NK cells from patients with cervical cancer and precursor lesions, in the context of HPV infection. Methods NKp30, NKp46, NKG2D, NKp80 and 2B4 expression was analyzed by flow cytometry on NK cells from 59 patients with cervical cancer and squamous intraepithelial lesions. NK cell cytotoxicity was evaluated in a 4 hour CFSE/7-AAD flow cytometry assay. HPV types were identified by PCR assays. Results We report here for the first time that NK cell-activating receptors NKp30 and NKp46 are significantly down-regulated in cervical cancer and high grade squamous intraepithelial lesion (HGSIL) patients. NCRs down-regulation correlated with low cytolytic activity, HPV-16 infection and clinical stage. NKG2D was also down-regulated in cervical cancer patients. Conclusion Our results suggest that NKp30, NKp46 and NKG2D down-regulation represent an evasion mechanism associated to low NK cell activity, HPV-16 infection and cervical cancer progression.


Background
Cervical cancer is the second most common female malignant neoplasm worldwide. Human papillomavirus (HPV) is a necessary but not sufficient cause of cervical cancer. Co-factors that increase the risk for cervical cancer among HPV-DNA positive women, include oral contraceptives, smoking, high parity, previous sexually transmitted disease and immunodeficiency [1]. Cervical carcinogenesis implies HPV infection, viral persistence, progression and invasion [2]. Both innate and adaptive immune responses play a complex role against HPV infection. Spontaneous regression of high grade squamous intraepithelial lesions due to HPV-16 infection is associated with HPV-16 E7 peptide-specific CD4+ T-cell response and with lymphoproliferative responses to E2 plus IFN-gamma production [3][4][5][6][7]. However, innate immune response acts directly or indirectly against viral agents, through TLRs activation, dendritic cell presentation and NK cell function at cervical tissue level [8][9][10][11].
NK cells represent the first line of defense against viral pathogens, killing infected cells or via secretion of cytokines and chemokines [12]. There is also accumulating evidence for the crucial role of NK cells in tumor immunosurveillance [13]. NK cell activation and tumor lysis occur through a complex interaction between triggering receptors such as NKp30, NKp44, NKp46 and NKG2D with tumor cell ligands, in fine balance with inhibitory receptors and co-receptors [14]. Recently, it has been reported that activating NK cell receptor ligands MICA (NKG2D ligand) and CD155 (DNAM-1 ligand) are differentially expressed during the progression to cervical cancer [15]. However, the expression of NKp30, NKp46, NKG2D (triggering receptors) and co-receptors (NKp80 and 2B4) in NK cells from patients with cervical cancer and precursor lesions remains unknown. The aim of the present work was to study NKp30, NKp46, NKG2D, NKp80 and 2B4 expression in NK cells from patients with cervical cancer and precursor lesions, in the context of HPV infection.

Activating receptors (NKp30, NKp46 and NKG2D) and coreceptors (CD80 and 2B4) evaluation
Peripheral blood mononuclear cells (PBMC) were isolated by gradient Lymphoprep (Oslo Norway Nycomed™). NK cells were obtained by immunomagnetic negative selection (NK cell isolation kit, Miltenyi Biotec) in accordance with the manufacturer's instructions. After immunomagnetic depletion, NK cell CD56 + were >95%, (confirmed by flow cytometry analysis). Cell concentration was adjusted to 1 × 10 5 and cells were incubated at 4°C for 30 min with proper dilution of anti-NKG2D (ECM217), anti-NKp30 (Z25); anti-NKp46 (BAB281), anti-NKp80 (MA252), or anti-2B4 (PP35) specific antibodies (kindly donated by Professor Alessandro Moretta, University of Genova, Italy). Cells were washed with a phosphate buffered saline (PBS) solution and incubated at 4°C for 30 min (in the dark) with FITC goat anti-mouse secondary Ab (IgG1). The cells were washed twice and incubated with 5 μL of PE-conjugated anti-CD56, PC5conjugated anti-CD3 mAbs or with control isotype and fixed with 0.05% formaldehyde solution (all reagents from Beckman Coulter). The percentages and Median Fluorescence Intensity (MFI) were determined with a proper protocol and controls to compensate electronically for overlapping signals, using an EPICS XL-MCL flow cytometer (Beckman Coulter™).

NK cell cytotoxicity assay
NK cell cytotoxicity against K562 cells was evaluated in a 4 hours CFSE/7-AAD flow cytometry assay [16]. NK cells were isolated from PBMC by cell sorting with anti-CD56 using FACSAria Cell Sorter (BD Bioscience). NK cell gate was set based on their forward/sideward light scatter and CD56/CD3 expression. The purity of the NK cells fraction was ≥ 98%. NK cells were labeled with 5-6-carboxyfluorescein diacetate succinimidyl ester (CSFE 200 nM) in PBS/1% BSA for 15 min at 37°C. CFSE-labeled NK cells were washed twice with PBS and seeded with a constant number of K562 cells (20,000) at different E:T ratios (1:1, 3:1, 10:1. 30:1). Target cells were incubated alone to measure basal cell death. Target cells and NK cells were incubated in complete medium for 4 hours in a 5% CO 2 atmosphere at 37°C. Cells were washed twice in PBS-1% BSA containing 0.1% sodium azide (NaN 3 ) and incubated in the same buffer plus 20 μL/mL to 7-amino actinomycin D (7-AAD, BD Biosciences) during 15 min at 4°C in darkness. Acquisition was performed with the FACS Diva Software (BD Bioscience). Cytotoxic activity was expressed as % Specific Lysis calculated by the following formula:

DNA Extraction
DNA extraction was done in cervical cells obtained by cytobrush. Pellets containing cervical cells were obtained by centrifugation at 10 000 rpm for 3 min, 200 μg of Proteinase K was added for 48 hours at 37°C. Proteinase K was inactivated at 94°C for 10 minutes. Aqueous supernatant was transferred to another fresh microtube. DNA was precipitated by adding 100% ethanol and 20 mg/mL glycogen (Sigma) for 30 min at 22°C. The pellet was washed twice with 70% ethanol, dried, resuspended in 200 μL distilled water, and measured spectrophotometrically at 260/ 280 nm. .

Statistical analysis
Statistical analysis was performed using the SPSS software package version 10.0 (SPSS, Inc Chicago, IL). Data were expressed as percentage, mean median fluorescence intensity (MFI) and analyzed by ANOVA. Significance was tested by non parametric test (Mann-Whitney U test). Differences were considered statistically significant when the p value was < 0.05. Spearman correlation was done to correlate activating receptors with HPV expression and Pearson correlation to cytotoxic activity.
We also found a significant lower expression of NKG2D on NK cells from cervical cancer patients in comparison with healthy women (p < 0.05). There was not a significant difference with HGSIL and LGSIL patients ( Figure 1).

Expression of co-receptors 2B4 and NKp80 in NK cells
Expression of co-receptors 2B4 and NKp80 (MFI) on NK cells was not significantly different between cervical cancer, HGSIL, LGSIL and healthy groups ( Figure 2).

Low cytotoxic activity in NK cells from cervical cancer and HGSIL patients
NK cell-mediated specific cell lysis was decreased according to the natural history of cervical cancer: healthy women > LGSIL > HGSIL > cervical cancer (Figure 3). NK cell activity was significantly lower in patients with cervical carcinoma at all E/T ratios in comparison with LGSIL patients and healthy women (p < 0.0001). NK cell activity was also significantly decreased in HGSIL patients at 10:1 and 30:1 E/T ratios (p < 0.02) in comparison with LGSIL patients and healthy women (Figure 3). Percentage of specific lysis was correlated with NKp30 and NKp46 MFI. We found a significant correlation with NKp30 at 10:1 ratio (r = 0.686, p < 0.001) and at 30:1 ratio (r = 0.749, p < 0.001).

Discussion
It has been proposed that immunosurveillance escape might be the seventh hallmark of cancer   In this study we also found that the NK cell-activating receptor NKG2D is significantly down-regulated in cervical cancer patients. This result is consistent with a previous study reporting a significant decrease in the number of NKG2D-expressing NK and T cells in both cervical cancer and precursor lesion patients. However, they only found a significant correlation between high soluble MICA levels and low NKG2D in T cells [24].
NCRs and NKG2D may be down-regulated by different molecules and mechanisms such as TGF-beta, indoleamine 2,3-dioxygenase, prostaglandin E2, corticosteroids, 17 beta-estradiol and reactive oxygen species [25-29]. We do not know yet the precise mechanism that underlies NKp30, NKp46 and NKG2D down-regulation in NK cells from cervical cancer patients; however, it has been reported that TGF-beta1 mRNA overexpression is associated with progression from LGSIL to HGSIL [30,31]. TGFbeta is also upregulated at least two-fold in lymph node cervical cancer micrometastases [32]. Indoleamine 2,3dioxygenase was also found at the invasion front of invasive cervical tumors and peritumoral stromal cells [33]. In the present work we found that NKp30 and NKp46 diminution was linked to HPV-16 infection.

Conclusion
Our results suggest that NKp30, NKp46 and NKG2D down-regulation represent an evasion mechanism associ-ated with low NK cell activity, HPV-16 infection and cervical cancer progression.