Mutated Von Hippel-Lindau-renal cell carcinoma (RCC) promotes patients specific natural killer (NK) cytotoxicity

The human renal cancer cell lines CAKI-1 (HLA-A*23:01/A*24:02; HLA-B*35:02/B*44:03; HLA-C*04/C*04new) and SN12C (HLA-A*03/A*24new; HLA-B*07/B*44; HLA-C*05/C*07:02) VHL wild-type (VHL-WT), A498 (HLA-A*02:01; HLA-B*08:01; HLA-C*07) and 786-O (HLA-A*03:01; HLA-B*07/B*44, HLA-C*05/C*07:02) VHL mutated (VHL-MUT) and the human leukemic cell line K562 were obtained from the NCI 60 cancer cell line collection [20, 21]. All cells were cultured in the recommended growth medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1% L-glutamine, 1% penicillin/streptomycin and maintained in 95% air 5% CO² at 37 °C.

VHL mutational status was determined by PCR amplification and direct sequencing. DNA was extracted from primary tumor tissues using Nucleon Genomic DNA extraction Kit (GE Healthcare). The three VHL coding exons, with exon-intron junctions, were PCR amplified using the following primers: exon 1, 5′-agcgcgttcc atcctctac-3′ (forward) and 5′-agttccccgtctgcaaaat-3′ (reverse); exon 2, 5′-aggacggtcttgatctcctg-3′ (forward) and 5′-gcccaaagtgcttttgagac-3′ (reverse); exon 3, 5′-ttgttggcaaagcctcttgt-3′ (forward) and 5′-tgcccctaaacatcacaatg-3′ (reverse). The thermal cycler (Applied Biosystems® 2720 Thermal Cycler) was programmed to first incubate the sample for 10 min for 95 °C followed by 35 cycles consisting of 95 °C for 45 s, 56 °C (exon1 and 3) and 60 °C (exon 2) for 45 s with final extension for 7 min at 72 °C. Purified PCR products were then sequenced by using the Big Dye terminators version 3.1 cycle sequencing kit (Applied Biosystems, Courtaboeuf, France) and the 3130 Genetic Analyzer (Applied Biosystems).

FITC-conjugated major histocompatibility complex (MHC) class I–specific antibody (IgG2a, W6/32, CBL139F, Cymbus Biotech, Hants, UK) and PE anti-human CD155/PVR (clone SKIL.4, Biolegend, Cat No 337609) were used for flow cytometry. FACSAria II Cell Sorter 8-colour flow cytometer with BD FACSDiva™ 8.0 Software (BD Biosciences, San Jose, CA) was daily calibrated with calibrite beads (Fitc, Pe, PerCP and APC) and compbeads (Pe-Cy7 and APC-Cy7; BD Biosciences). PerCP-Cy5.5 anti-CD3 (BD Biosciences Cat No 560835), FITC-conjugated anti-CD56 (BD Biosciences, Cat No 345811) and PE-conjugate anti-CD107a antibody (BD Bioscience, Cat No 555801) were used to identify degranulating NK-cells from peripheral blood. For intracellular staining PE-conjugated anti-IFN-γ antibody (BD Biosciences Cat No 554701) was used. Fluorochrome-labelled monoclonal antibodies for identification of tissue Treg cells were: FITC-anti-FOXP3 (BD Biosciences, Cat No 560047), Pe-Cy7-anti-CD25 (BD Biosciences, Cat No 557741) and APC-Cy7-anti-CD4 (BD Biosciences, Cat No 557871). Intracellular staining for FoxP3 was performed using a commercially available kit (BD Cytofix/Cytoperm; fixation and permeabilization kit; BD Pharmingen) according to the manufacturer’s instructions. A minimum of 100.000 events for each sample were collected.

CD56⁺NK cells were isolated from PBMCs of 4 RCC patients (1 VHL-WT, 3 VHL-MUT) after Ficoll gradient centrifugation using the NK cell isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany). Purified NK cells were cultured in RPMI 1640 medium (GIBCO Invitrogen) in the presence of 100 U/ml recombinant interleukin 2 (IL-2) overnight at 37 °C, 5%CO₂. Autologous tumor cells were isolated from tumor tissue with single cell sorting by FACS ARIAII post mechanical disruption and labelings with Antibody AbI RCC pure Mouse Dako (clone SPM314), 7AAD (Thermo Fischer) and PE anti-CD45 (BD Biosciences, cat No 555483) and AbII Goat anti-Mouse FITC IgG(H + L) (Caltag Laboratory) for indirect staining. Through the gate strategy 7AAD⁻/CD45⁻/RCC⁺ ~ 10,000 of tumor cells were isolate and cultured 24 h in 96-well plates with RPMI-1640 with 10% FBS medium.

Tumor CD4⁺CD25⁺ T regulatory cells (Tregs) and peripheral CD4⁺CD25⁻ T effector (Teff) cells were isolated using the Dynabeads Regulatory CD4⁺CD25⁺ T cell kit as previously described [22]. All purification steps were performed according to the manufacturer’s instructions (Invitrogen by Life Technologies) and collected cells were found to be > 95% pure by flow cytometry. Tregs function was assessed by evaluating the CFSE-labeled Teff cells by FACS analysis. Carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled autologous CD4⁺CD25⁻ T cells from peripheral blood (CellTrace CFSE Cell Proliferation Kit, Molecular Probes, by Life Technologies) were cultured with tumor CD4⁺CD25⁺ Tregs in 1:1 ratio. Cells were cultured (5 × 10³ cells/well) in U-bottom 96-well plates with RPMI medium (Thermo scientific, HyClone Laboratories, Inc) supplemented with 2-mM L-glutamine, 100 U/ml penicillin, 100 μg streptomycin, and 10% fetal bovine serum. Cells were stimulated for 5 days in the presence of Dynabeads Human T-Activator CD3/CD28 (Gibco by Life Technologies).

Lysosome-associated membrane protein LAMP-1 (CD107a) is a marker of NK-cell degranulation. CD107a expression was measured by flow cytometry as previously described [23, 24]. Briefly, 1 ml whole blood was diluted with one volume RPMI-1640 containing 10% heat-inactivated fetal bovine serum and incubated with IL-2 (100 U/ml) overnight at 37 °C, 5% CO₂. The cytotoxic activity of NK cells was tested against NK-sensitive cell line K562 (HLA class I-negative) [25], human renal cancer cell lines CAKI-1-VHL-WT, A498 and 786-OVHL-MUT. 200 μl of IL-2 activated blood was co-cultured with 2 × 10⁵ target cell lines in presence of PE-anti-CD107a antibody for 3-h at 37 °C in 5% CO₂. Alternatively, purified IL-2 activated NK cells from peripheral blood of RCC patients were used as effector cells. The autologous RCC tumor cells, A498-VHL-MUT and SN12C-VHL-WT cell lines were used as target cells. Assays were performed at the indicated effector-target ratio (10E:1 T; 5E:1 T). To detect spontaneous degranulation samples were incubated in the absence of target cells. Following 3-h culture, cells were stained with anti-CD56 and NK cells were identified as CD3⁻CD56⁺ in the lymphocyte gate. CD107a was analyzed on CD56⁺CD3⁻ NK. For intracellular staining of IFN-γ, 200 μl of IL-2 preactivated blood samples were incubated with 2 × 10⁵ K562 or RCC target cells for 6 h at 37 °C in a humidified 5% CO₂ incubator. The secretion inhibitor Brefeldin A (Sigma-Aldrich, Seelze, Germany) was added to the assay medium at a concentration of 10 μg/ml during the 6 h coincubation. After incubation, samples were placed on ice and stained with anti-CD56 and anti-CD3 mAbs, followed by erythrocyte lysis of blood samples with FACS Lysing Solution (BD Biosciences). Thereafter, cells were permeabilized using FACS permeabilizing solution (Perm2, BD Biosciences) and stained for intracellular IFN-γ with a PE-anti-IFN-γ antibody. Analysis was performed on a FACSAria II Cell Sorter 8-colour flow cytometer, blinded to patients’ clinical characteristics.

RNA was isolated from tumoral and peritumoral RCC tissues using the RNeasy Mini kit (Qiagen, Hilden, Germany), according to manufacturer’s instructions. cDNA was synthesized from 1 μg of total RNA using SuperScript III Reverse Transciptase (Invitrogen). Quantitative real-time PCR was performed using SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA) and data were collected and quantitatively analyzed on an ABI Prism 7000 System (Applied Biosystems). Primers sequences for NCAM-1, FCGR3a (CD16), DNAM-1, NKG2D, NKp46, NKp44, NKp30,KLRB1 and TGFB1 are detailed in Additional file 1: Table S1. Relative mRNA expression was normalized with GUSB gene expression.

Paraffin-embedded RCC tissue blocks, after heat induced epitope retrieval with citrate buffer (pH 6), were incubated overnight at 4 °C with the mouse monoclonal IgG2B, anti-human NKp46 antibody, Clone 195,314 (MAB1850 R&D Systems) diluted 1:50, followed by incubation with peroxidase-conjugated secondary antibodies according to the manufacturer’s instructions (EnVision+System, Dako North America, Inc., Carpinteria, CA). The NKp46 receptor demonstrates a high degree of lineage-specificity, being expressed almost exclusively in natural killer cells._. Five randomly selected areas were identified across the entire section from the center to the invasive margin (100× magnification). NKp46⁺ cells were counted in 5 consecutive non-overlapping high-power fields (HPF) 400x magnification (0.237 mm²/field), using an Olympus BX51 microscope (Olympus, Tokyo, Japan). The results were expressed as the mean of NKp46+/mm² out of 5 regions of interest. Immunostaining and scoring were evaluated by 4 independent observers (C.D., G.B., E.L. and A.A.) blinded to patients’ clinical characteristics.

IFN-γ was measured by ELISA on the culture supernatant collected on day 5 from suppression experiments. Cytokine concentration was assessed by Human IFN-gamma Instant ELISA (Bender MedSystems). Samples were acquired by LB 940 Multimode Reader Mithras (Berthold Technologies).

Clinical pathological features of patients are summarized in Table 1. The VHL status was assessed in 55 primary renal cancers by direct sequencing. As shown in Tables 1 and 2, VHL was mutated in 26/55 (47%) cases, with missense mutations in 7/26 (27%), nonsense in 3/26 (12%), splice site in 2/26 (7%), frameshift deletions in 11/26 (42%) and frameshift insertions in 3/26 (12%) cases. The mutations exon prevalence was: exon 1, 50%; exon 2, 30.7%; and exon 3, 19.3%.

IL-2 activated whole-blood-NK derived cytotoxicity (as CD107a externalization and intracellular IFNγ production) was evaluated in 51 available RCC patients (28 VHL-WT and 23 VHL-MUT) and 12 healthy donors (HD). IL-2 activated whole-blood samples were co-cultured with target cells K562, A498-VHL-MUT (containing a 4-nt deletion at nt 639–642 in VHL exon 2) [26] and CAKI-1-VHL-WT cell lines. As reported in Additional file 2: Figure S1 the target cells K562 are MHC-I negative, while A498 and CAKI-1 cell lines express high levels of MHC-I molecules (81 and 86.2% respectively). In Fig. 1 NKs from HD (A) and RCC patients, either VHL-WT (B) or –MUT (C), displayed comparable externalization of CD107a against K562 cells (average CD107a⁺NK cells: 15 ± 0.02% vs 15 ± 3% vs 17 ± 2.5% respectively); absence of CD107a⁺NK cells activity was detected evaluating HD blood samples after incubation with human RCC lines (Fig. 1A). Comparable CD107a⁺NK cells exposure was observed in VHL-WT-RCC patients after stimulation with human renal cells, A498 and CAKI-1 (average CD107a⁺NK cells: 3 ± 1.3% vs 3 ± 1% respectively, p = 0.84, Fig. 1B). Interestingly, high percentage of NK cells expressing CD107a was observed for blood derived from VHL-MUT-RCC patients against A498-VHL-MUT cells and not versus CAKI-1-VHL-WT cells (average CD107a⁺NK: 7 ± 2% vs 1 ± 0.41% respectively, p = 0.015, Fig. 1C). Similar results were obtained when IL-2 activated whole blood samples from VHL-MUT-RCC patients (n = 5) were evaluated toward another VHL-MUT RCC cell line, 786-O cells (MHC-I expressing cells, Additional file 2: Figures S1-S2A) carrying a single mutation in VHL exon 1 (1-nt deletion at nt 523) [26] different from A498 cells. Low expression of CD107a⁺NK cells was observed incubating RCC-VHL-WT blood samples (n = 9) with 786-O (Additional file 2: Figure S2B). The higher percent of CD107a⁺NK induced in VHL-MUT patients by the culture with A498 and 786-O VHL-MUT cell lines suggest that RCC-VHL mutated tumors displayed higher susceptibility to NK lysis .

Clinical-pathological features and the CD107a assay results from single patient were shown in Additional file 3: Table S2 (VHL-MUT-RCC patients) and Additional file 4: Table S3 (VHL-WT-RCC patients). NK derived-IFN-γ was evaluated in 8 HD (Fig. 1D) versus 13 VHL-WT (E) and 14 VHL-MUT (F) RCC patients in the presence of target cells (K562, A498 and CAKI-1). The highest IFN-γ production was registered with VHL-MUT blood versus A498 cells as compared to CAKI-1 cells (average IFN-γ⁺NK: 6.26 ± 3.4% vs 1.78 ± 0.9% respectively; p = 0.23 Fig. 1F). In addition autologous NK cell degranulation was evaluated in the presence of MUT-VHL tumors. Patients derived (1 VHL-WT, 3 VHL-MUT)-IL-2 activated NK cells were cultured in the presence of RCC cell lines or freshly isolated autologous tumor cells. As shown in Fig. 2, NKs derived from VHL-MUT patients (#24, #25, #26) displayed higher CD107a exposure versus RCC autologous tumor cells or A498-VHL-MUT cells as compared to SN12C-VHL-WT cells (average CD107a⁺NK: 4.7 and 2.7% vs 0.3% respectively at 10E:1 T ratio). Conversely, NKs derived from VHL-WT patient (#29) did not significantly increase CD107a exposure either toward RCC autologous tumor cells than toward SN12C or A498 cell lines. Thus we can speculate that NKs from VHL-MUT-RCC patients are more efficient toward human renal cancer VHL-MUT cells.

With the intent to correlate peripheral and tumoral NK status, NK cell infiltration was analyzed on paraffin-embedded primary RCC tissues by immunohistochemical staining using the NK cell–specific marker NKp46. 23 RCC-patients, 15/23 (65.2%) VHL-MUT and 8/23 (34.8%) VHL-WT were evaluated for the expression of NKp46⁺ cells. Although RCCs generally display low NK cell infiltration [27], NKp46 was expressed in the cytosol and on cell surface in lymphocytes (Fig. 3A). In VHL-WT-RCC the NKp46⁺ cells ranged from 0 to 5 per mm² (median 0.5 cells/mm²), (4/8 VHL-WT-RCC were totally negative for NKp46). In 15 VHL-MUT-RCCs NKp46⁺ cells ranged from 1 to 12 per mm² (median 6 cells/mm²); a significant higher number of NKp46⁺cells was detected in VHL-MUT-RCCs as compared to VHL-WT-RCCs (average NKp46⁺cells: 5.553 ± 0.78 vs 1.625 ± 0.78, p = 0.0041, Mann–Whitney test, Fig. 3B).

NKs function is regulated by activating receptors other than NKp46 that include NKp30, NKp44, C-type lectin receptors NKG2D, CD94/NKG2C, FcγRIIIa (CD16), activator KIRs, DNAX accessory molecule-1 (DNAM-1, CD226), and costimulatory receptors such as NCAM-1, and the main NK cell-activating ligands on A498 and CAKI-1 cells was recently reported [27‐29]. The expression of NKp30, NKp44, NKp46, NKG2D, DNAM-1, NCAM-1 and FcγRIIIa was evaluated by RT-PCR analysis in VHL-WT (n = 17) and VHL-MUT (n = 17) RCC samples. A significant high expression of NKp30 and NKp46 was detected in VHL-MUT-RCC as compared to VHL-WT-RCC samples (p = 0.04 and p = 0.017, respectively, Fig. 3C and D) and NKp44 and DNAM-1 were slightly overexpressed in VHL-MUT compared to VHL-WT patients. Conversely expression of NCAM-1, FcγRIIIa and NKG2D were comparable in the two groups (Additional file 2: Figure S3). In addition, NKp30, NKp44, NKp46 and FcγRIIIIa mRNA were highly expressed in VHL-MUT tumors as compared to surrounding unaffected tissue (Additional file 2: Figure S4A) while in the VHL-WT tumors no significant difference was retrieved (Additional file 2: Figure S4B). Thus VHL-MUT tumors were characterized by high density of NKp46⁺ cells infiltration and high expression of NK activating genes such as NKp30, NKp46, NKp44 and DNAM-1.

Since Tregs are supposed to suppress NK activity tumoral, Tregs (tTreg) identified as CD4⁺CD25^hiFoxp3⁺ cells, were evaluated in 26 RCC patients (13 VHL-WT and 13 VHL-MUT). tTregs were significantly lower in VHL-MUT-RCC as compared to VHL-WT-RCC tumors (1.84 ± 0.36% vs 3.79 ± 0.74%; p = 0.04, Fig. 4A). Treg function was evaluated ex vivo on patients T effectors (Teff) proliferation. Tregs isolated from VHL-MUT tumors were less efficient in inhibiting T-effectors proliferation (VHL-MUT: 6.7 ± 3.9% vs VHL-WT: 2.8 ± 1.1%, p = 0.14 Fig. 4B) as confirmed by increased IFN-γ production in co-colture of Tregs:Teff isolated from VHL-MUT-RCC patients (22.8 ± 2.8 vs 12.36 ± 3.9 pg/mL, p = 0.09 Fig. 4C). Few patients were also evaluated for TGF-β1 mRNA (6 VHL-WT/6 VHL-MUT peritumoral and tumoral tissues). Consistent with a reduction of Tregs function, TGF-β1 expression, though not significant, was lower in VHL-MUT RCC as compared to VHL-WT-RCC (2.5 ± 1.0 vs 4.9 ± 1.15, p = 0.16 Fig. 4D).