Prediction and identification of novel HLA-A*0201-restricted cytotoxic T lymphocyte epitopes from endocan

The human TAP-deficient T2 cell line, BB7.2 cell line producing mAb against HLA-A2, human glioma cell line U251 (HLA-A2+), microglia cell line BV2 (HLA-A2-) were purchased from the American Type Culture Collection (Manassas, VA). Cells were cultured in RPMI-1640 medium containing 10% FBS (Gibco, with endotoxin level < 10 IU/ml), penicillin (200 U/ml), and streptomycin (100 μg/ml). All cell lines mentioned previously were kept at 37 °C in a humidified atmosphere containing 5% CO₂. HLA-A*0201/Kb transgenic (Tg) mice, 8–12 weeks-old, were purchased from The Jackson Laboratory (USA). Mice were bred and maintained in specific pathogen-free (SPF) facilities. Animal experiments were performed in accordance with the guidelines of the Animal Care and Use Committee of Chongqing Medical University.

A panel of endocan specific peptides (9 mer) containing HLA-A* 0201-binding motifs were predicted by software BIMAS (Section of BioInformatics and Molecular Analysis, National Institutes of Health, Bethesda, MD, USA) and SYFPEITHI (Institute for Immunology, University of Tübingen, Tübingen, Germany). The two 9-mer-peptides that demonstrated the highest scores with both SYFPEITHI and BIMAS were utilized for further analysis. Peptides were synthesized by F-moc chemistry. Before use, HPLC-purified peptides (> 95% pure) were dissolved at 40 mg/ml in DMSO and diluted to 1 mg/ml with Iscove’s modified Dulbecco’s medium (IMDM) (Life Technologies, Grand Island, NY).

HLA-A*0201 Ag-processing defective T2 cell line was cultured in RPMI 1640 containing 10% FBS. Before use, the cells were incubated for 6 h at 37 °C in serum-free IMDM. Then, cells were washed once, suspended in serum-free IMDM containing 20 μM of 2-ME and 15 μg/ml of human β2-microglobulin (β2m) (Calbiochem, La Jolla, CA), and pulsed with 50 μM peptide. HLA-A2.1-restricted MAGE-2 CTL epitope KMVELVHFL (amino acid position in MAGE-2; 112–120) and Kb-restricted Hpa CTL epitope FSYGFFVI (amino acid position in Hpa; 519–526) served as positive and negative controls. After a 24 h incubation at 37 °C, T2 cells were washed once with cold PBS containing 0.5% BSA and 0.02% NaN₃. They were then stained directly with primary anti-HLA-A2 Ab derived from BB7.2 and FITC-labeled goat-antimouse IgG (BD Biosciences Pharmingen, USA) secondary antibody. The percentage of FITC-positive cells as well as their staining intensity (mean fluorescence intensity (MFI)) was determined on an Epics Profile II (Coulter, Hialeah, FL). The Δ MFI for a particular mAb was calculated by subtracting the MFI of either the isotype-matched control mAb or the second-step Ab from each MFI value. The fluorescence ratio (FR) was calculated using the following formula: FR = (Δ MFI of peptide-treated T2 cells)/(Δ MFI of nontreated T2 cells).

The tumor cells were homogenized using RNAiso Plus (Takara) and ceramic beads for 1 min in a speedmill plus according to the instructions of the manufacturer (Alytik Jena). RNA was isolated according to the instructions of the manufacturer and reverse transcripted to obtain cDNA using a PrimeScript™ RT Reagent Kit with gDNA Eraser (Takara). Real-time PCR was performed using cDNA samples with SYBR@Premix ExTaq™II (Takara, Tli RNaseH Plus) by the One-step Plus analyzer (ABI). We normalized the results for each individual gene using the housekeeping gene beta-actin. RT-PCR products were then run on a gel and visualized with ethidium bromide.

Proteins from cultured tumor cells were resolved using SDS-PAGE and transferred onto polyvinylidene fluoride membranes using electroblotting. The membranes were incubated with primary antibodies, all diluted to 1:1000 (Cell Signaling Technology), at 4 °C overnight. β-actin (1:200; Santa Cruz Biotechnology, Dallas, TX) was used as the loading control. The membranes were incubated with HRP-conjugated goat anti-rabbit secondary Abs (1:2500; Sigma-Aldrich, St. Louis, MO) at 25 °C for 1 h. Bound Abs were visualized using a chemiluminescence detection system. Protein levels were calculated as the ratio of the target protein value to the β-actin value.

In brief, PBMC were isolated from HLA-A0201+ donors by ficoll-hypaque density gradient centrifugation. The cells were allowed to adhere in culture flasks for 2 h at 37 °C in RPMI1640 with 10% FBS. Then, non-adherent cells were collected and frozen in freezing media (60% RPMI-1640, 30% FBS and 10% DMSO) for later use in CTL assays. Adherent cells were cultured in 6 mL of RPMI-1640 with 10% FBS containing 800 U/mL recombinant human granulocyte-macrophage colony- stimulating factor (rhGM-CSF, R&D Systems, McKinley Place NE, MN, USA) and 1200 U/mL recombinant human interleukin-4 (rhIL-4, R&D Systems). On days 3, 5 and 7, half of the media was refreshed without discarding any cells and fresh cytokine-containing (rhGM-CSF and rhIL-4) media was added. On day 8 of culture, 1000 U/mL of tumor necrosis factor-a (TNF-a, R&D Systems) was added to the media. On day 9, non-adherent cells obtained from these cultures were considered mature human PBMC-derived DC.

Mature human PBMC-derived DCs were pulsed with 100 lg/ml HLA-A0201- restricted CTL epitopes of endocan for 4 h. The DCs were then irradiated with 20 Gy to prevent outgrowth. Subsequently, the non-adherent autologous peripheral blood lymphocytes were co-cultured with the DC. After stimulation, 800 U/mL recombinant interleukin-2 (IL-2) was added. On day 7, and weekly thereafter, the autologous peripheral blood lymphocytes were restimulated with peptide-pulsed pulsed irradiated dendritic cells.

ELISPOT assay were utilized to analyze the inferferon-gamma (IFN-γ) production of CTLs. Briefly, effectors were plated in triplicate at a final concentration of 1 × 10⁵ cells/well in 96-well nitrocellulose plates. Effector cells were stimulated with candidate peptides at a final concentration of 30 μM. The plate was incubated at 37 °C, 5% CO₂ for 24 h. The plate was processed using a biotin labeled anti-mouse IFN-γ antibody, an enzyme labeling marker and an antimarker. Then, a freshly prepared developer was added and incubated in the dark at 37 °C for 8 min (Quick Spot Mouse IFN-γ Precoated ELISPOT kit, Dakewe). Spots were quantified using the ELISPOT reader (BioReader 4000 Pro-X, BIOSYS, Germany).

To evaluate levels of CTL activity, a standard 4-h ⁵¹ Cr-release assay was used. In the autologous lymphocytes lysis assay, the autologous lymphocytes activated by PHA were used as target cells. Briefly, target cells were incubated with ⁵¹ Cr (100 μCi per 1× 10⁶ cells; Amersham Biosciences Corp) for 2 h in a 37 °C water bath. After incubation with ⁵¹Cr, target cells were washed three times with PBS, resuspended in RPMI 1640, and mixed with effector cells at effector-to-target (E/T) ratios of 25:1, 50:1, or 100:1. Assays were performed in triplicate wells for each experiment at each ratio in a 96-well round-bottomed plate. After a 4-h incubation, the supernatants were harvested, and the amount of ⁵¹Cr released was measured with a γ-counter. The percentage of specific lysate was calculated as 100 × (experimental release - spontaneous release) / (maximum release - spontaneous release). Maximum release was determined from supernatants of cells that were lysed by the addition of 2% Triton X-100.

12 weeks old HLA-A*0201/Kb mice were immunized three times once a week, by subcutaneous injection in the back with 1 × 10⁶ syngeneic mature DC pulsed with the peptides. After 7 days, the spleens of the mice were removed and the splenocytes were harvested as effectors.

The high affinity peptides binding to HLA-A2.1 could enhance HLA-A2.1 expression of antigen processing-deficient T2 cells. Therefore, the binding affinity of the predicted peptides to HLA-A2.1 was determined by using antigen processing- deficient T2 cells. As shown in Table 2, the four peptides were bound to HLA-A2.1 molecules with various affinities. Of four peptides selected, endocan_4–12 and endocan_9–17 enhanced the HLA-A2.1 molecular expression and demonstrated high affinity to HLA-A2.1 molecule. However, endocan_6–14 and endocan_3–11 demonstrated low affinity to HLA-A2.1 molecule.

To explore endocan levels of target cells, we analyzed endocan mRNA and protein in several cell lines by RT-PCR and Western blot assays. As shown in Fig. 1, endocan mRNA and protein could be detected in U251 cell lines. However, endocan mRNA and protein could not be detected in BV2 cell line and autologous lymphocytes.

To detect whether predicted peptides could elicit CTL to produce IFN-γ, we analyzed IFN-γ-producing cells by ELISPOT assay. As shown in Fig. 2, endocan_4–12 and endocan_9–17 could elicit IFN-γ-producing cells. However, IFN-γ-producing cells could not be detected in endocan_6–14 and endocan_3–11 groups. These results suggested that.

endocan_4–12 and endocan_9–17 could effectively promote CTL response.

To detect whether predicted peptides could elicit CTL to lyze target cells, we utilized the peptides to elicit PBMCs from HLA-A2.1+ donors. As shown in Fig. 3, endocan_4–12 and endocan_9–17 could elicit specific CTL to lyse target cells expressing endocan and HLA-A2.1. However, the lysate could not be detected in endocan_6–14 and endocan_3–11 groups. These results suggested that endocan_4–12 and endocan_9–17 could effectively elicit CTL mediated cytotoxicity.

To further identify whether peptide induced effectors recognized target cells in an HLA-A2-restricted manner and whether the effectors were CD8+ T lymphocytes, HLA-A2 mAbs and CD8 mAbs were utilized to block recognition of target cells and effectors. In addition, HLA-B0702 mAbs and CD4 mAbs were served as negative controls. As Fig. 4 demonstrated, cytotoxicity of endocan_4–12 and endocan_9–17 specific CTLs was significantly attenuated after blocking by HLA-A2 mAbs and CD8 mAbs. However, cytotoxicity of endocan-specific CTLs could not been attenuated by negative controls. These results indicated that peptide induced effectors were HLA-A2.1- restricted and were mainly from CD8+ T lymphocytes.

Furthermore, we investigated whether peptide could induce immune response in vivo. HLA-A*0201/Kb mice were immunized by subcutaneous injection with syngeneic mature DC pulsed with the peptides. After 7 days, the splenocytes were harvested as effectors to analyze the cytotoxicity. As Fig. 5 demonstrated, endocan_4–12 and endocan_9–17 could induce CTL response to lyse endocan and HLA-A2.1 positive cells with high efficiency. These results suggested that the peptides also had immunogenicity in vivo.