Identification of new HLA-A*0201-restricted cytotoxic T lymphocyte epitopes from neuritin

Identification of cytotoxic T lymphocyte (CTL) epitopes from additional tumor antigens is essential for the development of specific immunotherapy of malignant tumors. Neuritin, a recently discovered antigen overexpressed in astrocytoma, is considered to be a promising target for biological therapy. In the present study, we predicted and identified HLA-A2-restricted CTL epitopes from neuritin by using the following four-step procedure: (1) computer-based epitope prediction from the amino acid sequence of neuritin; (2) peptide-binding assay to determine the affinity of the predicted peptide with HLA-A2.1 molecule; (3) stimulation of primary T cell response against the predicted peptides in vitro; and (4) testing of the induced CTLs toward target cells expressing neuritin and HLA-A2.1. The results demonstrated that effectors induced by peptides of neuritin containing residues 13–21, 121–129 and 4–12 could specifically-secrete interferon-γ and lyse target cells. Our results indicate that these peptides are new HLA-A2.1-restricted CTL epitopes, and may serve as valuable tools for astrocytoma immunotherapy.


Introduction
Astrocytoma is a common malignant primary brain tumor with a poor clinical prognosis in both adults and children [1][2][3]. The therapeutic options for the treatment of patients with astrocytoma are limited to three fundamental modalities: surgical resection, chemotherapy, and radiation therapy [4][5][6]. Cellular adoptive immunotherapy with specific CTLs has been recently used to treat malignant tumors [7][8][9].
Neuritin has been cloned and characterized as an important neurotrophin [10]. Recently, neuritin expression was found in astrocytomas and the expression level increased with astrocytoma pathologic grade, indicating that neuritin has an important role both in the promotion and in the progression of astrocytomas [11].
Anti-tumor vaccination is based on the existence of antigens, selectively or preferentially expressed by tumors, called tumor-associated antigens (TAAs) [12][13][14]. Vaccination with peptides derived from TAAs designed to stimulate specific T-cells is being a practicable approach evaluated in clinical trials [15][16][17]. To investigate the immune response elicited by neuritin CTL epitopes, the present study was undertaken to identify candidate CTL epitopes derived from neuritin.

Cell lines and animals
The human TAP-deficient T2 cell line, BB7.2 cell line producing mAb against HLA-A2, human glioma cell line U251 and U87 (HLA-A2?), human breast cancer cell line MCF-7 (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 B 10 EU/ml), penicillin (200 U/ml), and streptomycin (100 lg/ml). All cell lines mentioned previously were kept at 37°C in a humidified atmosphere containing 5 % CO 2 . 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 Third Military Medical University.

Peptide-binding assay
To determine whether the candidate epitopes can bind to HLA-A*0201 molecules, up-regulation of peptide-induced HLA-A*0201 molecules on T2 cells was examined. Briefly, according to previous reference, 1 9 10 6 T2 cells were incubated with 50 lM of the synthesized peptides in serum-free RPMI 1640 medium supplemented with b 2microglobulin (Sigma) at a concentration of 3 lg/ml for 16 h at 37°C, 5 % CO 2 [18]. Expression of HLA-A*0201 on T2 cells was then determined with the FACS Calibur flow cytometer (Becton-Dickinson, USA), by staining with primary anti-HLA-A2 Ab derived from BB7.2 and FITClabeled goat-antimouse IgG (BD Biosciences Pharmingen, USA) secondary antibody. The data were analyzed using Cell Quest software (Becton-Dickinson, USA). The Fluorescence index (FI) was calculated as follows: FI = (mean FITC fluorescence with the given peptide -mean FITC fluorescence without peptide)/(mean FITC fluorescence without peptide). Samples were measured in triplicate and then mean FI was calculated. An octapeptide mHpa (519-526) (FSYGFFVI) derived from mouse Hpa was served negative control.
Measurement of the peptide/HLA-A*0201 complex stability Briefly, T2 cells (10 6 /ml) were incubated overnight with the candidate peptides, respectively, at a concentration of 20 lg/ml in serum-free medium supplemented with b 2microglobulin at a concentration of 3 lg/ml at 37°C. Thereafter, they were washed four times to remove free peptides, incubated with Brefeldin A (10 lg/ml) for 1 h to block cell surface expression of newly synthesized HLA-A2.1 molecules, washed and incubated at 37°C for 0, 2, 4, 6, or 8 h. Subsequently, cells were stained with anti-HLA-A2 antibody from BB7.2 cells to evaluate the HLA-A2.1 molecule expression. For each time point, peptide induced HLA-A*0201 expression was evaluated by the formula mentioned above. Dissociation complex50 (DC50) was defined as the time required for the loss of 50 % of the HLA-A*0201/peptide complexes stabilized at time = 0.

RT-PCR analysis of neuritin expression
RT-PCR was used to analyze the expression of neuritin mRNA in cell lines. Total RNA was isolated from tumor cell lines using Tripure Isolation Regent Kit (Progema). Synthesis of cDNA was performed with 2 lg of total RNA with the aid of a reverse transcriptase Kit (Progema) and oligo(dT) primers. Two microliters RT product was amplified with PCR by using TaqDNA polymerase (Sangon, Shanghai) using standard procedures. The forward and reverse primer sequences were as follows: neuritin: sense primer, 5 0 -GTG CGA TGC AGT CTT TAA GTT-3 0 ; antisense primer, 5 0 -GGG CTT TTC AGA CTG TTT GTT-3 0 ; GAPDH: sense primer, 5 0 -GCA CCG TCA AGG CTG AGA AC-3 0 ; antisense primer, 5 0 -ATG GTG GTG AAG ACG CCA GT-3 0 . Thirty amplification cycles were run: 1 min at 94°C; 1 min at 60°C; and 1 min at 72°C. Cycling was ceased with a final extension of 10 min at 72°C. RT-PCR products were then run on a gel and visualized with ethidium bromide.

Western blot analysis of neuritin expression
For Western blot analysis, proteins in the cell extracts were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) through an 8 % polyacrylamide gel and were then transferred onto a nitrocellulose membrane. The membrane was incubated with 5 % non-fat milk in PBS and later with anti-neuritin MAb for 2 h at room temperature. After washing, the membranes were incubated with an alkaline phosphatase-conjugated goat anti-mouse IgG antibody (Amersham Biosciences, Buckinghamshire, England) for 1 h at room temperature.
Immunoreactive bands were detected using the ECL Western blot analysis system (Amersham Biosciences, Buckinghamshire, England).

Dendritic cell generation from human peripheral blood precursors
PBMCs were isolated from healthy HLA-A2? donors by Ficoll-Hypaque density gradient centrifugation (TBC company, Tianjing, China) and then seeded into culture flasks in RPMI-1640 medium supplemented with penicillin (100 U/ml), streptomycin (100 lg/ml), and 10 % FBS. After monocytes adhered (incubation for 2 h), the nonadherent cells were collected and frozen in freeze medium (60 % RPMI-1640 and 30 % FBS, 10 % DMSO) for later use in CTL assays. The adherent cells were cultured for 5 days in RPMI-1640 containing 1,000 U/ml of granulocyte-macrophage colony-stimulating factor (R&D Systems, Inc., Minneapolis, MN) and interleukin-4 (IL-4; R&D Systems, Inc.) and were the cultured for an additional 2 days in the presence of 1,000 U/ml of tumor necrosis factor a (R&D Systems, Inc.) to induce final maturation. After 7 days of culture, the mature DCs were harvested and analyzed for DC typical phenotypes by FACS analysis.
Induction of peptide-specific CTL with synthetic peptides Briefly, DCs were loaded with different peptides at a final concentration of 100 lg/ml for 4 h and were then irradiated with 20 Gy, which prevented all outgrowths in the control cultures. Autologous T cells were restimulated every 7 days with the previously mentioned peptide-pulsed DCs to generate peptide-specific CTLs. Recombinant interleukin 2 (IL-2) at a concentration of 20 U/ml was added to the culture medium on day 3 after every stimulation. Cytotoxic T lymphocyte activity was then assessed on day 23 by a 4 h 51 Cr release assay. Effectors generated from negative peptide-pulsed DCs were used as controls.
ELISPOT assay IFN-c secretion of effectors was assayed by enzyme-linked immunospot (ELISPOT). Multiscreen 96-well assay plates (Dakewe, Shenzhen, China) were precoated overnight at 4°C with anti-IFN-c antibody according to the manufacturer's instruction. After washing with PBST (PBS-0.05 % Tween 20), plates were blocked for 1 h at 37°C with PBS/ 1 % BSA. Cytotoxic T lymphocyte effectors from human HLA-A2? donors were plated in triplicate wells at a density of 1 9 10 5 /100 ll in RPMI-1640 medium. Plates were cultured overnight, washed extensively with PBST, and incubated with anti-IFN-c mAb for 1 h at 37°C. After washing, goat anti-biotin antibodies (Dakewe) were added, and the plates were incubated for 1 h at 37°C. Thirty microliters of activator solution (Dakewe) was added to develop spots, and after 10-30 min, the plates were washed with distilled water to stop the reaction. After being airdried, the number of spots in each well was counted using the Bioreader 4000 PRO-X (Bio-Sys; Germany).

Cytotoxicity assay
To evaluate the levels of CTL activity, a standard 4-h 51 Cr release assay was used. Briefly, target cells were incubated with 51 Cr (100 lCi per 1 9 10 6 cells) for 2 h in a 37°C water bath. After incubation with 51 Cr, target cells were washed three times with PBS, resuspended in RPMI-1640 medium, and mixed with effector cells at a 25:1, 50:1 or 100:1 of effector to target (E/T) ratio. Assays were performed in triplicate for each sample at each ratio in a 96-well round-bottomed plate. After a 4-h incubation, the supernatants were harvested, and the amount of released 51 Cr was measured with a gamma counter. The percent specific lysis was calculated according to the following formula: Specific lysis = (experimental release -spontaneous release)/(maximal release -spontaneous release) 9 100 % Analysis of in vivo immunogenicity HLA-A*0201/Kb mice were immunized with 100 lg of various peptides prepared in incomplete Freund's adjuvant (IFA) and boosted once a week for three times. As a control, mice were injected with an IFA emulsion without peptide. 7 days after immunization, splenocytes from injected animals were cultured and used as effector cells.

Statistical analysis
Results were expressed as mean ± SEM. Analysis of Student's t test were performed to determine effects of the treatments. A difference was considered significance level of P \ 0.05.

Prediction of putative CTL epitopes restricted with HLA-A*0201
To predict the HLA-A*0201-restricted CTL epitopes of neuritin, two programs (BIMAS and SYFPEITHI), were used to scan the complete amino acid sequence of this antigen. Four highest-scored 9-amino-acid peptides were chosen as candidates for further identification (Table 1). These peptides were chemically synthesized, purified, and identified. The molecular weight of each peptide determined by mass spectrometry assay was similar to its theoretical molecular weight, and the purities of these peptides were all [95 % (data not shown).

Expression of neuritin in target cells
The expression of neuritin mRNA and protein in cell lines in this study was analyzed by RT-PCR and Western blot. As shown in Fig. 1, neuritin mRNA and protein were detected in U251 and U87 cell lines. However, neuritin

Enzyme-linked immunospot (ELISPOT) assay for IFN-c
Since CTLs are known to produce the Th1 cytokine IFN-c, peptide-specific T cells were enumerated by measuring IFN-c-producing cells by ELISPOT assay. As shown in Fig. 2, neuritin [13][14][15][16][17][18][19][20][21] , neuritin 121-129 and neuritin 4-12 peptides were found to generate a strong peptide-specific T cell response by virtue of their ability to induce increased frequencies of IFN-c-producing T cells, as compared to the negative peptide control. These results suggest that neuritin peptide vaccines can increase IFN-c secretion by effectors and enhance the Th1 immune response.

Inhibition of the recognition of effectors by anti-HLA2 antibody
To determine whether the peptides induced effectors recognized target cells in an HLA-A2-restricted manner, the mAbs against HLA-A2 were used to block recognition by effectors. Our results showed that the anti-HLA-A2 antibody could significantly eliminate the cytotoxicity of the effectors against neuritin and HLA-A2 positive cells (Fig. 4), which implied that the induced effectors lysed target cells in an HLA-A2-restricted manner.

Discussion
Survival in the majority of high-grade astrocytoma (HGA) patients is very poor, with only a rare population of longterm survivors [20][21][22]. During the last decades, great progress has been made in the treatment of patients with astrocytoma [23][24][25]. Many astrocytoma can be cured or eliminated using the therapeutic options such as surgical resection, chemotherapy, and (or) radiation therapy.
Over the past few years, the analysis of spontaneous immune responses to autologous tumors in cancer patients has allowed the identification of several kinds of tumorassociated antigens that can be the targets for tumor specific immune responses based on the recognition of tumor antigen by CTLs in an MHC-class I/peptide complexrestricted manner [29][30][31]. Therefore, cancer-specific immunotherapy has become an attractive fourth-therapeutic approach against carcinomas. Among them, one of the most relevant for the development of tumor immunotherapy is peptide-based, cancer-specific immunotherapy using the group of the tumor antigens [32,33]. HLA-A*0201/Kb mice were immunized with 100 lg of various peptides prepared in incomplete Freund's adjuvant (IFA) and boosted once a week for three times. As a control, mice were injected with an IFA emulsion without peptide. 7 days after immunization, splenocytes from injected animals were cultured and used as effector cells. The cytotoxic activities of CTLs were determined against target cells at various E/T ratios using 51 Cr release assay. Experiments performed in triplicate showed consistent results. Compared with controls, P \ 0.05 Neuritin has been cloned and characterized as an important neurotrophin. The expression of neuritin is closely associated with the growth of afferent nerves and the development of dendrites, axons, and synapses [34]. Recently, neuritin expression was found in tissues besides nervous system and tumor tissues [35]. Moreover, the neuritin protein was highly expressed in astrocytomas and increased with pathologic grade, indicating that neuritin has an important role both in the promotion and in the progression of astrocytomas.