This study was approved under institutional guidelines for the use of human subjects in research. The patients and their families as well as healthy donors gave informed consent for the use of blood samples and tissue specimens in our research.
The osteosarcoma cell lines OS2000 and KIKU were established in our laboratory [11
]. The osteosarcoma cell lines U2OS, Saos-2 and HOS, human lymphoblastoid cell line T2, and erythroleukemia cell line K562 were purchased from ATCC (Manassas, VA). OS2000, KIKI, U2OS, Saos-2, HOS and K562 are PBF-positive [12
]. U2OS, Saos-2, and T2 are HLA-A*0201 positive. The HLA genotypes of the osteosarcoma cell lines were as follows: OS2000, A*2402, B*5502, B*4002, Cw*0102
; U2OS, A*0201, A*3201, B*4402, Cw*0501, Cw*0704
; Saos-2, A*0201, A*2402, B*1302, B*4402, Cw*0602, Cw*0704
; HOS, A*0211, B*5201, Cw*1202
; KIKU, A*0206, A*2402, B*4006, B*5201, Cw*0802
. Epstein-Barr virus-transformed B cell line NS-EBV-B was established from a healthy donor in our laboratory. Another Epstein-Barr virus-transformed B cell line, LCL-OS2000, was established from a patient with osteosarcoma [11
Autologous tumor cells were developed from fresh frozen biopsy specimens of osteosarcoma. The specimens were thawed in Iscove's modified Dulbecco's modified Eagle's medium containing 10% FCS at room temperature, minced into small pieces and filtrated with a 70 μm Cell Strainer (BD Biosciences, Bedford, MA). The cells were used immediately for cytotoxicity assay.
Design and synthesis of PBF-derived peptides
Based on the entire amino acid sequence of PBF, peptides with the ability to bind to HLA-A*0201 class I molecules were searched for through the World Wide Web site Bioinformatics and Molecular Analysis Section (BIMAS) HLA Peptide Binding Predictions http://www-bimas.cit.nih.gov/molbio/hla_bind/
]. Based on the binding scores, ten peptides were selected and synthesized [see Additional file 1
HLA class I stabilization assay
The affinity of peptides for HLA-A*0201 molecules was evaluated by T2 cell surface HLA class-I stabilization assay as described previously [20
]. An HLA-A*0201-binding influenza matrix protein-derived peptide (Inf-MP A2; GILGFVFTL) [22
] was used for positive control. Mouse H-2Kb-restricted peptide VSV8 (RGYVYQGL) [23
] was used for negative control. Assays were performed in triplicate. The affinity of each peptide for HLA-A*0201 molecules was evaluated by the percent mean fluorescence intensity (%MFI) increase of the HLA-A*0201 molecules detected by staining with an anti-HLA-A2 monoclonal antibody (BB7.2, purchased from ATCC) using the following calculation. %MFI increase: [(MFI with the given peptide – MFI without peptide)/(MFI without peptide)] × 100.
Limiting dilution/mixed lymphocyte peptide culture
Prior to frequency analysis and cytotoxicity assays, PBMC of patients were subjected to mixed lymphocyte peptide culture under limiting dilution conditions (LD/MLPC) according to the method described by Karanikas et al. [24
] with some modifications [17
]. LD/MLPC aims to seed at most one CTL precursor cell per well and induces proliferation of the precursor cell by subsequent mixed lymphocyte peptide culture. For this purpose, the appropriate number of PBMC and CD8+
cells per well is considered to be 1 × 105
–2 × 105
PBMCs were used as a source of responder cells in the initial five subjects (Patients 1 and 2 and three healthy donors) and CD8+
cells were used in the following three patients (Patients 3–5) [see Additional file 2
PBMC obtained from peripheral blood samples (50 ml) of Patients 1 and 2 and three healthy donors were suspended in AIM-V (Invitrogen Corp., Carlsbad, CA) supplemented with 1% human serum (HS). These cells were incubated for 60 min at room temperature with peptide PBF A2.2 (50 μg/ml). Peptide-pulsed PBMC were seeded at 2 × 105 cells/200 μl/well into round-bottom 96-microwell plates in AIM-V with 10%HS, IL-2 (20 U/ml; a kind gift from Takeda Chemical Industries, Ltd., Osaka Japan) and IL-7 (10 ng/ml; R&D Systems, Minneapolis, Minnesota, USA), and incubated. On day 7, half of the medium was replaced with fresh AIM-V containing IL-2, IL-7 and the same peptides. The cell cultures were maintained by adding fresh AIM-V containing IL-2. On days 14–21, they were subjected to tetramer-based frequency analysis.
PBMC obtained from Patients 3–5 were separated into CD8+ cells and CD8- cells using magnetic anti-CD8 microbeads (Miltenyi Biotec, Gladbach, Germany). CD8- cells were pulsed with the PBF A2.2 peptide for 60 min. Half of the CD8- cells were cryopreserved at -80°C for the second stimulation. CD8+ cells (1.0–2.1 × 105/well) and irradiated PBF A2.2 peptide-pulsed CD8- cells (1–5 × 105/well) were cocultured in 48-well cell culture plates in 500 μl of AIM-V with 10%HS, IL-2 and IL-7. On day 7, the second stimulation was performed by adding irradiated peptide-pulsed CD8- cells to each culture well in 500 μl of AIM-V with 10%HS, IL-2 and IL-7. On days 13–23, they were subjected to tetramer-based frequency analysis.
Tetramer-based frequency analysis
An FITC-conjugated HLA-A*0201/HIV tetramer (here termed the control tetramer) and a PE-conjugated HLA-A*0201/PBF A2.2 tetramer (A2/PBF A2.2 tetramer) were constructed by Medical & Biological Laboratories Co., Ltd. (Tokyo, Japan). PBMCs from patients were stimulated with the PBF A2.2 peptide by using the LD/MLPC procedure as described above. From each microwell containing 200 μl of the microculture pool, 100 μl was transferred to a V-bottom microwell and washed. To the spin-down pellets, the control tetramer and A2/PBF A2.2 tetramer (10 nM in 25 μl of PBS) were added in combination and incubated for 15 min at room temperature. Then a PE-Cy5-conjugated anti-CD8 antibody (eBioscience, San Diego, California, USA) was added (dilution of 1:30 in 25 μl of PBS containing the control tetramer and A2/PBF A2.2 tetramer) and incubated for another 15 min. The cells were washed in PBS twice, fixed with 0.5% formaldehyde, and analyzed by flow cytometry using FACScan and CellQuest software (Becton Dickinson, San Jose, California, USA). CD8+ living cells were gated and the cells labeled with the A2/PBF A2.2 tetramer were referred to as tetramer-positive cells. Tetramer-positive cells in each well are theoretically derived from a single CTL precursor, regardless of the number (percentage) of tetramer-positive cells. Accordingly, the number of tetramer-positive wells represents the number of CTL precursors. The frequency of anti-PBF A2.2 CTLs was evaluated using the following calculation: (number of tetramer-positive wells)/[(total number of tested wells) × (initial number of CD8+ cells per well)].
Development of CTL line and CTL clones
Attempts to establish CTL clones were made by a limiting dilution procedure and subsequent single-cell sorting procedures.
In the limiting dilution procedure, cells from a tetramer-positive T cell pool derived from Patient 4 were replated into a 96-well round-bottom microplate at one cell per well. In each well, a T cell was cocultured with irradiated A*0201+ NS-EBV-B cells (2 × 104) pulsed with the PBF A2.2 peptide and irradiated allogeneic PBMCs (8 × 104) in 200 μl of AIM-V containing 10%HS, IL-2 (200 U/ml) and IL-7 (10 ng/ml). On days 7, 14 and 21, the stimulation was repeated by adding irradiated peptide-pulsed NS-EBV cells (1 × 104), LCL-OS2000 cells (1 × 104), and allogeneic PBMCs (8 × 104) to each culture well in 100 μl of freshly replaced AIM-V with 10%HS, IL-2 and IL-7. On day 35, tetramer staining of all wells was performed. The tetramer-positive population was selected and further expanded. These cells were seeded at 2 × 103 per well with irradiated allogeneic PBMCs (1 × 105) in 100 μl of AIM-V containing 10% HS, IL-2 (200 U/ml) and phytohemagglutinin-P (PHA; 7.5 μg/ml, Wako Chemicals, Osaka, Japan) in a total of 192 wells of 96-well round-bottom microplates. On day 7, 100 μl of AIM-V containing 10% HS and IL-2 was added. On day 14, all proliferated cells were collected, washed and replaced with fresh AIM-V containing 10% HS and IL-2, followed by maintenance in a 48-well microplate at 0.5–1 × 106 cells per well. The established oligoclonal cell line was designated CTL 5A9.
Subsequently, a frozen stock of the oligoclonal CTL 5A9 was reactivated and subjected to single-cell sorting. In the reactivation procedure, thawed CTL 5A9 cells were cultured with allogeneic PBMCs in AIM-V containing 10% HS, IL-2 (200 U/ml) and PHA (7.5 μg/ml) for 27 days. The reactivated CTL 5A9 cells were stained by the A2/PBF A2.2 tetramer and the control tetramer. The tetramer-positive cells (0.82%) were sorted at one cell per well using FACS Aria II (Becton Dickinson) with allogeneic PBMCs (1 × 105) to each culture well in 200 μl of AIM-V with 10% HS, IL-2 (200 U/ml) and PHA (7.5 ug/ml) in a total of 384 wells of 96-well microplates. On days 7, 10 and 14, half of each medium was replaced with fresh medium without PHA. On days 20–34, tetramer staining was performed. Single-cell sorting was repeated until tetramer staining showed single clone populations.
CTL-mediated cytolytic activity was measured by a 6 h-51
Cr-release assay [25
]. Osteosarcoma cell lines (U2OS, OS2000, Saos-2, KIKU and HOS), K562, T2, and autologous osteosarcoma cells obtained from Patient 4 were used as target cells. T2 cells were treated with or without peptides at the indicated concentrations for 1 h at room temperature after 51
Cr-labeling. An HIV peptide (SLYNTVATL)[26
] was used as a negative control peptide. Target cells were labeled with 100 μCi of 51
Cr for 1 h at 37°C. The labeled target cells were suspended in RPMI without serum and seeded to microwells (2–5 × 103
CTL 5A9 and CTL clones were used as the effector cells. The effector cells were transferred to V-bottom microwells, suspended in AIM-V and mixed with the labeled target cells. After a 6 h incubation period at 37°C, the 51Cr level in the culture supernatant was measured using an automated gamma counter. The percentage of specific cytotoxicity was calculated as follows: the percentage of specific 51Cr release = 100 × (experimental release – spontaneous release)/(maximum release – spontaneous release).