Malignant glioma patients usually undergo debulking surgery followed by radiation and aggressive rounds of chemotherapy. Despite the majority of the tumors being removed or killed, no lasting and effective antitumor immunity is generated and the patients die from the tumor cells escaping or resisting those therapies. These failures lead many to believe that an aggressive combination of standard therapies, along with other biologically based therapies, is needed to successfully treat this cancer. Although the central nervous system (CNS), and tumors that arise therein, reside in an “immunologically privileged” site [1], some glioma patients were successfully treated by immunotherapy [2-7]. Adjuvant immunotherapy modalities may be particularly useful for treating gliomas located next to critical brain control regions (brain stem or the thalamus) where surgery or radiation cannot be used, provided inflammation can be controlled. The advantage of generating an immune response toward the cancer cells is that the immunized T-cells can now seek and destroy the remaining tumor cells that resisted the therapies or were localized to sites that were inaccessible to the traditional treatments. Therefore, immunotherapy for glioma is an attractive alternative treatment option.

Adoptive transfer of large numbers of tumor-specific cytotoxic T-lymphocytes (CTLs) is currently being developed to treat cancer by changing the relative balance between tumor load and the immune response [8,9]. CTLs recognize “processed” peptides that are derived from endogenous proteins and presented on the cell surface in association with major histocompatibility complex (MHC) class I molecules [10,11]. Peptides that bind to a MHC class I molecule have been shown to share common amino acid motifs, which are called major anchor motifs [11]. Hence, tumor-specific CTLs can recognize and select the antigenic peptides, then kill tumor cells in an antigenic peptide-specific fashion.

Survivin is a newly identified member of the inhibitor of apoptosis (IAP) gene family that has been implicated in suppression of apoptotic cell death and regulation of cell division [12,13]. Survivin expression has been found to be undetectable in normal adult tissues. However, it has been found to be abundantly expressed in fetal tissues and a wide variety of human malignancies [14]. The fact that survivin overexpression may provide a survival benefit for tumor cells and that its enhanced expression is almost completely restricted to malignant tissues makes survivin an interesting target for the development of immunotherapeutic strategies.

Recently, survivin has been reported to be abundantly overexpressed in malignant glioma [15,16]. The human leukocyte antigen (HLA)- A2.1-restricted CD8⁺ T-cell epitope ELTLGEFLKL (referred to as pSurvivin_95-104) derived from the tumor-associated antigen (TAA) survivin was identified [17]. In the present study, we used W6/32 antibody-mediated artificial antigen-presenting cells (aAPCs) made by coating HLA-A2/pSurvivin_95-104 tetramer mediated by W6/32 antibody, anti-CD28 antibody, 4-1BBL and CD83 molecules to cellsized latex beads to generate specific CTL against malignant glioma from the peripheral blood lymphocytes (PBLs) of healthy donors in vitro, in which these CTL lysed HLA-A2⁺ glioma cells that expressed pSurvivin_95-104 and could eliminate glioma cells in NOD/SCID mice.

Cell line and cell culture

T2 cells bear the HLA-A*0201 gene, but express a very low level of cell surface HLA-A2.1 molecules, and are unable to present endogenous antigens due to a deletion of most of the MHC class II region, including the transporter associated with antigen processing, and genes encoding immuno-proteasomal subunits. This cell line was maintained in Roswell Park Memorial Institute (RPMI)-1640 medium (Invitrogen, Carlsbad, USA) supplemented with 10% fetal bovine serum (FBS; HyClone, Logan, USA) and 1% antibiotics (penicillin / streptomycin; 100 U/ml). Glioma cell lines U251 (HLA-A2⁺, survivin⁺) and A172 (HLA-A2^-, survivin⁺) were cultured in our laboratory. These glioma cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM)-supplemented with 10% FBS and 1% antibiotics (penicillin / streptomycin; 100 U/ml). NOD/SCID mice, 6 to 8 weeks of age, were obtained from Vital River Laboratories (Beijing, China) and were maintained under a specific pathogen-free condition.

Synthetic peptide

Survivin-derived peptide pSurvivin_95-104 ELTLGEFLKL [17], and the control peptide HIV-Gag peptide SLYNTVATL (pHIV) [18] were used in this study; they were synthesized by standard solid-phase chemistry and characterized by mass spectrometry. The purity of the synthetic peptides was more than 95%, as indicated by analytical HPLC. Lyophilized peptides were dissolved in dimethylsulfoxide and stored at − 80ºC after dilution in phosphate-buffered saline (PBS).

HLA class I typing

HLA class I typing was performed with microcytotoxicity by Lambda antigen tray class I (One Lambda, Canoga Park, USA).

Detection of survivin gene expression in glioma cell lines by reverse transcription (RT)–polymerase chain reaction (PCR)

The expression of the survivin gene in glioma cell lines U251 and A172 was detected by RT-PCR. The primers used for amplification of human survivin and β2-microglobulin (β2m) were as follow: survivin, 5’-CGACCCCATAGAGGAACATAAA-3’ (sense), and 5’-GGAATAAACCCTGGAAGTGGTG- 3’ (anti-sense); β2m, 5’-GGGTTTCATCCATCCGACAT- 3’ (sense), 5’-GATGCTGCTTACATGTCTCGA-3’ (anti-sense). For the surviving and the β2m cDNA the PCR temperature profiles were as follows: 2 minutes pretreatment at 94°C and 30 cycles at 94ºC for 30 seconds, annealing at 59°C for 30 seconds and 72°C for 60 seconds with a final extension at 72°C for 7 minutes.

Preparation of HLA-A2-pSurvivin_95-104 tetramer

Synthesis of HLA-A2-pSurvivin_95-104 tetramer was carried out according to the protocol of Altman et al. [19]. Briefly, plasmids encoding HLA-A*0201 (heavy chain) molecules with a C-terminal biotinylation site and human β2m molecule were constructed by insertion of the target genes into pET28a. The heavy chain and β2m molecules were expressed in Escherichia coli, and purified from inclusion bodies, then refolded in the presence of excess pSurvivin_95-104 to form HLA-A2-pSurvivin_95-104 monomeric product. The folded product was then subjected to enzymatic biotinylation by BirA enzyme (Avidity, Denver, USA) at 25ºC for 12 h. This biotinylated HLA-A2- pSurvivin_95-104 monomer was used for the preparation of the HLA-A2- pSurvivin_95-104 tetramer, which was produced by mixing the purified biotinylated monomer with phycoerythrin (PE)-labeled streptavidin (Sigma, St. Louis, USA) at a molar ratio of 4:1. HLA-A2-pHIV tetramer was also prepared according to the protocol.

Generation of W6/32 antibody-mediated aAPC

The 4.5 μm s epoxy-activated magnetic beads (Dynal Biotech, Lake Success, NY) were incubated with a mixture of W6/32 (2 μg/107 beads; BD pharMingen), anti-human CD28-specific antibodies (1 g/107 beads; BD PharMingen , San Diego, CA,USA), 4-1BBL (0.5 μg/107 beads; BD PharMingen , San Diego, CA, USA) and CD83 (0.5 μg/107 beads; BD PharMingen , San Diego, CA, USA) for 1 h in 1 ml wash buffer per 107 beads at 4ºC on a rotator, washed twice with PBS, then blocked with 0.4% BSA for 15 min and washed with PBS. Finally, the beads were incubated with 20μg HLA-A2/pSurvivin_95-104 tetramer or HLA-A2/ pHIV tetramer for 24 h at 4ºC on a rotator and washed with PBS. The W6/32 antibody-mediated pSurvivin_95-104-specific aAPCs or W6/32 antibody-mediated pHIV-specific aAPCs were stored PBS at 4ºC.

Expansion of CTLs

PBLs from six healthy HLA-A2-positive donors (Informed consent was obtained from each donor). were separated using standard Ficoll- Hypaque (Sigma) gradient density centrifugation and PBL isolation kit (Miltenyi). The PBLs were stimulated with W6/32 antibody-mediated pSurvivin_95-104-specific aAPCs in vitro using the protocol adapted from previous studies [20]. Briefly, The PBLs were used as responder cells and stimulated with W6/32 antibody-mediated pSurvivin_95-104-specific aAPCs. Responder cells (3 × 10⁶ /well) were co-cultured with W6/32 antibody-mediated pSurvivin _95-104-specific aAPCs (3 × 10⁵/well) in 24- well plates in RPMI-1640 medium supplemented with 10% fetal bovine serum (1 ml/well). IL-7 (10 ng/ml, Pepro Tech EC, London, UK), IL-21 (10 ng/ml, Pepro Tech EC, London, UK) and IL-15 (10 ng/ml, Pepro Tech EC, London, UK) was added on day 1. IL-2 (50 units/ml, R&D, UK) was added on day 4. Medium, IL-2, IL-7, IL-21 and IL-15 were replenished twice a week. On day 7 and weekly thereafter, responder cells were collected, counted and re-plated at 3 × 10⁶ per well at the 24-well plate, together with 3 × 10⁵ W6/32 antibody-mediated aAPC per well in RPMI-1640 medium with 10% fetal bovine serum, 10 ng/ ml IL-7, 10 ng/ml IL-21, 10 ng/ml IL-15 and 50 units/ml IL-2. After four rounds of co-culture, pSurvivin_95-104-specific CTLs were isolated from the bulk cultures by fluorenscence activated cell sorter (FACS) using HLA-A2/pSurvivin_95-104 tetramer. After sorting, the pSurvivin95- 104-specific CTLs were restimulated and expanded with W6/32 antibody-mediated pSurvivin_95-104-specific aAPCs in the same fashion for tetramer staining, IFN-γ production, CTL reactivity and adoptive immunotherapy experiments. The pHIV-specific CTLs (as a control) were expanded by W6/32 antibody-mediated pHIV-specific aAPCs in the same fashion.

Tetramer staining

Tetramer staining was carried out as previously described [21,22]. In brief, 1 × 10⁶ cells were incubated in 100 μl fluorescence activated cell sorter (FACS) staining buffer (PBS supplemented with 1% BSA and 0.05% NaN₃) with 20 μg/ml HLA-A2-peptide tetramer at 37ºC for 30 min. Cells were washed with PBS and subsequently incubated with FITC labeled anti-CD8 antibody (BD PharMingen, San Diego, USA) at 4ºC for 30 min. All cells were washed with PBS twice after being stained, and then they were fixed in 1% formaldehyde. Stained cells were analyzed with FACScalibur (Becton Dickinson, Heidelberg, Germany).

Enzyme-linked Immuno-spot assay

To determine the frequency of T-cells capable of responding to a specific stimulus by secretion of interferon (IFN)-γ, an enzyme-linked immune-spot (ELISPOT) assay was carried out. The W6/32 antibodymediated pSurvivin_95-104-specific aAPCs, W6/32 antibody-mediated pHIV-specific aAPCs, Beads pulsed without HLA-A2/pSurvivin_95-104 tetramer (referred to uncoated aAPCs), K562, A172 (HLA-A2^-, survivin⁺), U251 (HLA-A2⁺, Survivin⁺) were used as stimulating target cells. Other target cells T2pSurvivin_95-104 (the pSurvivin_95-104-pulsed T2 cells) and T2pHIV (the pHIV-pulsed T2 cells) were prepared by incubating T2 cells with pSurvivin_95-104 (60 μg/ml) and pHIV (60 μg/ml) respectively for 3 h at 37ºC. T2 cells pulsed without peptide were used as the negative control. Responder cells were co-incubated with the target cells at a ratio of 1:10 (stimulator cell : responder cell) at 37ºC for 24 h in 96-well ELISPOT plates. The assay was performed and developed according to the manufacturer’s instructions (BD PharMingen).

Cytotoxicity assay

To analyze the cytotoxic activity of the expanded CTLs against various target cells, cytotoxic assays were performed by incubating 51Cr-labeled (500 μCi) target cells with effector cells at various effector : target ratios at 37°C for 4 h. Glioma cell lines U251 (HLA-A2⁺, survivin⁺) and A172 (HLA-A2^-, survivin⁺), T2pSurvivin_95-104, T2pHIV, K562 and T2 cells pulsed without peptide were selected as target cells. The percentage of ⁵¹Cr release was calculated according to the following formula: 100 × (experimental release - spontaneous release) / (maximum release - spontaneous release).

Inhibition of the cytotoxicity with HLA class I-specific monoclonal antibody

T2pSurvivin_95-104 and U251 target cells were incubated with anti- HLA class I monoclonal antibody (mAb) W6/32 (American Type Culture Collection [ATCC], http://www.atcc.org/) [18] and a control isotypic mAb of irrelevant specificity, immunoglobulin G2a (IgG2a; BD PharMingen) at a final concentration of 30 μg/ml for 40 min at 4°C before cytotoxicity assay. After incubation, the target cells were mixed with effector cells for the ⁵¹Cr release assay.

Adoptive immunotherapy experiments

NOD/SCID mice were inoculated with 4 × 10⁶ human glioma U251 cells in 0.5 ml PBS intravenously. Mice were divided into three groups (n = 10 each). For adoptive transfer experiments, mice were treated 3 times on days 5, 15, 25 after tumor inoculation with i.v. injection of induced pSurvivin_95-104-specific CTLs (up to 3×10⁶ cells per injection). Mice with injection of PBS or pHIV-specifc CTLs were served as the control groups. All mice received two injections of rhIL-2 (2 ×10⁵ IU/mouse) i.p. on days 5, 15, 25. Tumor volumes were calculated using Vernier calipers at 5-day intervals after tumor cells inoculation according to the formula: d1 × (d2)2 ×0.5 (d1 = largest diameter, d2 = perpendicular diameter). The survival time of each group of mice was monitored and recorded on a regular basis. They were sacrificed when any single or combined tumor linear measurement exceeded 20 mm. Three independent experiments were performed.

Statistical analysis

All data in this study were analyzed using version SPSS 11.0 software (SPSS, Chicago, USA). P<0.05 was considered as statistically significant.

Survivin gene expression and HLA class I typing in glioma cell lines

The mRNA expression of the survivin gene was detected in U251 and A172 cell lines. As shown in Figure 1, the expression level of the survivin gene was high in U251 and A172 cell lines, whereas there was no expression in PBMC. The results of HLA class I typing detection showed that U251 was an HLA-A2⁺ cell line and A172 was an HLA-A2- cell line.

Frequency of pSurvivin_95-104-specific CTLs as determined by tetramer staining

A flowcytometric analysis of PBLs was performed before or after coculture with the W6/32 antibody-mediated aAPC. Before stimulation using W6/32 antibody-mediated pSurvivin_95-104-specific aAPCs, the frequency of CD8⁺ T-cells stained with HLA-A2/pSurvivin_95-104 tetramers was 0.63%. However, after sorting and expansion, FACS analysis revealed that 95.1% of CD8⁺ T-cells were stained with HLA-A2/pSurvivin_95-104 tetramers, which was not observed when staining with the control tetramers (HLA-A2/pHIV tetramer) (Figure 2). The pSurvivin_95-104-specific CTLs expanded by W6/32 antibody- mediated aAPC from the other five normal donors showed similar results (Table 1).

Enzyme-linked immuno-spot assay

The Enzyme-linked immuno-spot assay was used to determine the frequency of individual antigen-specific IFN-γ producing T-cells. As shown in Figure 3, priming with U251 cells, T2pSurvivin_95-104 or W6/32 antibody-mediated pSurvivin_95-104-specific aAPCs resulted in the generation of peptide-specific IFN-γ-producing CD8⁺ T-cells after the stimulation. The average spot number of responding lymphocytes induced by U251 cells, T2pSurvivin_95-104 or W6/32 antibody-mediated pSurvivin_95-104-specific aAPCs (~ 95 peptide-specific spots / 10² cells) were significant higher than that of responding lymphocytes induced by K562, T2pHIV, T2, A172 or uncoated-aAPCs (0 peptide-specific spots / 10² cells) (P < 0.05).

Cytotoxicity of pSurvivin_95-104-specific CTLs

The cytotoxic activity of the expanded CTLs against various target cells was tested using the ⁵¹Cr-releasing assay. CTLs showed approximately 75% specific lysis against the T2pSurvivin_95-104 and U251 (HLA-A2+, survivin+) at an effector : target ratio of 8:1. However, CTL showed approximately 1% cytolysis against the T2pHIV, A172 (HLA-A2-, survivin+), K562 and T2 without pulsed peptide at any tested effector : target ratio (Figure 4). The specific killing activity of the CTLs expanded by the W6/32 antibody-mediated pSurvivin_95-104-specific aAPCs against specific target cells was much more obvious than in any other control group (P < 0.05). The specific CTLs for pSurvivin_95-104 expanded by the W6/32 antibody-mediated pSurvivin_95-104-specific aAPCs from the other five normal donors showed similar specific lysis ability (Table 1). These results showed that the cytotoxicity of the CTLs expanded by W6/32 antibody-mediated pSurvivin_95-104-specific aAPCs is pSurvivin_95-104-specific.

Inhibition of cytotoxicity of pSurvivin_95-104-specific CTLs by HLA class I specific mAb W6/32

To determine whether the CTLs expanded by W6/32 antibodymediated pSurvivin_95-104-specific aAPCs could recognize the specific target cells in an HLA class I-restricted manner, anti-HLA class I mAb W6/32 was utilized to block the cytotoxicity of the expanded CTLs. The cytotoxic activity against the T2pSurvivin_95-104 and U251 was significantly eliminated by W6/32. As shown in Figure 5, W6/32 inhibited target cell lysis, whereas mouse IgG2a, used as an isotype control, showed no effect in T2pSurvivin_95-104 and U251 cells. These results suggested that the expanded CTLs lysed the specific targets in an HLA class I-restricted manner.

Adoptive transfer of the expanded pSurvivin_95-104-specific CTLs can mediate effective therapy of human glioma in NOD/SCID mice

To evaluate the therapeutic effects of pSurvivin_95-104-specific CTLs expanded by W6/32 antibody-mediated pSurvivin_95-104-specific aAPCs, NOD/SCID mice bearing U251 cells were treated by adoptive transfer of the expanded CTLs. Tumor volumes of PBS group or pHIV-specific CTLs group expanded as determined by measuring every 5 days. In contrast, the rate of tumor growth in animals treated with pSurvivin95- 104-specific CTLs was significantly decreased as compared with that in animals treated with PBS or pHIV-specific CTLs (P <0.05) (Figure 6A). In addition, as shown in Figure 6B, none of treatment control mice survived longer than 40 days. However, treatment with the expanded pSurvivin_95-104-specific CTLs was effective and cured 100% of the mice (> 60 days). These results demonstrated that adoptive transfer of the expanded pSurvivin_95-104-specific CTLs can mediate effective therapy of human glioma in NOD/SCID mice.

Malignant gliomas are the most common tumors in the central nervous system. When treated with conventional therapy such as surgery, radiation, or chemotherapy, the prognosis for patients with malignant glioma is poor [22]. Clearly, novel therapeutic strategies are necessary.

The adoptive transfer of tumor-specific CTLs provides a promising approach to the immunotherapy of cancer. Previous attempts to determine the impact of adoptive transfer in tumor immunotherapy have been limited by the difficulty of isolating T-cells of known antigen specificity. Melanoma and renal cell carcinoma were the first tumors in humans to be treated with adoptive immunotherapy [23]. Infusions of polyclonal T-cell populations isolated from the tumor (tumorinfiltrating lymphocytes) and nonspecifically expanded in vitro with high concentrations of IL-2 suggested the therapeutic potential of this approach in cancer patients [23]. The recent characterization of glioma-associated antigens recognized by human CTLs has opened new possibilities for the adoptive transfer of T-cells in glioma immunotherapy.

Rimoldi et al. [24] were the first to document that melanoma associated antigen-specific CTL lines could recognize HLA-matched glioma cells in vitro. Then, Chi et al. [25], Scarcella et al. [26], and Sahin et al. [7] reported that gp100 and MAGE-1 mRNA was expressed in glioma tumor cells and tumor tissue by RT-PCR, and HER-2 was found in brain tumor by immunohistochemical staining [28,29]. Prins et al. [30] validated melanoma-associated antigen gp100 and TRP-2 as immunotherapeutic targets in a murine glioma model. Liu et al. [31] reported on TRP-2 as a CTL target in malignant glioma, which demonstrated that TRP-2 antigen can be naturally processed and recognized by TRP-2-specific CTLs. They also found that TRP- 2-specific cytotoxic T-cell activity was detected when PBMCs were stimulated with autologous DCs pulsed with irradiated GBM tumor cells in vitro and in patents’ PBMCs after DC-pulsed autologous tumor lysate vaccinations. Very importantly, IL-13 receptor α2 has been identified as a glioma-specific antigen [32], and a HLA-A2.1- restricted CTL epitope (WLPFGFILI) might serve as an attractive component of peptide-based vaccines to treat glioma [33,34]. Survivin is a member of the inhibitors of apoptosis family and is overexpressed in different types of malignancies [35]. Cytotoxic T-cells recognizing survivin epitopes can be elicited in vitro and by vaccination in patients with leukemia, breast cancer, and melanoma. Our results indicate that pSurvivin_95-104 epitope could be used as tumor antigen targets for surrogate assays for antigen-specific CTLs or to develop antigenspecific active immunotherapy strategies for glioma patients.

There are several methods currently in use to activate and expand CD8⁺ T-cells to the numbers required for a trial of adoptive immunotherapy. One of the most widely used approaches has been based on the use of autologous antigen-loaded dendritic cells (DCs) as antigen presenting cells (APCs). However, the generation and maintenance of DCs is expensive and cumbersome. Furthermore, variability in DC number and the quality of the DCs are affected by both pretreatment of the patient and the patient’s disease. Therefore, a number of different systems have been developed in which aAPC present antigen–HLA complexes in combination with the appropriate costimulatory molecules. The benefits of an aAPC system to expand T-cells include their consistency over time and over multiple donors, their “off-the-shelf” availability, and the lack of need for coculture of allogeneic or virally infected cells with the desired T-cells. Furthermore, the use of artificial APCs may be more attractive for clinical use because they are more amenable to manufacturing within the constraints of current Good Manufacturing Practices than are cellular APCs [36-38]. In the present study, we used W6/32 antibody-mediated pSurvivin95- 104-specific aAPCs to induce specific CTL against malignant glioma from the PBLs of healthy donors. Following this procedure, we found that the expanded CTLs, derived from PBLs in an HLA-A2⁺ healthy donor, could kill the HLA-A2⁺ pSurvivin_95-104⁺ glioma cells when expanded with W6/32 antibody-mediated pSurvivin_95-104-specific aAPCs in vitro. The specific killing activity of CTLs against specific target cells T2pSurvivin_95-104 and U251 was much more effective than that of any other control group. The results indicated the cytotoxicity of the CTLs expanded with W6/32 antibody-mediated pSurvivin_95-104- specific aAPCs is antigen-specific, that is, against the target cells bearing the corresponding HLA-A2-pSurvivin_95-104 complexes. Intravenous injection of the expanded pSurvivin_95-104-specific CTLs into nonobese diabetic–severe combined immunodeficiency (NOD/SCID) mice harboring glioma cells resulted in glioma cells elimination, whereas transfer of control T-cells was ineffective.

Proper cell signaling during the expansion phase is also an essential component in producing effective, functioning CTLs following infusion. Excessive stimulation and improper or absent co-stimulation could result in deficient cytolytic activity, lack of persistence or apoptosis. June et al. found the beads coated with HLA class II tetramers in an indirect fashion, via an anti-Class II antibody covalently bound to the bead, were more efficient activators of antigenspecific CD4⁺ T-cells than beads directly and covalently coated with HLA monomers or tetramers [39]. Our results show the beads coated with HLA class I tetramers via antibody W6/32 covalently bound to the bead, were more efficient activators of antigen-specific CD8⁺ T-cells than beads directly and covalently coated with HLA monomers or tetramers (data not shown). In addition, in this study, we used a combination of anti-CD28, 4-1BBL and CD83 for the generation of a costimulatory signal. Recent studies have demonstrated that combining B7.1/CD28 stimulation and 4-1BB signaling was required for optimal induction of CD25 and bcl-XL expression in CD8⁺ T-cells, increased CTL activity and protective anti-tumor immunity [40-42]. In addition, CD83 molecule is important in: (a) priming naive CD8+ T-cells; (b) driving their antigen-specific expansion; and (c) supporting their long-term survival and function when coupled to aAPCs [43,44]. As we learn more about the in vivo response and the effects of differing amounts and types of co-stimulation on CTL function post-infusion, future generations of the aAPC might prove to be essential because the bead can easily adapt to the new stimulatory requirements by adding or removing different co-stimulatory complexes.

In summary, we have presented data showing that the use of aAPC represents the state-of-the-art in generation of antigen-specific CTLs for adoptive immunotherapy. Thus, The W6/32 antibody-mediated artificial antigen-presenting cells (aAPCs) made by coating HLA-A2/ pSurvivin_95-104 tetramer mediated by W6/32 antibody, anti-CD28 antibody, 4-1BBL and CD83 molecules to cell-sized latex beads could provide a useful tool for the reproducible expansion of peptidespecific CTLs in vitro and significantly advance the field of adoptive immunotherapy. The W6/32 antibody-mediated pSurvivin_95-104-specific aAPCs could be used as a standardized, “off-the-shelf” reagent to enrich pSurvivin_95-104-specific CTLs for the treatment of patients with malignant glioma.

The authors have no financial conflict of interest.

This work was supported by the grants from the National Natural Science Foundation of China (No. 30801355, No. 81060183, No. 81072161 No. 30760248, No. 30660185) and Supported by Program for New Century Excellent Talents in University (No. NECT-06-0761) and International Cooperation Project of The Ministry of Science and Technology of the People’s Republic of China (No. 2008DFA31380).