Clinical Cancer Research CR Balducci AACR Membership
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
Clinical Cancer Research 13, 7329, December 15, 2007. doi: 10.1158/1078-0432.CCR-07-1025
© 2007 American Association for Cancer Research
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Jiang, X.
Right arrow Articles by Zhao, H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Jiang, X.
Right arrow Articles by Zhao, H.

Human Cancer Biology

HLA Tetramer–Based Artificial Antigen-Presenting Cells Efficiently Stimulate CTLs Specific for Malignant Glioma

Xiaobing Jiang1, Xiaoling Lu2, Ruen Liu3, Fangcheng Zhang1 and Hongyang Zhao1

Authors' Affiliations: 1 Department of Neurosurgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China; 2 Department of Immunology, Shanghai Medical College, Fudan University, Shanghai, China; and 3 Department of Neurosurgery, China-Japan Friendship Hospital, Beijing, China

Requests for reprints: Xiaobing Jiang, Department of Neurosurgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China. Phone: 86-27-8535-1616; E-mail: jxb917{at}126.com.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: The interleukin-13 receptor {alpha}2 (IL-13R{alpha}2) is a glioma-restricted cell-surface epitope not otherwise detected within the central nervous system. Here, we report a novel approach for targeting malignant glioma with IL-13R{alpha}2–specific CTLs.

Experimental Design: Artificial antigen-presenting cells (aAPC) were made by coating human leukocyte antigen (HLA)-A2/pIL-13R{alpha}2345-354 tetrameric complexes, anti-CD28 antibody, and CD83 molecules to cell-sized latex beads, and used to stimulate IL-13R{alpha}2–specific CTLs from the peripheral blood mononuclear cells of HLA-A2+ healthy donors. After multiple stimulations, the induced CTLs were analyzed for tetramer staining, IFN-{gamma} production, and CTL reactivity.

Results: Tetramer staining assay showed that the induced CTLs specifically bound HLA-A2/pIL-13R{alpha}2345-354 tetramers. The CTLs specifically produced IFN-{gamma} in response to the HLA-A2/pIL-13R{alpha}2345-354-aAPCs and exhibited specific lysis against T2 cells pulsed with the peptide pIL-13R{alpha}2345-354 and HLA-A2+ glioma cells expressing IL-13R{alpha}2345-354, whereas HLA-A2– glioma cell lines that express IL-13R{alpha}2345-354 could not be recognized by the CTLs. The peptide-specific activity was inhibited by anti–HLA class I monoclonal antibody.

Conclusion: The induced CTLs specific for IL-13R{alpha}2345-354 peptide could be a potential target of specific immunotherapy for HLA-A2+ patients with malignant glioma.

Despite significant advances in modern microsurgery, radiotherapy, and chemotherapy, the prognosis for patients with malignant glioma remains poor (1). Therefore, the development of a new treatment modality is needed. Although the central nervous system (CNS), and tumors that arise therein, reside in an "immunologically privileged" site (2), many data have successfully shown that effective anti-CNS tumor immune responses can be induced in preclinical models using syngeneic tumor- and dendritic cell–based vaccines (3–5). In addition, activated, antitumor immune cells have the potential to migrate into the CNS and to selectively destroy malignant cells that have infiltrated normal CNS tissues (6). Therefore, immunotherapy for glioma is an attractive alternative treatment option.

The induction of antigen-specific CTLs has been suggested to be highly efficacious in the prevention and treatment of various types of tumors (7). CTLs recognize "processed" peptides that are derived from endogenous proteins and presented on the cell surface in association with MHC class I molecules (7, 8). Peptides that bind to a given MHC class I molecule have been shown to share common amino acid motifs, which are called major anchor motifs (8). Hence, tumor-specific CTLs can recognize and select the antigenic peptides by scanning peptide sequences, and then kill tumor cells in an antigenic peptide-specific fashion. Interleukin-13 receptor {alpha}2 (IL-13R{alpha}2) chain has been reported to be abundantly and specifically overexpressed in malignant glioma and not otherwise presented in the normal brain (9–11). IL-13R{alpha}2–specific CTLs can be generated in vitro using peptide-pulsed autologous dendritic cells as antigen-presenting cells (APC; ref. 12). However, this method is time-consuming and expensive due to the limited availability of donor-derived dendritic cells. To date, several reports indicated effective stimulation of T cells by human leukocyte antigen (HLA)-peptide ligand coating on aAPCs, such as liposomes (13, 14) and microbeads (15–18). The aAPCs simulate the natural APCs (such as dendritic cells, macrophages, and B cells), which are able to provide the dual signals (the antigen-specific and costimulatory signals) for T-cell activation. These aAPCs can be prepared by coating the T-cell receptor ligand (peptide-MHC) and costimulatory molecules (such as B7) on a cell-sized bead, and it provides a practical and convenient approach to generating antigen-specific T cells for adoptive immunotherapy.

In this study, we explored a simple and efficient method to induce IL-13R{alpha}2345-354–specific CTLs from the peripheral blood mononuclear cells (PBMC) of HLA-A2+ normal donors using aAPCs made by coating HLA-A2/pIL-13R{alpha}2345-354 complexes, anti-CD28 antibody, and CD83 to cell-sized latex microbeads in vitro, in which these CTLs kept their antigen-specific cytolytic properties.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Cell line and cell culture. T2 cells were kindly provided by Prof. Nicholas Zavazava (Kiel University, Kiel, Germany). This cell bears the HLA-A*0201 gene, but expresses a very low level of cell-surface HLA-A2.1 molecules and is 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 for immunoproteasomal subunits. This cell line was maintained in RPMI 1640 (Invitrogen) supplemented with 10% fetal bovine serum (HyClone) and antibiotics. Glioma cell lines U251 (HLA-A2+, IL-13R{alpha}2+) and A172 (HLA-A2–, IL-13R{alpha}2+) were purchased from the American Type Culture Collection. These glioma cell lines were cultured in DMEM supplemented with 10% fetal bovine serum and antibiotics.

Synthetic peptides. The following HLA-A2–restricted peptides were used in this study: the IL-13R{alpha}2–derived peptide WLPFGFILI (referred to as pIL-13R{alpha}2345-354; 12) and the control peptide HIV-Gag peptide SLYNTVATL (pHIV; ref. 19). Peptides were synthesized by standard solid-phase chemistry and characterized by mass spectrometry. The purity of the synthetic peptides was >95% as indicated by analytical HPLC. Lyophilized peptides were dissolved in DMSO and stored at –80°C after dilution in PBS.

HLA class I typing. HLA class I typing was done with microcytotoxicity by Lambda Antigen Traye Class I (One Lamda, Inc.).

Detection of IL-13R{alpha}2 gene expression in glioma cell lines by reverse transcription-PCR. The expression of IL-13R{alpha}2 mRNA in glioma cell lines U251 and A172 was detected with reverse transcription-PCR. The primers used for amplification of human IL-13R{alpha}2 and GAPDH were as follows: IL-13R{alpha}2, 5'-TGGTCAGAAGTGTGCCTGTC-3' (S) and 5'-TCTGCCCAGGAACTTTGAAC-3' (AS); GAPDH, 5'-ACAGTCAGCCGCATCTTCTT-3' (S) and 5'-TTGATTTTGGAGGGATCTCG-3' (AS). The PCR reactions using Ex Taq DNA polymerase (Life Technologies) were subjected to 30 amplification cycles; conditions consisted of 94°C denaturation for 30 s, 57°C primer annealing for 30 s, followed by primer extension at 72°C for 1 min.

Preparation of HLA-A2/pIL-13R{alpha}2345-354 tetramers. Synthesis of HLA-A2/pIL-13R{alpha}2345-354 tetramers was carried out according to the protocol of Altman et al. (20). Briefly, plasmids encoding HLA-A*0201 (heavy chain) molecules with a COOH-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, purified from inclusion bodies, and then refolded in the presence of excess pIL-13R{alpha}2345-354 to form the HLA-A2/pIL-13R{alpha}2345-354 monomeric product. The folded product was then subjected to enzymatic biotinylation by BirA enzyme (Avidity) at 25°C for 12 h. This biotinylated HLA-A2/pIL-13R{alpha}2345-354 monomer was used for the preparation of the HLA-A2/pIL-13R{alpha}2345-354 tetramer. The HLA-A2/pIL-13R{alpha}2345-354 tetramer was produced by mixing the purified biotinylated monomer with FITC-labeled streptavidin (Sigma) at a molar ratio of 4:1. HLA-A2/pHIV tetramer was also prepared according to the manufacturer's protocol.

Generation of HLA-A2/pIL-13R{alpha}2345-354-aAPCs. The 5-µm sulfate polystyrene latex (interfacial Dynamics) was incubated sequentially with streptavidin (1 µg/107 beads; Sigma), CD28-specific antibody, and CD83 (1 µg and 1.5 µg/107 beads, respectively; BD PharMingen) for 30 min in 1-mL PBS per 107 beads at 4°C on a rotator. The beads were then incubated with biotinylated HLA-A2/pIL-13R{alpha}2345-354 complexes (2 µg/107 beads) for 1 h at 4°C on a rotator. After each incubation step, the beads were washed with PBS. The HLA-A2/pIL-13R{alpha}2345-354-aAPCs were stored in PBS at 4°C.

Induction of CTL. PBMCs from five healthy HLA-A2+ donors were separated using standard Ficoll-Hypaque (Sigma) gradient density centrifugation. The PBMCs were stimulated with HLA-A2/pIL-13R{alpha}2345-354-aAPCs in vitro using the protocol adapted from previous studies (21). Briefly, The PBMCs were used as responder cells and stimulated with HLA-A2/pIL-13R{alpha}2345-354-aAPCs. Responder cells (3 x 106 per well) were cocultured with HLA-A2/pIL-13R{alpha}2345-354-aAPCs (3 x 105 per well) in 24-well plates in RPMI 1640 supplemented with 10% fetal bovine serum (1 ml/well). Interleukin-7 (IL-7; 10 ng/mL; PeproTech EC) was added on day 1. IL-2 (50 units/mL, R&D Systems) was added on day 4. Medium, IL-2, and IL-7 were replenished twice a week. On day 7 and weekly thereafter, responder cells were collected, counted, and replated at 3 x 106 per well, together with 3 x 105 HLA-A2/pIL-13R{alpha}2345-354-aAPCs per well in RPMI 1640 with 10% fetal bovine serum, 10 ng/mL IL-7, and 50 units/mL IL-2. After a total of four rounds of stimulations in vitro, responder cells were tested for tetramer staining and cytotoxicity assay.

Tetramer staining. Tetramer staining was done as previously described (22, 23). In brief, 1 x 106 cells were incubated in 100-µL fluorescence-activated cell sorting staining buffer (PBS supplemented with 1% bovine serum albumin and 0.05% NaN3) with 20 µg/mL HLA-A2/peptide tetramer at 37°C for 30 min. Cells were washed with PBS and subsequently incubated with phycoerythrin-Cy5–labeled anti-CD8 antibody (BD PharMingen) at 4°C for 30 min. All cells were washed with PBS twice after being stained, and then fixed in 1% formaldehyde. Stained cells were analyzed with FACSCalibur (Becton Dickinson).

ELISA. To determine the frequency of T cells capable of responding to a specific stimulus by secretion of IFN-{gamma}, an enzyme-linked immunospot (ELISPOT) assay was done. The HLA-A2/pIL-13R{alpha}2345-354-aAPCs were used as stimulating target cells. Beads pulsed without HLA-A2/pIL-13R{alpha}2345-354 complexes were used as the negative control. Responder cells were coincubated with the target cells at a stimulator cell-to-responder cell ratio of 1:10 at 37°C for 24 h in 96-well ELISPOT plates. The assay was done and developed according to the manufacturer's instructions (BD PharMingen).

Cytotoxicity assay. To analyze the cytotoxic activity of the HLA-A2/pIL-13R{alpha}2345-354-aAPCs–induced CTLs against various target cells, cytotoxic assays were done 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+, IL-13R{alpha}2+) and A172 (HLA-A2–, IL-13R{alpha}2+), K562, and T2 cells pulsed without a peptide were selected as target cells. Other target cells, T2pIL-13R{alpha}2345-354 (pIL-13R{alpha}2345-354–pulsed T2 cells) and T2pHIV (pHIV-pulsed T2 cells), were prepared by incubating T2 cells with pIL-13R{alpha}2345-354 (50 µg/mL) and pHIV (50 µg/mL) for 3 h at 37°C. The percentage of 51Cr release was calculated according to the following formula: 100 x (experimental release – spontaneous release) / (maximum release – spontaneous release).

Inhibition of the cytotoxicity with HLA class I–specific monoclonal antibody. T2pIL-13R{alpha}2345-354 and U251 target cells were incubated with anti–HLA class I monoclonal antibody (mAb) W6/32 (American Type Culture Collection4; ref. 24) and a control isotypic mAb of irrelevant specificity IgG2 (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.

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


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
IL-13R{alpha}2 gene expression and HLA class I typing in glioma cell lines. IL-13R{alpha}2 mRNA expression was detected in U251 and A172 cell lines. The results of HLA class I typing detection showed that U251 was a HLA-A2+ cell line and A172 was a HLA-A2– cell line.

Growth kinetics of coculture bulk and phenotype of induced T cells. Following the first stimulation using HLA-A2/pIL-13R{alpha}2345-354-aAPCs, PBMCs were expanded continuously. After 4 weeks of coculture with HLA-A2/pIL-13R{alpha}2345-354-aAPCs, we could obtain as many as 1.5 x 108 to 1.8 x 108 T cells from an initial number of 3 x 106 cells, corresponding to a mean expansion of 50- to 60-fold. The phenotype of the expanded cells was measured by flow cytometry. The percentage of CD4+ cells gradually decreased whereas the CD8+ cells increased with the progression of the coculture with HLA-A2/pIL-13R{alpha}2345-354-aAPCs. The phenotypes of the CTLs for pIL-13R{alpha}2345-354 were CD8+, CD4–, CD3+, CD16–, and CD56–.

Frequency of pIL-13R{alpha}2345-354–specific CTLs increased by coculture with HLA-A2/pIL-13R{alpha}2345-354-aAPCs as determined by tetramer staining. A flow cytometric analysis of PBMCs was done before or after coculture with the HLA-A2/pIL-13R{alpha}2345-354-aAPCs. Before the four rounds of stimulation using HLA-A2/pIL-13R{alpha}2345-354-aAPCs, the frequency of CD8+ T cells stained with HLA-A2/pIL-13R{alpha}2345-354 tetramers was 0.11%. However, after stimulation, fluorescence-activated cell sorting analysis revealed that 14.7% of CD8+ T cells were stained with HLA-A2/pIL-13R{alpha}2345-354 tetramers, which was not observed when staining with the control tetramers (HLA-A2/pHIV tetramer; Fig. 1 ). The pIL-13R{alpha}2345-354–specific CTLs expanded by HLA-A2/pIL-13R{alpha}2345-354-aAPCs from the other four normal donors showed similar results (Table 1 ).


Figure 1
View larger version (39K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 1. Identification of the HLA-A2/pIL-13R{alpha}2345-354-aAPCs–induced specific CTLs by HLA-A2/peptide tetramer staining. Frequency of HLA-A2/peptide tetramer–binding CD8+ T lymphocytes before stimulation and after four rounds of stimulation with HLA-A2/pIL-13R{alpha}2345-354-aAPCs. The HLA-A2/pIL-13R{alpha}2345-354 tetramer and the control HLA-A2/pHIV tetramer were used. A, the frequency of HLA-A2/pHIV tetramer–binding CD8+ T lymphocytes before stimulation was 0.09%. B, the frequency of HLA-A2/pIL-13R{alpha}2345-354 tetramer–binding CD8+ T lymphocytes before stimulation was 0.11%. C, the frequency of HLA-A2/pHIV tetramer–binding CD8+ T lymphocytes after stimulation was 0.06%. D, the frequency of HLA-A2/pIL-13R{alpha}2345-354 tetramer–binding CD8+ T lymphocytes after stimulation was 14.7%. Representative results of many experiments, showing that the coculture of HLA-A2/pIL-13R{alpha}2345-354-aAPCs and HLA-A2+ PBMCs can expand the pIL-13R{alpha}2345-354–specific CTLs.

 

View this table:
[in this window]
[in a new window]

  		Table 1. Induction of CTLs specific for pIL-13R{alpha}2345-354 in four HLA-A2+ healthy donors

 
ELISPOT assay. The ELISPOT assay was used to determine the frequency of individual antigen-specific IFN-{gamma} producing T cells. As shown in Fig. 2 , priming with HLA-A2/pIL-13R{alpha}2345-354-aAPCs resulted in the generation of peptide-specific IFN-{gamma}–producing CD8+ T cells after the stimulation. The average spot number of responding lymphocytes induced by HLA-A2/pIL-13R{alpha}2345-354-aAPCs (164 ± 35 peptide-specific spots/105 cells) was significant higher than that of responding lymphocytes induced by beads without HLA-A2/pIL-13R{alpha}2345-354 (41 ± 8 peptide-specific spots/105 cells; P < 0.05).


Figure 2
View larger version (9K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 2. Detection of the HLA-A2/pIL-13R{alpha}2345-354-aAPCs–induced specific CTLs by ELISPOT assay. The CTLs induced by the HLA-A2/pIL-13R{alpha}2345-354-aAPCs were tested for ELISPOT assay. The effector cells were cocultured with HLA-A2/pIL-13R{alpha}2345-354-aAPCs or beads pulsed without HLA-A2/pIL-13R{alpha}2345-354 (as a control). After 24 h of incubation, the frequencies of individual antigen-specific IFN-{gamma}–producing T cells were measured with an ELISPOT assay kit.

 
Cytotoxicity of pIL-13R{alpha}2345-354–specific CTLs. The cytotoxic activity of the HLA-A2/pIL-13R{alpha}2345-354-aAPCs–induced CTLs against various target cells was tested with the 51Cr release assay. The CTLs exhibited ~70% specific lysis against T2pIL-13R{alpha}2345-354 and U251 (HLA-A2+, IL-13R{alpha}2+) at an effector/target ratio of 50:1. However, the CTLs showed an ~10% cytolysis against T2pHIV, A172 (HLA-A2–, IL-13R{alpha}2+), K562, and the T2 without a pulsed peptide at the same effector/target ratio (Fig. 3A ). The specific killing activity of the CTLs induced by the HLA-A2/pIL-13R{alpha}2345-354-aAPCs against specific target cells was much more obvious than in any other control group (P < 0.05). The specific CTLs for pIL-13R{alpha}2345-354 induced by HLA-A2/pIL-13R{alpha}2345-354-aAPCs from the other four normal donors showed similar specific lysis (Table 1). This result shows that the cytotoxicity of the HLA-A2/pIL-13R{alpha}2345-354-aAPCs–induced CTL is pIL-13R{alpha}2345-354 specific.


Figure 3
View larger version (20K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 3. Cytotoxic activity of HLA-A2/pIL-13R{alpha}2345-354-aAPCs–induced CTLs. The CTLs were induced with HLA-A2/pIL-13R{alpha}2345-354-aAPCs for 28 d. The cytotoxic activity of one individual's CTLs is shown. The cytotoxic activity of the CTLs was assessed against U251 (HLA-A2+, IL-13R{alpha}2+) and A172 (HLA-A2–, IL-13R{alpha}2+), T2 cells pulsed without a peptide, T2pHIV (pHIV-pulsed T2 cells), T2pIL-13R{alpha}2345-354 (pIL-13R{alpha}2345-354–pulsed T2 cells), and K562 at various effector/target ratios (A). In addition, anti–HLA class I or a control antibody (mouse IgG2) was added to the T2pIL-13R{alpha}2345-354 and U251 targets, respectively, and incubated for 40 min at 4°C. After incubation, the target cells were mixed with effector cells for 51Cr release assay. The CTL assays were done at various effector/target ratios. Points, mean from triplicate wells; bars, SE. T2pIL-13R{alpha}2345-354 (B) and U251 (HLA-A2+, IL-13R{alpha}2+; C) were used as target cells. Results show that the cytotoxicity of the HLA-A2/pIL-13R{alpha}2345-354-aAPCs–induced CTLs is pIL-13R{alpha}2345-354 specific and the CTLs lysed the specific targets in a HLA class I–restricted manner.

 
Inhibition of cytotoxicity of HLA-A2/pIL-13R{alpha}2345-354-aAPCs–induced T cells by HLA class I–specific mAb (W6/32). To determine whether the induced CTLs could recognize the specific target cells in a HLA class I–restricted manner, anti–HLA class I mAb W6/32 was used to block the cytotoxicity of the HLA-A2/pIL-13R{alpha}2345-354-aAPCs–induced CTLs. The cytotoxic activity against T2pIL-13R{alpha}2345-354 and U251 was significantly eliminated by W6/32. As shown in Fig. 3B and C, incubation of T2pIL-13R{alpha}2345-354 and U251 target cells with W6/32 led to the inhibition of the targeted cells lysis, whereas mouse IgG2, used as an isotype control, showed no effect. These results suggested that the HLA-A2/pIL-13R{alpha}2345-354-aAPCs–induced CTLs lysed the specific targets in a HLA class I–restricted manner.


   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
Malignant gliomas represent a significant class of CNS tumors derived from the glial lineage. Despite recent advances in traditional treatment options, the prognosis for these patients has not changed appreciably. The 5-year survival rate for patients harboring the most common class of gliomas, glioblastoma multiforme, is <2% (25). In an effort to improve the outcome of patients with respectable brain tumors, there have been attempts to give adjunctive therapies consisting of radiation with or without chemotherapy. Thus far, research over three decades has failed to provide definitive evidence of improved outcome (i.e., overall survival and disease-free survival) in patients. Clearly, modalities other than radiation and/or chemotherapy must be explored in an adjuvant setting. Immune-based treatments represent a promising new class of therapy designed to harness the immune system to specifically eradicate malignant cells. However, immunotherapy for tumors located in the CNS has generally not achieved the results seen for peripherally located tumors (26–29). These previous findings have historically been ascribed to the "immune privilege" of the CNS (30). In reality, however, the immune privilege of the brain is not absolute but was originally used to describe the observation that tissue and tumor grafts survived better in the CNS than in other peripheral sites (31). Effective anti-CNS tumor immune responses have been generated by immune-based treatments such as adoptive T-cell transfer (32–37), GAA-pulsed dendritic cells (3–5, 38), and cytokine-secreting gliomas or fibroblasts (39–41), lending further credence to the idea that the efficient induction of a cellular antitumor immune response can be targeted to antigens within the CNS.

Tumor-specific CD8-positive CTLs constitute the most important effector cells for antitumor responses (8). It has previously been shown that in vitro specific CTLs can be generated using peptide-pulsed autologous dendritic cells as APCs (42, 43). However, CTL expansion to clinically relevant amounts requires multiple leukophoreses to obtain enough autologous dendritic cells. Variability in both quantity and quality of obtained dendritic cells, which presumably relate to underlying diseases and the pretreatment of the patients, also significantly affects the viability of dendritic cell–based therapeutics. For these reasons, use of dendritic cells has been a limiting step in ex vivo expansion of T cells (18). Strategy of aAPC offers a promising way to overcome the disadvantages of natural APCs. CTLs generated by artificial stimulation protocols can kill both peptide-loaded and natural target cells (18, 44, 45). The presented data showed that CTLs specific for pIL-13R{alpha}2345-354 could be induced in vitro with the aAPCs coating HLA-A2/pIL-13R{alpha}2345-354, anti-CD28 antibody, and CD83 molecule to cell-sized latex beads. The HLA-A2+ PBMCs were induced to generate pIL-13R{alpha}2345-354–specific CTLs in vitro by coculture with the HLA-A2/pIL-13R{alpha}2345-354-aAPCs, and specificity of the aAPC-induced CTLs was confirmed by both their binding to HLA-A2/pIL-13R{alpha}2345-354 tetramers and killing activity against HLA-A2/pIL-13R{alpha}2345-354–bearing cells. Following this procedure, we found that the induced CTLs, derived from PBMCs in a HLA-A2+ healthy donor, could kill the HLA-A2+ pIL-13R{alpha}2345-354+ glioma cell lines when stimulated with HLA-A2/pIL-13R{alpha}2345-354-aAPCs in vitro. The specific killing activity of CTL induced by the HLA-A2/pIL-13R{alpha}2345-354-aAPCs against specific target cells T2pIL-13R{alpha}2345-354 and U251 was much more effective than that of any other control group. The results indicated that the cytotoxicity of the aAPCs-induced CTL is antigen specific (i.e., against the target cells bearing the corresponding HLA-A2/pIL-13R{alpha}2345-354 complexes). In addition, it is possible to increase the percentage of antigen-specific CTL by sorting the cells after staining with tetramer and isolating the positive cells with magnetic beads loaded with anti-FITC antibody (Miltenyi System). Many groups have applied this approach to obtain higher percentages of antigen-specific CTLs that could be further expanded with aAPC or beads coated with anti-CD3/CD28 mAb (46). Therefore, this technique can be extrapolated to expand enough T cells for clinical use.

For clinical studies, HLA tetramer–based aAPCs have several distinct advantages over cellular APCs including dendritic cells. One of these is ease of preparation, which is not required for sterile cell culture and cytokines, thereby reducing both the variability and expense associated with ex vivo expansion. The variability is particularly important when considering therapies for cancer because there have been reported defects in dendritic cells obtained from patients with malignancies (47). Another advantage is the good stability of aAPCs, unlike the biological variability and patient-to-patient variation when producing cellular APCs such as dendritic cells. In addition, aAPCs can present defined combinations of MHC alleles and peptides and be easily adapted using other MHC alleles and/or peptides, so that immunodominant or subdominant epitopes can be expanded preferentially. When cellular APCs are used, an array of MHC molecules is used and a broad but uncontrolled MHC restricted response is generated. The last advantage is the ability to control the combination of costimulatory complexes associated with aAPCs, unlike cellular APCs that may participate in the T cell-APC interaction in such a way that it promotes tolerance or anergy. For example, on T-cell activation, B7 binding to CTLA-4 instead of CD28 would limit T-cell expansion (48). It is convenient to prepare aAPCs coated with anti-CD28, which binds specifically to CD28, avoiding the binding of B7 to CTLA-4. In this study, we used a combination of anti-CD28 and CD83 for the generation of a costimulatory signal because 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 (49). In contrast to other types of aAPCs previously engineered by others (50), there is no need for additional molecules like intercellular adhesion molecule-1 or lymphocyte function-associated antigen-3. The antigen-specific CTL can be successfully induced with the HLA-A2/pIL-13R{alpha}2345-354-aAPCs in vitro. Further studies need to compare the efficiency of this approach with those of other techniques such as aAPCs with a combination of other costimulatory molecules.

The use of aAPC represents the state-of-the-art generation of antigen-specific CTLs for adoptive immunotherapy. Thus, HLA tetramer–based aAPCs coated with a HLA/peptide complex, anti-CD28 antibody, and CD83 molecule could provide a useful tool for the reproducible expansion of specific CTLs in vitro and significantly advance the field of adoptive immunotherapy. HLA-A2/pIL-13R{alpha}2345-354-aAPCs could be used as a standardized, "off-the-shelf" reagent to enrich pIL-13R{alpha}2345-354–specific CTLs for the treatment of patients with malignant gliomas.


   Acknowledgments
 
We thank Drs. Wen-Hua Wu and Guo-An Chen in the Wuhan Blood Center for their kind assistance with this work.


   Footnotes
 
Grant support: National Natural Science Foundation of China grant 30500522.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: X. Jiang and X. Lu contributed equally to this work.

4 http://www.atcc.org/ Back

Received 4/30/07; revised 7/25/07; accepted 8/ 8/07.


   References
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

  1. Wen PY, Kesari S. Malignant gliomas. Curr Neurol Neurosci 2004;4:218–27.[CrossRef]
  2. Walker PR, Calzascia T, De Tribolet N, Dietrich PY. T-cell immune responses in the brain and their relevance for cerebral malignancies. Brain Res Brain Res Rev 2003;42:97–122.[CrossRef][Medline]
  3. Ashley DM, Faiola B, Nair S, Hale LP, Bigner DD, Gilbao E. Bone marrowgenerated dendritic cells pulsed with tumor extracts or tumor RNA induce antitumor immunity against central nervous system tumors. J Exp Med 1997;186:1177–82.[Abstract/Free Full Text]
  4. Okada H, Tahara H, Shurin MR, et al. Bone marrow derived dendritic cells pulsed with a tumor specific peptide elicit effective anti-tumor immunity against intracranial neoplasms. Int J Cancer 1998;78:196–201.[CrossRef][Medline]
  5. Liau LM, Black KL, Prins RM, et al. Treatment of intracranial gliomas with bone marrow-derived dendritic cells pulsed with tumor antigens. J Neurosurg 1999;90:1115–24.[Medline]
  6. Holladay FP, Heitz T, Chen YL, Chiga M, Wood GW. Successful treatment of a malignant rat glioma with cytotoxic T lymphocytes. Neurosurgery 1992;31:528–33.[Medline]
  7. Melief CJ, Kast WM. T-cell immunotherapy of tumors by adoptive transfer of cytotoxic T lymphocytes and by vaccination with minimal essential epitopes. Immunol Rev 1995;145:167–77.[CrossRef][Medline]
  8. Rammensee HG, Friede T, Stevanovic S. MHC ligands and peptide motifs: first listing. Immunogenetics 1995;41:178–228.[Medline]
  9. Murata T, Obiri NI, Debinski W, Puri RK. Structure of IL-13 receptor: analysis of subunit composition in cancer and immune cells. Biochem Biophys Res Commun 1997;238:90–4.[CrossRef][Medline]
  10. Mintz A, Gibo DM, Slagle-Webb B, Christensen ND, Debinski W. IL-13R{alpha}2 is a glioma-restricted receptor for interleukin-13. Neoplasia 2002;4:388–99.[CrossRef][Medline]
  11. Husain SR, Puri RK. Interleukin-13 receptor-directed cytotoxin for malignant glioma therapy: from bench to bedside. J Neurooncol 2003;65:37–48.[CrossRef][Medline]
  12. Okano F, Storkus WJ, Chambers WH, Pollack IF, Okada H. Identification of a novel HLA-A*0201 restricted cytotoxic T lymphocyte epitope in a human glioma associated antigen, interleukin-13 receptor 2 chain. Clin Cancer Res 2002;8:2851–55.[Abstract/Free Full Text]
  13. van Rensen AJ, Wauben MH, Grosfeld-Stulemeyer MC, van Eden W, Crommelin DJ. Liposomes with incorporated MHC class II/peptide complexes as antigen presenting vesicles for specific T cell activation. Pharm Res 1999;16:198–204.[CrossRef][Medline]
  14. Prakken B, Wauben M, Genini D, et al. Artificial antigen-presenting cells as a tool to exploit the immune ‘synapse’. Nat Med 2000;6:1406–10.[CrossRef][Medline]
  15. Curtsinger J, Deeths MJ, Pease P, Mescher MF. Artificial cell surface constructs for studying receptor-ligand contributions to lymphocyte activation. J Immunol Methods 1997;209:47–57.[CrossRef][Medline]
  16. Tham EL, Jensen PL, Mescher MF. Activation of antigen-specific T cells by artificial cell constructs having immobilized multimeric peptide-class I complexes and recombinant B7-Fc proteins. J Immunol Methods 2001;249:111–9.[CrossRef][Medline]
  17. Maus MV, Riley JL, Kwok WW, Nepom GT, June CH. HLA tetramer-based artificial antigen presenting cells for stimulation of CD4+ T cells. Clin Immunol 2003;106:16–22.[CrossRef][Medline]
  18. Oelke M, Maus MV, Didiano D, June CH, Mackensen A, Schneck JP. Ex vivo induction and expansion of antigen-specific cytotoxic T cells by HLA-Ig-coated artificial antigen-presenting cells. Nat Med 2003;9:619–24.[CrossRef][Medline]
  19. Parker KC, Bednarek MA, Hull LK, et al. Sequence motifs important for peptide binding to the human MHC class I molecule, HLA-A2. J Immunol 1992;149:3580–87.[Abstract]
  20. Altman JD, Moss PA, Goulder PJ, et al. Phenotypic analysis of antigen-specific T lymphocytes. Science 1996;274:94–6.[Abstract/Free Full Text]
  21. Molldrem JJ, Lee PP, Wang C, Champlin RE, Davis MM. A PR1-human leukocyte antigen-A2 tetramer can be used to isolate low-frequency cytotoxic T lymphocytes from healthy donors that selectively lyse chronic myelogenous leukemia. Cancer Res 1999;59:2675–81.[Abstract/Free Full Text]
  22. Rees W, Bender J, Teague TK, et al. An inverse relationship between T cell receptor affinity and antigen dose during CD4+ T cell responses in vivo and in vitro. Proc Natl Acad Sci USA 1999;96:9781–86.[Abstract/Free Full Text]
  23. Qian L, Qian GX. In vivo CTL immunity can be elicited by OVA-linker-β2m fusion protein. Acta Biochim Biophys Sin 2002;34:547–52.[Medline]
  24. Maziarz RT, Fraser J, Srominger JL, Burakoff SJ. The human HLA-specific monoclonal antibody W6/32 recognizes a discontinuous epitope within the {alpha}2 domain of murine H-2Db. Immunogenetics 1986;249:206–8.
  25. Surawicz TS, Davis F, Freels S, Laws ER, Menck HR. Brain tumor survival: results from the National Cancer Data Base. J Neurooncol 1998;40:151–60.[CrossRef][Medline]
  26. Graf MR, Merchant RE. Interleukin-6 transduction of a rat T9 glioma clone results in attenuated tumorigenicity and induces glioma immunity in Fischer F344 rats. J Neurooncol 1999;45:209–18.[CrossRef][Medline]
  27. Graf MR, Prins RM, Merchant RE. IL-6 secretion by a rat T9 glioma clone induces a neutrophil-dependent antitumor response with resultant cellular, antiglioma immunity. J Immunol 2001;166:121–9.[Abstract/Free Full Text]
  28. Ram Z, Walbridge S, Heiss JD, Culver KW, Blaese RM, Oldfield EH. In vivo transfer of the human interleukin-2 gene: negative tumoricidal results in experimental brain tumors. J Neurosurg 1994;80:535–40.[Medline]
  29. Tjuvajev J, Gansbacher B, Desai R, et al. RG-2 glioma growth attenuation and severe brain edema caused by local production of interleukin-2 and interferon-{gamma}. Cancer Res 1995;55:1902–10.[Abstract/Free Full Text]
  30. Barker CF, Billingham RE. Immunologically privileged sites. Adv Immunol 1977;25:1–54.[Medline]
  31. Prins RM, Liau LM. Immunology and immunotherapy in neurosurgical disease. Neurosurgery 2003;53:144–53.[CrossRef][Medline]
  32. Barba D, Saris SC, Holder C, Rosenberg SA, Oldfield EH. Intratumoral LAK cell and interleukin-2 therapy of human gliomas. J Neurosurg 1989;70:175–82.[Medline]
  33. Merchant RE, Grant AJ, Merchant LH, Young HF. Adoptive immunotherapy for recurrent glioblastoma multiforme using lymphokine activated killer cells and recombinant interleukin-2. Cancer 1988;62:665–71.[Medline]
  34. Plautz GE, Miller DW, Barnett GH, et al. T cell adoptive immunotherapy of newly diagnosed gliomas. Clin Cancer Res 2000;6:2209–18.[Abstract/Free Full Text]
  35. Sankhla SK, Nadkarni JS, Bhagwati SN. Adoptive immunotherapy using lymphokine-activated killer (LAK) cells and interleukin-2 for recurrent malignant primary brain tumors. J Neurooncol 1996;27:133–40.[Medline]
  36. Lillehei KO, Mitchell DH, Johnson SD, McCleary EL, Kruse CA. Long-term follow-up of patients with recurrent malignant gliomas treated with adjuvant adoptive immunotherapy. Neurosurgery 1991;28:16–23.[CrossRef][Medline]
  37. Quattrocchi KB, Miller CH, Cush S, et al. Pilot study of local autologous tumor infiltrating lymphocytes for the treatment of recurrent malignant gliomas. J Neurooncol 1999;45:141–57.[CrossRef][Medline]
  38. Heimberger AB, Archer GE, Crotty LE, et al. Dendritic cells pulsed with a tumor-specific peptide induce long-lasting immunity and are effective against murine intracerebral melanoma. Neurosurgery 2002;50:158–64.[CrossRef][Medline]
  39. Glick RP, Lichtor T, Mogharbel A, Taylor CA, Cohen EP. Intracerebral versus subcutaneous immunization with allogeneic fibroblasts genetically engineered to secrete interleukin-2 in the treatment of central nervous system glioma and melanoma. Neurosurgery 1997;41:898–906.[CrossRef][Medline]
  40. Glick RP, Lichtor T, de Zoeten E, Deshmukh P, Cohen EP. Prolongation of survival of mice with glioma treated with semiallogeneic fibroblasts secreting interleukin-2. Neurosurgery 1999;45:867–74.[CrossRef][Medline]
  41. Sampson JH, Archer GE, Ashley DM, et al. Subcutaneous vaccination with irradiated, cytokine-producing tumor cells stimulates CD8+ cell-mediated immunity against tumors located in the "immunologically privileged" central nervous system. Proc Natl Acad Sci U S A 1996;93:10399–404.[Abstract/Free Full Text]
  42. Paczesny S, Banchereau J, Wittkowski KM, Saracino G, Fay J, Palucka AK. Expansion of melanoma-specific cytolytic CD8+ T cell precursors in patients with metastatic melanoma vaccinated with CD34+ progenitor-derived dendritic cells. J Exp Med 2004;199:1503–11.[Abstract/Free Full Text]
  43. Hirschowitz EA, Foody T, Kryscio R, Dickson L, Sturgill J, Yannelli J. Autologous dendritic cell vaccines for non-small-cell lung cancer. J Clin Oncol 2004;22:2808–15.[Abstract/Free Full Text]
  44. Oelke M, Schneck JP. HLA-Ig-based artificial antigen-presenting cells: eetting the terms of engagement. Clin Immunol 2004;110:243–51.[CrossRef][Medline]
  45. Salter RD, Howell DN, Gresswell P. Genes regulating HLA class I antigen expression in T-B lymphoblast hybrids. Immunogenetics 1985;21:235–46.[CrossRef][Medline]
  46. Oelke M, Moehrle U, Chen LJ, et al. Generation and purification of CD8+ Melan-A-specific cytotoxic T lymphocytes for adoptive transfer in tumor immunotherapy. Clin Cancer Res 2000;6:1997–2005.[Abstract/Free Full Text]
  47. Salgaller ML, Lodge PA, Mclean JG, et al. Report of immune monitoring of prostate cancer patients undergoing T-cell therapy using dendritic cells pulsed with HLA-A2-specific peptides from prostate-specific membrane antigen (PSMA). Prostate 1998;35:144–51.[CrossRef][Medline]
  48. Walunas TL, Lenschow DJ, Bakker CY, et al. CTLA-4 can function as a negative regulator of T cell activation. Immunity 1994;1:405–13.[CrossRef][Medline]
  49. Hirano N, Butler MO, Xia ZN, et al. Engagement of CD83 ligand induces prolonged expansion of CD8+ T cells and preferential enrichment for antigen specificity. Blood 2006;107:1528–36.[Abstract/Free Full Text]
  50. Butler MO, Lee JS, Ansen S, et al. Long-lived antitumor CD8+ lymphocytes for adoptive therapy generated using an artificial antigen-presenting cell. Clin Cancer Res 2007;13:1857–67.[Abstract/Free Full Text]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Jiang, X.
Right arrow Articles by Zhao, H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Jiang, X.
Right arrow Articles by Zhao, H.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online