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Acta Biochim Biophys Sin (2009): 980 – 990 | ª The Author 2009. Published by ABBS Editorial Office in association with Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. DOI: 10.1093/abbs/gmp092. Advance Access Publication 12 November 2009

Discovery of YB-1 as a new immunological target in neuroblastoma by vaccination in the context of regulatory T cell blockade Jin Zheng1*, Weiqing Jing2, and Rimas J. Orentas2 1

The Key Laboratory of Biomedical Information Engineering of Ministry of Education, Xi’an Jiaotong University, Xi’an 710061, China Department of Pediatrics, Medical College of Wisconsin and the Children’s Research Institute, Children’s Hospital of Wisconsin, 8701 Watertown Plank Rd, Milwaukee, WI 53226, USA *Correspondence address. Tel: þ86-29-82655429-812; Fax: þ86-29-82655499; E-mail: [email protected] 2

Neuroblastoma is one of the most common solid tumors in infancy and early childhood. Using the A/J mouse and a syngeneic neuroblastoma cell line AGN2a, we induced a strong anti-neuroblastoma cellular immune response when AGN2a transfected to express costimulatory molecules (CD80/CD86/CD54/CD137L) was used as a vaccine in the context of regulatory T cell blockade. Strong humoral immunity was induced by AGN2a-4p immunization in the context with regulatory T cell blockade. Serum from treated mice was used to screen an AGN2a cDNA expression library that was constructed with l ZAP express vector in order to identify tumor-associated antigens by SEREX. Twentyone clones were identified by sequencing and comparative analysis of gene pools. Most transcripts play some roles in the neuronal differentiation, cell metabolism, or have previously been identified as transcripts that are over-expressed in other malignancies. The most commonly identified tumor-associated antigen, using serum from AGN2a-4p immunization with Treg blockade mice, was YB-1 protein that also induced a T cell response. These results indicated that potential neuroblastoma-associated antigens were found by the sera from mice immunized with tumor cells expressing costimulatory molecules with regulatory T cell function blockade. The identification of YB-1 as tumor-associated antigens capable of eliciting a T cell response validates our experimental approach and argues for the antigens we have identified here to be evaluated as targets of effector immunity and as vaccine candidates.

Keywords neuroblastoma; costimulatory molecule; Treg blockade; SEREX Received: June 20, 2009

Accepted: October 9, 2009

Acta Biochim Biophys Sin (2009) | Volume 41 | Issue 12 | Page 980

Neuroblastoma originated in the primitive neural crest cells, often appears in the adrenal medulla, retroperitoneal, mediastinal, spinal adjacent pelvic, neck and lower abdomen and other parts [1]. It is the most common extracranial solid cancer in childhood and the most common cancer in infancy, accounting for 8–10% of malignant tumors in children. The annual incidence rate of neuroblastoma is (0.3–5.5)/10 million, just below the leukemia and central nervous system tumors. Eighty percent of neuroblastoma cases occurs in children under 5 years old [2]. Currently, no effective clinical treatments are available for advanced neuroblastoma. It has been confirmed that early clinical observation coupled with a bone marrow transplant may help neuroblastoma treatment [3]. In the mouse model, tumor cells expressing soluble cytokines or immune co-stimulatory molecules as vaccines could induce host anti-tumor immune response [4 – 6]. Our work has also confirmed that neuroblastoma cell lines AGN2a expressing a variety of immune costimulatory molecules as a vaccine in mice could induced host cells to produce a strong immune response [7,8]. Based on the experimental results, neuroblastoma tumor cell vaccine has already begun its phase I clinical trial [9]. In the experimental system, we also found that the host anti-neuroblastoma immune response could be enhanced by the inhibition of regulatory T cell function with anti-CD25 antibodies [10]. It is well known that regulatory T cells can inhibit the immune response, especially by the inhibition of autologous antigeninduced immune responses, including tumor autoantigen. As a result, a new type of human malignant tumor immunization strategy study, blocking the inhibitory effect of regulatory T cells to disrupt the tolerance of host against body’s self-tumor antigen, has become a

Discovery of new immunological target in neuroblastoma by vaccination

major highlight of the study [11,12]. As a result of blocking the regulatory T cell function, the ‘self’ antigen-induced immunity could be identified. So, the application of anti-CD25 antibody under the conditions of tumor cells as the vaccine may lead to a strong antineuroblastoma immune response [13]. The technique of SEREX (serological expression of cDNA expression libraries), which was established by Sahin and Tureci [14,15], has been proved for the identification of T cell antigens as well as B cell antigens. SEREX technology has long been used in the clinical research, and is proved to be used to identify antigens recognized by immune system cells. The finding of NY-ESO-1 antigen is a typical example [16]. We have already identified DEK as a neuroblastoma tumorassociated antigen by using sera from mice immunized with tumor cells (expressing CD80 and CD86) in the context of Treg inhibition [17]. In the present study, by similar strategy, we used the sera from mice immunized with modified tumor cells expressing CD80, CD86, CD54 and CD137L (4-1BBL), and anti-CD25 antibody treatment to identify new tumor-associated antigens from neuroblastoma models by SEREX technology.

100 ml of nucleofector solution R (Amaxa Biosystems, Koeln, Germany) and 1 mg of each of expression plasmids containing CD80, CD86, CD54 and CD137L coding sequences. Nucleofection was performed using the Nucleofector system (Amaxa Biosystems, South San Francisco, USA) with program setting T-20. Transfected cells were then cultured in regular culture medium containing 10% serum for 18 h prior to harvest.

Materials and Methods

Preparation of immune serum A/J mice were divided into six groups randomly, and each group has five mice. The immunization process was carried out as described in Table 1. In brief, A/J mice were given two subcutaneous (s.c.) injections of 2 106 irradiated AGN2a or AGN2a-4p cells (CD80/CD86/ CD54/CD137L-AGN2a) in 1-week interval. Before each experiment, the AGN2a-4p cells were analyzed by flow cytometry to confirm high-level expression of the costimulatory molecules. For the blockade/depletion of T regulatory cells, mice received 250 mg of anti-CD25 monoclonal antibody PC61 generated by Integra CL 1000 bioreactor (Integra Biosciences, Chur, Switzerland), by intraperitoneal injection 3 days prior to the first vaccination. Blood was collected 5 days after the second vaccination, incubated at 378C for 30 min, centrifuged at 800 g for 10 min, and stored at 2808C.

Mice and tumor cell lines A/J mice, 6–8 weeks of age, were purchased from Jackson Laboratories (Bar Harbor, Maine, USA). Mice were housed in the Medical College of Wisconsin Biomedical Resource Center (AALAC accredited) and all protocols were approved by the MCW Institutional Animal Care and Use Committee. AGN2a, an aggressive clone of Neuro2a, was derived from successive in vivo passage, and AGN2a-4p transfectants that expressed CD80, CD86, CD54 and CD137L were prepared [8]. In brief, 5 106 AGN2a cells were resuspended with

Flow cytometric analyses To analyze AGN2a-4p cells for the cell surface expression of immune stimulatory molecules, the cells were incubated with combinations of FITC-anti-CD80 (clone 16-10A1), PE-anti-CD86 (clone GL1), PE-anti-CD54 (clone 3E2), and FITC-anti-4-1BBL (clone TKS-1). All antibodies were from BD Biosciences Pharmingen (San Diego, USA). Cell samples were incubated with antibody at 48C for 15 min, and then washed with PBS. The stained samples were

Table 1 The schedule of immunization/no treatment

Group

Time (days) 0

I II III IV V VI

– 250 mg PC61 – 250 mg PC61 – 250 mg PC61

3

10

15

– – 2 106 AGN2a 2 106 AGN2a 2 106 AGN2a-4p 2 106 AGN2a-4p

– – 2 106 2 106 2 106 2 106

Harvester serum Harvester serum Harvester serum Harvester serum Harvester serum Harvester serum

AGN2a AGN2a AGN2a-4p AGN2a-4p



run through a Becton Dickinson FACScan flow cytometer, and the resulting data were analyzed using Flow-Jo software (Tree Star Inc, San Carlos, USA). Before the first immunization, regulatory T cells (CD4þ/CD25þ) from mice peripheral blood were checked by flow cytometry. FITC-anti-CD4 and PE-anti-CD25 antibodies were from BD Biosciences Pharmingen. Blood cells were incubated with antibodies at 48C for 15 min; then the red blood cells were lysed and analyzed by FACScan flow cytometer. The antibody titer of serum from each group was checked by flow cytometry on day 15 when the bloods were harvested. AGN2a cells were incubated with serum from each group at 48C for 15 min. After being washed with PBS, the cells were incubated with PE-labeled goat anti-mouse IgG antibody (Jackson Immuno Research), and then analyzed by FACScan flow cytometer.

Construction of cDNA expression library The AGN2a cDNA library was constructed as described before [17]. In brief, total RNA was isolated from AGN2a cells using Trizol (Invitrogen, Carlsbad, USA) and mRNA was purified using the Oligotex mRNA kit (Qiagen, Valencia, USA). Purified polyA mRNA (5 mg) was used to construct a cDNA library using the ZAP Express cDNA synthesis kit and ZAP Express cDNA Gigapack III gold cloning kit (Stratagene Inc, La Jolla, USA). cDNA fragments were cloned into the l ZAPII Express vector (Stratagene), packaged into phage particles, and transfected Escherichia coli, resulting in at least 1.25 105 primary recombinants. Immunoscreening of the AGN2a cDNA library Proteins encoded by the cDNA expression library were probed with sera from different groups as described in “Materials and Methods”. Recombinant phages at a concentration of 5000 pfu per plate (150 mm2) were amplified for 4 h at 428C until plaques were visible and then transferred to nitrocellulose membranes pre-wetted with 10 mM isopropyl-b-D-thiogalactopyranoside (IPTG) (Invitrogen) for an additional 3.5 h at 378C. Membranes were then washed three times with TBST (20 mM Tris– HCl, 150 mM NaCl, 0.05% Tween 20, pH 7.5), blocked with 1% bovine serum albumin (Sigma-Aldrich, St. Louis, USA) in TBS, and then incubated with a 1:250 dilution of immune serum, which had been preadsorbed with E. coli phage lysate following the manufacturer’s protocol (Stratagene). Bound antibody was detected by incubation with alkaline phosphatase (AP)-conjugated rabbit anti-mouse IgG (HþL) (Abcam, Acta Biochim Biophys Sin (2009) | Volume 41 | Issue 12 | Page 982

Cambridge, USA) and visualized by staining with 4-nitro blue tetrazolium chloride/5-bromo-4-chloro-3indoyl-phosphate (NBT/BCIP) in picoBLUE immunoscreening kit (Stratagene). Positive clones were subcloned and re-screened as above. In vivo excision was carried out with plaques that were positive upon secondary screening in order to generate the pBK-CMV phagemid containing the cloned insert. Phagemid was isolated with QIAprep columns (Qiagen) and the size of the cDNA insert was analyzed by XhoI/EcoRI restriction digest. Inserts were sequenced using T7 and T3 primers by ABI3100 automated DNA sequencing (MCW Protein and Nucleic Acid Facility, Milwaukee, USA).

Expression of YB-1 protein Full-length YB-1 (GenBank No. BC061634) was cloned from AGN2a cDNA using the following primers: 5 0 -GGAATTCCATATGAGCAGCGAGGCCGAG-3 0 (forward), 5 0 -CGGGATCCTTACTCAGCCCCGCCCTG-3 0 (reverse). NdeI and BamHI restricted PCR fragments were ligated into pET-15b (Novagen, Madison, USA) and recombinant plasmid with inserted sequence (pET-15b/YB-1) was verified by DNA sequencing. Plasmid was transformed into E. coli BL21(DE3) and gene expression was induced with 0.8 mM IPTG. Expressed proteins were purified using a Ni-NTA purification system (Invitrogen) and analyzed by SDS-PAGE and western blotting. Bacterial lysates were lysed in reducing loading buffer (NuPAGE system, Invitrogen), proteins resolved by SDS-PAGE, and transferred to PVDF membranes (Invitrolon, 0.45 mm) (Invitrogen) using a NuPAGE Bis-Tris electrophoresis system (Invitrogen). Blots were probed with anti-human YB-1 (ARP, Belmont, USA) and anti-His antibody (Serotec, Raleigh, USA) at a 1:1000 dilution, followed by AP-conjugated rabbit antimouse IgG (HþL) at a 1:2500 dilution. AP detection was performed using the picoBLUE immunoscreening kit. Vaccination and immune assays A/J mice were immunized by s.c. injection of 2 106 irradiated (5000 rad) AGN2a-4p cells with PC61 treatment (as above). Five days after the second immunization, splenocytes were collected, and CD8þ T cells purified using the CD8a (Ly-2) Microbead kit (Miltenyi Biotech) on an AutoMACS device (Miltenyi Biotech, Auburn, USA). To enumerate CD8þ IFN-g-producing cells, ELISPOT analysis was carried out using the BD ELISPOT mouse IFN-g Set and 96-well PVDF membrane plates (Millipore, Bedford, USA) according to the manufacturer’s protocols. Peritoneal exudate cells (PEC)


were used as antigen-presenting cells. 1 105 PEC from naive A/J mice were placed in ELISPOT wells and loaded with protein by incubating in 100 ml media containing recombinant YB-1 or EGFP at 25 mg/ml for 4 h at 378C. 1 105 CD8þ T cells (100 ml) were added to each well and incubated for 18 h to test antigen recognition. Spots were counted using an Immunospot 3 automated reader (C.T.L., Ltd, Cleveland, USA). YB-1-specific IgG was detected by coating 96-well plates (EIA/RIA, Corning, USA) with YB-1 or EGFP (1 mg per well) in carbonate buffer (45.3 mM NaHCO3, 18.2 mM Na2CO3, pH 9.6). Diluted sera were added to blocked wells and then wells were detected with AP conjugated rabbit antimouse IgG (HþL) (Abcam) and developed with NBT/BCIP (Stratagene).

Results Efficient expression of CD80, CD86, CD54 and CD137L on AGN2a cell surface One exciting aspect of nucleofection technology is that several plasmid vectors encoding immune stimulatory molecules can be transfected simultaneously, allowing the preparation of a tumor cell that more closely resembles an antigen-presenting cell. AGN2a cells were nucleofected with plasmids containing genes for CD80 and CD86 (each at 1 mg per 106 cells) and CD54 and CD137L (each at 2 mg per 106 cells). Cells were recovered 18 h after nucleofection. When analyzed by flow cytometry, each molecule was expressed on more than 95% of the cells (Fig. 1), which was important for the successful immununization of the animals. Strong humoral immunity was induced by AGN2a-4p immunization in the context of regulatory T cell blockade We have already identified that CD25þ regulatory T cell blockade could enhance vaccine-induced immunity to neuroblastoma [10]. So in order to improve the host

immunity against AGN2a, the CD25þ regulatory T cells of Groups II, IV and VI were blocked with anti-CD25 monoclonal antibody (PC61) 3 days before the immunization (Fig. 2). Besides Groups I and II, the other four groups of mice were immunized with AGN2a or AGN2a-4p twice weekly. All mice sera were collected 5 days after the second immunization. Before screening the cDNA library, the anti-AGN2a IgG was detected by the sera from all groups. It was shown that sera of the immunized mice contained higher titer of anti-AGN2a IgG compared with sera from Group I (negative control group) and Group II (regulatory T cell blockade alone group). Specially, Group V (AGN2a-4p immune group) and Group VI (AGN2a-4p immunized combined with the regulatory T cells blockade group) were significantly higher than Group III (AGN2a immune group) and Group IV (AGN2a immunized combined with the regulatory T cells blockade group), indicating that the expression of costimulatory molecules on AGN2a cell surface could induce an effective immune response against AGN2a. We also found that the antibody titer of Group IV was higher than Group III and that of Group VI was higher than Group V (Fig. 3). These findings further confirmed that CD25þ regulatory T cell inhibition could enhance vaccine-induced immunity to neuroblastoma.

Screening AGN2a cDNA library The modification of syngeneic tumor cell lines with immune co-stimulatory molecules can transform lethal tumor cell lines into effective vaccines. The immunization of A/J mice with a cell-based vaccine, AGN2aCD80/CD86/CD54/CD137L, has been demonstrated to induce a strong cellular immune response [18]. This immune response is enhanced when the vaccine is given in the context of anti-CD25 antibody (clone PC61) as a means to block regulatory T cells. We sought to explore the specificity of the immunoglobulin response to this cell-based vaccine, in order to find new tumor-associated

Figure 1 High-level expression of four different immune stimulatory molecules (CD80, CD86, CD54 and CD137L) on AGN2a cell surface. Acta Biochim Biophys Sin (2009) | Volume 41 | Issue 12 | Page 983


Figure 2 Flow cytometric analysis of regulatory T cells from mice treated with/without anti-CD25 monoclonal antibody (PC61) after 3 days (A) The regulatory T cells ratio of Groups I, III, V (from left to right) in the peripheral blood. (B) The regulatory T cells ratio of Groups II, IV, VI (from left to right) in the peripheral blood.

Figure 3 Flow cytometric analysis of anti-AGN2a IgG of different groups From left to right was Groups I, II, III, IV, V and VI in order.

antigens. To do so, we constructed a cDNA library using mRNA purified from the AGN2a cell line and expressed proteins encoded by tumor-derived mRNA in E. coli using the l ZAPII phage system. To identify clones expressing antigenic epitopes recognized by IgG present in the immune sera, a total of 2 105 clones were screened. Twenty-six and 28 unique plaques were identified as expressing proteins that could be recognized by IgG antibody when sera from Groups V and VI were screened at a dilution of 1:250, respectively (Fig. 4). When pooled sera (n ¼ 5 for each group) from Groups I, II, III or IV were used to screen the cDNA library, 0, 0, 2, or 3 unique clones were identified, respectively. Acta Biochim Biophys Sin (2009) | Volume 41 | Issue 12 | Page 984

Figure 4 Positive clones of the primary screening on the nitrocellulose membranes (A) Sera from Group V (AGN2a-4p immune group). (B) Sera from Group VI (AGN2a-4p immunized combined with regulatory T cells blockade group). Magnification, 200.

Genetic analysis of positive clones Antibody reactive plaques identified by serum from Groups V and VI mice were picked and used to reinfect E. coli, and screened once more serologically (secondary screening/primary screening) (Fig. 5). In vivo excision was performed with helper phage and phagemid DNA isolated. Each phagemid insert DNA was sequenced using both T7 and T3 primers and the sequence was used to search on-line databases. The 54 clones identified by immune serum-SEREX screening encoded 21 individual proteins, as listed in Tables 2 and 3. Primers


Figure 5 Positive clones of the secondary screening on the nitrocellulose membranes Magnification, 200.

Table 2 SEREX-identified neuroblastoma antigens from AGN2a-4p immunized mice

Gene

Accession No.

Major characters

Matr3

NM_010771

Slc25a4 Elovl6

XM_991099 NM_130450

Gmeb-1

AF210433

Vps54 Acbd3(PAP7)

BC024789 BC060602

Sfrs2ip Pdha1

BC055486 BC007142

Dnajc2(ZRF1)

BC052027

Agrn

NM_021604

Mus musculus matrin 3: the protein encoded by this gene is localized in the nuclear matrix. It may play a role in transcription or may interact with other nuclear matrix proteins to form the internal fibrogranular network [18]. M. musculus solute carrier family 25: similar to ADP/ATP translocase 1 [19]. M. musculus ELOVL family member 6: a long-fatty acid elongase, contributes to de novo synthesis of fatty acids and regulates hepatic insulin sensitivity [20]. M. musculus glucocorticoid modulatory element binding protein 1: GMEB1 is a trans-acting DNA-binding protein that may mediate the demonstrated functions of GME as a positive modulator of the glucocorticoid response [21]. M. musculus vacuolar protein sorting 54 is known to be involved in intracellular protein sorting [22]. M. musculus acyl-Coenzyme A-binding domain containing 3: highly expressed in the gonads, adrenal, hippocampus, and distinct brain neuronal and glial populations. Over-expression of the full-length PAP7 increased the hCG-induced steroid production. PAP7 is a functional element of the hormone-induced signal transduction cascade leading to steroidogenesis [23]. M. musculus splicing factor, arginine/serine-rich 2, interacting protein [24]. M. musculus pyruvate dehydrogenase E1 alpha 1: the pyruvate dehydrogenase complex is a nuclear-encoded mitochondrial matrix multienzyme complex that provides the primary link between glycolysis and the tricarboxylic acid (TCA) cycle by catalyzing the irreversible conversion of pyruvate into acetyl-CoA [25]. M. musculus DnaJ (Hsp40) homolog, subfamily C, member 2, transcript variant 2: this gene is a member of the M-phase phosphoprotein family [26]: The protein is capable of forming a heterodimeric complex that associates with ribosomes, acting as a molecular chaperone for nascent polypeptide chains as they exit the ribosome. This protein was identified as a leukemia-associated antigen and expression of the gene is upregulated in leukemic blasts [27]. Also, chromosomal aberrations involving this gene are associated with primary head and neck squamous cell tumors [28]. Agrn (M. musculus agrin): agrin is a neuronal aggregating factor that induces the aggregation of acetylcholine receptors and other postsynaptic proteins on muscle fibers and is crucial for the formation of the neuromuscular junction [29].



Table 3 SEREX-identified neuroblastoma antigens from AGN2a-4p immunized mice with PC61 treatment

Gene

Accession No.

Major characters

YB-1(Nsep1)

BC061634

Prep

BC012869

Nufip1

NM_013745

Cbx5(HP1)

XM_001004073

Nup88

XM_993678

Sfrs6(SRp55)

BC012039

Gars

BC021747

Eea1

NM_001001932

Cabin1

NM_172549

Eif4g2

BC043034

mCBP

X75947

M. musculus Y box protein 1: nuclease sensitive element binding protein 1. YB-1 is an important regulator of PTP1B expression. A correlation between expression of PTP1B and YB-1 in several human cancer cell lines [30]. M. musculus prolyl endopeptidase: the protein encoded by this gene is a cytosolic prolyl endopeptidase that cleaves peptide bonds on the C-terminal side of prolyl residues within peptides that are up to approximately 30 amino acids long. Prolyl endopeptidases have been reported to be involved in the maturation and degradation of peptide hormones and neuropeptides [31]. M. musculus nuclear fragile X mental retardation protein interacting protein 1: NUFIP1 (nuclear FMRP interacting protein 1) is a nucleocytoplasmic shuttling protein associated with active synaptoneurosomes [32]. M. musculus chromobox homolog 5: similar to chromobox protein homolog 5 (heterochromatin protein 1 homolog alpha) (HP1 alpha): The family of heterochromatin protein 1 (HP1) are highly conserved adapter molecules, which have important functions in the cell nucleus. These functions include gene repression by heterochromatin formation, transcriptional activation, regulation of binding of cohesion complexes to centromere, sequesteration of genes to nuclear periphery, transcriptional arrest, maintenance of heterochromatin integrity, gene repression at single nucleosome level and gene repression by heterochromatization of euchromatin [33,34]. M. musculus nucleoporin 88: similar to nuclear pore complex protein Nup88:Nup88 mRNA over-expression is associated with high aggressiveness of breast cancer; localization of NUP88 to the NPC is dependent on CAN binding [35,36]. M. musculus splicing factor, arginine/serine-rich 6. The protein encoded by this gene is involved in the mRNA splicing and may play a role in the determination of alternative splicing [37]. M. musculus glycyl-tRNA synthetase: the Gars gene provides instructions for making an enzyme called glycyl-tRNA synthetase. This enzyme is found in all cell types and plays an important role in the production (synthesis) of proteins. M. musculus early endosome antigen 1: the Rab5 effector EEA1 is essential for fusion of early endosomes. The protein encoded by this gene binds phospholipid vesicles containing phosphatidylinositol 3-phosphate and participates in endosomal trafficking [38]. M. musculus calcineurin binding protein 1: the protein encoded by this gene binds specifically to the activated form of calcineurin and inhibits calcineurin-mediated signal transduction. The encoded protein is found in the nucleus and contains a leucine zipper domain as well as several PEST motifs, sequences that confer targeted degradation to those proteins that contain them [39]. M. musculus eukaryotic translation initiation factor 4, gamma 2: translation initiation is mediated by specific recognition of the cap structure by eukaryotic translation initiation factor 4F (eIF4F), which is a cap-binding protein complex that consists of three subunits: eIF4A, eIF4E and eIF4G. The protein encoded by this gene shares similarity with the C-terminal region of eIF4G that contains the binding sites for eIF4A and eIF3; eIF4G, in addition, contains a binding site for eIF4E at the N-terminus. This gene product functions as a general repressor of translation by forming translationally inactive complexes. In vitro and in vivo studies indicate that translation of this mRNA initiates exclusively at a non-AUG (GUG) codon [40]. M. musculus mCBP mRNA: the mouse poly(C)-binding protein exists in multiple isoforms and interacts with several RNA-binding proteins, and has a function in RNA metabolism [41].



were generated for each clone identified, and expression of each was verified by PCR analysis of AGN2a-derived cDNA (data not shown). The most notable thing was the frequency with which YB-1 was detected. While all other antigens listed in Tables 2 and 3 were detected once from a single positive clone, the YB-1 antigen was detected 12 separate times. Careful inspection of the cDNA library clones demonstrates that most transcripts seem to have some role in the neuronal differentiation, cell cycle control, or have previously been identified as transcripts that are overexpressed in other malignancies.

Expression of YB-1 protein Recombinant YB-1 was produced in the pET-15b bacterial vector encoding an N-terminal 6-His sequence to facilitate purification on a nickel affinity column. Proteins containing the 6-His tag purified from bacterial lysates using a Ni-NTA column. When columns were eluted with imidazole, a distinct band of 42 kDa corresponding to YB-1 was readily resolved by SDS-PAGE [Fig. 6(A)]. We then tested whether the purified protein was recognized by anti-His antibody and anti-human YB-1 antibody. Western blot analysis demonstrated that the prepared mouse YB-1 was recognized by anti-His antibody and anti-human YB-1 antibody [Fig. 6(B)].

Figure 6 SDS-PAGE and western blot analysis of purified YB-1 (A) Ni-NTA column purified protein was diluted in reducing sample loading buffer, resolved by SDS-PAGE and stained with Coomassie blue. Lanes were loaded with: 1, molecular weight marker; 2, purified protein from control bacteria; 3 –5, purified YB-1. (B) Proteins were resolved by SDS-PAGE and then transferred to PVDF membrane using a Bis-Tris electrophoresis buffer system for western blot analysis. Lanes 1 and 3, control bacteria lysate; 2 and 4, purified YB-1. Blot (a) was probed with anti-His antibody. Blot (b) was probed with anti-YB-1 monoclonal antibody.

YB-1 induced humoral and cellular immune response To further prove that the phage-expressed YB-1 cDNA was genuine immune targets, we tested serum from AGN2a-4p vaccinated mice in the context of PC61

Figure 7 ELISA analysis of immune sera for YB-1 and EGFP reactivity 96-well plates were coated with 10 mg/ml recombinant YB-1 and EGFP. All proteins were produced in bacterial vectors. One hundred microliters of test serum (as described on the x-axis) diluted 1:100, and were added to each well. Sera used for this assay were the same sera used for cDNA library screening, and were pooled from immunized mice.

treatment for the presence of YB-1 reactive immunoglobulin by ELISA. Using polystyrene plates coated with recombinant YB-1 and EGFP, YB-1-reactive IgG could be readily detected in serum from mice immunized with AGN2a-4p with PC61 treatment (Fig. 7). To investigate the CD8þ T cell response against YB-1 in mice that had received a cell-based cancer vaccine with PC61 treatment, A/J mice were immunized twice with 2 106 irradiated AGN2a-4p cells with Treg blocked. Splenocytes were collected 5 days after the second vaccination, and CD8þ T cells purified by immunomagnetic selection. T cell responses to YB-1 were detected in IFN-g ELISPOT assays by using peritoneal exudate cells (PECs), which are primarily composed of macrophages, as the antigen-presenting cells. Control cultures containing T cells plus non-protein-loaded PECs, or T cells plus EGFP loaded PECs, had similar low numbers of IFN-g-producing cells. In contrast, significant anti-YB-1 reactivity was seen in T cells from mice immunized with AGN2a-4p in the context of PC61 treatment (Fig. 8). These assays demonstrated that anti-YB-1 CD8þ T cell responses are induced by AGN2a-4p with PC61 treatment immunization protocol. This also validates that our serologic cDNA screening assay, based on the use of immune serum rather than tumor-onset serum, could identify important T cell epitopes.

Discussion The study of tumor immune recognition has been mostly concentrated on the T cell-mediated immunity, Acta Biochim Biophys Sin (2009) | Volume 41 | Issue 12 | Page 987


Figure 8 IFN-g ELISPOT analysis of CD8 1 T cells from immunized mice CD8þ T cells from immunized and control mice were purified by immunomagnetic sorting and co-incubated with the indicated APCs ( protein-loaded PECs) for 18 h. 1 105 syngeneic PEC loaded with buffer (no protein), recombinant EGFP, or recombinant YB-1 were co-cultured with 1 105 CD8þ T cells from the following groups of mice: naive (stripes), PC61-treated mice (gray), PC61 treated plus AGN2a-4p vaccinated (black). Data are representative of three separate experiments. Each experiment consisted of T cells purified and pooled from five mice. Error bars represent the standard deviation calculated from triplicate ELISPOT wells.

particularly on CTL against tumor cells in vitro. As cellular and humoral immunity play a coordinated role in the anti-tumor immune responses, the tumor antigen not only could induce cellular immune responses, but also could induce specific antibodies in vivo. SEREX can help to find tumor-associated antigen in many malignant tumors [16]. The major immune mechanism of SEREX are sera from cancer patients containing anti-tumor antigen antibody induced by tumor antigen, so the screening of the tumor-derived cDNA library with sera from tumor patients, is an ideal way to find tumor-associated antigens. However, the immune system of most tumor patients is in a state of tolerance, and the immune response against tumor antigen is very low, so there may be only a low level or no anti-tumor antibody induced. This is a major obstacle in the area of finding tumor-associated antigen. In a sub-clinical neuroblastoma treatment model, we have applied a new type of tumor cell vaccine for the treatment of neuroblastoma and found that AGN2a transfected with costimulatory molecules as vaccine could induce strong immune response [8]. Moreover, immunization with a genetically modified tumor cell line AGN2a-4p is even more effective when administered in the context of Treg blockade with the anti-CD25 monoclonal antibody PC61. Therefore, in this experiment, we use AGN2a expressed four types of immune costimulatory molecules (CD80, CD86, CD54 and CD137L) as Acta Biochim Biophys Sin (2009) | Volume 41 | Issue 12 | Page 988

vaccines with PC61 treatment to block Treg function. The results showed that the use of tumor cells expressing costimulatory molecules as a vaccine and the regulatory T cell function blockade could significantly enhance the host immune response. We finally identified 21 clones by sequencing and comparative analysis of gene pools. Careful inspection of the cDNA library clones revealed that most transcripts seem to have some role in the neuronal differentiation, cell metabolism, or have previously been identified as transcripts that are over-expressed in other malignancies as show in Tables 2 and 3. The most commonly identified tumor-associated antigen, using serum from AGN2a-4p immunized in the context of regulatory T cell blockade mice, was YB-1 protein that also induced a T cell response. YB-1 protein is a 42-kDa multifunctional cellular protein that expressed in various cancers [30]. It is localized in the cytoplasm as well as in the nucleus. In particular, YB-1 is localized in the nuclear compartment following cellular stress, such as radiation, drug treatment, hyperthermia, or viral infection [42]. Within the nucleus, YB-1 can act as a transcription factor, and it is involved in the regulation of important cancer-associated genes. For example, YB-1 triggers the expression of Her-2 and estrogen receptor alpha (ERalpha) in breast cancer. Thus, nuclear YB-1 appears to be a potential target for the inhibition of Her-2 and ERalpha-dependent proliferation signals, particularly with regard to resistance to Her-2-targeting drugs such as trastuzumab [43]. In some cancers, such as nasopharyngeal cancer, YB-1 is a promising predictive marker of radioresistance and chemoradioresistance [44]. Furthermore, YB-1 is involved in the replication of adenovirus type 5, a commonly used vector in the gene therapy. Thus, YB-1 can trigger an ‘oncolytic’ effect in YB-1 nuclear positive cancer cells treated with adenoviruses [45]. Besides its impact as a prognostic factor, in the future the diagnostics of cellular YB-1 status may provide the basis for a virotherapy or a gene therapy incorporating adenoviruses. Otherwise, no overlapping clones were found when sera from AGN2a-4p immunized group and AGN2a-4p immunized in the context of Treg blocked group were screened. It has been already identified that in vivo anti-CD25 mAb treatment of mice can enhance neuroblastoma vaccine-induced immunity. The effect of anti-CD25 mAb treatment seems to be due to the combined partial depletion of natural CD4þFoxp3þ Treg cells and modulation/blockade of CD25 molecules on


the surface of the Treg cells [10]. But it is also known that neuroblastoma cells were transiently transfected to simultaneously express the co-stimulatory molecules CD54, CD80, CD86, and CD137L could generate strong antitumor immunity in mice [8]. It was unclear which group’s clones were more closely associated with neuroblastoma. In brief, we successfully separated neuroblastomaassociated tumor antigens by using SEREX. The most important thing is that we first found YB-1 protein is neuroblastoma-related tumor antigen that could induce host immune response against the tumor. So, we are going to study these 2 clones systematically, trying to find out the possibility of using these antigens as neuroblastoma immunotherapy molecular targets. However, to extrapolate these findings to human disease, serologybased antigen discovery should be carried out not with the onset or diagnostic serum (which is most commonly banked), but with the serum derived from treated patients in which antigen loading into the immune system is optimal and the Treg effects are minimal.

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Funding

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This work was supported by the Midwest Athletes Against Childhood Cancer (MACC Fund, Inc., Milwaukee, WI).

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