Tumor Rejection Antigen Research Articles

Phase I Clinical Trial of Autologous Ascites-derived Exosomes Combined With GM-CSF for Colorectal Cancer

Exosomes are small membrane vesicles that are secreted by a multitude of cell types. The exosomes derived from dendritic cells (Dex), tumor cells (Tex), and malignant effusions demonstrate immunomodulatory functions, and are even under clinical trial for cancer treatments. In this study we report the phase I clinical trial of the ascites-derived exosomes (Aex) in combination with the granulocyte–macrophage colony-stimulating factor (GM-CSF) in the immunotherapy of colorectal cancer (CRC). The Aex isolated by sucrose/D2O density gradient ultracentrifugation are 60–90-nm vesicles that contain the diverse immunomodulatory markers of exosomes and tumor-associated carcinoembryonic antigen (CEA). Totally 40 patients (HLA-A0201+CEA+) with advanced CRC were enrolled in the study, and randomly assigned to treatments with Aex alone or Aex plus GM-CSF. Patients in both groups received a total of four subcutaneous immunizations at weekly intervals. We found that both therapies were safe and well tolerated, and that Aex plus GM-CSF but not Aex alone can induce beneficial tumor-specific antitumor cytotoxic T lymphocyte (CTL) response. Therefore, our study suggests that the immunotherapy of CRC with Aex in combination with GM-CSF is feasible and safe, and thus can serve as an alternative choice in the immunotherapy of advanced CRC.

Mechanisms of Immunization Against Cancer Using Chimeric Antigens

Successful approaches to tumor immunotherapy must overcome the physiological state of tolerance of the immune system to self-tumor antigens. Immunization with appropriate variants of syngeneic antigens can achieve this. However, improvements in vaccine design are needed for efficient cancer immunotherapy. Here we explore nine different chimeric vaccine designs, in which the antigen of interest is expressed as an in-frame fusion with polypeptides that impact antigen processing or presentation. In DNA immunization experiments in mice, three of nine fusions elevated relevant CD8(+) T-cell responses and tumor protection relative to an unfused melanoma antigen. These fusions were: Escherichia coli outer membrane protein A (OmpA), Pseudomonas aeruginosa exotoxin A, and VP22 protein of herpes simplex virus-1. The gains of immunogenicity conferred by the latter two are independent of epitope presentation by major histocompatibility complex class II (MHC II). This finding has positive implications for immunotherapy in individuals with CD4(+) T-cell deficiencies. We present evidence that antigen instability is not a sine qua non condition for immunogenicity. Experiments using two additional melanoma antigens identified different optimal fusion partners, thereby indicating that the benefits of fusion vectors remain antigen specific. Therefore large fusion vector panels such as those presented here can provide information to promote the successful advancement of gene-based vaccines.

Proteomic approaches to the analysis of atopic dermatitis and new insights from interactomics

In this review, we summarized the recent findings regarding atopic dermatitis (AD) skin disease based on proteomic studies. AD is a chronic relapsing inflammatory skin disease typically characterized by a distribution of eczematous skin lesions with lichenification, pruritic excoriations and dry skin with wide varieties of pathophysiological aspects. We summarized the alterations of the protein expressions in the primary cultured AD cells from the patients'-biopsy samples that were mostly analyzed by 2-D PAGE and MALDI-TOF. Further, we also conducted protein-protein interaction mapping according to the obtained candidate proteins. As a result, we found that several hub proteins, i.e. heat shock 70-kDa protein 2, heat shock 70-kDa protein 9, tumor rejection antigen-1 (gp96), spermatogenesis-associated factor, protein kinase C inhibitor 1, vimentin, tenascin, semaphorin 4f (SEMA4F), complement component C1r deficiency (C1R) and apolipoprotein A (LPA), respectively, could receive important consideration in future studies. Since the mechanism of AD disease has been shown to be complex, our results may provide new clues to aid understanding of AD.

ITRAQ-FACILITATED PROTEOMIC ANALYSIS OF HUMAN PROSTATE CANCER CELLS IDENTIFIES PROTEINS ASSOCIATED WITH PROGRESSION

The unpredictable behavior of prostate cancer presents a major clinical challenge during patient management. In order to gain an insight into the molecular mechanisms associated with prostate cancer progression, we employed the shot-gun proteomic approach of isobaric tags for relative and absolute quantitation (iTRAQ), followed by 2D-LC-MS/MS, using the poorly metastatic LNCaP cell line and its highly metastatic variant LNCaP-LN3 cell line as a model. A total number of 280 unique proteins were identified (≥95% confidence), and relative expression data was obtained for 176 of these. Ten proteins were found to be significantly up-regulated (≥1.50 fold), while 4 proteins were significantly down-regulated (≥−1.50 fold), in LNCaP-LN3 cells. Differential expression of brain creatine kinase (CKBB), soluble catechol- O-methyltransferase (S-COMT), tumor rejection antigen (gp96), and glucose regulated protein, 78 kDa (grp78), was confirmed by Western blotting or independent 2D-PAGE analysis. Additionally, iTRAQ analysis identified absence of the lactate dehydrogenase-B (LDH-B) subunit in LNCaP-LN3 cells, confirming our published data. The clinical relevance of gp96 was assessed by immunohistochemistry using prostate tissues from benign ( n = 95), malignant ( n = 66), and metastatic cases ( n = 3). Benign epithelium showed absent/weak gp96 expression in the basal cells, in contrast to the moderate/strong expression seen in malignant epithelium. Furthermore, there was a statistically significant difference in the intensity of gp96 expression between benign and malignant cases ( p < 0.0005, Mann–Whitney U). Our study is the first to report the application of iTRAQ technology and its potential for the global proteomic profiling of prostate cancer cells, including the identification of absent protein expression.

Corpora amylacea from multiple sclerosis brain tissue consists of aggregated neuronal cells.

In this report, we describe proteomic analysis of corpora amylacea collected by postmortem laser microdissection from multiple sclerosis (MS) brain lesions. Using low level protein loads (about 30 microg), a combination of two-dimensional electrophoresis with matrix-assisted laser desorption/ionization-time of flight mass spectrometry and database interrogations we identified 24 proteins of suspected neuronal origin. In addition to major cytoskeletal proteins like actin, tubulin, and vimentin, we identified a variety of proteins implicated specifically in cellular motility and plasticity (F-actin capping protein), regulation of apoptosis and senescence (tumor rejection antigen-1, heat shock proteins, valosin-containing protein, and ubiquitin-activating enzyme E1), and enzymatic pathways (glyceraldehyde-3-dehydrogenase, protein disulfide isomerase, protein disulfide isomerase related protein 5, lactate dehydrogenase). Samples taken from regions in the vicinity of corpora amylacea showed only traces of cellular proteins suggesting that these bodies may represent remnants of neuronal aggregates with highly polymerized cytoskeletal material. Our data provide evidence supporting the concept that biogenesis of corpora amylacea involves degeneration and aggregation of cells of neuronal origin.

ITRAQ-Facilitated Proteomic Analysis of Human Prostate Cancer Cells Identifies Proteins Associated with Progression

The unpredictable behavior of prostate cancer presents a major clinical challenge during patient management. In order to gain an insight into the molecular mechanisms associated with prostate cancer progression, we employed the shot-gun proteomic approach of isobaric tags for relative and absolute quantitation (iTRAQ), followed by 2D-LC-MS/MS, using the poorly metastatic LNCaP cell line and its highly metastatic variant LNCaP-LN3 cell line as a model. A total number of 280 unique proteins were identified (> or =95% confidence), and relative expression data was obtained for 176 of these. Ten proteins were found to be significantly up-regulated (> or =1.50 fold), while 4 proteins were significantly down-regulated (> or = -1.50 fold), in LNCaP-LN3 cells. Differential expression of brain creatine kinase (CKBB), soluble catechol-O-methyltransferase (S-COMT), tumor rejection antigen (gp96), and glucose regulated protein, 78 kDa (grp78), was confirmed by Western blotting or independent 2D-PAGE analysis. Additionally, iTRAQ analysis identified absence of the lactate dehydrogenase-B (LDH-B) subunit in LNCaP-LN3 cells, confirming our published data. The clinical relevance of gp96 was assessed by immunohistochemistry using prostate tissues from benign ( n = 95), malignant ( n = 66), and metastatic cases ( n = 3). Benign epithelium showed absent/weak gp96 expression in the basal cells, in contrast to the moderate/strong expression seen in malignant epithelium. Furthermore, there was a statistically significant difference in the intensity of gp96 expression between benign and malignant cases ( p < 0.0005, Mann-Whitney U). Our study is the first to report the application of iTRAQ technology and its potential for the global proteomic profiling of prostate cancer cells, including the identification of absent protein expression.

Induction of hsp70-Mediated Th17 Autoimmunity Can Be Exploited as Immunotherapy for Metastatic Prostate Cancer

A close connectivity between autoimmune and tumor rejection responses is known to exist in the case of melanoma immunotherapy. However, relatively little is known about self-antigens on other types of normal cells, their relation to the development of autoimmune disease, and their possible coexistence as potential tumor rejection antigens on associated tumors. In the current study, we induced inflammatory killing of normal prostate tissue in situ using a fusogenic membrane glycoprotein along with the immune adjuvant hsp70. We show here that, in the prostate, hsp70 induces interleukin (IL)-6, which triggers a CD4- and CD8-dependent progressive autoimmune reactivity, associated with IL-17 expression. This autoimmune response was also able to induce the rejection of established prostate tumors, but not other histologic types of tumors, growing elsewhere in the animal. These data show that the intimate connectivity between autoimmune and tumor rejection responses extends beyond the classic melanoma paradigm and may be clinically valuable for the treatment of established metastatic disease of the prostate.

A T cell receptor associated with naturally occurring human tumor immunity

The onconeural antigens appear to serve as tumor rejection antigens in the paraneoplastic neurologic disorders. Here, we used an unbiased peptide binding screen, followed by studies in HLA-A2.1 transgenic mice to identify naturally processed HLA-A2.1 restricted epitopes of the paraneoplastic cerebellar degeneration breast/ovarian cancer antigen cdr2. These mice were used to clone high-avidity cdr2-specific CD8(+) T cells that recognize human tumor cells presenting endogenously loaded MHC class I-cdr2 peptide. T cells with this specificity were detected in the peripheral blood of two HLA-A2.1(+) paraneoplastic cerebellar degeneration patients. We cloned T cell receptor (TCR) alpha and beta genes from cdr2-specific T cells; electroporation of RNA encoding this TCR turned nonreactive donor T cells into efficient killers of human cdr2-expressing tumor cells. Cloned cdr2-specific TCR genes provide a clinically relevant means for immunologic targeting of human gynecologic cancers.

Pathway for Differentiation of Human Embryonic Stem Cells to Vascular Cell Components and Their Potential for Vascular Regeneration

We demonstrated previously that mouse embryonic stem (ES) cell-derived vascular endothelial growth factor receptor-2 (VEGF-R2)-positive cells can differentiate into both vascular endothelial cells and mural cells. This time, we investigated kinetics of differentiation of human ES cells to vascular cells and examined their potential as a source for vascular regeneration. Unlike mouse ES cells, undifferentiated human ES cells already expressed VEGF-R2, but after differentiation, a VEGF-R2-positive but tumor rejection antigen 1-60 (TRA1-60)-negative population emerged. These VEGF-R2-positive but tumor rejection antigen 1-60-negative cells were also positive for platelet-derived growth factor receptor alpha and beta chains and could be effectively differentiated into both VE-cadherin+ endothelial cell and alpha-smooth muscle actin+ mural cell. VE-cadherin+ cells, which were also CD34+ and VEGF-R2+ and thought to be endothelial cells in the early differentiation stage, could be expanded while maintaining their maturity. Their transplantation to the hindlimb ischemia model of immunodeficient mice contributed to the construction of new blood vessels and improved blood flow. We could identify the differentiation process from human ES cells to vascular cell components and demonstrate that expansion and transplantation of vascular cells at the appropriate differentiation stage may constitute a novel strategy for vascular regenerative medicine.

Clonal T‐cell response against autologous pleomorphic malignant fibrous histiocytoma antigen presented by retrieved HLA‐A*0206

Towards the goal of identifying tumor-rejection antigens on eradicated tumors in bone and soft tissue sarcomas, we evaluated the immune response against antigens presented by lost HLA class I molecules. Tumor specimens and peripheral blood samples were obtained from a 70-year-old woman with pleomorphic malignant fibrous histiocytoma. Over 1-year culture, a tumor cell line (MFH2004) was established. A B-cell line infected with Epstein-Barr virus (B2004-EBV) was developed from the blood samples. HLA genotypes of B2004-EBVcells were A*0206/2402, B*4006/4601, and C*0102/0801, whereas MFH2004 cells were defective for A*0206, B*4006, and C*0102. Loss of HLA-A2 expression was also proved immunohistochemically in the primary tumor tissues. Lost HLA-A2 in MFH2004 cells was retrieved by transfection of HLA-A*0206 cDNA to develop MFH2004-A2. Attempts to induce CTLs by mixed culture with autologous T cells and MFH2004 cells resulted in failure. In contrast, those with MFH2004-A2 induced CTL clones CTL2004-c6 and CTL2004-c17. These CTL clones specifically killed MFH2004-A2 but not MFH2004 or B2004-EBV in an HLA-A2-restricted manner. These findings suggest that CTL2004-c6 and CTL2004-c17 recognize autologous tumor-rejection antigens presented by HLA-A*0206, which may have been expressed by tumor cells that had been eradicated by the host's immunosurveillance system.

Leukemia Inhibitory Factor-Expressing Human Embryonic Lung Fibroblasts as Feeder Cells for Human Embryonic Germ Cells

A robust culture system is critical for maintaining both proliferation and the developmental potential of human embryonic germ (hEG) cells. Here, we use human embryonic lung fibroblasts (hELF) overexpressing leukemia inhibitory factor (LIF) as feeder cells to support the self-renewal of hEG cells. We examine the morphology, gene expression, and developmental potential of hEG cells grown on a feeder layer of LIF-expressing hELF (hELF/lif) cells. hEG cells were positive for alkaline phosphatase (AP), stage-specific embryonic antigen (SSEA)-1, SSEA-4, tumor rejection antigen (TRA)-1–60, and TRA-1–81. In addition, hEG cells maintained on hELF/lif expressed higher levels of pluripotency genes such as Oct4 and Nanog. In addition, hEG cells maintained on hELF/lif cells gave rise to differentiated tissues when grown as embryoid bodies, consistent with the broad developmental potential of the starting population. Our results suggest that a hELF/lif feeder layer can support the proliferation of hEG cells, and that LIF signaling plays an essential role in this process. This human-derived culture system provides an attractive alternative to more commonly used mouse-derived feeder layers for use in clinical applications.

PROTEIN PROFILING OF MOUSE OOCYTES MATURED IN VITRO DURING THE FIRST WAVE OF FOLLICULOGENESIS

During the first month after birth, synchronous follicular growth occurs in the ovaries of immature mice (the first wave). Previously, we showed that mouse oocytes during the first wave were more competent developmentally in older females, although the numbers of mature oocytes did not differ with female age (17, 18, and 24 days old). In this study, we profiled the proteins of oocytes matured in vitro during the first wave using two-dimensional electrophoresis (2-DE) as a first step in search of proteins involved in the maturation of mouse oocytes. Oocytes collected from ovarian follicles of B6D2F1 females at nine ages (from 17 to 25 days of age) without hormonal treatment were matured in Waymouth medium (Sigma) supplemented with pyruvate (0.23 mM), antibiotics, polyvinylpyrrolidone (3 mg mL−1), and recombinant human FSH (0.5 iu mL−1, Serono). After culturing for 17 h at 37°C in an atmosphere of 5% CO2, 5% O2, and 90% N2, oocytes whose germinal vesicles had broken down (mature oocytes) were dissolved in rehydration/sample buffer (8 M urea, 2% CHAPS, 50 mM DTT, 0.2% Bio-Lyte 3/10, and 0.001% BPB; Bio- Rad). In the first dimension, 4–10 μg of proteins from 200–400 mature oocytes were separated by isoelectric focusing using IPG ReadyStrip pH 3– 10 (7 cm in length, Bio-Rad) with a Protean IEF Cell (Bio-Rad). After alkylation with iodoacetamide, the proteins were separated in the second dimension using SDS-PAGE gradient gels (ReadyGel J 10–20%, gel size: 80[W] x 73[L] mm, Bio-Rad). The protein spots on the gels were visualized using SYPRO Ruby stain (Invitrogen). Approximately 200 protein spots were detected on each gel. We identified six commonly detected proteins on 2-DE gels throughout the first wave using peptide mass fingerprinting and a Mascot search against the NCBI nonredundant protein sequence database to identify protein spots that can be used as protein landmarks for future 2-DE analyses. The most prominent proteins were lactate dehydrogenase B (37 kDa, pI=5.70) and ubiquitin carboxy-terminal hydrolase L1 (25.2 kDa, pI=5.33). The other proteins were tumor rejection antigen gp96 (92.7 kDa, pI=4.74), a fragment of zona pellucida glycoprotein 2 (~18 kDa, pI ~4; the amino acid sequence was confirmed by MS/MS analysis), pyruvate kinase M (58.4 kDa, pI=7.58), and peroxiredoxin 1 (22.4 kDa, pI=8.26). Our results provide a basis for further 2-DE analyses of the proteins involved in the maturation of mouse oocytes. This work was supported by a grant from the Ministry of Health, Labor, and Welfare of Japan. (poster)

Tumor immunotherapy targeting WT1 (Wilms’ tumor gene) peptide for bone and soft-tissue sarcomas: A preliminary report

10042 Background: WT1 (Wilms’ tumor gene) is originally isolated as a tumor-suppressor gene in a subset of Wilms’ tumors, however, recent studies have indicated its oncogenic activity in various types of cancers, and demonstrated that WT1-specific cytotoxic T lymphocytes specifically kill WT1-expressing cancer cells, thus WT1 protein can be a molecular target for cancer immunotherapy as an attractive tumor rejection antigen. However, WT1 expression in human bone and soft-tissue sarcomas has been poorly understood. Methods: 1) The expression levels of WT1 gene were examined in 36 cases of various types of human bone and soft-tissue sarcomas using quantitative RT-PCR method. They included 12 MFH (11 soft-tissue, 1 bone), 9 osteosarcomas, 6 synovial sarcomas, 4 myxoid liposarcomas, 3 MPNST, one each case of angiosarcoma and clear cell sarcoma. 2) Then, we conducted a phase I/II clinical trial of tumor-specific immunotherapy targeting WT1 peptide for various kinds of cancers including 12 patients with locally advanced and/or metastatic sarcoma (bone 2, soft-tissue 10) which showed WT1 expression and HLA -A*2402. Patients were intracutaneously injected with 3.0mg of HLA- A*2402-restricted natural/or modified 9-mer WT1 peptide emulsified with Montanide ISA51 adjuvant weekly. After 12-times injection, clinical response was evaluated by RECIST. Results: 1) 28 (78%) out of 36 cases of various types of bone and soft-tissue sarcomas overexpressed the WT1 gene, and its expression at the protein level was confirmed by immunohistochemical analysis, suggesting a possibility of immunotherapy using WT1 peptide vaccine for sarcomas. 2) The clinical responses included 4 SD and 7 PD with a case of long-standing SD over 1.5 years. One patient discontinued treatment with patient's decision. There were no patients with objective response (PR/CR). Conclusion: The present preliminary study suggested that tumor immunotherapy using WT1 peptide vaccine can be a new treatment strategy for patients with bone and soft-tissue sarcomas. No significant financial relationships to disclose.

Identification ofNTN4, TRA1, andSTC2as Prognostic Markers in Breast Cancer in a Screen for Signal Sequence Encoding Proteins

In a previous screen using a signal-trap library, we identified a number of secreted proteins up-regulated in primary tumor cells isolated from invasive breast cancers. The purpose of this study was to assess the expression of these genes in human invasive breast tumors and to determine the significance of their expression for prognosis in breast cancer. A tissue microarray containing 245 invasive breast tumors from women treated with curative surgery followed by anthracycline-based chemotherapy and hormone therapy for the estrogen receptor (ER)-positive tumors was screened by in situ hybridization with probes against thrombospondin 3 (TSP3), insulin-like growth factor binding protein 7 (IGFBP7), tumor rejection antigen 1 (TRA1), stanniocalcin 2 (STC2), and netrin 4 (NTN4). Correlations between categorical variables were done using the chi(2) test and Fisher's exact test. Cumulative survival probabilities were calculated using the Kaplan-Meier method and multivariate survival analysis was done with Cox hazard model. A series of breast cancers were also stained with NTN4 antibodies. All five genes examined were expressed in invasive breast tumor cells. NTN4 protein expression was also confirmed by immunohistochemistry. Together, these data validate the design and screening of the signal-trap library. Univariate survival analysis revealed that expressions of TRA1, STC2, and NTN4 are correlated with longer disease-free survival and that TRA1 and NTN4 are associated with longer overall survival. Multivariate analysis showed that NTN4 is an independent prognostic factor of overall survival. This article describes the identification of three secreted proteins, NTN4, TRA1, and STC2, as potential novel prognostic markers in breast cancer.

Spontaneous and vaccine induced AFP-specific T cell phenotypes in subjects with AFP-positive hepatocellular cancer.

We are investigating the use of Alpha Fetoprotein (AFP) as a tumor rejection antigen for hepatocellular carcinoma (HCC). We recently completed vaccination of 10 AFP+/HLA-A2.1+ HCC subjects with AFP peptide-pulsed autologous dendritic cells (DC). There were increased frequencies of circulating AFP-specific T cells and of IFNgamma-producing AFP-specific T cells after vaccination. In order to better understand the lack of association between immune response and clinical response, we have examined additional aspects of the AFP immune response in patients. Here, we have characterized the cell surface phenotype of circulating AFP tetramer-positive CD8 T cells and assessed AFP-specific CD4 function. Before vaccination, HCC subjects had increased frequencies of circulating AFP-specific CD8 T cells with a range of naïve, effector, central and effector memory phenotypes. Several patients had up-regulated activation markers. A subset of patients was assessed for phenotypic changes pre- and post-vaccination, and evidence for complete differentiation to effector or memory phenotype was lacking. CD8 phenotypic and cytokine responses did not correlate with level of patient serum AFP antigen (between 74 and 463,040 ng/ml). Assessment of CD4+ T cell responses by ELISPOT and multi-cytokine assay did not identify any spontaneous CD4 T cell responses to this secreted protein. These data indicate that there is an expanded pool of partially differentiated AFP-specific CD8 T cells in many of these HCC subjects, but that these cells are largely non-functional, and that a detectable CD4 T cell response to this secreted oncofetal antigen is lacking.

DNA Vaccines Encoding Ii-PADRE Generates Potent PADRE-specific CD4+ T-Cell Immune Responses and Enhances Vaccine Potency

DNA Vaccines Encoding Ii-PADRE Generates Potent PADRE-specific CD4+ T-Cell Immune Responses and Enhances Vaccine Potency

Hydrodynamic Limb Vein Delivery of a Xenogeneic DNA Cancer Vaccine Effectively Induces Antitumor Immunity

Tumor-associated antigens (TAA) are typically poorly immunogenic "self" antigens. An effective strategy to break tolerance and induce antitumor immunity is by genetic vaccination, employing the orthologous TAA-sequence from a different species. We recently developed a clinically relevant approach for intravascular hydrodynamic limb vein (HLV) delivery of nucleic acids to skeletal muscle. Using the human gp100 xenogeneic TAA in the murine B16 melanoma model, we show that genetic vaccination of mice by HLV plasmid DNA delivery was highly effective at breaking tolerance against the homologous murine gp100 (mgp100) TAA and induced prophylactic antitumor protection. HLV vaccination resulted in an anti-hgp100 humoral and cellular response, with 4-5% of CD8(+) T cells being gp100(25-33)-epitope-specific. Vaccinated animals demonstrated in vivo cytolytic activity against human and mgp100(25-33) peptide-pulsed targets. Antitumor immunity could be adoptively transferred by splenocytes from human gp100-vaccinated animals. Furthermore, a durable antitumor memory response was established as approximately 3% of CD8(+) T cells were gp100(25-33) antigen-specific in mice 6 months after vaccination. Following a single HLV human gp100 DNA boost, this level increased to approximately 17% and protected animals from subsequent B16 tumor rechallenge. Our results warrant further consideration of HLV as a clinically relevant method for cancer gene therapy.

Cytotoxic T-Lymphocyte Responses to Human Papillomavirus Type 16 E5 and E7 Proteins and HLA-A*0201-Restricted T-Cell Peptides in Cervical Cancer Patients

Previously, we found that human papillomavirus type 16 (HPV-16) E5 protein is a tumor rejection antigen and can induce cytotoxic T-lymphocyte (CTL) activity. Therefore, in this study, human leukocyte antigen A*0201 (HLA-A*0201)-restricted human CTL epitopes of HPV-16 E5 protein were identified using a bioinformatics approach, and the abilities of these predicted peptides to induce an immune response in HLA-A*0201 transgenic mice were confirmed by assaying E5-specific CTLs and in vitro-generated CTLs from normal peripheral blood T lymphocytes of HLA-A2-positive human donors. Second, the CTL responses to HLA-A*0201 CTL epitopes (E5 63-71 and E7 11-20) were examined in HPV-16-infected patients with HLA-A2. Third, the effect of HLA-A-type alleles on CTL activities in response to the entire E5 and E7 proteins was examined in cervical cancer patients. E5 and E7 peptides (but not the whole proteins) stimulated E5- and E7-specific CTL recall responses in HPV-16- and HLA-A2-positive cervical cancer patients, and HPV-16 E5 and E7 proteins stimulated naïve T cells in HPV-16-negative cervical cancer patients with HLA-A11 and -A24 haplotypes. In summary, this is the first demonstration that E5 63-71 is an HLA-A*0201-restricted T-cell epitope of HPV-16 E5.

166 EXPRESSION OF PLURIPOTENCY-DETERMINING FACTORS IN IN VITRO-PRODUCED BUFFALO EMBRYOS

Embryonic stem cells (ESCs) are derived from the inner cell mass (ICM) of blastocysts. These are pluripotent cells that retain the ability to differentiate into all cell types. Various cell surface antigens, the expressions of which have been widely used as markers to monitor the pluripotency of ESCs, include Oct-4, stage-specific embryonic antigens (SSEAs) such as SSEA-1, SSEA-3, and SSEA-4, and tumor rejection antigens (TRAs) such as TRA-1-60 and TRA-1-81. In this study, the cell surface expression patterns of these markers were examined in in vitro-produced buffalo embryos at the 2-, 4-, 8- to 16-cell, morula, and blastocyst stages using immunofluorescence microscopy. Oocytes obtained from slaughterhouse buffalo ovaries were subjected to IVM and IVF, following which the cleaved embryos were cultured for 9 days for production of embryos at different stages (n = 246). The embryos were fixed in 4% paraformaldehyde in Dulbecco's phosphate-buffered saline (DPBS) for 30 min, permeabilized by treatment with 0.1% Triton X-100 in DPBS for 30 min, and incubated first with the blocking solution (4% normal goat serum) for 30 min and then with the primary antibody (Oct-4: clone 9E3; SSEA-1: MC-480; SSEA-3: MC-631; SSEA-4: MC-813-70; TRA-1-60: clone TRA-1-60; and TRA-1-81: clone TRA-1-81, Chemicon� Inc., Temecula, CA, USA) at a dilution of 1:10 to 1:20 for 1 h. After being washed with DPBS, the embryos were incubated with appropriate FITC-labeled second antibody (anti-rat IgM or anti-mouse IgG or IgM, diluted 1:100 to 1:200) for 1 h and then examined under a fluorescence microscope. Oct-4 expression was detected at all embryonic stages starting from the 2-cell to the blastocyst stage, in which ICM, but not trophectoderm cells, exhibited a strong expression. SSEA-4 signal was found to be strongest at the 2-cell stage, with continued expression at all intermediate stages until the blastocyst stage in which there was a strong expression in ICM cells. In contrast, all of the embryonic stages were found to be negative for SSEA-3 expression. The SSEA-1 signal was present at all of the embryonic stages but was very weak. Expression of TRA-1-60 and TRA-1-81, which was detected only on the inner surface of the zona pellucida and in the perivitelline space in early embryonic stages, was absent in morulae and blastocysts. The results of this study indicate that the pluripotency-determining markers are differentially expressed in buffalo embryos and that the pattern of their expression is distinct from that of murine and human embryos but resembles to some extent that of goat embryos. Comparison of the expression pattern of these markers needs to be done between embryonic cells and ESCs for a better understanding of their developmental regulation.

290 A CYNOMOLGUS MONKEY EMBRYONIC STEM CELL LINE DERIVED FROM A SINGLE BLASTOMERE

The establishment of most embryonic stem (ES) cell lines requires the destruction of embryos. Some ES cell lines in mice and humans are currently derived from a single blastomere, so that remaining blastomeres can still develop into fetuses. However, the procedures currently in use for establishing these lines are very complicated, and other ES cell lines from the same species are needed (Chung et al. 2006 Nature 439, 216–219; Klimanskaya et al. 2006 Nature 444, 481–485). The objective of this study was to devise a method simpler than those previously described for establishing ES cell lines from a single blastomere in the cynomolgus monkey. Controlled ovarian stimulation and oocyte recovery have been described previously by Torii et al. (2000 Primates 41, 39–47). Cumulus-free mature oocytes were fertilized by intracytoplasmic sperm injection (ICSI), and then cultured at 38�C in 5% CO2, 5% O2 for 2 days. The zona pellucida of 4- to 5-cell-stage embryos was disrupted using acidic Tyrode's solution, and individual blastomeres were separated from the denuded embryos using trypsin. These blastomeres were cultured on mitomycin-C-treated mouse embryonic fibroblasts and ES medium containing adrenocorticotropic hormone (ACTH) (Ogawa et al. 2004 Genes to Cells 9, 471–477). After the formation of initial outgrowths, half of the medium was changed every other day until the outgrowths reached approximately 100 cells. Passage of putative monkey ES cells was performed by mechanical dispersion of the colonies and transfer to fresh feeders every 3–4 days until there were enough cells for enzymatic dispersion. One stable ES cell line was obtained from two 4- or 5-cell-stage embryos using ES medium containing ACTH. The morphology of this ES cell colony was consistent with the monkey ES cell colony previous described by Suemori et al. (2001 Dev. Dynamics 222, 273–279). The ES cell line was passaged more than 17 times, and the morphology of the ES cell colony did not differ between the first and seventeenth passages. The ES cells showed normal karyotype and retained pluripotency markers for primate ES cells including octamer-binding protein 4 (Oct-4), stage-specific embryonic antigen (SSEA)-4, tumor-rejection antigen (TRA)-1-60, and TRA-1-81. We are presently confirming whether this ES cell line possesses potencies to differentiate in all three embryonic germ layers using both an in vitro assay and teratoma formation. Here we showed that cynomolgus monkey ES cells can be derived from a single blastomere, without co-culturing another ES cell line, as has been done in previous studies on mice and humans. This method allows the establishment of ES cell lines from a single blastomere, leaving the other blastomeres available for embryo transfer. Thus, the method described here is simpler than previously described methods and alleviates some ethical concerns.