Progressive changes in the protein expression profile of alveolar septa in early-stage lung adenocarcinoma.

BACKGROUND: Adenocarcinomas show a stepwise progression from atypical adenomatous hyperplasia (AAH) through adenocarcinoma in situ (AIS) to invasive adenocarcinoma (IA). Immunoglobulin superfamily containing leucine-rich repeat (ISLR) is a marker of tumor-restraining cancer-associated fibroblasts (CAFs), which are distinct from conventional, strongly α-smooth muscle actin (αSMA)-positive CAFs. Fibroblast activation protein (FAP) has been focused on as a potential therapeutic and diagnostic target of CAFs. METHODS: We investigated the changes in protein expression during adenocarcinoma progression in the pre-existing alveolar septa by assessing ISLR, αSMA, and FAP expression in normal lung, AAH, AIS, and IA. Fourteen AAH, seventeen AIS, and twenty IA lesions were identified and randomly sampled. Immunohistochemical analysis was performed to evaluate cancer-associated changes and FAP expression in the pre-existing alveolar structures. RESULTS: Normal alveolar septa expressed ISLR. The ISLR level in the alveolar septa decreased in AAH and AIS tissues when compared with that in normal lung tissue. The αSMA-positive area gradually increased from the adjacent lung tissue (13.3% ± 15%) to AIS (87.7% ± 14%), through AAH (70.2% ± 21%). Moreover, the FAP-positive area gradually increased from AAH (1.69% ± 1.4%) to IA (11.8% ± 7.1%), through AIS (6.11% ± 5.3%). Protein expression changes are a feature of CAFs in the pre-existing alveolar septa that begin in AAH. These changes gradually progressed from AAH to IA through AIS. CONCLUSIONS: FAP-positive fibroblasts may contribute to tumor stroma formation in early-stage lung adenocarcinoma, and this could influence the development of therapeutic strategies targeting FAP-positive CAFs for disrupting extracellular matrix formation.

Prediction for oxaliplatin-induced liver injury using patient-derived liver organoids.

BACKGROUND: Liver injury associated with oxaliplatin (L-OHP)-based chemotherapy can significantly impact the treatment outcomes of patients with colorectal cancer liver metastases, especially when combined with surgery. To date, no definitive biomarker that can predict the risk of liver injury has been identified. This study aimed to investigate whether organoids can be used as tools to predict the risk of liver injury. METHODS: We examined the relationship between the clinical signs of L-OHP-induced liver injury and the responses of patient-derived liver organoids in vitro. Organoids were established from noncancerous liver tissues obtained from 10 patients who underwent L-OHP-based chemotherapy and hepatectomy for colorectal cancer. RESULTS: Organoids cultured in a galactose differentiation medium, which can activate the mitochondria of organoids, showed sensitivity to L-OHP cytotoxicity, which was significantly related to clinical liver toxicity induced by L-OHP treatment. Organoids from patients who presented with a high-grade liver injury to the L-OHP regimen showed an obvious increase in mitochondrial superoxide levels and a significant decrease in mitochondrial membrane potential with L-OHP exposure. L-OHP-induced mitochondrial oxidative stress was not observed in the organoids from patients with low-grade liver injury. CONCLUSIONS: These results suggested that L-OHP-induced liver injury may be caused by mitochondrial oxidative damage. Furthermore, patient-derived liver organoids may be used to assess susceptibility to L-OHP-induced liver injury in individual patients.

Potent CTLs can be induced against tumor cells in an environment of lower levels of systemic MFG-E8.

The direction and magnitude of immune responses are critically affected when dead cells are disposed of. Milk fat globule-epidermal growth factor-factor 8 (MFG-E8) promotes the engulfment of apoptotic normal and cancerous cells without inducing inflammation. We have previously reported that a certain proportion of the cancer cells express abundant MFG-E8, and that such expression is associated with the shorter survival of patients with esophageal cancer who had received chemotherapy before surgery. However, the influence of tumor-derived and systemically existing MFG-E8 on antitumor immune responses has not yet been fully investigated. Herein, we showed that CTL-dependent antitumor immune responses were observed in mice with no or decreased levels of systemic MFG-E8, and that such responses were enhanced further with the administration of anti-PD-1 antibody. In mice with decreased levels of systemic MFG-E8, the dominance of regulatory T cells in tumor-infiltrating lymphocytes was inverted to CD8+ T cell dominance. MFG-E8 expression by tumor cells appears to affect antitumor immune responses only when the level of systemic MFG-E8 is lower than the physiological status. We have also demonstrated in the clinical setting that lower levels of plasma MFG-E8, but not MFG-E8 expression in tumor cells, before the treatment was associated with objective responses to anti-PD-1 therapy in patients with non-small cell lung cancer. These results suggest that systemic MFG-E8 plays a critical role during the immunological initiation process of antigen-presenting cells to increase tumor-specific CTLs. Regulation of the systemic level of MFG-E8 might induce efficient antitumor immune responses and enhance the potency of anti-PD-1 therapy.

A Method for Extending Target Regions of Genomic Profiling by Combining a Custom Probe Pool with a Commercial Targeted Panel.

BACKGROUND: Next-generation sequencing (NGS)-based genomic profiling is becoming widespread in determining treatment policies for patients with tumors. Commercially available gene panels for pan-tumor targets comprise hundreds of tumor-related genes but frequently lack genes of interest in specific tumor types. In this study, we demonstrate a method for extending target regions of genomic profiling by combining a custom probe pool with a commercial targeted panel. METHODS: We used TruSight Oncology 500 (TSO500) as a commercial targeted panel and a custom probe pool designed for all exons of the SMARCA2 gene. Sequencing libraries of custom targets were constructed using a portion of the TSO500 library solution before the hybridization-capture process. After hybridization capture, both libraries were combined and sequenced using a next-generation sequencer. RESULTS: Sequencing results showed that >96.8% and 100% of the target exons were covered at a depth of over 100× using the TSO500 and custom panels, respectively. The custom panels had slightly better median exon coverage than the TSO500. The combined libraries of the custom and TSO500 panels showed a mapped read ratio close to the mixing ratio. Analysis of mutation-free regions showed similar accuracies between the TSO500 and custom panels regarding variant calling. CONCLUSIONS: Our devised method easily and affordably extends the targets beyond a ready-made panel. This method provides a valuable solution until the widespread adoption of whole-exome sequencing, which is costly for large target sizes.

Metabolic clogging of mannose triggers dNTP loss and genomic instability in human cancer cells.

Mannose has anticancer activity that inhibits cell proliferation and enhances the efficacy of chemotherapy. How mannose exerts its anticancer activity, however, remains poorly understood. Here, using genetically engineered human cancer cells that permit the precise control of mannose metabolic flux, we demonstrate that the large influx of mannose exceeding its metabolic capacity induced metabolic remodeling, leading to the generation of slow-cycling cells with limited deoxyribonucleoside triphosphates (dNTPs). This metabolic remodeling impaired dormant origin firing required to rescue stalled forks by cisplatin, thus exacerbating replication stress. Importantly, pharmacological inhibition of de novo dNTP biosynthesis was sufficient to retard cell cycle progression, sensitize cells to cisplatin, and inhibit dormant origin firing, suggesting dNTP loss-induced genomic instability as a central mechanism for the anticancer activity of mannose.

In order to grow and divide, cells require a variety of sugars. Breaking down sugars provides energy for cells to proliferate and allows them to make more complex molecules, such as DNA. Although this principle also applies to cancer cells, a specific sugar called mannose not only inhibits cancer cell division but also makes them more sensitive to chemotherapy. These anticancer effects of mannose are particularly strong in cells lacking a protein known as MPI, which breaks down mannose. Evidence from honeybees suggests that a combination of mannose and low levels of MPI leads to a build-up of a modified form of mannose, called mannose-6-phosphate, within cells. As a result, pathways required to release energy from glucose become disrupted, proving lethal to these insects. However, it was not clear whether the same processes were responsible for the anticancer effects of mannose. To investigate, Harada et al. removed the gene that encodes the MPI protein in two types of human cancer cells. The experiments showed that mannose treatment was not lethal to these cells but overall slowed the cell cycle ­ a fundamental process for cell growth and division. More detailed biochemical experiments showed that cancer cells with excess mannose-6-phosphate could not produce the molecules required to make DNA. This prevented them from doubling their DNA ­ a necessary step for cell division ­ and responding to stress caused by chemotherapy. Harada et al. also noticed that cancer cells lacking MPI did not all react to mannose treatment in exactly the same way. Therefore, future work will address these diverse reactions, potentially providing an opportunity to use the mannose pathway to search for new cancer treatments.

Establishment of organoids using residual samples from saline flushes during endoscopic ultrasound-guided fine-needle aspiration in patients with pancreatic cancer.

Background and study aims In patients with pancreatic cancer (PC), patient-derived organoid cultures can be useful tools for personalized drug selection and preclinical evaluation of novel therapies. To establish a less invasive method of creating organoids from a patient's tumor, we examined whether PC organoids can be established using residual samples from saline flushes (RSSFs) during endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA). Methods Five patients with PC who underwent EUS-FNA were enrolled in a prospective study conducted at our institution. RSSFs obtained during EUS-FNA procedures were collected. An organoid culture was considered as established when ≥ 5 passages were successful. Organoid-derived xenografts were created using established organoids. Results EUS-FNA was performed using a 22- or 25-gauge lancet needle without complications. Patient-derived organoids were successfully established in four patients (80.0 %) with the complete medium and medium for the selection of KRAS mutants. Organoid-derived xenografts were successfully created and histologically similar to EUS-FNA samples. Conclusions Patient-derived PC organoids were successfully established using EUS-FNA RSSFs, which are produced as a byproduct of standard manipulations, but are usually not used for diagnosis. This method can be applied to all patients with PC, without additional invasive procedures, and can contribute to the development of personalized medicine and molecular research.

Atezolizumab with bevacizumab, paclitaxel and carboplatin was effective for patients with SMARCA4-deficient thoracic sarcoma.

SMARCA4-deficient thoracic sarcoma (DTS) is a recently noted progressive thoracic malignancy. We recently experienced three cases of SMARCA4-DTS who were treated with atezolizumab in combination with bevacizumab, paclitaxel and carboplatin (ABCP) as the first-line therapy. Immunohistopathological analysis revealed absent expression of SMARCA4 in all cases. The tumor mutational burden was over 11/Mb and mutations in SMARCA4 and TP53 were detected in all three cases. Partial response to ABCP treatment was observed in all three cases, with a progression-free survival of approximately 6 months or longer and a continuous response of 1 year or longer in one case. The first-line ABCP treatment demonstrated durable efficacy in SMARCA4-DTS regardless of the degree of PD-L1 expression.

Lay abstract Lung cancer is the leading cause of cancer-related death worldwide. Among them, SMARCA4-deficient thoracic sarcoma (DTS), which lacks SMARCA4 expression and exhibits an undifferentiated carcinoma histology, is a recently identified subtype of lung cancer. It tends to occur in younger people with heavy smoking status and has been reported to recur quickly and have a poor prognosis even after chemotherapy, radiation therapy or surgery. There is no effective molecularly targeted agent for SMARCA4-DTS and the identification of an effective therapy is required. Here, we report the clinical features and genomic information of three SMARCA4-DTS cases in which atezolizumab with bevacizumab, paclitaxel and carboplatin treatment was effective. This report suggests the efficacy of atezolizumab with bevacizumab, paclitaxel and carboplatin treatment compared with conventional chemotherapy.

Production of an antibody Fab fragment using 2A peptide in insect cells.

Antibody Fab fragments consist of heavy chain (Hc) and light chain (Lc) polypeptides assembled with a disulphide bond. The production of a recombinant Fab fragment requires the simultaneous expression of two genes encoding both an Hc and an Lc in the same host cell. In the present study, we investigated the production of Fab fragments in lepidopteran insect cells using a bicistronic plasmid vector carrying the Hc and Lc genes linked with a 2A self-cleaving peptide sequence from the porcine teschovirus-1. We also examined the arrangement of a GSG spacer sequence and a furin cleavage site sequence with the 2A sequence. Western blot analysis and enzyme-linked immunosorbent assay (ELISA) of culture supernatants showed that Trichoplusia ni BTI-TN-5B1-4 (High Five) cells transfected with a plasmid in which the Hc and Lc genes were joined by the 2A sequence successfully secreted Fab fragments with antigen-binding activity after self-cleavage of the 2A peptide. The GSG linker enhanced 2A cleavage efficiency, and the furin recognition site was useful for removal of 2A residues from the Hc. Transfection with a single plasmid that contained sequences for GSG, the furin cleavage site, GSG, and the 2A peptide between the Hc and Lc genes exhibited a higher productivity than co-transfection with a set of plasmids separately carrying the Hc or Lc gene. These results demonstrate that bicistronic expression with the appropriate combination of a furin recognition site, GSG linkers, and a 2A peptide may be an effective way to efficiently produce recombinant antibody molecules in insect cells.

Immunoregulatory influence of abundant MFG-E8 expression by esophageal cancer treated with chemotherapy.

Milk fat globule-epidermal growth factor factor 8 (MFG-E8) is secreted from macrophages and is known to induce immunological tolerance mediated by regulatory T cells. However, the roles of the MFG-E8 that is expressed by cancer cells have not yet been fully examined. Expression of MFG-E8 was examined using immunohistochemistry in surgical samples from 134 patients with esophageal squamous cell carcinoma. The relationships between MFG-E8 expression levels and clinicopathological factors, including tumor-infiltrating lymphocytes, were evaluated. High MFG-E8 expression was observed in 23.9% of the patients. The patients with tumors highly expressing MFG-E8 had a significantly higher percentage of neoadjuvant chemotherapy (NAC) history (P < .0001) and shorter relapse-free survival (P = 0.012) and overall survival (OS; P = .0047). On subgroup analysis, according to NAC history, patients with high MFG-E8 expression had significantly shorter relapse-free survival (P = .027) and OS (P = .0039) only when they had been treated with NAC. Furthermore, tumors with high MFG-E8 expression had a significantly lower ratio of CD8+ T cells/regulatory T cells in tumor-infiltrating lymphocytes (P = .042) only in the patients treated with NAC, and those with a lower ratio had a shorter OS (P = .026). High MFG-E8 expression was also found to be an independent prognostic factor in multivariate analysis. The abundant MFG-E8 expression in esophageal squamous cell carcinoma might have a negative influence on the long-term survival of patients after chemotherapy by affecting T-cell regulation in the tumor microenvironment.

Negative influence of programmed death-1-ligands on the survival of esophageal cancer patients treated with chemotherapy.

The programmed death-1/programmed death-1 ligands (PD-1/PD-L) pathway plays an important role in immunological tumor evasion. However, the clinical significance of the PD-L (L1 and L2) expression in esophageal cancer treated with chemotherapy has not been fully investigated. We examined the expression of PD-L of the primary tumors obtained from 180 esophageal cancer patients who underwent radical resection with or without neoadjuvant chemotherapy (NAC) using immunohistochemical staining. The relationship between the expression patterns and clinico-pathological characteristics was examined. In the present study, 53 patients (29.4%) and 88 patients (48.3%) were classified into positive for PD-L1 and PD-L2 expression, respectively. In all the patients examined, overall survival rates of the patients with tumors positive for PD-L1 or PD-L2 were significantly worse than those with tumors negative for PD-L1 or PD-L2 (P = 0.0010 and P = 0.0237, respectively). However, subgroup analysis showed that these tendencies are only found in the patients treated with NAC, and not in those without NAC. The patients with positive PD-L1 expression had a significantly higher rate of NAC history (P = 0.0139), but those with positive PD-L2 expression did not have a significantly high rate of NAC history (P = 0.6127). There is no significant relationship between PD-L1 expression and response to chemotherapy (P = 0.3118), but patients with positive PD-L2 expression had significantly inferior responses to chemotherapy (P = 0.0034). The PD-1/PD-L pathway might be an immunological mechanism associated with the long-term effectiveness of chemotherapy in esophageal cancer patients. Further investigation into the roles of PD-1 pathway in chemotherapy could lead to the development of better treatment options for this disease.

Prolongation of overall survival in advanced lung adenocarcinoma patients with the XAGE1 (GAGED2a) antibody.

PURPOSE: The cancer/testis antigen XAGE1 (GAGED2a) is expressed in approximately 40% of advanced lung adenocarcinomas. We investigated the clinical relevance of the XAGE1 (GAGED2a) immune responses in patients with advanced lung adenocarcinoma. EXPERIMENTAL DESIGN: The XAGE1 (GAGED2a) antigen expression and EGFR mutation were determined with tumor tissues. The XAGE1 (GAGED2a) antibody and T-cell immune responses, as well as immune cell phenotypes, were analyzed with blood samples. Patients with EGFR wild-type (EGFRwt) tumors were treated with conventional platinum-based doublet chemotherapy and patients with EGFR-mutated (EGFRmt) tumors were treated with EGFR-TKI and conventional chemotherapy. The overall survival (OS) rates of the antibody-positive and -negative patients were investigated. RESULTS: The results showed that the OS of antibody-positive patients was prolonged significantly compared with that of antibody-negative patients with either XAGE1 (GAGED2a) antigen-positive EGFRwt (31.5 vs. 15.6 months, P = 0.05) or EGFRmt (34.7 vs. 11.1 months, P = 0.001) tumors. Multivariate analysis showed that the presence of the XAGE1 (GAGED2a) antibody was a strong predictor for prolonged OS in patients with XAGE1 (GAGED2a) antigen-positive tumors and in patients with either EGFRwt or EGFRmt tumors. On the other hand, XAGE1 (GAGED2a) antigen expression was a worse predictor in patients with EGFRmt tumors. Phenotypic and functional analyses of T cells indicated immune activation in the antibody-positive patients. CONCLUSIONS: The findings suggest that production of the XAGE1 (GAGED2a) antibody predicts good prognosis for patients with lung adenocarcinoma as an immune biomarker and the protective effect of this naturally occurring immune response supports the concept of immunotherapy.

Vaccination with NY-ESO-1 overlapping peptides mixed with Picibanil OK-432 and montanide ISA-51 in patients with cancers expressing the NY-ESO-1 antigen.

We conducted a clinical trial of an NY-ESO-1 cancer vaccine using 4 synthetic overlapping long peptides (OLP; peptides #1, 79-108; #2, 100-129; #3, 121-150; and #4, 142-173) that include a highly immunogenic region of the NY-ESO-1 molecule. Nine patients were immunized with 0.25 mg each of three 30-mer and a 32-mer long NY-ESO-1 OLP mixed with 0.2 KE Picibanil OK-432 and 1.25 mL Montanide ISA-51. The primary endpoints of this study were safety and NY-ESO-1 immune responses. Five to 18 injections of the NY-ESO-1 OLP vaccine were well tolerated. Vaccine-related adverse events observed were fever and injection site reaction (grade 1 and 2). Two patients showed stable disease after vaccination. An NY-ESO-1-specific humoral immune response was observed in all patients and an antibody against peptide #3 (121-150) was detected firstly and strongly after vaccination. NY-ESO-1 CD4 and CD8 T-cell responses were elicited in these patients and their epitopes were identified. Using a multifunctional cytokine assay, the number of single or double cytokine-producing cells was increased in NY-ESO-1-specific CD4 and CD8 T cells after vaccination. Multiple cytokine-producing cells were observed in PD-1 (-) and PD-1 (+) CD4 T cells. In conclusion, our study indicated that the NY-ESO-1 OLP vaccine mixed with Picibanil OK-432 and Montanide ISA-51 was well tolerated and elicited NY-ESO-1-specific humoral and CD4 and CD8 T-cell responses in immunized patients.

Production of NY-ESO-1 peptide/DRB1*08:03 tetramers and ex vivo detection of CD4 T-cell responses in vaccinated cancer patients.

We established CD4 T-cell clones, Mz-1B7, and Ue-21, which recognized the NY-ESO-1 121-138 peptide from peripheral blood mononuclear cells (PBMCs) of an esophageal cancer patient, E-2, immunized with an NY-ESO-1 protein and determined the NY-ESO-1 minimal epitopes. Minimal peptides recognized by Mz-1B7 and Ue-21 were NY-ESO-1 125-134 and 124-134, respectively, both in restriction to DRB1*08:03. Using a longer peptide, 122-135, and five other related peptides, including either of the minimal epitopes recognized by the CD4 T-cell clones, we investigated the free peptide/DR recognition on autologous EBV-B cells as APC and peptide/DR tetramer binding. The results showed a discrepancy between them. The tetramers with several peptides recognized by either Mz-1B7 or the Ue-21 CD4 T-cell clone did not bind to the respective clone. On the other hand, unexpected binding of the tetramer with the peptide not recognized by CD4 T-cells was observed. The clone Mz-1B7 did not recognize the free peptide 122-135 on APC, but the peptide 122-135/DRB1*08:03 tetramer bound to the TCR on those cells. The failure of tetramer production and the unexpected tetramer binding could be due to a subtly modified structure of the peptide/DR tetramer from the structure of the free peptide/DR molecule. We also demonstrated that the NY-ESO-1 123-135/DRB1*08:03 tetramer detected ex vivo CD4 T-cell responses in PBMCs from patients after NY-ESO-1 vaccination in immunomonitoring.

TLR4 and NLRP3 inflammasome activation in monocytes by N-propionyl cysteaminylphenol-maleimide-dextran (NPCMD).

BACKGROUND: N-propionyl cysteaminylphenol-maleimide-dextran (NPCMD) is a toxic tyrosinase substrate developed to treat melanoma. OBJECTIVE: We investigated the effect of NPCMD on innate immune responses in monocytes. METHODS: CD14⁺ monocytes and a monocytic cell line, THP-1, were stimulated with NPCMD in vitro. Cytokines in the culture supernatants were determined by ELISA and flow cytometry. RESULTS: NPCMD stimulated CD14⁺ monocytes and THP-1 cells to secrete TNFα, IL-6 and IL-8, but not IL-10 or IL-12. TNFα secretion from THP-1 cells stimulated with NPCMD was inhibited by addition of an anti-TLR4 mAb in culture. Moreover, NPCMD stimulated production of pro-IL-1ß in CD14⁺ monocytes and monocytic cell line THP-1 cells and activated the NLRP3-inflammasome, resulting in production of mature IL-1ß. Use of ASC and NLRP3-deficient THP-1 cell lines established involvement of the NLRP3 inflammasome in an IL-1ß secretion in treatment with NPCMD. Inhibition of IL-1ß secretion by an endocytosis inhibitor, cytochalasin B, and a lysosomal enzyme cathepsin B inhibitor, CA-074 Me, suggested the involvement of lysosomal rupture and leakage of cathepsin B into the cytosol in NLRP3 activation by NPCMD. CONCLUSION: The immunopotentiating effect of NPCMD mediated by TLR4 and NLRP3 inflammasome activation could be useful for eliciting effective adaptive immune responses against melanoma and other tumors.

Spontaneous antibody, and CD4 and CD8 T-cell responses against XAGE-1b (GAGED2a) in non-small cell lung cancer patients.

The spontaneous immune responses against XAGE-1b (GAGED2a) were analyzed in non-small cell lung cancer (NSCLC) patients. An antibody response against XAGE-1b (GAGED2a) was observed in 10% (20/200) of NSCLC patients and in 19% (13/69) of stage IIIB/IV lung adenocarcinoma patients. A CD4 T-cell response was detected in 88% (14/16) and a CD8 T-cell response in 67% (6/9) in the XAGE-1b (GAGED2a) antibody-positive patients examined. Frequent antibody responses and CD4 and CD8 T-cell responses in XAGE-1b (GAGED2a) antibody-positive patients indicate the strong immunogenicity of the XAGE-1b (GAGED2a) antigen in NSCLC patients. We established T-cell clones from PBMCs of antibody-positive patients and determined the DRB1*04:05-restricted XAGE-1b (GAGED2a) 18-31 peptide (14-mer) as a CD4 T cell epitope and the A*02:06-restricted XAGE-1b (GAGED2a) 21-29 peptide (9-mer) as a CD8 T cell epitope. As for peptide recognition, CD4 and CD8 T-cell clones responded to naturally processed antigen. The CD4 T-cell clone recognized DCs pulsed with the synthetic protein or a lysate from XAGE-1b-transfected 293T cells. The CD8 T-cell clone showed cytotoxicity against a tumor expressing XAGE-1b (GAGED2a) and the appropriate HLA class I allele. These findings establish XAGE-1b (GAGED2a) as a promising target for a lung cancer vaccine.

Three novel NY-ESO-1 epitopes bound to DRB1*0803, DQB1*0401 and DRB1*0901 recognized by CD4 T cells from CHP-NY-ESO-1-vaccinated patients.

Three novel NY-ESO-1 CD4 T cell epitopes were identified using PBMC obtained from patients who were vaccinated with a complex of cholesterol-bearing hydrophobized pullulan (CHP) and NY-ESO-1 protein (CHP-NY-ESO-1). The restriction molecules were determined by antibody blocking and using various EBV-B cells with different HLA alleles as APC to present peptides to CD4 T cells. The minimal epitope peptides were determined using various N- and C-termini truncated peptides deduced from 18-mer overlapping peptides originally identified for recognition. Those epitopes were DRB1*0901-restricted NY-ESO-1 87-100, DQB1*0401-restricted NY-ESO-1 95-107 and DRB1*0803-restricted NY-ESO-1 124-134. CD4 T cells used to determine those epitope peptides recognized EBV-B cells or DC that were treated with recombinant NY-ESO-1 protein or NY-ESO-1-expressing tumor cell lysate, suggesting that the epitope peptides are naturally processed. These CD4 T cells showed a cytokine profile with Th1 characteristics. Furthermore, NY-ESO-1 87-100 peptide/HLA-DRB1*0901 tetramer staining was observed. Multiple Th1-type CD4 T cell responses are beneficial for inducing effective anti-tumor responses after NY-ESO-1 protein vaccination.