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Dendritic cell-based cancer vaccines (DC vaccines) have been proved efficient and safe in immunotherapy of various cancers, including melanoma, ovarian and prostate cancer. However, the clinical responses were not always satisfied. Here we proposed a novel strategy to prepare DC vaccines. In the present study, a fusion protein SNU containing a secretin-penetratin (SecPen) peptide, NY-ESO-1 and ubiquitin was designed and expressed. To establish the DC vaccine (DC-SNU), the mouse bone marrow-derived DCs (BMDCs) were isolated, pulsed with SNU and maturated with cytokine cocktail. Then peripheral blood mononuclear cells (PBMCs) from C57BL/6 mice inoculated intraperitoneally with DC-SNU were separated and cocultured with MC38/MC38
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Objective@#To detect the expression of New York esophageal squamous cell carcinoma antigen 1 (NY-ESO-1) in common types of mesenchymal myxoid tumors, and to investigate its significance in the diagnosis and differential diagnosis of myxoid liposarcoma.@*Methods@#A total of 43 formalin-fixed paraffin-embedded samples of mesenchymal myxoid tumors from the Affiliated Hospital of Qingdao University and Qingdao Municipal Hospital ranging between 2010 and 2017 were selected. NY-ESO-1 expression was detected by immunohistochemical staining. DDIT3 gene status was detected by fluorescence in situ hybridization (FISH). NY-ESO-1 mRNA was detected by reverse transcription-PCR (RT-PCR).@*Results@#Histopathology and FISH results confirmed that there were 11 cases of myxoid liposarcoma and 32 other types (including 7 cases of well-differentiated liposarcoma, 1 dedifferentiated liposarcoma, 3 lipomas, 2 lipoblastomas and 19 non-adipocytic tumors). Immunohistochemical staining showed that the positive expression propotion of NY-ESO-1 in myxoid liposarcoma was 11/11, and the positive location was the cytoplasm and nucleus of lipoblast cells. The expression intensity is higher in regions with round cell differentiation. Among the 32 cases of other mesenchymal myxoid tumors, only one well-differentiated liposarcoma showed positive immunoreactivity for NY-ESO-1. RT-PCR confirmed that 7 cases of myxoid liposarcoma (7/11) and one well-differentiated liposarcoma (1/7) had NY-ESO-1 mRNA expression.@*Conclusions@#NY-ESO-1 is positively expressed in myxoid liposarcoma. It can be served as a useful marker for the diagnosis and differential diagnosis of myxoid liposarcoma.
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Objective:To exptore the potential of autologous dendritic cells (DCs) pulsed with caner/ testis antigen NY-ESO-1 peptides in inducing specific cytotoxic T lymphocyte (CTLs) response and an tineoplastic immune function of specific CTLs.Methods:Fifteen patients with Ⅱ to Ⅲ stage positive HLA-A0201 + and NY-ESO-1 + were enrolled in the Cancer Hospital Chinese Academy of Medical Sciences on the basis of preclinical experiments from November 2014 to October 2015,and their peripheral blood mononuclear cells (PBMCs) and peripheral blood lymphocytes (PBLs) were isolated.The PBMCs were induced into DCs and pulsed with NY-ESO-1 peptide.The phenotypes of DCs were stained with antibodies against HLA-DR+ CD11c +,CD80 +,CD83 + and CD86 +,and subsequently analyzed by multichannel flow cytometry (FCM).The killing effects of CTLs pulsed with HLA-A0201-binding peptide NY-ESO-1 and the potential of autologous DCs pulsed with NY-ESO-1 peptides in inducing specific cytotoxic T lymphocytes (CTLs) responses were determined.The patients were administered two infusions of autologous CTLs for 1 time every two weeks.The total infusion was with 2 times.The immunological responses and clinical responses were examined in 1 week after the final administration.Results:The immunophenotype of DCs pulsed with NY-ESO-1 peptide was analyzed,HLA-DR+ CD11c+ cells (93.6％ ± 1.2％),CD80 + cells (87.3％ ± 3.6％),CD83 + cells (82.8％ ± 2.5％) and CD86 + cells (93.4％ ± 6.4％).PBLs isolated from patients primed by DCs pulsed with NY-ESO-1 peptide proliferated continuously and the proliferation index (PI) of the PBLs were analyzed.There was significant difference between the DCs loaded with polypeptides and those unloaded,though it could promote the proliferation of PBLs,but the PI was significantly lower than that of the DCs loaded with NY-ESO-1 peptide (P ＜ 0.05).The average percentage of special CTLs primed by DCs pulsed with NY-ESO-1 peptides was significantly higher than that in the control group (5.2％ ± 1.2％ vs.0.4％ ± 0.1％).CTLs induced by NY-ESO-1 pulsed DCs exerted a stronger killing effect on T2 cell line pulsed with NY-ESO-1 peptide than that in the control group at the ratio of E (effect) to T (target) as 30 ∶ 1,P ＜ 0.05.The cytokine levels in the patients' sera such as IFN-γ,IL-2 and IL-12 were increased after treatments [(132.9 ± 10.2) μg/Lvs.(46.4±3.1) μg/L;(101.3 ±6.4) μg/Lvs.(26.7 ±1.2) μg/L;(51.3 ±2.6) μg/L vs.(26.4 ± 1.1) μg/L;all P ＜ 0.05],and the percentages of antigen-specific CD8 + IFN-γ + increased in these patients (P ＜0.01).Conclusion:Auto-DCs pulsed with NY-ESO-1 peptides can in duce the proliferation of allogenic CTLs,which elicit specific immune responses ex vivo or in vivo,and boost anticancer immunity markedly.
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Background Cell immunologic therapy for retinoblastoma(RB)is becoming a hot research topic.Cancer-testis antigen is a human immunogenic protein and is used to treat some tumors.However,its effect on RB has not been investigated.Objective The present study was to discuss the antigen specific anti-tumor effect of cytotoxic T lymphocytes(CTL)induced by the cancer-testis antigen,NY-ESO-1-sensitized dendritic cells(DCs),on human RB.Methods PCR was performed to amplify target gene fragments from the NY-ESO-1 plasmids,and then the target gene fragments were digested with the restriction enzymes SalI and EcoRI.Harvested fragments were inserted into the pDC316 plasmid to construct the recombinant plasmid pDC316/NY-ESO-1.The expression of NY-ESO-1 protein in human RB cells strain,HXO-RB44,was detected by immunofluorescence and Western blot.Monocytes were isolated from 60 ml of peripheral blood from a healthy donor using Ficoll density-gradient centrifugation with a cell density of 1 × 107/ml.DCs isolated from blood were stimulated with recombinant human granulocyte-macrophage colony stimulating factor(rhGM-CSF)and recombinant human interleukin-4(rhIL-4).The recombinant plasmid pDC316/NY-ESO-1 was transfected into DCs and the DCs were co-cultured with T lymphocytes.The resultant CTL were used as effector cells.The growth of the CTL was detected by MTT assay.The CTL were then added into the growth medium used for culturing HXO-RB44 cells and the vitality of the HXO-RB44 cells was assayed by MTT assay.Results The sequence of the cloned DNA fragment of the recombinant plasmid pDC316/NY-ESO-1 was conforms with the sequence of the NY-ESO-1 gene.The expression of the NY-ESO-1 protein in HXO-RB44 cells was tested by immunofluorescence and Western blot.DCs were successfully induced with rhIL-4 and rhGM-CSF from PBMC.The recombinant expression plasmid pDC316/NY-ESO-1 was successfully transferred into DCs.These DCs had high expression of surface molecules such as HLA-DR(42.1％),CD80(54.2％),CD83(39.7％)and CD86 (94.8％).The CTL that was induced by DCs-sensitized with NY-ESO-1 specifically killed HXO-RB44 cells.CTL induced by the sensitized DCs had a stronger cytotoxic effect against HXO-RB44 cells compared with un-sensitized DCs and CTL un-induced with DCs,as shown by MTT asssay(P＜0.05).The anti-tumor activity was highest when the ratio of effector to target was 75∶1(P＜0.05).Conclusions DCs transfected by the recombinant plasmid pDC316/NY-ESO-1 can induce the proliferation of allogenic CTLs,which showed a specific anti-tumor effect against HXO-RB44 cells.These results present a new type of immunotherapy for the treatment of RB.
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Objective To detect the expression of Cancer-Testis antigen NY-ESO-1 in human laryngeal squamous carcinoma (LSC) and to explore its significance in immunotherapeutic application. Methods The expressions of NY-ESO-1 protein in the LSC and in the pathologically positive lymph nodes were detected by PV-9000 Immunohistochemistry. Western blotting was also employed to measure the expressions of NY-ESO-1 in the tumor core region(TC), the tissues at the sites of 0.5cm, 1.0cm away from LSC periphery and the distant normal larynx tissues. Results NY-ESO-1 protein expression was positive in 30 out of 69 (43.48 %) cases of LSC. The expression level of NY-ESO-1 protein were found to significantly decrease by tums in TC and corresponding adjacent tissues (P ＜0.01). None of the nine normal larynx tissues expressed NY-ESO-1 protein.It did not display an obvious correlation between the expression of NY-ESO-1 with T staging, pathological grading and lymph node metastasis (P ＞0.05). Its positive expression was found in pathologically positive cervical lymph nodes, which were significantly lower than that in the primary site (P ＜0.05). Conclusion NY-ESO-1 protein express at high level in human laryngeal squamous carcinoma, and they may play a role in genesis and development of tumors, which suggests that NY-ESO-1 gene might be used as target antigens for immunotherapy of LSC and the further research is necessary.