Objective:To develop a novel artificial antigen-presenting system for efficiently inducing melanoma-specific CD8+CD28+ cytotoxic T lymphocyte（CTL） responses.Methods:Cell-sized Dynabeads? M-450 Epoxy beads coated wit...Objective:To develop a novel artificial antigen-presenting system for efficiently inducing melanoma-specific CD8+CD28+ cytotoxic T lymphocyte（CTL） responses.Methods:Cell-sized Dynabeads? M-450 Epoxy beads coated with H-2Kb:Ig-TRP2(181111K and anti-CD28 antibody were used as artificial antigen-presenting cells（aAPCs） lo induce melanoma-specific CD8*CD28’ CTL responses with the help of IL-2I and IL-I5.Dimer staining,proliferation,ELISPOT,and cytotoxicity experiments were conducted to evaluate the frequency and activity of induced CTLs.Results:Dimer staining demonstrated that the new artificial antigen-presenting system efficiently induced melanoma TRP2-specific CD8CD28' CTLs.Proliferation and ELISPOT assays indicated that the induced CTLs rapidly proliferate and produce increased IFN- y under the slimulalion of H-2K:Ig-TRP2-aAPCs,TL-15,and IL-21.In addition,cytoloxicily experiments showed lhat induced CTLs have specific killing activity of target cells.Conclusions:The new artificial antigen-presenting system including aAPCs plus IL-21 and IL-15 can induce a large number of antigen-specific CD8+CD28+ CTLs against the melanoma.Our study provides evidence for a novel adoptive immunotherapy against tumors.展开更多
In this study, norcanthridin （NCTD）-encapsulated liposomes were modified with a novel murine anti-human CD19 monoclonal antibody 2E8 （2E8-NCTD-liposomes） and the targeting efficiency and specific cytotoxicity of 2...In this study, norcanthridin （NCTD）-encapsulated liposomes were modified with a novel murine anti-human CD19 monoclonal antibody 2E8 （2E8-NCTD-liposomes） and the targeting efficiency and specific cytotoxicity of 2E8-NCTD-liposomes to CD19^＋ leukemia cells were evaluated. BALB/c mice were injected with 2E8 hybridoma cells to obtain 2E8 monoclonal antibody （mAb）. NCTD-liposomes were prepared by using film dispersion method. 2E8 mAbs were linked to NCTD-liposomes using post-incorporation technology. Flow cytometry showed that the targeting efficiency of purified 2E8 mAbs on CD19＋ Nalm-6 cells was 99.93%. The purified 2E8 mAbs were conjugated with NCTD-liposomes to prepare 2E8-NCTD-liposomes whose targeting efficiency on CD19^＋ Nalm-6 was also 95.82%. The average size of 2E8-NCTD-liposomes was 118.32 nm in diameter. HPLC showed that the encapsulation efficiency of NCTD was 46.51%. When the molar ratio of 2E8/Mal-PEG2000-DSPE reached 1：50, we obtained the liposomes with 9 2E8 molecules per liposome. The targeting efficiency of 2E8-NCTD-liposomes on CD19^＋ leukemia cells was significantly higher than that on CD19-1eukemia cells. Similarly, the targeting efficiency of the immtmoliposomes was also higher than that of the NCTDAiposomes on CD 19^＋ leukemia cells. Those results were consistent with those observed by laser scanning confocal microscopy. 3-（4, 5-Dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide （MTT） assay demonstrated that 2E8-NCTD-liposomes specifically killed Nalm-6 cells in a dose- and time-dependent manner. The viability of Nalm-6 cells treated by 2E8-NCTD-liposomes was significantly lower than that of Molt-3 cells and it was also significantly lower than that of Nahn-6 cells treated with the same concentration of NCTD-liposomes or free NCTD. We are led to concluded that 2E8 antigen can serve as a specific targeting molecule of B lineage hematopoietic malignancies for liposome targeting, and 2E8-NCTD-liposomes can be used as a new and effective means for the treatment of B lineage hematopoietic malignancies.展开更多
基金supported,in part,by grants from the Program for New Century Excellent Talents in University(NECT-10-0098)the National Natural Scientific Foundation of China(Nos.81072161.81000769.81172139.and 81060183)+3 种基金Programs for Changjiang Scholars and Innovative Research Team in University(No. IRT1119)Innovative Research Team in Guangxi Natural Science Foundation (No.2011-18-5)Fund for Distinguished Young Scholars in Guangxi Natural Science Foundation(2012jjFA40005)Project of science and technology of Guangxi (1140003A-17)
文摘Objective:To develop a novel artificial antigen-presenting system for efficiently inducing melanoma-specific CD8+CD28+ cytotoxic T lymphocyte（CTL） responses.Methods:Cell-sized Dynabeads? M-450 Epoxy beads coated with H-2Kb:Ig-TRP2(181111K and anti-CD28 antibody were used as artificial antigen-presenting cells（aAPCs） lo induce melanoma-specific CD8*CD28’ CTL responses with the help of IL-2I and IL-I5.Dimer staining,proliferation,ELISPOT,and cytotoxicity experiments were conducted to evaluate the frequency and activity of induced CTLs.Results:Dimer staining demonstrated that the new artificial antigen-presenting system efficiently induced melanoma TRP2-specific CD8CD28' CTLs.Proliferation and ELISPOT assays indicated that the induced CTLs rapidly proliferate and produce increased IFN- y under the slimulalion of H-2K:Ig-TRP2-aAPCs,TL-15,and IL-21.In addition,cytoloxicily experiments showed lhat induced CTLs have specific killing activity of target cells.Conclusions:The new artificial antigen-presenting system including aAPCs plus IL-21 and IL-15 can induce a large number of antigen-specific CD8+CD28+ CTLs against the melanoma.Our study provides evidence for a novel adoptive immunotherapy against tumors.
基金supported by a grant from the National Natural Sciences Foundation of China (No. 30170391)the Zhejiang Provincial Fund of Natural Sciences (No:Z205166)the Zhejiang Provincial Fund of Science and Technology Bureau (No:2007C23007)
文摘In this study, norcanthridin （NCTD）-encapsulated liposomes were modified with a novel murine anti-human CD19 monoclonal antibody 2E8 （2E8-NCTD-liposomes） and the targeting efficiency and specific cytotoxicity of 2E8-NCTD-liposomes to CD19^＋ leukemia cells were evaluated. BALB/c mice were injected with 2E8 hybridoma cells to obtain 2E8 monoclonal antibody （mAb）. NCTD-liposomes were prepared by using film dispersion method. 2E8 mAbs were linked to NCTD-liposomes using post-incorporation technology. Flow cytometry showed that the targeting efficiency of purified 2E8 mAbs on CD19＋ Nalm-6 cells was 99.93%. The purified 2E8 mAbs were conjugated with NCTD-liposomes to prepare 2E8-NCTD-liposomes whose targeting efficiency on CD19^＋ Nalm-6 was also 95.82%. The average size of 2E8-NCTD-liposomes was 118.32 nm in diameter. HPLC showed that the encapsulation efficiency of NCTD was 46.51%. When the molar ratio of 2E8/Mal-PEG2000-DSPE reached 1：50, we obtained the liposomes with 9 2E8 molecules per liposome. The targeting efficiency of 2E8-NCTD-liposomes on CD19^＋ leukemia cells was significantly higher than that on CD19-1eukemia cells. Similarly, the targeting efficiency of the immtmoliposomes was also higher than that of the NCTDAiposomes on CD 19^＋ leukemia cells. Those results were consistent with those observed by laser scanning confocal microscopy. 3-（4, 5-Dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide （MTT） assay demonstrated that 2E8-NCTD-liposomes specifically killed Nalm-6 cells in a dose- and time-dependent manner. The viability of Nalm-6 cells treated by 2E8-NCTD-liposomes was significantly lower than that of Molt-3 cells and it was also significantly lower than that of Nahn-6 cells treated with the same concentration of NCTD-liposomes or free NCTD. We are led to concluded that 2E8 antigen can serve as a specific targeting molecule of B lineage hematopoietic malignancies for liposome targeting, and 2E8-NCTD-liposomes can be used as a new and effective means for the treatment of B lineage hematopoietic malignancies.
