[In vitro anti-tumor effect of CTL induced by HSP70-Id complex-modified dendritic cells].
OBJECTIVE
To prepare the tumor antigen peptide complex (HSP70-1d) of HSP70 and idiotype (Id) from SmIg ScFv fragment in patients with Chronic B cell leukemia (B-CLL), and to study the anti-tumor effect of cytotoxic T lymphocyte (CTL) induced by HSP70-Id complex-modified dendritic cell (DC) in vitro and explore their immune mechanism.


METHODS
Purified HSP70 was combined into peptide complex (HSP70-Id) with the prepared Id-ScFv from B-CLL cells in vitro by using biochemical technique. The plastic-adherent monocytes from human peripheral blood were cultured and induced into DC with rhGM-CSF and rhIL-4 using cell culture and separation technique. The cultured DC were harvested and pulsed by HSP70-Id complex. DC morphology was observed under converted phase microscope and its phenotype was characterized by FCM on 8th day as well as their secreting cytokines were measured. Host lymphocytes were stimulated by DC loaded with HSP70-Id complex and co-cultured in the medium containing IL-2. The activation and proliferation of lymphocytes were examined by MTr test, which was also used to assay cytotoxicity of CTL elicited by modified DC to Daudi, K562 and HepG2 tumor cells, and FCM analyzed the changes of T lymphocyte subsets.


RESULTS
Mature DCs were obtained successfully, showing typical morphology and phenotypic properties, the expression ratio of cellular surface molecules, CD1a was 20% - 30%, CD83 was more than 72% , both CD86 and HLA-DR over-expressed obviously in the complex-loaded DC group secreting cytokines of Thl type, IL-12 and TNF-alpha. The culturing lymphocytes that were activated by modified DC could more effectively and specifically kill Daudi (71. 24%), but not K562 and HepG2 tumor cells. Results of FCM assay demonstrated that percentage of CD4+ and CD8+ T lymphocytes cocultured with complex-modified DC increased notably to 56.51% and 70.21%, respectively. CD4+ T/ CD8+ T proportion was changed from 1.49 to 0.81. The dose of peptide would be reduced to 1/50 if specific CTL induced by complex-modified DC instead of directly by peptide complex.


CONCLUSIONS
DCs modified by HSP70-Id complex exhibit powerful biological activities, and could induce CTL to specific cytotoxicity against carcinoma cells. It might be produced by cooperation of CD4+ T, CD8+ T lymphocytes and DC. The results also suggested that DC modified by HSP70-Id complex can present antigen and induce CTL with high efficacy and specificity.
Wang
Zhi-Hua
ZH
Department of Urology, Tongii Hospital, Tongji Medical College of Huazhong University of Science and Technology, Wuhan 430030, China.
Ye
Qing
Q
Hu
Zhi-Quan
ZQ
Ye
Zhang-Qun
ZQ
Yu
Xiao
X
Shen
Guan-Xin
GX
D045744:Cell Line, Tumor; D002478:Cells, Cultured; D003602:Cytotoxicity, Immunologic; D003713:Dendritic Cells / Q000166:cytology / Q000187:drug effects / Q000276:immunology*; D005434:Flow Cytometry; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000494:pharmacology; D018840:HSP70 Heat-Shock Proteins / Q000494:pharmacology; D006801:Humans; D007130:Immunoglobulin Idiotypes / Q000494:pharmacology; D007135:Immunoglobulin Variable Region / Q000494:pharmacology; D015847:Interleukin-4 / Q000235:genetics / Q000494:pharmacology; D020014:K562 Cells; D008213:Lymphocyte Activation; D009000:Monocytes / Q000166:cytology / Q000187:drug effects / Q000276:immunology; D011994:Recombinant Proteins / Q000494:pharmacology; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*
D018840:HSP70 Heat-Shock Proteins; D007130:Immunoglobulin Idiotypes; D007135:Immunoglobulin Variable Region; D011994:Recombinant Proteins; D015847:Interleukin-4; D016178:Granulocyte-Macrophage Colony-Stimulating Factor