The Preparation Of Leukemia Cells Vaccine Expressing BCG HSP70and Anti-leukemia Therapeutic Effect

The preparation of HL-60cells vaccine expressing BCG HSP70and anti-leukemia therapeutic effectObjective To construct the recombinant eukaryotic expression vector of heat shock protein70of BCG (BCG HSP70), and to prepare the HL-60cells vaccine expressing the protein onto the cell surface after gene transfection, so as to study its anti-tumor effect and mechanism.Methods (1) Construction of the recombinant eukaryotic expression vector of BCG HSP70: The whole BCG HSP70gene was amplified from BCG genome by polymerase chain reaction (PCR). The PCR primers, including Bgl II and Sma I restriction endonuclease sites, were designed. The HSP70gene was sub-cloned into the polyclone endonuclease sites in pDisplay. The recombinant vector of pDisplay-HSP70was verified by sequencing.(2) Preparation of the HL-60cells vaccine expressing HSP70onto its surface:Lipofectamine2000was used to transfect the pDisplay-HSP70plasmid into HL-60cells. The expression of HSP70on the cell surface was detected by fluorescene microscope.(3) Detection of the immunogenicity of HL-60cells expressing HSP70:The test groups were divided into three subgroups, wide-type HL-60cells (HL60-wt), pDisplay cells (HL60-pDisplay), and HSP70cells (HL60-HSP70) respectively. The mitomycin C-treated HL-60cells of different groups were cultured with allogeneic peripheral blood T cells at the radio of1:10for72h. The proliferation indices of T cells were assayed by CFSE-staining method, and the contents of cytokine (IFN-y) were tested by enzyme-linked immunosorbent assay (ELISA). The mitomycin C-treated HL-60cells of different groups were cultured with allogeneic peripheral blood T cells at the radio of1:10for48h. The wild-type HL-60cells were added (at the different ratios of CTL:HL60=10:1,20:1,40:1,80:1) and continued to culture for another12h. Cytotoxicity assay was measured by LDH release.Results1Vector construction:The fragment of BCG HSP70was consistent with Mycobacterium tuberculosis HSP70gene published in GeneBank. DNA sequencing showed that the recombinant vector of pDisplay-HSP70was correctly constructed.2Gene transfection:After BCG HSP70gene transfection, the yellow-green fluorescence on the HL-60cells surface was observed under the confocal microscope.3Detection of the immunogenicity:(1) Allogenic T cell proliferation:The most significant T cell proliferation was observed in the group of HSP70-transfected HL-60cells (p<0.05). There was no difference between the HL60-wt group and HL60-pDisplay group (p>0.05).(2) The contents of cytokine:The contents of IFN-y were (100.23±3.55)pg/ml,(102.68±3.23)pg/ml, and (146.01±2.98)pg/ml, respectively. The HSP70-HL60group was the highest and there was no difference between the HL60-wt group and HL60-pDisplay group.(3) LDH release:T he inhibiting activity of CTLs on HL-60cells in the group of HSP70-transfected HL-60cells was more significant, in comparison to that of wide-type HL-60cells and pDisplay--transfected ones. And with the increase of the ratio from10:1to80:1, the inhibiting activity of CTL in the HSP70-HL60group was rising.Conclusion1. The recombinant eukaryotic expression vector of BCG HSP70, which was named pDisplay-HSP70, was successfully constructed.2. The HL-60cells vaccine expressing BCG HSP70onto its surface was successfully prepared.3. The HL-60cells vaccine expressing BCG HSP70onto its surface could promote the proliferation and secretion of allogeneic lymphocytes, as well as enhance the killing activity. So, it can indicate that gene transfection of BCG HSP70can significantly enhance the immunogenicity of HL-60cells. Part ⅡThe preparation of leukemia cells vaccine expressing BCG HSP70and anti-leukemia therapeutic effectObjective To culture the acute leukemia cells in vitro, and to prepare cancer vaccine expressing heat shock protein70of BCG (BCG HSP70) onto the cell surface, so as to study its anti-tumor effect and mechanism.Methods (1) Culture of fresh acute leukemia cells in vitro:Fifteen cases of acute myeloid leukemia (AML) cells were collected and cultured in a serum-free Stemspan(?) culture supplemented with cytokines (SCF、FL、IL-3and IL-6) in vitro; Fifteen cases of B-lineage acute lymphoblastic leukemia (B-ALL) cells were collected and cultured in a serum-free IMDM culture supplemented with cytokines (SCF、FL、IL-3and IL-7) in vitro. Cell morphology observed by microscopy, cell staining and immunophenotype determination were used to vertify the biological characteristics of acute leukemia cells after culture.(2) Preparation of the leukemia cells vaccine expressing HSP70onto its surface:Lipofectamine2000was used to transfect the pDisplay-HSP70plasmid into acute leukemia cells. The expression of HSP70on the cell surface was detected by fluorescene microscope.(3) Detection of the immunogenicity of the leukemia cells expressing HSP70:The test groups were divided into three subgroups, wide-type acute. leukemia cells (wt-LC), pDisplay-leukemia cells (pDisplay-LC), and pDisplay-HSP70-leukemia cells (HSP70-LC) respectively. The mitomycin C-treated leukemia cells of different groups were cultured with autologous peripheral blood T cells at the radio of1:10for72h. The proliferation indices of T cells were assayed by CFSE-staining method, and the contents of cytokine (IFN-y) were tested by enzyme-linked immunosorbent assay (ELISA). The mitomycin C-treated leukemia cells of different groups were cultured with autologous peripheral blood T cells at the radio of1:10for48h. The fresh acute leukemia cells were added (at the different ratios of CTL: leukemia cells=10:1,20:1,40:1,80:1) and continued to culture for another12h. Cytotoxicity assay was measured by LDH release. Results1Culture of fresh acute leukemia cells:Short-term culture in vitro, the leukemia cells were in colony-like suspension and maintain the proliferation characteristics. The cell proliferation was rapid cultured for about10days and then gradually slowed down. But there was no difference between the day10and day0in the expressions of CD13and CD33of fifteen cases of AML cells (p>0.05). Equally, there was no difference between the day10and day0in the expressions of CD19, CD10and CD22of fifteen cases of B-ALL cells (p>0.05).2Gene transfection:After BCG HSP70gene transfection, the yellow-green fluorescence on the leukemia cells surface was observed under the confocal microscope3Detection of the immunogenicity:(1) Autologous T cell proliferation:The most significant T cell proliferation was observed in the group of HSP70-transfected leukemia cells (p<0.05). There was no difference between the wt-LC group and pDisplay-LC group (p>0.05).(2) The contents of cytokines:HSP70-transfected leukemia cells stimulated autologous T cells to secret the highest cytokine level (IFN-y), compared to those of wide-type acute leukemia cells and pDisplay--transfected ones.(3) LDH release:The killing rate in HSP70-transfected leukemia cells was significantly higher than that of wide-type acute leukemia cells and pDisplay--transfected ones. And with the increase of the ratio from10:1to80:1, the inhibiting activity of CTL in the HSP70-LC group was rising.Conclusion1. Fresh acute leukemia cells can be successfully cultured in vitro. Short-term culture can significantly increase the number of leukemia cells, but has little effect on surface antigen expression. So, the biological characteristics of the leukemia cells can be maintained.2. The leukemia cells vaccine expressing BCG HSP70onto its surface was successfully prepared.3. The leukemia cells vaccine expressing BCG HSP70onto its surface could promote the proliferation and secretion of autologous lymphocytes, as well as enhance the killing activity. So, it can indicate that gene transfection of BCG HSP70can significantly enhance the immunogenicity of leukemia cells. Part ⅢBCG HSP70gene transfection and its effects on immunogenicity of murine lymphocytic leukemiaObjective To assess the effects of heat shock protein70of BCG (BCG HSP70) gene transfection on tumorigenicity and immunogenicity of murine lymphocytic leukemia cells (L1210).Methods BCG HSP70gene was transfected onto the surface of murine lymphocytic leukemia cells (L1210) by lipofectamine2000, and then the positive clone (L1210-HSP70) highly expressing HSP70was selected. As the tumor vaccine, the following studies were done.(1) Tumorigenicity experiments in nude mice and syngeneic mice:L1210-HSP70cells, L1210-neo cells and their parental cells were inoculated into BALB/c nude mice and syngeneic DBA/2mice. Tumor growth was monitored, mean survival time was calculated and the number of L1210specific Thl cells was detected by IFN-y ELISPOT assay. Tumor tissues from the treated mice were subjected to histopathological examination and immunohistochernistry.(2) For therapeutic experiments, DBA/2mice were first injected s.c. with parental L1210cells, and then were treated by s.c. injection with PBS, L1210cells, L1210-neo cells and L1210-HSP70cells respectively. Tumor growth was monitored and mean survival time was calculated.(3) DBA/2mice were firstly immunized and then were s.c. inoculated with parental L1210cells. Tumor size was measured and mean survival time was calculated in order to investigate the immunoprotective effects.Results The expression of BCG HSP70on the L1210cells surface was detected by confocal microscopy.(1) Tumorigenicity experiments in nude mice:L1210-HSP70cells had the same tumorigenicity as the parental L1210cells, and the tumor formation rates were100%.(2) Tumorigenicity experiments in syngeneic mice:In L1210-HSP70group, tumor growth was slow or without the formation of tumor, and the mice survival time was significantly prolonged or long-time survival. IFN-γ ELISPOT assay indicated that the treatment of the vaccine L1210-HSP70showed a marked stimulating effect on L1210specific Thl cells when compared with L1210-neo or L1210. Tumor-bearing mice treated with the L1210-HSP70cells showed thorough coagulation necrosis and abundant CD8+T lymphocyte infiltration.(3) Antitumor therapeutic efficacy:In L1210-HSP70group, the tumor growth was significantly inhibited, tumor diameter was markedly reduced and the survival time of tumor-bearing DBA/2mice was further prolonged.(4) Immune protection effect:In all groups, the strongest inhibition on tumor growth was observed in the group treated with L1210-HSP70. Accordingly, the mice vaccinated with the L1210-HSP70vaccine displayed the longest survival duration of all.Conclusion1. BCG HSP70gene transfection could effectively improve the immunogenicity of tumor cells, activate specific T cells and enhance the anti-tumor immunity in vivo,2. BCG HSP70gene transfection could enhance the host anti-tumor immunity.