Preparation of leukemia cell vaccine expressing Bacille Calmette-Guérin heat shock protein 70 and its anti-leukemia therapeutic effect

Abstract

Objective To culture the acute leukemia cells in vitro, and to prepare cancer vaccine expressing heat shock protein 70 (HSP70) of Bacille Calemette-Geerin(BCG) onto the cell surface, so as to study its anti-tumor effect and mechanism. Methods Acute myeloid leukemia (AML) cells were cultured in a serum-free Stemspan® culture supplemented with cytokines[stem cell factor(SCF), flt-3 ligand(FL), interleukin(IL)-3 and IL-6] in vitro.And B-lineage acute lymphoblastic leukemia (B-ALL) cells were cultured in a Iscove modified medium(IMDM) culture supplemented with cytokines (SCF, FL, IL-3 and IL-7) in vitro.Cellular morphology was observed by the microscopy and immunophenotype determination was used to verify the biological characteristics of acute leukemia cells after culture.Lipofectamine 2000 was used to transfect the pDisplay-HSP70 plasmid into acute leukemia cells.The expression of HSP70 on the cell surface was detected by fluorescene microscope.Then the immunogenicity of the leukemia cells expressing HSP70 were detected.The experimental groups were divided into 3 subgroups: the wide-type acute leukemia cells (wt-LC group), the pDisplay-leukemia cells (pDisplay-LC group), and the pDisplay-HSP70-leukemia cells (HSP70-LC group), respectively.The leukemia cells in different groups were cultured with autologous peripheral blood T cells for 72 hours.The proliferation indices of T cells were assayed by carboxyfluorescein diacetate succinimidyl ester (CFSE)-staining method, and the contents of interferon-γ(IFN-γ) were tested by enzyme-linked immunosorbent assay (ELISA). The leukemia cells in different groups were cultured with autologous peripheral blood T cells, and after 6 days, the fresh acute leukemia cells were added[in the different ratios of cytotoxicity T lymphocyte (CTL): leukemia cells were 10: 1, 20: 1, 40: 1 and 80: 1] and continued to be cultured for another 12 hours.Cytotoxicity assay was measured by lactate dehydrogenase(LDH) release. Results After short-term culture in vitro, the leukemia cells were in colony-like suspension and maintain the proliferation characteristics were maintained.The cell proliferation was rapidly cultured for about 10 days and then was gradually slowed down.But there was no difference between the day 10 and day 0 in the expressions of CD13 and CD33 in fifteen cases of AML cells (P>0.05). Equally, there was no difference between the day 10 and day 0 in the expressions of CD19, CD10 and CD22 in fifteen cases of B-ALL cells (P>0.05). After BCG HSP70 gene transfection, the yellow-green fluorescence on the leukemia cells surface was observed under the confocal microscope.Detection of the immunogenicity: (1)Autologous T cell proliferation: the most significant T cell proliferation was observed in the group of HSP70-transfected leukemia cells (t=17.89, 19.58, all P 0.05). (2)The contents of cytokines: the IFN-γ level in the group of HSP70-transfected leukemia cells was higher than those of wide-type acute leukemia cells and the pDisplay-transfected ones (t=24.72, 24.81, all P<0.05). (3)Cytotoicity of CTL: the killing rate in HSP70-transfected leukemia cells was significantly higher than those of wide-type acute leukemia cells and pDisplay––transfected ones(F=13.66, P<0.05). And with the increase of the ratio from 10: 1 to 80: 1, the inhibiting activity of CTL in the HSP70-LC group was raising(F=19.69, P<0.05). Conclusions 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.The leukemia cells vaccine expressing BCG HSP70 onto its surface was successfully prepared, and gene transfection of BCG HSP70 can significantly enhance the immunogenicity of leukemia cells. Key words: Bacille Calmette-Guerin; Heat shock protein 70; Leukemia cells; Cancer vaccine