The preparation of HL-60 cells vaccine expressing BCG heat shock protein 70 and detection of its immunogenicity in vitro

Abstract

Gene-modified cell vaccines are the best way to achieve the immunotherapy for all types of acute leukemia. In this study, the recombinant eukaryotic expression vector (pDisplay-HSP70) of heat shock protein 70 (HSP70) of Bacille Calmette-Guérin (BCG) was constructed by amplifying the whole BCG HSP70 gene using polymerase chain reaction (PCR) and sub-cloning into the polyclone endonuclease sites in pDisplay. Then the HL-60 cell vaccine expressing the protein onto the cell surface was prepared by lipofectamine transfection and its anti-tumor effect and mechanism were further studied. Results showed that the fragment of BCG HSP70 was consistent with Mycobacterium tuberculosis HSP70 gene published in GeneBank. DNA sequencing showed that the recombinant vector was correctly constructed and named pDisplay-HSP70. After BCG HSP70 gene transfection, the yellow-green fluorescence on the HL-60 cells surface was observed under a fluorescence microscope. The immunogenicity of HSP70-transfected HL-60 cells exhibited upregulated proliferation of lymphocytes, increased cytokine secretion (IFN-γ) and enhanced killing activity. These results suggested that gene transfection of BCG HSP70 could significantly enhance the immunogenicity of HL-60 cells. It may be used as a suitable candidate gene-modified cell vaccine for cancer immunotherapy.

Keywords: BCG; HL-60; Heat shock protein 70; cancer vaccine; gene transfection.

Figure 1. Agarose gel electrophoresis of PCR product. Lane 1: PCR product of HSP70; Lane 2: DNA marker (DL2000). The whole BCG HSP70 gene was amplified by using PCR and an approximate 1880bp fragment was consistent with Mycobacterium tuberculosis HSP70 gene released by GeneBank.

Figure 2. Identification of recombinant plasmid pDisplay-HSP70. Lane 1: DNA marker (DL2000); Lane 2: PCR product of HSP70; Lane 3: plasmid pDisplay digested with BglII and SmaI; Lane 4: HSP70 digested with BglII and SmaI; Lane 5: recombinant plasmid pDisplay-HSP70 digested with BglII and SmaI; Lane 6: PCR product from recombinant plasmid pDisplay-HSP70; Lane 7: DNA marker (DL15000). The two ways, restriction endonuclease digestion and PCR amplification, were used to identify that HSP70 gene was sub-cloned into the polyclone endonuclease sites in pDisplay.

Figure 3. Fluorescene microscopy of HL60-HSP70 cells after staining with heat shock pritein 70 mAb and secondary Ab conjugated with FITC. The yellow-green fluorescence on the HL-60 cells surface was observed on the basis of the nucleus stained blue and it showed that HSP70 was successfully transfected into HL-60 cells surface.

Figure 4. T cell proliferation. Purified T cells were co-cultured with the mitomycin C-treated different HL-60 cells at the radio of 10:1 for 72h. HSP70-transfected HL-60 cells stimulated the most significant T cell proliferation, compared with that of the HL60-wt group and HL60-pDisplay group. p < 0.05.

Figure 5. Regulation of T cells function (IFN-γ). Purified T cells were co-cultured with HL60-wt, HL60-pDisplay and HL60-HSP70 cells at the radio of 10:1. Cytokine production of IFN-γ from T cells was measured by ELISA. The highest cytokine level (IFN-γ) was stimulated to secret in the group of HSP70-transfected HL-60 cells. And with the increase of time, the level of IFN-γ was rising. p < 0.05.

Figure 6. The inhibiting activity on HL60 cells by CTL cells (at the different ratio of E:t = .A (10:1), B (20:1), C (40:1), D (80:1)). The inhibiting activity of CTLs on HL-60 cells in the group of HSP70-transfected HL-60 cells was more significant, in comparison to that of wild-type HL-60 cells and pDisplay–transfected ones. And with the increase of the ratio from 10:1 to 80:1, the inhibiting activity of CTL in the HSP70-HL60 group was rising. p < 0.05.