Study Of Specific Immune Responses Induced By BCR-ABL-SEA DNA Vaccine In BALB/c Mice

Objective:To evaluate the immune response induced by a DNA vaccine expressing both BCR-ABL and SEA peptides in mice which was expected to provide basic data for immunotherapy in CML;To measure the expression of signaling transduction factor T lymphocyte-CD3ζchain gene in cord blood mononuclear cells which stimulated by K562 cells or SEA or both of them.Methods:(1) The vaccine plasmids of BCR-ABL-pIRES-SEA,which expressed both BCR-ABL and SEA peptides was constructed previously in our laboratory was intramuscularly injected into BALB/c mice at 14-d intervals for 3 cycles. The plasmids of BCR-ABL-pIRES and SEA-pIRES,which were only expressed either BCR-ABL peptide or SEA peptide,were also used in the same procedure for comparison. The cytotoxicity of T cells from mouse spleen against K562 cell lines was examined by Cell Counting Kit-8(CCK-8). The ratio of CD4+ and CD8+ surface markers on the harvested T cells was detected by flow cytometry (FCM).The serum level of interferon-y and IL-4 were measured by ELISA.The antibodies against BCR-ABL were detected by the immunofluorescence. The transcription and expression of BCR-ABL and SEA in injection site were detected by RT-PCR and immunohistological methods. (2) Real-Time PCR with SYBR GreenⅠtechnique was used for detecting CD3ζchain expression level in cord blood mononuclear cells from four normal individuals, which were induced by the anti-CD3 antibodies,K562 cells, SEA or both of K562 cells and SEA respectively at the beginning or for 48 hours. Relative changes in CD3ζchain expression level were indicated by the 2-△△Ct method between each group and the control.β2-microglobulin gene (β2M) was used as an endogenous reference.Results:(1) Seven weeks after immunization,the specific cytotoxicity of T cells against K562 cells and INF-y in co-expression of BCR-ABL-pIRES-SEA group were significantly higher than those in mono-expression of BCR-ABL-pIRES group and SEA-pIRES group respectively (P<0.05). The ratio of CD4+ and CD8+ surface markers on the harvested T cells and the level of IL-4 in vaccinated mouse serum were not different between groups (P>0.05). The specific antibody against BCR-ABL was detected in BCR-ABL-pIRES-SEA group and BCR-ABL-pIRES group but not in SEA-pIRES group by indirect immunofluorescene analysis.The BCR-ABL/SEA mRNA and protein could be identified in the injection site of BCR-ABL- pIRES-SEA vaccinated mice. (2) The expression level of CD3ζchain detected were 4.52±0.96,1.65±0.26,1.43±0.44,3.41±0.30 in four groups of the anti-CD3 antibodies,K562 cells,SEA,both of SEA and K562 cells respectively.This show that the activation of T cells in each group was induced.The expression level of CD3ζchain induced by both SEA and K562 was significantly higher than that of the K562 or SEA group (P<0.01, P<0.01).Conclusion:The recombinant BCR-ABL- pIRES-SEA plasmids can induce specific humoral and the cellular immune responses in vivo. It might be have the common feature of DNA vaccine and may be farther used in the research as BCR-ABL-SEA DNA vaccine. The superantigen (SEA) could enhance T cell activation stimulated by the K562 cells in vitro.