The Construction of HA-1-DC Nucleic-Acid Vaccine and Induction of Specific Cytotoxic T Lymphocytes.

The hematopoiesis-restricted minor histacompatibility antigens (mHags) HA-1 expressed on malignant cells of the recipient may serve as target antigens for alloreactive donor T cells. HA-1-DC nucleic-acid vaccine was constructed and cause anti-leukemia effect after allogeneic hematopioetic stem cell transplantation (allo-HSCT). The mononuclear cells were isolated from normal human peripheral blood to culture DCs. After 2 hours, nonadherent cells were removed and adherent cells were cultured for 7 days to induce DC differentiation with GM-CSF and IL-4. The cell populations were analyzed with CD80, CD86, CD83 or CD14 by flow cytotometry. Mix lymphocyte reaction was performed to study the effect of DCs to stimulate proliferation of allogenic lymphocytes, taken syngenic peripheral blood monocytes (PBMCs) as control. In vitro the eukaryotic expression vector pcDNA3.1/hisA-HA-1^H encoding HA-1^H gene was recombinated. Through electroporating DCs, the DC nucleic-acid vaccine was constructed. The plasmid containing enhanced green fluorescent protein (EGFP) gene was transfected as control. Under the fluorescence microscope, the percentage of GFP⁺ cells could reflect the transduction efficiency of DCs. The expression of HA-1 protein after transfecting 48h could be detected by using Western blot. The tansfected DCs and syngenic lymphocytes were cultured together with IL-2 to induce specific cytotoxic T lymphocytes (CTLs). The cytotoxity of CTLs was detected by the lactate dehydrogenase(LDH) assay. A large number of suspended cells could been harvested from PBMCs being cultured with GM-CSF and IL-4 for 7 days. By flow cytotometry, these cells highly expressed CD80 and CD86, on 88% and 93% respectively. The marker of mature DCs CD83 also highly expressed on 85%, while the specific marker of PBMCs CD14 lowly expressed on 13%. All the CD molecules accorded with the phenotype of DCs. The capacity of DCs to stimulate proliferation of the allogenic lymphocytes was increased in comparison with the control group(P<0.05). In vitro digesting recombinated plasmid pcDNA3.1/hisA-HA-1^H with the restriction enzyme XbaI and BamHI, the same length of target gene could be obtained as digesting the plasmid pcDNA-HA-1^H. Sequencing identification proved the successful recombination. The plasmid encoded the 358bp cDNA of HA-1^H which contained the geneic information of the HA-1 T lymphocyte epitope VLHDDLLEA. Through observing the expression of GFP, the transduction efficiency of DCs was about 15%. After electroporating 48h, the expression of HA-1 protein was detected by using Western blot. The DCs and the syngenic lymphocytes were cultured together with IL-2 for 5 days. They could induce specific CTLs. In vitro, K562 cells transfected HA-1^H gene were taken as target cells. The cytotoxity of the CTLs to target cells was 40.51% higer than the control group(P<0.05) whose cytoxity was 13.61%. In vitro DCs were successfully induced from PBMCs. DC nucleic-acid vaccine containing mHags HA-1 induce the production of specific CTLs which could be against leukemia after allo-HSCT.