Effect Of Immune Response Induced By APL Cells And PML-RARα Vaccine In Vitro And In Vivo

Objective: To investigate the specific T-cell immune response to acute promyelocytic leukemia (APL) associated antigen and evaluate the in vivo immune effect induced by PML-RARαvaccines in mice, which was expected to provide basic data for immunotherapy in APL.Methods: (1) The complementary determining region 3 (CDR3) of TCR Vα29 subfamily genes in peripheral blood mononuclear cells from 9 APL patients were amplified using RT- PCR. The positive products were further analyzed to identify the clonality of T cells by Genescan technique; (2) T cells from cord blood were induced and amplified by APL cell in vitro by MLTC (mixed lymphocyte-tumor culture). to detect the usage and clonality feature of TCR Vαand Vβsubfamilies. These induced cells in different time points were detected for the specific cytotoxicity by LDH release assay; (3) the reconstructed plasmids (PML-RARα-hIL-2) which were developed previous in our laboratory were injected into BALB/C mice at 14-d intervals for three cycles. The transcription and expression of PML-RARαand hIL-2 in injection site were detected by RT-PCR and immunohistological methods; the specific cytotoxicity of spleen cells from the vaccinated mice were detected by LDH release assay; interferon-γsecretion were measured by ELISA; antibodies against human PML-RARαwere detected with specific FITC by Immunofluorescene; the T-cell receptor rearrangement excision circles (TRECs) in DNA of thymic cells were evaluated by real-time quantitative RT-PCR using TaqMan probe.Results: (1) One to seven of TCR Vαsubfamilies could be detected in peripheral blood T cells from 9 cases with APL, the frequent expression of Vαsubfamilies predominated in Vα3 and Vα19. Clonal expanded T cells could be detected in 8 APL patients, which predominant used Va3, Va26 or Va27 (3 out of 8 cases) ;(2) A part of TCR Vαand Vβrepertoires were detected in cord blood T cells after cultured with APL cells. There are oligoclonal tendency T cells in TCR Vα10, TCR Vβ5 and TCR Vβ15 subfamilies. Cytotoxicity on APL cells of induced cells is much higer than the uninducded cells from CB ;( 3)The PML-RARα/ hIL-2 mRNA and protein could be identified in the injection site of PML-RARα-hIL-2 vaccinated mice; time dependent antibody production and an increase in INF-γcould be detected in vaccinated mice serum. TRECs level and specific cytotoxicity on NB4 cells were significantly higher than that from control groups.Conclusion: Specific immune response could be induced by APL assocaited antigen, which displayed the predominant usage and clonal expansion of TCR Vαand Vβsubfamily T cells, and specific cytotoxity on APL cells.The recombinant PML-RARαplasmids 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 PML-RARαDNA vaccine.