Specific Anti-leukemic Cell Effect Mediated By Dendritic Cells Pulsed With Acute Myelogenous Leukemia Antigen In Vitro

Objective: To explore the effective method for culture of the dendritic cells from bone marrow mononuclearcell of acute myelocytic leukemia patient and the specific anti-leukemic cell effect mediated by dendritic cells pulsed with acute myelogenous leukemia antigen in vitro and to provide a theoretic basis of AML immunotherapy.Method: Bone marrow mononuclearcells isolated with lymphocytes separation medium from 10 cases of AML patients were cultured with recombinate granulocyte-macrophage colony stimulating factor and calcium ionophore A23187 for 6-day to induce to dendritic cells. Induced dendritic cells were pulsed with major histocompatibility complex class I peptides isolated by acid elution assay and acute myelogenous leukemia antigen isolated by destroy assay with 0.4% KCl ,respectively. After the AML-DC had been pulsed by the AML antigen for 6-day, they were cocultured with T lymphocyte derived from healthy peripheral blood which had been cultured with IL-2 for 7 days to producing cytotoxic T lymphocyte. Then, the differences of the inhibition ratio of CTL producing with different method to AML cells were compared by methyl thiazolyl tetrazolium(MTT)assay, the morphology changes of AML-DC were observed with inverted microscope, the phenotype changes of CD_1α,CD₈₃ on AML-DC and the expression of CD₁₅₂ after the T lymphocyte stimulated were detected with flow cytometry.Result: Bone marrow mononuclearcell isolated from AML patients had been successfully induced to dendritic cells with 100ng/ml recombinate granulocyte-macrophage colony stimulating factor and 500ng/ml calcium ionophore A23187.It had typical morphology of dendritic cells when observed with inverted microscope. The expression of CD_1α and CD₈₃ before inducing were 1.16±0.15% and 7.28±0.21% respectively. After inducing for 48 hours, CD₈₃ had the highest expression of 59.69±2.56%(P<0.01,48h vs 0h).The highest expression of CD_1α was 22.29±0.82% after inducing for 96 hours(P<0.01,96h vs 0h). The inhibition ratio to AML cells of CTLs induced by AML-DC pulsed with major histocompatibility complex class I peptides, by AML-DC pulsed with supernatant and sediment of AML cells destroyed by 0.4% KCl, by AML-DC unpulsed and by T cells just cultured with IL-2 (not induced by AML-DC) were 76.65±12.62%, 35.22±7.56%, 38.31±8.85%, 29.63±5.49% and 10.27±2.84% respectivly. The CTLs induced by AML-DC pulsed with major histocompatibility complex class I peptides had the most significantly activity than others(P<0.01).Conclusion: Bone marrow mononuclearcell isolated from AML patients can be successfully induced to dendritic cells with recombinate granulocyte-macrophage colony stimulating factor and calcium ionophore A23187. The CTL induced by AML-DC pulsed with peptides isolated by acid elution can acquire the stronger cytotoxic activity.