Effect Of BCG-PSN On Anti-leukemia Effect Mediated By Dentritic Cells Derived From Children With Leukemia

Effect of BCG-PSN on Anti-leukemia Effect Mediated by Dentritic Cells derived from children with leukemiaObjective To investigate the effect of polysaccharide nucleic acid fraction of Bacillus Calmette Guerin (BCG-PSN) on anti-leukemia effect mediated by leukemia antigen-loading Dentritic Cells (DCs) derived from children acute leukemia(AL) peripheral blood in vitro. Expect to enhance anti-1 eukemia effect of children with acute leukemia.Methods 12 acute lymphoblastic leukemia (ALL) children in complete remission were detected, in which peripheral blood mononuclear cells (PBMC) samples were obtained by density gradient centrifugation method and divided into control group and test group respectively. The control group was cultured with rhGM-CSF+rhIL-4+rhTNF-α(GIT), while the test groups were cultured with rhGM-CSF+rhIL-4+rhTNF-α+HL-60 cells freeze thawing antigens (GITA),rhGM-CSF+rhIL-4+rhTNF-α+HL-60 cells freeze thawing antigens +BCG-PSN (GITAB) respectively. The growth of DCs was observed under a invert microscope. We detected the immunephenotypes of DCs (CD1a,CD83,HLA-DR) by flow cytometry on the 8th day after cultured, and collected ripe DCs on the 8th day. We mixed the different groups'DCs with T cells derived from peripheral blood of the very patient. Cultured and rhIL-2 for 96 hours, we mixed the different groups'cytotoxic T cells (CTLs) with HL-60 cells to culture for 6 hours. Cytotoxicity assay was measured by LDH method. Also, we test the contents of IL-12 on the 8th day of DCs'culture, and IFN-y of CTL inducing for 96h by ELISA.Results①All groups attained a certain amount of typical DCs;②The rate difference of CD1a+cells in the test groups and the control group was not significant (t=1.120,1.812, P >0.05), and the GITAB group was lower than the GITA group(t= 4.078, P<0.05); and the rates of CD83+cells in the test groups were higher the control group (t= 4.578,8.670, P＜0.01), the GITAB group was higher than the GITA group (t=4.695, P<0.05), the difference was significant; the rates of HLA-DR+ cells in the test groups were higher the control group (t 2.174,5.279, P<0.05), the GITAB group was higher than the GITA group (t= 2.454, P<0.05), the difference was significant;③The result of LDH method showed that CTL killing activity on HL-60 cells in test groups were higher than the control group(t= 2.561,3.651, P<0.05), and in test groups, killing activity on HL-60 cells of CTLs in the GITAB group was dramaticly higher than the GITA group(t= 2.222, P<0.05), the difference was significant;④Also, the contains of IL-12,IFN-γin supernatants in test groups were higher than the control group(IL-12:t= 4.086,7.011; IFN-γ:t=2362,5.170, P<0.05),and the GITAB group were higher than the GITA group(IL-12:t= 2.180; IFN-γ:t= 2.817, P<0.05), the difference was significant.Conclusion①DCs loaded with freeze-thawed leukemia antigen from HL-60 cells are much riper than the unloaded, which derive from children with leukemia in complete remission. They can raise the secretion of IL-12, promote the differentiation of Th1 cells, particularly induce special CTL to develop special anti-leukemic immune effect. Also, they can promote the secretion of IFN-γto reinforce the anti-leukemic immune effect.②BCG-PSN can cooperate with rhGM-CSF, rhIL-4 and rhTNF-αin promoting the maturity of DCs and raising the secretion of IL-12 to activate T cells to raise the the anti-leukemia cytotoxicity of CTL. Accordingly, BCG-PSN can enhance the anti-leukemic immune effect of DCs loaded with leukemia antigen which derived from children acute leukemia peripheral blood.