Effect Of TLR3 Agonist Combined With TLR9 Agonist On Antitumor Immunity Of Dendritic Cell

Objective In this study,we explore the effect of Toll like receptor agonist as adjuvant on DC loaded with leukemia stem / progenitor cells;Investigate the effects of TLR agonists on the morphology and immunophenotype of DC loaded with LSC.Using TLR3 agonist(poly I:C)in combination with TLR9 agonist(Cp G ODN)to stimulate DC loaded with CD34+ leukemia cells,we investigate the effects of TLR agonists on the morphology and immunophenotype of DC loaded with CD34+ leukemia cells,provide a basis for studying the function of DC,and further explore the enhancement of immunogenicity of leukemia cells in vitro,It provides a theoretical basis for the possibility of immune clearance of leukemia cells in vivo.Methods After signing the informed consent with the patient or the authorized person of the patient,the fresh bone marrow anticoagulant blood was collected from the patients diagnosed as acute myeloid leukemia(excluding M3,according to FAB classification).Peripheral blood mononuclear cells(PBMC)were isolated by density gradient centrifugation.CD34+cells were separated by immunomagnetic beads and frozen for further study.The peripheral blood mononuclear cells(PBMC)of CD14 were obtained by density gradient centrifugation and immunomagnetic bead sorting,and then the granulocyte colony stimulating factor(GM-CSF)、interleukin(IL)-4,、tumor necrosis factor-α(TNF-α)was added to the peripheral blood mononuclear cells(PBMC)from patients with acute myeloid leukemia(AML).DC was obtained in vitro and then DC was fused with apoptotic CD34+ leukemia cells for 2 h.Different combinations of Toll-like receptor(TLR)agonists(TLR-3 agonists combined with TLR-9 agonists) were used to further mature DC to obtain functional DC,observing its cellular morphology and immunophenotype.Results(1)Under inverted microscope,mature DC cells became larger in volume,semi-suspended,irregular in shape,and had multiple spiny protuberances on the surface with typical morphological features of mature DC.(2)The expression of CD11 c was increased and the expression of CD14 was down-regulated by flow cytometry,indicating the transformation of DC.(3)Poly I: C combined with TLR9 agonist(Cp G ODN)induced the maturation of DC loaded with LSC.The positive expression of CD11 c,CD86 on the surface of DC was significantly higher than that of single drug group and blank control group,and DC maturation could be further enhanced in combined stimulation group.Thus,the anti-tumor effect was enhanced.Conclusion(1)TLR3 agonist(Poly I: C)combined with TLR9 agonist(Cp G ODN)can promote the maturation of DC and enhance the activation of CTL cells stimulated by DC,thus exerting an anti-tumor immune effect.(2)The DC tumor vaccine prepared in this study can effectively ingest,present antigen and induce tumor-specific CTL,producing high efficiency and specific anti-tumor effect,has potential clinical application prospect.