Regulatory Factors Of In Vitro Generation Of Dendritic Cells Derived From Cord Blood And Study Of The Biological Functions

Dendritic cells (DCs), the most potent professional antigen presenting cells, are efficient in phagocytosing,processing and presenting antigens to T cells,up-regulating the expression of costimulatory molecules, stimulating the proliferation and activation of naive T cells via two signals, ultimatly inducing immune response.Cord blood enriches CD34~+ hematopoietic progenitor cells(CD34~+HPC) and generates DCs in combination of cytokines, playing an important role in anti-tumor,transplantation immune responses. DCs undergo a complex and exquisite differentiation and maturation procedure. Therefore, regulatory factors of In vitro generation of dendritic cells derived from cord blood and study of the biological functions contribute to clinical applications of DCs based vaccination protocols. Our research were divided into three sections as the followings:In the first section our research focused on the expansion and function of DCs derived from human cord blood CD34~+ HPC combinating a cocktail cytokines,in order to find the optimum scheme of DCs derived from CD34~+ HPC in vitro. The second section were the expressions and interactions of costimulatory molecules during the procedure of the human cord blood CD34~+ HPC derived dendritic cells (CD34-DCs) differentiation and maturation, comparison the different biological effects between CD40 , TNF-α and 4-1BBL stimulated CD34-DCs, and involved mechanism of signal transduction. Costimulatory signals' effects on tumor specific CTL and the associated mechanisms were also investigated. The third section focused on expression of costimulatory molecules during the procedure of the human cord blood adherent cells derived DCs differentiation and maturation, comparing the different biological effects between CD40 and TNF-α stimulated DCs in vitro. These studies all contributed to explore the field of cord bloodderived DCs on malignant tumor and transplantation in clinic.Part I: Expansion and function of dendritic cells derived from human cord blood CD34+ hematopoietic progenitor cellsObject: Our study aimed to probe the expansion and function of dendritic cells(DCs)derived from human cord blood CD34+ hematopoietic progenitor cells, in order to find the optimum scheme of DCs derived from CD34+HPC in vitro.Methods: The isolation of CD34+ HPC from human cord blood used with mini-MACS;Concentrations of IL-12 in supernatant of DCs determined by ELISA; Expression of costimulator molecular detected by FCM; The proliferation of T cells activated by allo-DCs measured with 3H-TdR incorporation.Results: CD34+ hematopoietic progenitor cells significantly expanded in combinationwith SCF,FL,GM-CSF and IL-4 and generated typical DCs which released a large of IL-12 under stimulated by CD40 or TNF-a. The proliferation of allo-T cells stimulated by CD40-DCs was higher than those groups.Conclusions: The combination of SCF,FL,GM-CSF and IL-4 is the optimal plan of expansion of CD34-DCs.IL-4 cooperated the differentiation of CD34+ hematopoietic cells with other cytokines, and CD40 signal is critical in the maturation of human CD34-DCs.Part II :The biological character of dendritic cells derived from CD34+ hematopoietic progenitor cells and the regulator effection of costimulatormolecularObjective: Our study aimed to compare the different biological effects between CD40and TNF-ct stimulated CD34-DCs, including expressions of costimulatory molecules during the procedure of human cord blood CD34-DCs differentiation and maturation and involved mechanism of signal transduction, and to investigate activation of tumor specific CTL by CD40 or TNF-a or 4-1BBL stimulated CD34-DCs combining a cocktail of cytokines.Methods: DCs ultrastructures were observed by transmission electron microscopy;Expressions of costimulatory molecules on CD34-DCs were analyzed by FCM. PD-L1, PD-L2,4-IBB and 4-1BBL mRNA transcription were detected by RT-PCR and Real time-PCR. Concentrations of IL-10 and IL-12 in supernatant of DCs were determined by ELISA, same to the levels of IL-2 and IFN-y secreted by T lymphocytes. Expression of NF-kB molecules were detected by western-blotting. 3H-thymidine incorporation test and MTT analysis were used to detect the proliferation and activation of CTL stimulated by DCs in vitro.Results: CD40,CD86 and PD-L1 were constitutivly expressed in CD34+ hematopoieticprogenitor cells . Expression of PD-L2 and 4-IBB was low, and 4-1BBL was no detected. During the process of differentiation, 4-IBB and 4-1BBL were co-expressed in CD34-imDCs,and PD-L1 was up-regulated. Expressions of PD-L2 and 4-1BBL were higher in CD40 activated CD34-DCs than TNF-a stimulated CD34-DCs (PO.05). Expression of NF-kB (particularly RelB and P50) was up-regulated on CD34-DCs activated by CD40 or TNF-a. CTL activated by CD40-DCs secreted higher level of IFN-y and IL-2, and induced Thl type of immune response.Conclusion: CD40 signal was critical in the differentiation and function of human CD34-DCs. CD40-DCs combining a cocktail of cytokines could generate and activate tumor specific CTL which had the most efficient cytotoxic activity, induced Thl type of immune response. Part El: Expression of costimulatory molecules of cord blood adherent cells derived dendritic cells and the related biological effectsObjective: Our study aimed to detect expressions of costimulatory molecules during the procedure of the human cord blood adherent cells derived DCs differentiation and maturation, compare the different biological effects between CD40 and TNF-a stimulated DCs, Contributing to explore the field of cord blood derived DCs on malignant tumor and transplantation in clinic application.Methods: DCs configuration were observed by light microscopy; Expressions ofcostimulatory molecules on adherent cells derived DC were analyzed by FCM. PD-L1, PD-L2, 4-IBB and 4-lBBL mRNA transcription were detected by RT-PCR and Real-time PCR. Concentrations of IL-10 and IL-12 in supernatant of DCs were determined by ELISA; 3H-thymidine incorporation test were used to detect the proliferation of T cells stimulated by DCs in vitro.Results: CD14 mainly expressed on cord blood adherent cells;PD-Ll expression was moderate on adherent cells derived DCs; PD-L2 and 4-lBBL expression were high on CD40 activated DCs, and releasing a large of IL-12,compared with TNF-a stimulation (P<0.05).Conclusion: CD14+monocytes can be obtained by the way of adherent cells, and CD40 could promote the maturation and biological functions of adherent cells derived DCs.