Generation of murine dendritic cells in vitro using Flt3-L and their characteristics
Introduction. Currently, cell vaccines based on myeloid dendritic cells (mDCs) are being actively developed to modulate an adaptive immune response in order to correct certain human diseases. However, there is still little data on the effectiveness of the use of plasmacytoid dendritic cells (pDCs) for the same purposes. Aim of the study. Development of the protocol for in vitro generation of mice pDCs using recombinant Flt3-L and description of the phenotypic and functional properties of the obtained dendritic cells (DCs) to address the possibility of their use in the development of cell vaccines Material and methods. DCs were generated from mouse bone marrow cells using Flt3-L. Frequency and phenotype of the DCs were assessed by flow cytometry. DC's morphology was assessed using fluorescent microscopy. Treg frequency and CD4<sup>+</sup>-lymphocytes proliferation induced by DCs were assessed in mixed lymphocyte culture. The expression of CCR9 on DCs was assessed using flow cytometry. Results. It was shown that the use of Flt3-L provides in vitro generation of bone marrow-derived cultures with a relatively high content of B220<sup>+</sup>CD11c<sup>+</sup>-pDCs and Sirpa<sup>+</sup>CD11c<sup>+</sup>-mDCs. The B220<sup>+</sup>CD11c<sup>+</sup>-pDCs and the Sirpa<sup>+</sup>CD11c<sup>+</sup>-mDCs obtained differed in morphology and degree of maturity. In a mixed lymphocytes culture, the dendritic cells were able to generate an increased amount of Treg and cause a reduced proliferation of CD4<sup>+</sup>-lymphocytes, thus manifesting an immature phenotype. Further maturation of the DCs via CpG DNA stimulation led to an increased immunogenicity, according the Treg reduction and enhanced CD4<sup>+</sup>-lymphocytes proliferation in mixed lymphocytes culture. We found that a relatively small number of B220<sup>+</sup>CD11c<sup>+</sup>-pDCs and Sirpa<sup>+</sup>CD11c<sup>+</sup>-mDCs carried the CCR9 chemotaxis receptor on their surface, however, the number of B220<sup>+</sup>CD11c<sup>+</sup>-pDCs and the Sirpa<sup>+</sup>CD11c<sup>+</sup>-mDCs carrying intracellular CCR9 tended to 100 %. Thus, the use of Flt3-L provides the opportunity of in vitro generation of B220<sup>+</sup>CD11c<sup>+</sup> pDCs and Sirpa<sup>+</sup>CD11c<sup>+</sup>-mDCs that can be useful in the field of cell vaccine development to direct an adaptive immune response decreasing undesirable immune reactions or increasing desirable.