The Roles Of Flt3 And 4-1BBL On Monocytes And Monocytic Leukemia Cells And The Underlying Mechanisms

Flt3 is expressed on multipotential stem cells, normal hematopoietic cells, including myeloid and B-lymphoid lineage, and monocytes from PBMCs. Flt3 plays important role in differentiation and renewing of hematopoietic cells. Flt3 is also expressed at high levels in a spectrum of hematologic malignancies and involved in pathogenesis of leukemia and becoming a target of treatment of leukemia. 4-1BBL/4-1BB is an important member of TNF/TNFR superfamily. 4-1BBL is expressed on activated APCs and T cells, as well as on some types of malignancies, such as a fraction of leukemia cells.4-lBBL also plays an important role in survival and proliferation of leukemia cells. Monocytes isolated from PBMCs and monocytic leukemia cell line SHI-1 are selected to study the role of Flt3 signaling and 4-1BBL reverse signaling of monocytes and monocytic leukemia cells. We successfully cloned the full length cDNA of human Flt3 and constructed the Flt3 transgenic cells BaF-Flt3 and L-Flt3. Two strains of hybridoma secreting mAbs against human Flt3 were obtained. We investigated the effects and mechanisms of Flt3 signaling and 4-1BBL reverse signaling to monocytes . as well as the monocytic leukemia cells. Part I. Flt3 gene cloning and Flt3 transgenic cell construction 1. Flt3 gene cloning and construction of recombinant FltS retrovirus expressing vector constructingOverlapped cDNA fragments were cloned from human bone marrow cDNA library, and then the fragments were linked to the full length Flt3 gene.2. Construction of Flt3 transgenic cells with "kill two birds with one stone" strategyFull length Flt3 gene were subcloned to PstI and BamHI restriction sites of retrovirus expressing vector pGEZ and named as pGEZ-Flt3. After being verified by DNA Sequencing, the derived recombinant expressing vector pGEZ-Flt3, together with helper virus expressing vectors pHIT456 and pHIT60, was transfected into packed cell (293T cell) by liposomes to produce recombinant retrovirus, which was used to transfect the parent cells. L929 cells and BaF cells were cultured in the same 24 well-plate with the mixture of the pGEZ-Flt3 recombinant retrovirus and supernatants of WEHI containing the murine IL-3. And then L929 cells and BaF cells were successfully separated because BaF cell was IL-3 dependent suspending cell and L929 was IL-3 independent adhensive cell. Transgenic cells L-Flt3 and BaF-Flt3 stably expressing Flt3 were obtained by Zeocine selection and flow cytometry screen. Our "kill two birds with one stone" strategy provided an effective solution to the low efficiency of transgenic cell construction of the suspending cell. Part II. Preparation and characterization of anti-Flt3 mAbs 1. Preparation of anti-Flt3 mAbsBaF-Flt3 and L-Flt3 were used to immunize Balb/c mice. The immunized spleencytes were fused with mouse myeloma cells (SP2/0) by using polyethylene glycol (PEG), and then they were cultured in HAT selection medium. With BaF-Flt3 cells, the hybridomas secreting specific mAbs were screened by immunoflurescence assay. Through repeatedly cloning and screening, two hybridoma cell line(10A5 and 1 OG6) continuously and steadily secreting specific anti -human 4-IBBL were obtained. These two hybridoma cells grew well after long-term culture in vitro and storage in liquid nitrogen.MAb 10A5 and mAb 10G6 were produced by in-vivo procedures in mouse peritoneal cavity. The ascitic titer of 10G6 was over 1:1000 dilution and that of 10A5 was over 1:5000 by flow cytometry assay. Every 1*106 cells required 0.25~2.00microgram purified 10G6 mAb. Fast-strip method analyses displayed that mAb 10A5 belonged to mouse IgM and mAb 1 OG6 belonged to mouse IgGl . 2. The recognition ability of the mAb 10 A5 and mAb 10G6To further elucidate the recognition ability of the mAbs against Flt3 molecule, the phenotype analysis was utilized by flow cytometry assay. The result indicated that the mAb could recognize Flt3 molecule expressed on 8226, U266, HL60 and SHI-1 cells as the commercialized anti-human Flt3 mAb SF 1.340,Partlll. The effects of...