Characterization Of Human BAFF Transfected Cells And Mouse Anti-BAFF Monoclonal Antibody

BAFF (B cell-activating factor belong to the TNF family) belong to the TNF superfamily mainly expressed on the monocytes, macrophages, dendritic cells, activated T lymphocytes and neutrophils, which can bind to receptors, such as BAFF-R, TACI and BCMA, all of these receptors are the members of the TNFR superfamily and expressed on the T and B lymphocytes. BAFF is an important factor to the development, differentiation of B lymphocytes, and to the isotype transition of Ig. And it can costimulate to T lymphocyte. The abnormity of BAFF signal is correlated with SLE, RA and SS, with chronical B lymphocyte leukaemia, non-Hodgkin lymphoma and multiple myeloma, and also with allotransplantation rejection of heart transplantation et al.To deeply investigation on the BAFF signal, the full length cDNA of human BAFF was successfully cloned and the BAFF transfected cells L929-BAFF was constructed. Then the biology effect of L929-BAFF on the tonsil B cells was studied. One cell strain of hybridoma secreting mAbs against human BAFF were obtained, and its characteristic was studied. Then the BAFF expression pattern on the T lymphocytes of healthy PBLs and malignant pleural effusion was studied, and the significance of the expression was investigated too. Meanwhile the full length cDNA of human TACI was also successfully cloned, and the TACI-Fc transfected cells which secrected soluble TACI-Fc fusion protein was constructed. The biology function of TACI-Fc fusion protein was investigated too. Part I The construction of BAFF transfected cells and its effects on B lymphocytes in vitro.1. Cloning of BAFF gene and construction of recombinant BAFF retrovirus expressing vectorBAFF cDNA was cloned from human peripheral mononuclear cells by RT-PCR, then the fragment was linked to the retrovirus expressing vector pSFVl.2. Construction of the transfected cell L929-BAFF by lipofectamineThe recombinant expressing vector pSIVl-BAFF, together with helper virus expressing vectors pHIT456 and pHIT60, was transfected into packed cell (293T cell) by lipofectamine to produce recombinant retrovirus, which was used to transfect parent cells(L929). Transgenic cells L929-BAFF stably expressing BAFF was obtained by G418 selection and flow cytometry screen.3. Effect of L929-BAFF on the B lymphocytesIn the PWM-driven tonsil B cells culture system, BAFF-transfected L929 cells could promote the proliferation of B cells by 3H-TdR analysis. In the other B cell culture system driven by immobilized anti- P antibody, L929-BAFF cells could promote the secretion of IgG by ELISA assay.Part II The expression and characterization of human TACI-Fc fusion protein 1. Expression of human TACI-Fc fusion proteinTACI cDNA was cloned from freshly isolated tonsil cells by RT-PCR. The extracellular fragment of TACI cDNA added one signal peptide on its 5'-end was amplified by PCR. Then this fragment was inserted into eukaryotic expressing vector pcDNA3.1. The recombinant vector pcDNA3.1-TACI was obtained. Then the human IgGlFc fragment was inserted into the 3'-end of TACI in the vector pcDNA3.1-TACI by directed gene recombination method, so the recombinant vector pcDNA3.1-TACI-Fc was obtained, which could express soluble TACI-Fc fusion protein.The TACI-Fc transgenic L929 cells was obtained by lipofectamine and G418 selection, conformed by RT-PCR and ELISA analysis. Collected the supernant of L929-TACI-Fc cells, concentrated and purified, the TACI-Fc fusion protein was obtained. The concentration of fusion protein was about 10 u g/ml.2. Effect of human TACI-Fc fusion protein on the multiple myeloma cell line XG6 in vitroRhBAFF was added into the XG6 culture system which had been in starvation (deprived of IL-6) for one week, and rhBAFF could survive and promote the growth of XG6. The TACI-Fc fusion protein were added into this culture system for 4 days, the number of lived cells were counted, and the concentration with best blocked effects was 50 u g/ml. The proliferation rate of XG6 of different groups was tested by 3H-TdR, and the apoptosis rate of XG6 was investigated by Annexin V/PI. The results demonstrated that the TACI-Fc fusion protein could block the effects of BAFF on the promotion of prolifetation and survival, and promote apoptosis. Part III The preparation and characterization of anti-BAFF mAbs 1. Preparation of anti-BAFF mAbs ~BAFF transfected L929 cells (L929-BAFF), after pretreatment with mitomycin, were used as immunogen to immunize Balb/c mice to prepare anti-BAFF mAbs. According to the hybridoma technique of B lymphocytes, splenocytes harvested from the immunized mouse were fused with SP2/0, mice MM cells. After repeated screening by L929-BAFF (as positive screening) and Jurket cell line (as negative control), and multiple cloning in HAT conditioned culture, one cell strain of hybridoma (4F7) secreting anti-BAFF mAbs continuously and steadily was obtained, and designated as 4F7. The hybridoma grew well after long-term storage in the liquid nitrogen and in vitro culture.Ascites, induced in pristine-primed Balb/c mice by intraperitineal injected with well-grown hybridomas, was harvested during 10 to 12 days. Concentration of mAbs purified from ascites reached 0.8-1.0 mg/ml. Fast-strip analysis showed that 4F7 belonged to mouse IgGl. 2. Identification of BAFF 4F7 mAbs characteristicsCompetitive binding experiments was conducted to identify the antigen determinants of 4F7 mAbs. The results indicated that 4F7 mAbs might recognize different antigen epitope from standard Bafly-1 mAb.To further elucidate the recognition ability of the mAbs against BAFF molecule, the phenotype analysis was utilized by flow cytometry assay. The results indicated that the4F7 mAb could recognize BAFF molecule lowly expressed on XG2 and XG6, while highly expressed on HL60. The monocytes of peripheral blood mononuclear cells (PBMC) from healthy volunteer lowly expressed BAFF. Afer treated with IFN- y, the monocytes could express BAFF more highly. The CD14+ cells of PBMC almost all express BAFF. While BAFF could not be found on the CD19+ and untreated CD3+ cells , and when CD3+ cells were treated with PHA, it could highly express BAFF. Part IV Expression of BAFF on T lymphocytesOne paper reported that BAFF mRNA was expressed in the T lymphocytes of peripheral blood, and could be more highly expressed in the activated CD4+and CD8+T lymphocytes in 1999. But others could not repeat it. To 2004, one lab demonstrated that BAFF protein was expressed in the T lymphocytes by western-blot. This study obtained one cell stain secreting ant-BAFF mAb 4F7, and experiments demonstrated that it could recognize BAFF molecule expressed on human T lymphocyte of PBLs. So the expression patterns of BAFF on the untreated and treated normal T cells with different stimulaters were studied. At the same time, the expression pattern of BAFF on the T cells from malignant pleural effusions were analysed. The influence of BAFF on the viability of T cells was investigated by Annexin V/PI kits, and the concentration of IL-2, IL-10 and IFN- Y in the supernatant of different treated groups was tested by ELISA assay.BAFF was not expressed on the naive T lymphocytes from healthy volunteer, but expressed on the different phase of activated T cells stimulated PHA or IL-2, and the level of expression is related with activation state of T cells. And BAFF was expressed on the normal T cells pulsed by DCs which was loaded by apoptotic A549. BAFF was expressed on almost all of CD3+ cells, including of CD4+ and CD8+ cells, and expressed on partly of CD69+ cells, and almost all of CD25+ cells.But after washed three times with culture medium 1640 (derived of its activated condition), the activated T cells from healthy voluteers were cultured in normal culture medium 1640 for 3 days, BAFF was not detected on these T cells. This phenomenon was different from the T cells of pleural effusions.BAFF was highly and sustainingly expressed on the these T cells, almost all of CDS+ cells, including of CD4+ and CD8+ cells. BAFF was expressed on the partly CD69+ cells and almost all of the CD25+ cells. The BAFF expression pattern on T cells of pleural effusions was not varied after T cells was stimulated with PHA. These T cells in the pleural effusions was in the activated state.FACS analysis results indicated that, BAFF-R was highly expressed on the T cells from pleural effusions than T lymphocytes from healthy volunteer. While the expression pattern of TACI was different between the T cells from pleural effusions and healthy volunteer, the BAFF expression rate rised on the CD4+T cells, while descend on the CD8+ T cells.The viability assays indicated that rhBAFF could promote the survival rate of T cells from pleural effusions, and TACI-Fc fusion protein could promote the apoptosis of T cells. The blocked anti-IL-2R a chain mAb could promote apoptose also, cooperated with TACI-Fc fusion protein.In the pleural effusions, there is low level of IL-10, and no IL-2 and IFN- y. But when the T cells isolated from pleural effusions were cultured in vitro, the level of IL-10 in the supernant of T cells became lower. And the concentration of IL-2 was significantly high with the prolong of culture, and the level of IL-10 and IFN- Y increased also. The level of IFN- Y in supernant of the group of TACI-Fc fusion protein treated resembled as the control group, but became high significantly in the group of the rhBAFF treated.