Preparation and Function of PD-L1 Transfectant Cells and Functional Mouse Anti-human PD-L1 Monococlonal Antibody

Abstract: The B7 family of molecules provides signals that are critical for both stimulating and inhibiting T cell activation. Recently, a new member of the B7 family, PD-Ll (B7-H1), has been identified to be the ligand for PD-1 and lead to new road of co-stimulating pathway. Through ITIM of PD-1 molecule, PD-1/PD-Ll pathway functions as co-inhibitor in T, B cells proliferation. Recent researches indicate that PD-Ll has another unknown receptor to enhance T cells proliferation. Also this pathway plays an important role in autoimmune diseases and tumor escape. So we could block this pathway and enhance CTLs to kill cancer cells through specific anti-PD-Ll mono-coclonal antiboby or its soluble inhibitor. Now our research have prepared PD-Ll transfectant cells PD-L1/L929 and functional mouse anti-human PD-Ll mAb, next we wanted to find some relationships of this molecule with signal transduction, autoimmune diseases and tumor escaping. Details of our research are as follow.1. Cloning of human PD-Ll gene and constructing of PD-Ll transfectant cell line PD-L1/L929. cDNA fragment encoding human PD-Ll was obtained from the total RNA of human heart and inserted into retrovirus expression vector pEGZ with gene cloning technique. The recombinant plasimad, pEGZ-PD-Ll, was transferred into mouse fibroblast cell line L929 with lipofectamine and screened with Zeocin and cultured for more than thirty generations. We obtained PD-Ll transfectant cell line PD-Ll/L929, which was highly expressed and stable.2. The effect of PD-Ll signal on the function of activated human T cells in vitro.To investigate PD-Ll regulatory effect on purified human T cells derived from peripheralIVblood stimulated with PHA. Co-culture of transgenic cells PD-L1/L929 and T cells stimulated with 10)j.g/ml PHA, immunophenotypic analysis,3H-TdR incorporation and quantitative measurement of IL-2, IFN-y were performed. We finded with the stimulation of 10|ig/ml PHA, transgenic cells PD-L1/L929 could significantly down-regulate the stimulatory phenotype and proliferation of T cells, which resulted from cell cycle arrest of T cells in phase GO/G1, and the significantly decreased levels of IL-2 and IFN-y secretion. Meanwhile, it was found that PD-L1 signal could inhibit apoptosis of T cells stimulated with 10jj,g/ml PHA, which was reported for the first time. PD-L1 signal showed a negatively regulatory effect on PHA-activated human T cells in vitro.3. Establishment and property of a hybridoma cell line specifically secreting monoclonal antibody against PD-L1. Based on obtaining mock/L929 and PD-L1/L929, we immunized BALB/c mices with PD-L1/L929 cell line as immunogen. The immunized spleencoytes were fused with mouse myeloma cell line (SP2/0), by using polyethy-leneglycol(PEG) and cultured in HAT selection medium. Hybridoma cells were screened with mock/L929 and PD-L1/L929 cells and subcloned more than 5 times. Then one hybridoma cell lines, named 2H11 was obtained, which was able to secret specific mAb against human PD-L1 continuously and stably. The hybridoma grew well after long-term culture in vitro and storage in liquid nitrogen.Further studies showed that its isotype is mouse IgGl and it belongs to K chain, has a not completely the same binding site as compared with PE labeled PD-Ll.For additional analysis, western blotting was performed with PD-L1 membrane protein extracted from PD-L1/L929 cell line. MAb 2H11 identified a protein with approximately 30 kDa, importantly this mAb also can recognize cell surface molecule and be used for immune-staining.4. Function of mAb 2H11. We found that 2H11 mAb could induce T cell activation and proliferation in a dose-dependent way with pre-coating agonist anti-CD3 mAb andvcould up-regulate secretion of IL-10 significantly, but only down-regulate secretion of IL-2 and IFN-y weakly. Meanwhile, 2H11 mAb could inhibitor the maturation of DC and revise expression of co-stimulating molecule on DCs, and subsequently affect the function of DCs. More important and for the first time, we indicated tha…
Key words: PD-1; PD-L1; monocolonal antibody; reverse signal; DC; T cells

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