title: Expression of Human Soluble CD40 Ligand in Pichia pastoris and Its Effects on Dendritic Cells and Malignant B Cells.
abstract: CD40 ligand (CD40L) is a member of the tumor necrosis factor (TNF) superfamily and is expressed primarily on the activated CD4( )T lymphocytes. The CD40 molecule, the cognate receptor of CD40L presents on many immunocytes such as B lymphocytes, dendritic cells (DCs) as well as on some neoplastic cells. Triggering of CD40 through CD40L plays a central role in the initiation and regulation of the human immune response. In order to further investigate the possible biological roles of CD40 signaling triggered by CD40L, we subcloned the DNA fragment encoding the extracellular region of human CD40L into the pSK plasmid. After being sequenced, the target fragment was introduced into the pPICZalphaA plasmid to construct the pPICZalphaA-sCD40L expressing vector which was then transduced into Pichia pastoris GS115 cells by electroporation. The tansformant expressed sCD40L in culture supernatants with a maximum yield of about 35 mg/L. Furthermore, we found that the recombinant human soluble CD40 ligand (rhsCD40L) could effectively induced human peripheral blood monocytes(PBMCs) in vitro in the absence of TNFalpha into dendritic cells (DCs) with the typical morphology and special surface markers of dendritic cells including CD1a, CD80, CD83, and HLA-DR etc. To our surprise, the rhsCD40L also could inhibit directly in vitro proliferation of the CD40-positive multiple myeloma cell line XG-2 and the B lymphoma cell line Daudi significantly at an optimal concentration from 2.5 to 15.0 mg/L, while CD40 negative ovarian carcinoma cell lines, SKB and SKR, were not effected by either high or low concentration of rhsCD40L. Moreover, rhsCD40L had the same effects as CD40L-transfected cell in inducing XG2 cell apoptosis. Our results demonstrated that functional human soluble CD40L could be successfully expressed in the Pichia pastoris system and that the recombinant human soluble CD40L might be a potential immune adjuvant and a new powerful molecule for tumor bio-therapy.
authors.affiliation: Department of Immunology, Suzhou Medical College, Suzhou 215007, China. smbxuegz@publicl.sz.js.cn
authors.forename: Hong-Zhen
authors.initials: HZ
authors.lastname: Shi
authors.forename: Wei-Da
authors.initials: WD
authors.lastname: Huang
authors.forename: Yue-Dan
authors.initials: YD
authors.lastname: Wang
authors.forename: Yan
authors.initials: Y
authors.lastname: Lu
authors.forename: Yiang
authors.initials: Y
authors.lastname: Jiang
authors.forename: Xue-Guang
authors.initials: XG
authors.lastname: Zhang