Expression of Human
Soluble CD40 Ligand in Pichia pastoris and Its
Effects on Dendritic Cells and Malignant B Cells
SHI Hong-Zhen, HUANG Wei-Da1, WANG Yue-Dan, LU
Yan, JIANG Yiang, ZHANG Xue-Guang*
( Department of Immunology, Suzhou Medical College, Suzhou 215007,
China；1Laboratory of Biochemistry &
Molecular Biology, Fudan University, Shanghai 200433, China )
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
pPICZαA plasmid to construct the pPICZαA-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 TNFα 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. �お�
Key words CD40 ligand；Pichia pastoris；dendritic cell；malignant B cell
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