ABSTRACT
T cell plays a pivotal role in immune system. Not only direct antigen stimulation, but also costimulation signal transducted through cell/cell membrane molecule interactions are needed in the procedure of T cell activation. Besides B7/CD28, CD40/CD40L signal is another important costimulatory pathway, and plays critical roles in many physiologic processes. The disorders of CD40/CD40L costimulatory pathway always lead to serious pathologic phenomena. In this article, the role of CD40/CD40L interactions and therapeutic potentials of blockade of it are discussed.
ABSTRACT
CD40/CD40L, besides B7/CD28, is an alternative important costimulation signal transduction pathway. It plays a pivotal role in T cell activation. Moreover, it may play a critical role at many levels of sensitization and effector phases of allograft rejection. In order to get the fusion protein of human CD40 extracelluar region and IgG 1 Fc fragment, and investigate the potential role of blocking CD40/CD40L costimulation pathway in immunotherapy, total RNA was extracted from human lymphoma cell line Daudi, and CD40 gene extracelluar region was amplified by RT-PCR. The PCR products were inserted into pGEM T Easy vector, and the cloning vector pGE40 was obtained. The DNA sequence was analyzed by automatic DNA sequencer. After sequencing, the transient expressing vector was constructed by inserting correct fragment into pIG vector, which contains the genomic human IgG1 Fc (hinge, CH2 and CH3) gene. Hence the recombinant fusion expression vector was constructed successfully, and named after pIG/40 Ig. Then, COS-7 cells were transfected through DEAE-Dextran/chloroquine method. The CD40-Ig fusion protein expressed in COS-7 cell culture supernatant was identified by sandwich ELISA and Western blot. Result showed that the CD40-Ig fusion protein can be detected by sandwich ELISA in the cell culture supernatant. Western blot analysis also showed that it could react with McAbs of mouse anti-human CD40 G28-5 and mouse anti-human Ig gamma chain. There is only one obvious band at the position of relative molecular weight 50 kD, and it is equivalent to the expected value. Above all, the recombinant fusion expression vector pIG/40 Ig was constructed, and CD40-Ig fusion protein gene was expressed in COS-7 cells successfully. It could be laid a foundation to investigate the potential role of CD40/CD40L pathway as the target of GVHD prevention and therapy.
ABSTRACT
To establish murine graft versus host disease (GVHD) model, in order to evaluate the efficacy of human CD40 Ig fusion protein treatment. To establish murine GVHD model, 2 5?10 7 or 5 0?10 7 /L spleen cells of male C57BL/6 mice were intravenously injected into female BALB/c mice, as receipt, respectively, after sub lethally irradiation by 60 Co source. After the induction of GVHD, human CD40 Ig fusion protein was intravenously injected three times at a dose of 50?g, 150?g and 450?g on day 0, 2 and 4, respectively. The results showed that the typical expressions of GVHD were observed 4 or 5 days after the injection of donor spleen cells, and specific male Y chromosome fragment was amplified by genomic PCR in female BALC/c receipts. The mean survival time (MST) of GVHD induced mice was significantly prolonged by the treatment of human CD40 Ig fusion protein. It is suggested that Murine GVHD model successfully established can be used in evaluating the effects of anti GVHD therapies.
