| ICOS (the inducible costimulator), the newest member of the CD28 family, is predominately expressed on activated and memory T cells. Its binding with its specific ligand GL50, which is constitutively expressed on B cells, macrophages and DCs or induced on fibroblast, plays a pivotal role in inflammation, autoimmune diseases, tumor immunity and transplantation immunity, thus discerning a broad prospect of this newly found couple for potential clinic application. However, the molecular mechanism by which GL50-ICOS are involved in immuno-regulation is not clearly elucidated yet. Furthermore, studies so far on GL50-ICOS were mainly focused on mice. These prompted us to explore how GL50-ICOS function in human immune responses, which will present a clearer picture for the clinic application of GL50-ICOS. We successfully cloned the full-length sequence of ICOS and ICOS cDNA; expressed functional ICOS recombinant protein in E.coli and constructed ICOS-transfected cells. Two strains of hybridoma secreting anti-ICOS mAbs were obtained by irrimunizing Balb/c mice with ICOS-transfected cells. We first investigated the role of ICOS recombinant protein in the maturation of DCs and in humoral immunity as well as the effect of anti-ICOS mAbs and ICOS recombinant proteinon the biological behavior of MM in vitro and in vivo.Part I. human ICOS Expression and its biological functions1. ICOS gene cloning, expression of recombinant proteinThe published cDNA sequence for ICOS was used for designing primer. Full-lengthof ICOS was amplified through RT-PCR from activated human T cells and the cDNA fragment was cloned into T-vector plasmid. The derived plasniid (named as T-ICOS) was verified by DNA sequence analysis, and then used as template for PCR to obtain the cDNA fragment encoding extra-cellular domain of ICOS. The production of PCR was cloned into expression vector pET-28a to construct recombinant expression vector pET-ICOS. After confirmed by DNA sequencing, the recombinant expression vector pET-ICOS was transformed into BL-21 E. coll strain. Induced by l-5mmol/L IPTG, ICOS recombinant protein was expressed in BL-21 E. coli in forms of inclusion bodies. ICOS recombinant protein with bioactivity was obtained through denaturation, renaturation and purification by FPLC.2. Construction of ICOS cDNA transfected L929 cellThe specific primers with BamHI and EcoRI site were designed to amplify the ICOS full-length cDNA by PCR. The PCR production was then cloned into retrovirus expression vector pGEZ. After verified by DNA sequence analysis, the derived recombinant expression vector (named as pGEZ-ICOS) together with helper virus expression vector pHIT60 and pHIT456 were transfected into packed cell by liposomes to produce recombinant retrovirus, which was used to infect L929 cells. The transfectant L929 cell expressing ICOS stably was obtained through Zeocin selection and /or being sorted by flow cytometry.3. Alterations of the biological behavior of Daudi cells after ICOS stimulationHigh-level expression of GL50 molecule on Daudi cells and HL60 cells has been verified by FCM, so we focused our attention on investigating whether ICOS protein could induce these malignant B cells to apoptosis. Daudi cells were cultured with rhsICOS or membrane-type ICOS (ICOS-transfected L cell). Cell growth curve, early apoptosis as well as membrane surface molecules such as CD54, CD95, and CXCR4 expressed on Daudi cells were all analyzed. Our results showed that rhsICOS and L929-ICOS stimulation resulted in not only significant growth inhibition, but also apoptosis induction of Daudi in vitro. The apoptosis rate for Daudi was 89% (rhsICOS) and 76% (L929-ICOS). Phenotype analysis showed that CD54, CXCR4 were down- regulated, while CD95 up-regulated.In conclusion, we successfully expressed rhsICOS in E.coli and constructed ICOS-transfected L929 cells. ICOS protein significantly inhibited the growth andeffectively prompted the apoptosis of Daudi cells in vitro.Part II. Effect of ICOS protei...
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