CD70–CD27 ligation between neural stem cells and CD4+ T cells induces Fas–FasL-mediated T-cell death

1. Introduction : Neural stem cells (NSCs) are among the most promising candidates for cell replacement therapy in neuronal injury and neurodegenerative diseases. One of the remaining obstacles for NSC therapy is to overcome the alloimmune response on NSCs by the host. 2. Methods : To investigate the mechanisms of immune modulatory function derived from the interaction of human NSCs with allogeneic T cells, we examined the immune regulatory effects of human NSCs on allogeneic T cells in vitro. 3. Results : Significantly, NSCs induced apoptosis of allogeneic T cells, in particular CD4+ T cells. Interaction of CD70 on NSCs and CD27 on CD4+ T cells mediated apoptosis of T cells. Thus, blocking CD70–CD27 interaction prevented NSC-mediated death of CD4+ T cells. 4. Conclusions : We present a rational explanation of NSC-induced immune escape in two consecutive stages. First, CD70 constitutively expressed on NSCs engaged CD27 on CD4+ T cells, which induced Fas ligand expression on CD4+ T cells. Second, CD4+ T-cell apoptosis was followed by Fas–Fas ligand interaction in the CD4+ T cells.This work was supported by the Ministry of the Knowledge Economy (grants 2009-67-10033838) and a grant from Hanwha Chemical Corporation (Project No. 0411–20070011).Peer Reviewe

Up-regulation of fibrinogen-like protein 2 in porcine endothelial cells by xenogeneic CD40 signal

Acute humoral xenograft rejection (AHXR), characterized by thrombin generation and endothelial cell activation, should be overcome for the success of xenotransplantation. Fibrinogen-like protein 2 (fgl2) expressed on endothelial cells can convert prothrombin to thrombin directly, which indicates that the induced fgl2 expression in activated endothelial cells can contribute to thrombosis. In xenotransplant condition, the interaction between human CD40L and porcine endothelial CD40 can activate endothelial cells. In this study, we investigated the effect of endothelial cell activation through the interaction between human CD40L and porcine CD40 on fgl2 expression and its function as a direct prothrombinase. We found that CD40 stimulation up-regulated fgl2 expression as well as its enzymatic activity in porcine endothelial cells. Moreover, functional studies using knock-down system showed that the major factor converting human prothrombin to thrombin is fgl2 protein expressed on porcine endothelial cells. Overall, this study demonstrates that fgl2 expression can be induced by xenogeneic CD40 signal on endothelial cells and contribute to thrombin generation

Fgl2 induction in porcine endothelial cells through xenogeneic CD40-CD40L interaction

Background: Fibrinogen-like protein 2 (fgl2) is a direct prothrombinase that generates thrombin from prothrombin in the absence of a classical prothrombinase complex. Fgl2 is known to be involved in experimental xenograft rejection by mediating coagulation, fibrin deposition and microthrombus formation, leading to classical pathological changes of acute vascular rejection. We tried to investigate whether interaction between CD40 on porcine endothelial cells (PECs) and CD40L on human cells mediates induction of fgl2 on PECs. Methods: Fgl2 mRNA expression was assessed using reverse-transcriptase polymerase chain reaction (RT-PCR). Protein expression and enzymatic activity of fgl2 in PECs were assessed using western blot and fgl2 thrombin generation assay, respectively. Both porcine fgl2 and CD40 knockdowned stable cell lines were established using sifgl2 and siCD40 RNAi system. Results: Fgl2 mRNA expression was induced in PECs at 30 min after PECs had been cultured with Jurkat D1.1 and its expression was suppressed by anti-CD40L antagonistic antibody treatment. Protein expression of fgl2 in PECs was up-regulated at 4 h after treatment of anti-CD40 agonistic antibody as well as TNF-a. The enzymatic activity of fgl2 on PECs was increased by about twofold at 6 h after stimulation of anti-CD40 agonistic antibody. Stable cell lines for either sifgl2 or siCD40 were established. The enzymatic activity of fgl2 was diminished by both sifgl2 and siCD40, but not affected by a control siEGFP. Conclusion: Fgl2 can be up-regulated and mediate coagulation response in activated PECs through xenogeneic CD40-CD40L interaction. Therefore, it might be helpful for the successful xenotransplantation to control fgl2 induction through development of porcine CD40 knockout pigs or antagonistic anti-CD40 treatment.

Expression analysis of combinatorial genes using a bi-cistronic T2A expression system in porcine fibroblasts.

In pig-to-primate xenotransplantation, multiple transgenic pigs are required to overcome a series of transplant rejections. The generation of multiple transgenic pigs either by breeding or the introduction of several mono-cistronic vectors has been hampered by the differential expression patterns of the target genes. To achieve simultaneous expression of multiple genes, a poly-cistronic expression system using the 2A peptide derived from the Thosea asigna virus (T2A) can be considered an alternative choice. Before applying T2A expression system to pig generation, the expression patterns of multiple genes in this system should be precisely evaluated. In this study, we constructed several bi-cistronic T2A expression vectors, which combine target genes that are frequently used in the xenotransplantation field, and introduced them into porcine fibroblasts. The proteins targeted to the same or different subcellular regions were efficiently expressed without affecting the localization or expression levels of the other protein. However, when a gene with low expression efficiency was inserted into the upstream region of the T2A sequences, the expression level of the downstream gene was significantly decreased compared with the expression efficiency without the insertion. A small interfering RNA targeting one gene in this system resulted in the significant downregulation of both the target gene and the other gene, indicating that multiple genes combined into a T2A expression vector can be considered as a single gene in terms of transcription and translation. In summary, the efficient expression of a downstream gene can be achieved if the expression of the upstream gene is efficient

Generation and characterization of human heme oxygenase-1 transgenic pigs.

Xenotransplantation using transgenic pigs as an organ source is a promising strategy to overcome shortage of human organ for transplantation. Various genetic modifications have been tried to ameliorate xenograft rejection. In the present study we assessed effect of transgenic expression of human heme oxygenase-1 (hHO-1), an inducible protein capable of cytoprotection by scavenging reactive oxygen species and preventing apoptosis caused by cellular stress during inflammatory processes, in neonatal porcine islet-like cluster cells (NPCCs). Transduction of NPCCs with adenovirus containing hHO-1 gene significantly reduced apoptosis compared with the GFP-expressing adenovirus control after treatment with either hydrogen peroxide or hTNF-α and cycloheximide. These protective effects were diminished by co-treatment of hHO-1 antagonist, Zinc protoporphyrin IX. We also generated transgenic pigs expressing hHO-1 and analyzed expression and function of the transgene. Human HO-1 was expressed in most tissues, including the heart, kidney, lung, pancreas, spleen and skin, however, expression levels and patterns of the hHO-1 gene are not consistent in each organ. We isolate fibroblast from transgenic pigs to analyze protective effect of the hHO-1. As expected, fibroblasts derived from the hHO-1 transgenic pigs were significantly resistant to both hydrogen peroxide damage and hTNF-α and cycloheximide-mediated apoptosis when compared with wild-type fibroblasts. Furthermore, induction of RANTES in response to hTNF-α or LPS was significantly decreased in fibroblasts obtained from the hHO-1 transgenic pigs. These findings suggest that transgenic expression of hHO-1 can protect xenografts when exposed to oxidative stresses, especially from ischemia/reperfusion injury, and/or acute rejection mediated by cytokines. Accordingly, hHO-1 could be an important candidate molecule in a multi-transgenic pig strategy for xenotransplantation

The quantitative analysis of each gene by multi-cistronic vectors used in this study.

<p>The quantitative analysis of each gene by multi-cistronic vectors used in this study.</p

The downstream gene expression levels are correlated with the transcriptional efficiency of the upstream gene.

<p><b>A:</b> Porcine fibroblasts were transfected with each indicated vector, and the mean fluorescent intensity (MFI) of EGFP was analyzed by flow cytometry. Summarized bar graphs show means ± SE of three independent replications. <b>B:</b> The expression levels and the subcellular localization of EGFP were analyzed using a fluorescent microscope. Hoechst 33342 was used for nuclear staining (original magnification X200). <b>C:</b> After the removal of the residual plasmid DNA with DNase treatment, the RNA extracts were subjected to RT-PCR for EGFP. These results are representative of three independent experiments.</p

The expression levels of downstream genes are extremely low in the bi-cistronic IRES-Ex constructs.

<p><b>A:</b> Schematic diagram of the bi-cistronic IRES-Ex vector. <b>B:</b> Porcine fibroblasts were transfected with the vector containing the EGFP-IRES-(HA)HO1 or (HA)HO1-IRES-EGFP sequences. The cells were analyzed using flow cytometry at 24 h after transfection. For (HA)HO1 detection, the cells were incubated with the mouse anti-HA Ab, followed by the APC-conjugated goat anti-mouse IgG Ab, according to the intracellular staining protocol. Mock transfected cells were used as control (filled line). Summarized bar graphs show means ± SE of three independent replications. <b>C:</b> The cell lysates were subjected to immunoblotting with either the mouse anti-GFP or mouse anti-HA Abs, followed by HRP-conjugated goat anti-mouse IgG. These results are representative of three independent experiments.</p