The Role Of IRF-4 Binding Protein (IBP) In T Cell Apoptosis

IBP (IRF-4 binding protein) is a novel molecule associated with formation of immunological synapses, activation of T cells and polarization of Th1/Th2 cells. It was demonstrated that loss of IBP in mice leads to the spontaneous development of a systemic autoimmune disorder like human SLE, characterized by the significant elevation of serum IgG levels, autoantibody production, and accumulation of effector/memory T cells, but the precise role of depressed IBP expression resulting in autoimmune disorder is still unclear. Since cell apoptosis plays an important role in multicellularis development, homeostasis maintenance, immunoprotection and immunoregulation and the abnormal apoptosis accounts for many autoimmune diseases, in order to explore the potential role of IBP in T cell apoptosis, serial experiments have been done. Primary results indeed show that IBP is highly involved in T cell apoptosis. The following is the summary of the serial experiments.Methods and results1. Construction of the IBP siRNA stable expression vectorsiRNA sequences targeted for IBP and the negative control were subcloned into the GeneBuster vector to obtain the recombinant plasmids. For detecting the efficiency of depression of IBP expression, the IBP gene was put on the N terminal of pTRE-firefly luciferase gene, and Dual luciferase reporter gene assay kit (DLR) was used to check the RNAi efficiency. At last, an effective sequence named siRNA 178 was obtained and the efficiency of depression is 78%.2. Stable transfected cells with siRNA vector(1) Cell transfection: leukemia T cell line Jurkat was transfected by IBP siRNA 178 recombinant plasmid and negative control vector. With the neo resistance, G418 was used to kill untransfected cells so as to get stable transfected cells.(2) Quantitative method for mRNA level of IBP: The standard preparation of objective gene IBP and house-keeping geneβ-actin were used to get standard curve of real time PCR. (3) Quantitative method for protein level of IBP: Prokaryotic expression recombinant plasmid PET-32a/IBP and PGEX-KG/IBP were constructed, 6×his-IBP fusion protein was used as immunogen to immunize rabbits and GST-IBP was used as screening antigen so as to get the polyclonal antibody, then western blotting could be done to detect the expression of IBP in cells.When detected by two quantitative methods, the stable transfected cell clones were proved workable with an efficiency of depression about 73%.3. Role of IBP in T cell apoptosis(1) Effect on cell proliferation of IBP gene silenced cells upon TCR stimulation: We named the Jurkat cells transfected with IBP siRNA expression vector as J.IBP-, while the negative-vector transfected cells as J.NC. Then cells were stimulated with plate-bound anti- CD3 and anti-CD28 antibodies (5μg/ml), DNA synthesis was measured by assessing the incorporation of 3H-thymidine. As a result, the proliferation of J.NC was modestly decreased under the stimulation while the J.IBP- was normally.(2) Effect on apoptosis of IBP gene silenced cells upon TCR stimulation: Cells were activated with identical TCR signals, subsequently, apoptosis was determined by staining with annexin V/PI. It is showed that the J.NC undergoes apoptosis upon this stimulation, while J.IBP- is opposite. We think that the activation induced cell death (AICD) may happen, but J.IBP- are able to resist it. So we conclude that IBP could take part in the normal apoptosis after cell activation. (3) Investigation apoptosis pathway associated with IBP: J.IBP- was co-cultured with the hepatoma carcinoma cell SW480 which expresses FasL intensively, since SW480 has the ability to induce co-cultured lymphocytes apoptosis through FasL-Fas pathway, apoptosis was measured by staining the cells with PI After co-culture for 24h. The result shows that J.IBP- could block the Fas-apoptosis pathway. So we infer that IBP participates normal apoptosis through the Fas-associated pathway, thus plays a role in regulating the immunological homeostasis. Besides, HEK293T was transfected with pEGFP-C1/hIBP containing full IBP, then stimulated with PMA and ionomycin, we observed that IBP translocate to nucleus in some cells, which suggests that IBP might possess transcription factor activity and perhaps contribute to transcriptional control of Fas or other related genes. Conclusively, we constructed the IBP gene silencing T cell model by siRNA technology, and found that T cell with depressed expression of IBP could resist the Fas-mediated apoptosis pathway, which shows that IBP contribute to the T cell apoptosis through Fas-mediated apoptosis. Fas-mediated apoptosis plays an important role in immunological homeostasis and immunoregulation, making the immune response and activation of immunocyte to certain extent so as to avoid excessive damagement and allowing immune system keep balance. So IBP is highly involved in immunological homeostasis. We think the possible mechanism may be that IBP translocates to nucleus as the transcription factor to regulate the expression of Fas or Fas related genes. This study has important implication for understanding the mechanism of IBP associated with immunological homeostasis and development of new prevention and treatment about autoimmune diseases.