Abstract
Abstract 2769
In order to analyze the regulation of differentiation and proliferation of the BCL11B gene in human T cells, as well as the related molecular mechanism, BCL11B (B cell lymphoma/leukemia11B, BCL11B) recombinant plasmid and small interfering RNA (siRNA) were transferred into naïve T cells (CD3+CD45RA+ T cells) from healthy individual respectively by gene transfer and RNA interference techniques. The eukaryotic expression plasmid pIRES2-BCL11B-EGFP (pBCL11B) and BCL11B-siRNA-935(si-935), obtained by chemosynthesis, were transferred into different cell lines by using transfection techniques. The transfection efficiencies were examined by fluorescence microscopy and FCM. The expression of the mRNA and the corresponding protein levels of BCL11B were detected by real-time quantitative PCR with TaqMan technique and Western blotting. The pBCL11B plasmid and si-935 were transferred into naïve T cells from healthy individual using the Nucleofector™ technique. The microscopic morphologies were detected by atomic force microscopy (AFM), the expression pattern and clonality of TCR Vβ subfamily genes were analyzed by RT-PCR and genescan techniques, the effect of BCL11B in T cells proliferation in vitro were evaluated by the capability of CFU-T, the changes of T cells immune phenotypes (CD3, CD4, CD8, CD45RA, CD45RO, CD25 and CD86) were examined by FCM. The expression patterns of BCL11B were up-regulated or down-regulated respectively, then, the global gene expression profiling was analyzed by the Affymetrix HG U133 Plus 2.0 Gene Chips. Some of the differentially expressed genes were verified by various techniques. The up- or down-regulated BCL11B expression of mRNA and protein levels could be confirmed in naïve T cells. The AFM images revealed that the surface of naïve T cells treated with pBCL11B showed more signs of expansion than others, and were coated with a larger number of extracellular polymers on the outer layer. Seventeen Vβ subfamily T cells displayed polyclonality could be detected in naïve T cells in up-regulated BCL11B manner. The more effective of capability of CFU-T was also confirmed in the same naïve T cells group. The Th subset was obviously changed after up-regulated of BCL11B in naïve T cells, while the ratio of CD4/CD8 was doubled increased. By contrast, in si-935 group, all of the 24 Vβ subfamilies were unable to detect, the number of CFU-T decreased substantially, and the ratio of CD4/CD8 showed no significant difference in compared with control group. Global gene expression profiling analysis showed that up-regulated genes were found in 95 probe sets, while 124 genes were down-regulated in naïve T cells transfected with BCL11B gene, while up-regulated 302 genes and down-regulated 209 genes were detected in naïve T cells from si-935 group. The up- or down-regulation genes were mainly involved in T cells activation and proliferation, which were associated with the high levels of CD3 and IL-2. BCL11B could promote the Th cells differentiation selectively owing to the increased expression of CXCL10 and CXCL11. Suppression BCL11B expression might inhibit the proliferation and effectively induce the apoptosis, which related to the changes of CFLAR-CASP8-CASP10 in mitochondrial pathways. The changes of expression levels of these genes were confirmed by real-time PCR and ELISA. In conclusions, This is the first report providing a detailed analysis of the regulation of the BCL11B gene in T cell differentiation and proliferation, and the possible mechanism of this gene. It was shown that BCL11B was required for the survival of T cells. Up-regulated BCL11B expression could increase the abilities of T cells activation, proliferation, and Th cells differentiation effectively. However, suppression BCL11B expression might inhibit the proliferation and induce the apoptosis.
Disclosures:
Li: The study was supported by grants from the National Natural Science Foundation (30771980) and the Guangdong Science & Technology Project (2009B050700029): Research Funding.
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