Background Recent studies have suggested that mature T cells can change their specificity through reexpression of recombination-activating genes （RAG） and RAG-mediated V（D）J recombination. This process is named receptor revision and has been observed in mature peripheral T cells from transgenic mice and human donors. However, whether the receptor revision in mature T cells is a random or orientated process remains poorly understood. Here we used the Jurkat human T cell line, which represents a mature stage of T cell development, as a model to investigate the regulation of T cell receptor （TCR） gene recombination. Methods TCR Dβ-Jβ signal joint T cell receptor excision DNA circles （sjTRECs） were determined by nested and seminested PCR. Double-strand DNA breaks at recombination signal sequences （RSSs） in the TCRVβ chain locus were detected by ligation-mediated-PCR. Further analysis of the complementarity-determining region 3 （CDR3） size of the TCRVβ chain was examined by the TCR GeneScan technique. Results RAG1, RAG2, and three crucial components of the nonhomologous DNA end-joining （NHEJ） pathway were readily detected in Jurkat. Characteristics of junctional diversity of Dβ2-Jβ2 signal joints and ds RSS breaks associated with the Dβ2 5＇ and Dβ 2 3＇ sites were detected in DNA from Jurkat cells. CDR3 size and the gene sequences of the TCRVβ chain did not change during cell proliferation. Conclusions RAG1 and RAG2 and ongoing TCR gene recombination are coexpressed in Jurkat cells, but the ongoing recombination process may not play a role in modification of the TCR repertoire.However, the results suggest that Jurkat could be used as a model for studying the regulation of RAGs and V（D）J recombination and as a ＂special＂ model of the coexistence of TCR gene rearrangements and ＂negative＂ receptor revision.
ZOU Hong-yunMA LiMENG Min-jieYAO Xin-shengLIN YingWU Zhen-qiangHE Xiao-weiWANG Ju-fangWANG Xiao-ning
Objective To study the activities of interleukin （IL）-2 and granulocyte-macrophage colony-stimulating factor （GM-CSF） （hlL-2/mGM-CSF）. Methods SOE PCR was used to change the linker of the fusion protein for higher activities. The fusion protein was expressed in Escherichia coli （E. coil） BL21 （DE3） in inclusion body （IB） form. After IB was extracted and clarified, it was denatured and purified by affinity chromatography. The protein was refolded by dilution in a L-arginine refolding buffer and refined by anion chromatography. The protein activity was detected by cytokine-dependent cell proliferation assay. Results The expression of hIL-2/mGM-CSF in E. coli yielded approximately 20 mg protein/L culture and the purity was about 90%. The specific activities of IL-2 and GM-CSF were 5.4×10^6 IU/mg and 7.1×10^6 IU/mg, respectively. Conclusion This research provides important information about the anti-tumor activity of hIL-2/mGM-CSF in vivo, thus facilitating future clinical research on hlL-2/mGM-CSF used in immune therapy.
