Construction and Expression of Eukaryotic Expression Plasmids Containing TCR Vα6 and TCR Vβ13 Genes Specific for DLBL-Associated Antigen

The adoptive immunotherapy of antigen specific T cell receptor (TCR)-modified T cells is considered as a new and promising treatment. Recently, successful transfer TCR genes specific for a variety of virus-specific and tumor-associated antigens, such as MART-1/WT1 TCR-modified T cells were showed the specific cytotoxicity on melanoma or leukemia cells. Little is known the TCR gene-modified T cells specific for lymphomaassociated antigen. Our previous study have found some TCR gene sequences associated with diffuse large B-cell lymphomas (DLBL) and had submitted them to GenBank (Accession numbers: EU369627,EU368854). In the present study, we used different approaches to construct different recombinant plasmids containing the TCRVα6- and TCRVβ13-chain genes specific for DLBL-associated antigen and to transfect both K293 and Molt-4 cell line. TCRVα6- and Vβ13-chain genes were either introduced into two individual eukaryotic expression vectors (TCRVα6+TCRVβ13), or introduced into one eukaryotic expression vector in which the TCRVα6- and Vβ13-chain genes linked by an internal ribosomal entry site (IRES). As it is known that the gene inserted downstream to the IRES is expressed at significantly lower levels than the gene introduced into the upstream position, and the two chains maybe play nonequivalent roles in antigen selection by extensive kinetic and structural analyses, we integrated TCRα either into the IRES 5′ position (TCRVα6-IRES-Vβ13) or the 3′ position (TCRVβ13-IRES-Vα6), and generated two kinds of recombinant plasmids. Subsequently their sequences were confirmed by restriction enzyme digestion analysis and sequencing. Then K293 cells (a TCR-deficient cell line) and Molt-4 cells (a TCR Vβ2 -bearing cell line) were transfected with the respective TCR-encoding expression plasmids using Lipofectamine^™ 2000. The mRNA expression of TCR Vα6- and Vβ13- chain genes were detected by RT-PCR and real-time PCR. In K293 cells, all recombinant plasmids expressed equally well TCRVα6- and Vβ13- chain genes by Laser Confocal Microscopy (LCM) and the transfection efficiency was about 80% at 72 h after transfection, while transfection efficiency (20%) in a TCR-bearing cell line was lowered than that in the TCR-deficient cell; And the mRNA expression of TCRVα6- and Vβ13-chain genes in K293 cells was superior to that in Molt-4 cells as shown by real-time PCR. The results suggested that the transferred TCR maybe have to compete with endogenous TCR chains in a TCR-bearing cell line. However, there are no difference of transduction efficiency among three kinds of transfer modes and no significant differences in the mRNA expression of both TCR Vα6 and Vβ13 genes by realtime PCR detection. In conclusions, three kinds of eukaryotic expression plasmids of TCR Vα6-IRES-Vβ13, TCR Vβ13-IRES-Vα6 and TCR Vα6+Vβ13 specific for DLBL were constructed successfully in this study and could express well in vitro after transfecting both K293 cells and Molt-4 cells. Further study need to transfect human primary T cells and to compare the ability of TCR-redirected T cells to mediate a specific cytotoxicity. It is expected to establish the specific anti-DLBL T cell clone by TCR gene transfection.