Generation Of DLBL-associated Antigen-specific T Cells By TCR Gene Transfer

Objective:In order to generate DLBL-associated antigen-specific T cells by T cell receptor (TCR) gene transfer,clonally expanded TCR Vαand Vβrepertoire were analyzed, antigen-specific full-length TCRαandβgenes were amplified and cloned,the eukaryotic expression plasmids harboring TCRαandβgenes specific for DLBL-associated antigens were constructed and transferred into T cells from healthy individuals,the biological feature and the specific cytotoxicity from TCR gene-modified T cells were analyzed.The study was expected to provide the basic data for targeted adoptive cellular immunotherapy in DLBL.Methods:The clonality of TCR Vαand Vβsubfamily T cells was identified by analysis of TCR CDR3 size using RT-PCR-genescan in three patients with DLBL,the clonally expanded TCR Vαand Vβsubfamily products were directly sequenced.The full-length TCR genes were amplified for the monoclonally expanded T cells and their amino acid sequences were deduced by Primer Premier 5.0 analytical software.Three-dimensional spatial structures of the TCR harboring conserved motif in CDR3 region and the full-length TCR were modeled by SWISS-MODEL,an automated protein homology-modeling server. The full-length TCRαandβgenes were cloned into the eukaryotic expression vectors pIRES2-EGFP and pIRES,respectively.Based on the different transgene cassettes,we constructed different vectors,in which TCRα- andβ-chain genes were introduced into two individual expression vectors to obtain TCRαor TCRβsingle gene recombinant plasmid(α-IRES-EGFP,β-IRES-EGFP),or linked by an internal ribosomal entry site (IRES) to construct two different bicistronic eukaryotic expression plasmids(α-IRES-β,β-IRES-α) with either theα- or theβ-chain gene in the 5' position and the other gene in the 3' position.Then,different vectors were transferred into cell lines by gene transfer techniques.The expression of the mRNA and the corresponding proteins of antigen-specific TCRαandβgenes were detected by real-time PCR and Western Blot. Subsequently,the recombinant vectors containing different TCRαandβgenes were transferred into healthy individual T cells(HLA-A2 restricted) using Nucleofector^TM technique,and the expression of targeted genes and protein in modified cells was detected by real-time PCR,immunophenotyping analysis using TCR Vβantibody,and flow cytometry(FCM),and Western Blot,respectively.The specific cytotoxicity of TCR gene-transferred T cells in vitro was estimated,and the expression level of TCRζchain was detected on both actived and unactived TCR gene-modified T cells,respectively.The difference of gene expression profiling was compared between TCR gene-transduced T cells and untransduced T cells by cDNA microarray technique.Results:Clonally expanded TCR Vαand Vβsubfamily T cells in the peripheral blood from 3 patients with DLBL were identified.Thirty-one TCR-CDR3 region sequences(including 21 TCR ct genes and 10 TCRβgenes) of clonally expanded T cells from peripheral blood mononuclear cells(PBMCs) in patients with DLBL were characterized,among which 23 unique TCR sequences had been submitted to GenBank database of NCBI.Conserved amino acid motifs were displayed in the CDR3 region of the TCR Vαand TCR Vβsubfamily T cell clones by TCR-CDR3 region sequence comparative analysis.For TCRα-chain,the TCR of Vα8S1-Jα34 and Vα14S1-Jα34 clones generated from one patient (P1) contained not only the conserved motif "RSxY-NTDKLI" of CDR3 region in the primary structure,but also the high homology in the three-dimensional spatial structures, and displayed the same model(PDB ID:2ak4D) by the SWISS-MODEL homology-modeling server analysis.Compared with the motifs in the CDR3 region of TCRα-chain,the motifs in the CDR3 region of TCRβ-chain genes were relatively conserved,in which the motifs of "GT" and "RG" were obviously conserved.And the TCR of Vβ9S1-Jβ2.1-Cβ2(P1) and Vβ15S1-Jβ2.1-Cβ2(P3) clones in which CDR3 region harboring conserved motifs "SxGTG" also used an identical model(PDB ID:3dx9D).Additionally,full-length TCR genes in 6 of 31 identified TCR genes were amplified(including TCR Vα23 from P1,TCR Vα6 and TCR Vβ13 from P2,TCR Vα6, TCR Vα23 and TCR Vβ13 from P3) and had been applied for national patent. Different recombinant plasmids were constructed successfully and verified using restriction enzyme digestion and nucleotide sequencing,which included 2 TCRαorβgene recombinant plasmid:TCR Vα6-IRES-EGFP(P2),TCR V1313-IRES-EGFP(P2);4 different bicistronic recombinant plasmid harboring TCRαandβgenes:TCR Vα6-IRES-TCR Vβ13 and TCR Vβ13-IRES-TCR Vα6(P2);TCR Vβ13-IRES-TCR Vα6 and TCR Vβ13-IRES-TCR Vα23(P3).The mRNA and corresponding protein of antigenspecific TCRαandβgenes were expressed in the same effectivity in the transduced cell lines,and there was no difference among three kinds of transgene cassettes.The DLBL-specific cytotoxic T lymphocyte(CTL) could be inducted by transducing each kind of bicistronic recombinant plasmids to healthy T cells.The percentage of Vβ13⁺T cells in TCR gene-transduced cells was 20～50%by laser confocal microscopy(LSM) and FCM detection using Vβ13 antibody.And the expression of mRNA and protein levels of exogenous TCRαandβgenes could be detected in the transduced cells.Antigen-specific TCR gene-modified T cells acquired the ability of DLBL-specific cytotoxicity,and the best effectivity was found in TCR Vβ13-IRES-TCR Vα6(P2 and P3) recombinant plasmid transferred T cells,while the effectivity in which T cells transferred withα-IRES-βtransgene cassette(P2) and TCR Vβ13-IRES-TCR Vα23(P3) was limited.The expression level of TCRζchain on the actived TCR gene-transduced T cells was superior to that on the unactived TCR gene-transduced T cells and untransduced T cells.GO analysis showed the differential genes in TCR gene-modified T cells were closely related with the biological processs of taxis,defense response,immune response,inflammatory response,receptor binding,cellular calcium ion homeostasis,and the molecular functions of chemokine activity,G-protein-coupled receptor binding,MHC protein binding,signal transducer activity,peptide antigen binding and so on.And these genes was mainly innolved in several pathways,such as Ras signaling in the CD4+ TCR pathway,TCR signaling in na(?)ve CD8+ T cells,calcineurin-regulated NFAT-dependent transcription in lymphocytes,p38 MAPK signaling pathway,IL-23-mediated signaling events,and classâ… PI3K signaling events by Pathway analysis,in which the expression of the involved genes were mostly up-regulated.Conclusions:Conserved motifs of CDR3 region of clonally expanded TCR Vαand Vβsubfamily T cells was a common feature in the patients with DLBL,the TCR had high homology in the three-dimensional spatial structures,suggested that these clonally expanded T cells were derived from an identical antigenic epitope stimulation in the PB from patients with DLBL.There was successfully on cloning of full-length TCRαandβgenes specific for DLBL-associated antigens,constructing of different bicistronic eukaryotic expression plasmids harboring TCRαandβgenes and transferring of TCR genes into T cells.The TCR gene-transferred T cells displayed the ability of DLBL-specific cytotoxicity.The transgene cassette of TCR Vβ13-IRES-TCR Vα6 showed superiority to others in the function of TCR-redirected T cells.Up-regulation of genes related to several signaling pathway were identified by cDNA microarray technology in antigen-specific TCR gene-transduced T cell.It is,to our knowledge,the first description of DLBL-associated antigen-specific TCR gene-modified T cells to acquire specific cytotoxicity.These findings will definitively benefit to better understand the specific adoptive immunotherapy for DLBL.