T-cell Immune Reconstitution After Transplantation Based On TCR Gene Rearrangement And The Basic Research Of Immunotherapy

Objective To analyze the status of T-cell immune reconstitution after hematopoietic stem cell transplantation (HSCT) and determine the relationship among the factors correlated with transplantation and immune reconstitution for evaluating T-cell immune reconstitution after HSCT more precisely.To construct the Epstein Barr virus (EBV) specific cytotoxic T lymphocyte (CTL) by specific T cell receptor (TCR) gene transduction and make approach to the adoptive immunotherapy for EBV-associated concurrent disease after transplantation.Methods Peripheral blood preparation were sampled from patients received HSCT therapy for different types of blood diseases (sampled at pre-HSCT and few weeks intervals post-HSCT). Clinical observation of concurrent disease after transplantation (including graft versus host disease (GVHD), EBV-associated post transplant lymphoproliferative disease (EBV-PTLD) and so on) and the relapse of primary disease were performed during the research period. Peripheral blood preparation of donors and normal controls were sampled at the same time. Peripheral blood mononuclear cells (PBMNCs) were obtained from peripheral blood samples of patients, donors and normal controls. The positive ratios of CD45RA~+ cells and CD45RO~+ cells were detected by indirect immunofluorescence method. The detection of TCR rearrangement excision circles (TRECs) levels in DNA of PBMNCs was preformed by real-time quantitative PCR. TCR Vαand Vβrepertoire expression analysis, T-cell clonality analysis and nucleotide sequence analysis were preformed in 5 cases with GVHD.TCR Vαand Vβrepertoire expression analysis and T-cell clonality analysis were observed in EBV specific CTL clone. The gene sequence of oligoclonal expansion of TCR Vα15 and Vβ1 repertoire were determined by sequencing analysis. Gene fragments of TCR Vα15 gene and TCR Vβ1 gene amplified by PCR were cloned into the eukaryotic co-expression vector pIRES to construct the the bicistronic recombinant plasmid TCR Vα-pIRES-TCR Vβ. Then A549 cell line, Molt4 cell line and normal T cell were transfected with this recombinant plasmid by transgenic technology. The specific TCR gene expression in transfected cells and the specific cytotoxicity of transfected cells were detected in vitro.Results The TRECs average level of patients at pre-HSCT (1.3673±2.1022copies per 1000 PBMNCs) was significantly lower than the TRECs average level of normal controls (3.0108±0.8384 copies per 1000 PBMNCs). Early after allogeneic HSCT (within 8 weeks), the CD45RO~+ cells that expand were predominated. TRECs levels in PBMNCs went up a little in 4 weeks post HSCT, then went down soon and rose steadily until 8～12 weeks post HSCT, but the TRECs levels at the week 12 post HSCT were still lower than the TRECs levels at pre-HSCT. There was no statistically correlation between TRECs levels of recipient at pre-HSCT and TRECs levels within 12 weeks post HSCT. TRECs levels of donors had positive linear correlation with TRECs levels of recipient within 8～12 weeks post HSCT. T-cell immune reconstitution of peripheral blood stem cell transplantation group was faster than bone marrow transplantation group; the recovery of recent thymic output function with allogeneic HSCT was slower than autologous HSCT. The patients with acute GVHD or chronic GVHD histories had profoundly reduced TRECs levels during the first year post HSCT. Compared with normal controls, the different expression patterns of TCR Vαand Vβrepertoire of patients with cGVHD could be detected and the normal polyclonal expression pattern was changed to oligoclonal or oligoclonal trend pattern. Oligoclonal expanded T cells were identified in TCR Vα2, 3, 6, 10, 12, 14, 15, 25, 26 subfamilies and TCR Vβ1, 3, 7～9, 13, 17, 19, 20 subfamilies. One patient undergoing immune treatment for cGVHD experienced a rise in TRECs levels, but the TCR Vβrepertoire failed to normalize even after 4 years post HSCT. TRECs could be a relevant prognostic factor for HSCT; low TREC values in PBMNCs could help predict the early relapse after HSCT.The recombinant plasmid TCR Vα-pIRES-TCR Vβwas analyzed and verified by restriction enzyme digestion and nucleotide sequencing analysis. The increased mRNA levels of TCR Vα15 gene and TCR Vβ1 gene could be detected in transfected cell, and moreover, TCR Vα15 and TCR Vβ1 protein could express in vitro. The EBV specific CTL could be obtained by transfecting recombinant plasmid to normal T cells, and had the EBV specific cytotoxity.Conclusions Combined the analysis of T-cell subsets, T-cell repertoire and recent thymic output function together to monitor the status of T-cell immune reconstitution after HSCT may evaluate immune reconstitution after HSCT more precisely, also help to give the immunologic intervention treatment at the right moment and accelerate T-cell immune reconstitution after transplantation.The bicistronic eukaryotic expression plasmid TCR Vα-pIRES-TCR Vβassociated with EBV specific CTL clone was constructed successfully, and the co-expression of TCR Vα15 gene and TCR Vβ1 gene could be detected in vitro. The EBV specific cytotoxity was confirmed in the EBV specific CTL that obtained by transfecting recombinant plasmid to normal T cells. This study lays a foundation of new adoptive immunotherapy for EBV-associated concurrent disease after transplantation.