Integration of retroviral vectors induces minor changes in the transcriptional activity of T cells from ADA-SCID patients treated with gene therapy

Blood ◽  
2009 ◽  
Vol 114 (17) ◽  
pp. 3546-3556 ◽  
Author(s):  
Barbara Cassani ◽  
Eugenio Montini ◽  
Giulietta Maruggi ◽  
Alessandro Ambrosi ◽  
Massimiliano Mirolo ◽  
...  
Keyword(s):  
Gene Therapy ◽  
T Cell ◽  
Gene Transfer ◽  
T Cell Receptor ◽  
Cell Receptor ◽  
Retroviral Vectors ◽  
Hematopoietic Stem ◽  
T Cell Clones ◽  
Cell Clones ◽  
Integration Sites

Abstract Gene transfer into hematopoietic stem cells by γ-retroviral vectors (RVs) is an effective treatment for inherited blood disorders, although potentially limited by the risk of insertional mutagenesis. We evaluated the genomic impact of RV integration in T lymphocytes from adenosine deaminase-deficient severe combined immunodeficiency (ADA-SCID) patients 10 to 30 months after infusion of autologous, genetically corrected CD34+ cells. Expression profiling on ex vivo T-cell bulk population revealed no difference with respect to healthy controls. To assess the effect of vector integration on gene expression at the single-cell level, primary T-cell clones were isolated from 2 patients. T-cell clones harbored either 1 (89.8%) or 2 (10.2%) vector copies per cell and displayed partial to full correction of ADA expression, purine metabolism, and T-cell receptor-driven functions. Analysis of RV integration sites indicated a high diversity in T-cell origin, consistently with the polyclonal T-cell receptor-Vβ repertoire. Quantitative transcript analysis of 120 genes within a 200-kb window around RV integration sites showed modest (2.8- to 5.2-fold) dysregulation of 5.8% genes in 18.6% of the T-cell clones compared with controls. Nonetheless, affected clones maintained a stable phenotype and normal in vitro functions. These results confirm that RV-mediated gene transfer for ADA-SCID is safe, and provide crucial information for the development of future gene therapy protocols. The trials described herein have been registered at http://www.clinicaltrials.gov as #NCT00598481 and #NCT00599781.

The expansion and selection of T cell receptor αβ intestinal intraepithelial T cell clones

1996 ◽  
Vol 26 (4) ◽  
pp. 914-921 ◽  
Author(s):  
Armelle Regnault ◽  
Jean-Pierre Levraud ◽  
Annick Lim ◽  
Adrien Six ◽  
Christiane Moreau ◽  
...  
Keyword(s):  
T Cell ◽  
T Cell Receptor ◽  
Cell Receptor ◽  
T Cell Clones ◽  
Cell Clones ◽  
Selection Of

scholarly journals Diversification of the antigen-specific T cell receptor repertoire after varicella zoster vaccination

2016 ◽  
Vol 8 (332) ◽  
pp. 332ra46-332ra46 ◽  
Author(s):  
Qian Qi ◽  
Mary M. Cavanagh ◽  
Sabine Le Saux ◽  
Hong NamKoong ◽  
Chulwoo Kim ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Receptor ◽  
Chronic Infection ◽  
Cell Receptor ◽  
Control Mechanisms ◽  
Cell Repertoire ◽  
T Cell Clones ◽  
Varicella Zoster ◽  
Cell Clones

Diversity and size of the antigen-specific T cell receptor (TCR) repertoire are two critical determinants for successful control of chronic infection. Varicella zoster virus (VZV) that establishes latency during childhood can escape control mechanisms, in particular with increasing age. We examined the TCR diversity of VZV-reactive CD4 T cells in individuals older than 50 years by studying three identical twin pairs and three unrelated individuals before and after vaccination with live attenuated VZV. Although all individuals had a small number of dominant T cell clones, the breadth of the VZV-specific repertoire differed markedly. A genetic influence was seen for the sharing of individual TCR sequences from antigen-reactive cells but not for repertoire richness or the selection of dominant clones. VZV vaccination favored the expansion of infrequent VZV antigen–reactive TCRs, including those from naïve T cells with lesser boosting of dominant T cell clones. Thus, vaccination does not reinforce the in vivo selection that occurred during chronic infection but leads to a diversification of the VZV-reactive T cell repertoire. However, a single-booster immunization seems insufficient to establish new clonal dominance. Our results suggest that repertoire analysis of antigen-specific TCRs can be an important readout to assess whether a vaccination was able to generate memory cells in clonal sizes that are necessary for immune protection.

Diverse usage of human T-cell receptor gene segments in HLA-DR1 allospecific T-cell clones

1996 ◽  
Vol 49 (2) ◽  
pp. 122-129 ◽  
Author(s):  
Masao Ota ◽  
Mary Jane Geiger ◽  
Sandra Rosen-Bronson ◽  
Carolyn Katovich Hurley ◽  
David D. Eckels
Keyword(s):  
T Cell ◽  
T Cell Receptor ◽  
Receptor Gene ◽  
Cell Receptor ◽  
T Cell Clones ◽  
Human T Cell ◽  
Cell Clones ◽  
T Cell Receptor Gene ◽  
Cell Receptor Gene

Human T-cell receptor TCRAV, TCRBV, and TCRAJ sequences newly found in T-cell clones reactive with allogeneic HLA class II antigens

1993 ◽  
Vol 38 (1) ◽  
pp. 67-70 ◽  
Author(s):  
Fumiya Obata ◽  
Misao Tsunoda ◽  
Takehisa Kaneko ◽  
Koichi Ito ◽  
Ichiro Ito ◽  
...  
Keyword(s):  
T Cell ◽  
T Cell Receptor ◽  
Cell Receptor ◽  
Class Ii ◽  
Hla Class Ii ◽  
T Cell Clones ◽  
Human T Cell ◽  
Cell Clones ◽  
Class Ii Antigens

Efficient Identification of T-Cell Clones Associated with Graft-Versus-Host Disease (GvHD) in Target Tissue for Subsequent Detection in Peripheral Blood.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2243-2243
Author(s):  
Rose C. Beck ◽  
Marcin Wlodarski ◽  
Karl S. Theil ◽  
Ralph Tuthill ◽  
Brian Bolwell ◽  
...  
Keyword(s):  
T Cell ◽  
Cell Receptor ◽  
Unrelated Donor ◽  
Cell Transplant ◽  
Target Tissue ◽  
Distribution Analysis ◽  
Hematopoietic Stem ◽  
T Cell Clones ◽  
Cell Clones ◽  
Skin Biopsies

Abstract We hypothesized that after allogeneic hematopoietic stem cell transplant (HSCT), GvHD-affected tissues harbor expanded “immunodominant” T-cell clones, and characterization of these clones can be used to develop markers of disease. A multiplex PCR was used to detect T-cell receptor variable beta (VB) chain rearrangements in target tissue. Molecular analysis of the amplified VB CDR3 sequences allowed for identification and quantitation of putative disease-associated “clonotypes” and for the development of clone-specific PCR. We studied 5 HSCT patients for the presence of signature clonotypes in 10 skin biopsies taken during diagnostic GvHD work-up. Size distribution analysis of VB PCR products showed a skewed peak pattern in 9 biopsies; immunodominant clones (per definition frequency ≥30%) were detected in 6/7 biopsies with histologically confirmed GvHD, consistent with the presence of expanded clonotypes and the oligoclonal nature of the tissue-specific alloresponse. Immunodominant clones were also found in 2 of 3 biopsies not diagnostic for GvHD but obtained based on strong clinical suspicion, raising the possibility that they were associated with early evolving GvHD not distinguishable by histology. For example, when serial skin biopsies were analyzed, a GvHD-positive post-transplant d63 biopsy contained an immunodominant clone (frequency 60%), which was also detected in a subsequent biopsy positive for GvHD (frequency 33%). Similar results were seen in another patient, in whom serial biopsies taken on d214 (not diagnostic) and d217 (GvHD-positive) showed an identical immunodominant clone, not present in a d13 GVHD-negative biopsy. This finding suggests that the d214 biopsy might have contained early GvHD that was not detectable morphologically. In a patient who rejected an initial MUD graft (Tx 1) and then received a MUD SCT (Tx 2) from a different, unrelated donor, immunodominant clones were identified in GvHD-positive biopsies following each transplant, that were distinct for each graft. To examine whether immunodominant clonotypes derived from biopsies could be used as markers of disease, clonotypic PCR was developed for an immunodominant biopsy-derived clonotype for each transplant. The Tx 1 clonotype was detected in blood and skin following Tx 1, but not in tissue or blood taken after Tx 2. Specificity and correct size of the clonotypic PCR product were confirmed by both Genescan analysis and sequencing. Clonotypic PCR designed for an immunodominant clonotype from the Tx 2 donor detected the putative allospecific clonotype in serial samples after the second engraftment. Neither clonotype could be found in either donor, indicating that the disease-associated clones expanded to detectable levels following transplant. These results indicate that clonotypic PCR can distinguish distinct GvHD-associated clonotypes from different donors in both blood and tissue following transplant. Monitoring of the relative frequency of disease-associated clones in recipient blood indicated a significant peripheral expansion of disease-associated clones at the time of active GvHD. Our results demonstrate an efficient method for identification of disease-associated clonotypic markers, which can be used to aid diagnosis and monitoring of GvHD.

ROR1 Specific T Cell Clones from Healthy Individuals Show Common T Cell Receptor Motifs

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3364-3364
Author(s):  
Falk Heidenreich ◽  
Elke Ruecker-Braun ◽  
Juliane S. Stickel ◽  
Anne Eugster ◽  
Denise Kühn ◽  
...  
Keyword(s):  
Amino Acids ◽  
T Cells ◽  
T Cell ◽  
Mhc Class I ◽  
T Cell Receptor ◽  
Cd8 T Cells ◽  
Cd8 T Cell ◽  
Cell Receptor ◽  
T Cell Clones ◽  
Cell Clones

Abstract Background Immunotherapy for CLL with new antibodies or T-cells with modified TCR relies on attractive targets. ROR1 is such a promising target since it is highly overexpressed in CLL. Chimeric antigen receptor engineered T cells and antibodies directed against the extracellular part of ROR1 have already been developed and tested in vitro or in animal models, but still there is no MHC-class I presented peptide serving as target structure for CD8+ T cells (with or without a genetically modified T cell receptor) available. Aim The aim of this study was (1) to identify an immunogenic MHC-class I presented ROR1 peptide, (2) to generate respective ROR1 peptide specific CD8+ T cell clones, and (3) to analyze the nucleotide sequence of the CDR3 region of the expressed alpha and beta T cell receptor chain. Results In mass spectrometric-based analyses of the HLA-ligandome a HLA-B*07 presented ROR1 peptide was identified in primary CLL cells of two patients. Six T cell clones specific for this particular ROR1-peptide were generated from single CD8+ T cells from 2 healthy individuals with 3 T cell clones generated from each donor. Functionality and specificity of those T cell clones were tested in cytotoxicity assays. All 6 dextramer+ CD8+ T cell clones lysed peptide loaded and HLA-B*07+ transduced K562 cells (kindly provided by Lorenz Jahn, [Jahn et al., Blood, 2015 Feb 5;125(6):949-58]). Two selected clones (XD8 and XB6) were tested for their cytotoxic potential against 2 ROR1+ HLA-B*07+ tumor cell lines (with the ROR1 peptide identified by mass spectrometry for both of them) and against 2 primary CLL cell samples. Tested clones showed a significant lysis of the respective target cells. CDR3 regions of the alpha and beta T cell receptor chain were sequenced on a single cell level. The CDR3 alpha region from each of the 3 ROR1 specific T cell clones from donor A showed some similarities to T cell clones derived from donor B (Table 1). Conclusion For the first time a MHC-class I presented ROR1 peptide antigen is reported. ROR1 positive CLL cells can be targeted by specific HLA-B*07 restricted CTLs. Respective CD8+ T cell clones with anti-leukemic activity from 2 donors share some amino acid motifs of the CDR3 alpha and beta regions. In conclusion, this information provides the possibility of generating ROR1 specific CD8+ T cells with genetically modified T cell receptors for immunotherapy and for tracking those cells after administration with next generation sequencing in peripheral blood samples of patients. Furthermore, data suggest the existence of public TCR motifs in leukemia antigen specific CTLs, which needs to be proven in follow-up experiments with larger cohorts of donors and patients. Finally, the presented strategy to identify leukemia specific peptide antigens for CD8+ T cells might be an attractive method for similar projects. Table 1 Amino acid sequences of CDR3 alpha and beta regions of the TCR of ROR1 specific CD8+ T cell clones. When comparing two clones, matching amino acids are depicted in red. The aromatic amino acids phenylalanine (F) and tyrosine (Y) are shown in blue when situated at the same position. Gaps inserted during the sequence alignment process are indicated by a hyphen '-'. Table 1. Amino acid sequences of CDR3 alpha and beta regions of the TCR of ROR1 specific CD8+ T cell clones. When comparing two clones, matching amino acids are depicted in red. The aromatic amino acids phenylalanine (F) and tyrosine (Y) are shown in blue when situated at the same position. Gaps inserted during the sequence alignment process are indicated by a hyphen '-'. Disclosures Middeke: Sanofi: Honoraria. Schetelig:Sanofi: Honoraria.

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