Primer sets for cloning the human repertoire of T cell Receptor Variable regions

Boria, Ilenia; Cotella, Diego; Dianzani, Irma; Santoro, Claudio; Sblattero, Daniele

doi:10.1186/1471-2172-9-50

Cited by 21 publications

(27 citation statements)

References 22 publications

(29 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…But rare morphological changes of apoptosis were found in unspecific activated CTL or PBS group (white arrow indicate pyknosis). In IHC analysis, the infiltration of CD3 positive T cells in the transgenic CTL group were significantly more than that in DCs induced CTL group and unspecific activated CTL group dramatically and facilitate the creation of more diverse TCR library in our study [23].…”

Section: Discussionmentioning

confidence: 96%

Engineered cytotoxic T lymphocytes with AFP-specific TCR gene for adoptive immunotherapy in hepatocellular carcinoma

et al. 2015

View full text Add to dashboard Cite

Alpha-fetoprotein (AFP) is overexpressed in hepatocellular carcinoma (HCC) and could serve as a tumor-associated antigen (TAA) and potential target for adoptive immunotherapy. However, low frequency and severe functional impairment of AFP-specific T cells in vivo hamper adoptive infusion. TAA-specific T cell receptor (TCR) gene transfer could be an efficient and reliable alternation to generate AFP-specific cytotoxic T lymphocytes (CTLs). Autologous dendritic cells (DC) pulsed with AFP158-166 peptides were used to stimulate AFP-specific CTLs. TCR α/β chain genes of AFP-specific CTLs were cloned and linked by 2A peptide to form full-length TCR coding sequence synthesized into a lentiviral vector. Nonspecific activated T cells were engineered by lentivirus infection. Transgenetic CTLs were evaluated for transfection efficiency, expression of AFP158-166-specific TCR, interferon (IFN)-γ secretion, and specific cytotoxicity toward AFP+ HCC cells in vitro and in vivo. Flow cytometry revealed the AFP158-166-MHC-Pentamer positive transgenetic CTLs was 9.86 %. The number of IFN-γ secretion T cells and the specific cytotoxicity toward HpeG2 in vitro and in tumor-bearing NOD/SCID mice were significantly raised in transgenetic CTLs than that of AFP158-166-specific CTLs obtained by peptide-pulsed DCs or control group. TCR gene transfer is a promising strategy to generate AFP158-166-specific CTLs for the treatment of HCC.

show abstract

Section: Discussionmentioning

confidence: 96%

Engineered cytotoxic T lymphocytes with AFP-specific TCR gene for adoptive immunotherapy in hepatocellular carcinoma

et al. 2015

View full text Add to dashboard Cite

show abstract

“…The RACE approach avoids the potential bias associated with the use of the multiple primer sets required to amplify from all TRBV sequences (Boria et al 2008) and takes advantage of the conserved sequences offered by TRBC1 and TRBC2 (96% nucleotide sequence identity). Reverse transcription to generate cDNA was performed using a primer specific for the TRBC genes (Ozawa et al 2008) as well as a template-switching primer (Peters et al 1999;Douek et al 2002) to provide a 59 anchor for subsequent PCR.…”

Section: Experimental Strategymentioning

confidence: 99%

Profiling the T-cell receptor beta-chain repertoire by massively parallel sequencing

Freeman

Warren²,

Webb³

et al. 2009

Genome Res.

356

336

View full text Add to dashboard Cite

T-cell receptor (TCR) genomic loci undergo somatic V(D)J recombination, plus the addition/subtraction of nontemplated bases at recombination junctions, in order to generate the repertoire of structurally diverse T cells necessary for antigen recognition. TCR beta subunits can be unambiguously identified by their hypervariable CDR3 (Complement Determining Region 3) sequence. This is the site of V(D)J recombination encoding the principal site of antigen contact. The complexity and dynamics of the T-cell repertoire remain unknown because the potential repertoire size has made conventional sequence analysis intractable. Here, we use 59-RACE, Illumina sequencing, and a novel short read assembly strategy to sample CDR3 b diversity in human T lymphocytes from peripheral blood. Assembly of 40.5 million short reads identified 33,664 distinct TCR b clonotypes and provides precise measurements of CDR3 b length diversity, usage of nontemplated bases, sequence convergence, and preferences for TRBV (T-cell receptor beta variable gene) and TRBJ (T-cell receptor beta joining gene) gene usage and pairing. CDR3 length between conserved residues of TRBV and TRBJ ranged from 21 to 81 nucleotides (nt). TRBV gene usage ranged from 0.01% for TRBV17 to 24.6% for TRBV20-1. TRBJ gene usage ranged from 1.6% for TRBJ2-6 to 17.2% for TRBJ2-1. We identified 1573 examples of convergence where the same amino acid translation was specified by distinct CDR3 b nucleotide sequences. Direct sequence-based immunoprofiling will likely prove to be a useful tool for understanding repertoire dynamics in response to immune challenge, without a priori knowledge of antigen.

show abstract

“…For this paper only the TRAV1-2 primer is relevant as either the cells used were FACS sorted using a TRAV1-2-specific antibody (TRAV1-2 dataset) or only the TRAV1-2 sequences were used (AMC dataset). The TRAV1-2-specific primer is 5′-GGACAAARCMTTGASCAGCC-3′ (7). The Vα primers are tailed with the primer B sequence of the LibA system (Titanium protocol) (5′-CTATGCGCCTTGCCAGCCCGCTCAG-3′) (Roche/454).…”

Section: Methodsmentioning

confidence: 99%

“…After amplification, the amplicons were purified using AMPURE SPRI beads (Agencourt) according to the manufacturers instructions using equal amounts of amplicon volume and bead volume. In the second step, a PCR is performed using primer B sequence of the Roche 454 LibA system as forward primer and a Cα specific primer 5′-TCTCAGCTGGTACACGGCAG-3′ (7) tailed with both a genetic barcode and the primer A sequence of the LibA system (Titanium protocol) (5′-CGTATCGCCTCCCTCGCGCCATCAG-3′) (Roche/454). Amplification conditions are equal to the linear amplification step.…”

Section: Methodsmentioning

confidence: 99%

Discovery of Invariant T Cells by Next-Generation Sequencing of the Human TCR α-Chain Repertoire

Schaik

Klarenbeek

Doorenspleet

et al. 2014

The Journal of Immunology

View full text Add to dashboard Cite

During infection and autoimmune disease, activation and expansion of T cells takes place. Consequently, the T cell receptor (TCR) repertoire contains information about ongoing and past diseases. Analysis and interpretation of the human TCR repertoire is hampered by its size and stochastic variation and by the diversity of antigens and antigen presenting molecules encoded by the Major Histocompatibility Complex (MHC), but is highly desirable and would greatly impact fundamental and clinical immunology. A subset of the TCR repertoire is formed by invariant T cells. Invariant T cells express interdonor-conserved TCRs and recognize a limited set of antigens, presented by non-polymorphic antigen presenting molecules. Discovery of the three known invariant T cell populations has been a tedious and slow process, identifying them one by one. Because conservation of the TCR α chain of invariant T cells is much higher than the β chain, and because the TCR α chain variable (V) gene segment TRAV1-2 is used by two of the three known invariant TCRs, we employed next generation sequencing of TCR α chains that contain the TRAV1-2 gene segment to identify 16 invariant TCRs shared among many blood donors. Frequency analysis of individual clones indicates these T cells are expanded in many donors, implying an important role in human immunity. This approach extends the number of known interdonor-conserved TCRs, suggests that many more exist, and that these TCR patterns can be used to systematically evaluate human antigen exposure.

show abstract

Primer sets for cloning the human repertoire of T cell Receptor Variable regions

Cited by 21 publications

References 22 publications

Engineered cytotoxic T lymphocytes with AFP-specific TCR gene for adoptive immunotherapy in hepatocellular carcinoma

Engineered cytotoxic T lymphocytes with AFP-specific TCR gene for adoptive immunotherapy in hepatocellular carcinoma

Profiling the T-cell receptor beta-chain repertoire by massively parallel sequencing

Discovery of Invariant T Cells by Next-Generation Sequencing of the Human TCR α-Chain Repertoire

Contact Info

Product

Resources

About