Rearrangements of T- and B-cell receptor (TCR and BCR) genes are useful markers for clonality assessment as well as for minimal residual disease (MRD) monitoring during the treatment of haematological malignancies. Currently, rearrangements of three out of four TCR and all BCR loci are used for this purpose. The fourth TCR gene, TRA, has not been used so far due to the lack of a method for its rearrangement detection in genomic DNA. Here we propose the first high-throughput sequencing based method for the identification of clonal TRA gene rearrangements at the DNA level. The method is based on target amplification of the rearranged TRA locus using an advanced multiplex polymerase chain reaction system and high-throughput sequencing, and has been tested on DNA samples from peripheral blood of healthy donors. Combinations of all functional V- and J-segments were detected, indicating the high sensitivity of the method. Additionally, we identified clonal TRA rearrangements in 57 out of 112 tested DNA samples of patients with various T-lineage lymphoproliferative disorders. The method fills the existing gap in utilizing the TRA gene for a wide range of studies, including clonality assessment, MRD monitoring and clonal evolution analysis in different lymphoid malignancies.

• MeSH
• DNA nádorová genetika   MeSH
• genová přestavba - alfa řetězec receptoru antigenů T-buněk * MeSH
• hematologické nádory genetika   MeSH
• lidé MeSH
• lymfoproliferativní nemoci genetika   MeSH
• multiplexová polymerázová řetězová reakce * MeSH
• vysoce účinné nukleotidové sekvenování * MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Monitoring the T cell receptor (TCR) repertoire in health and disease can provide key insights into adaptive immune responses, but the accuracy of current TCR sequencing (TCRseq) methods is unclear. In this study, we systematically compared the results of nine commercial and academic TCRseq methods, including six rapid amplification of complementary DNA ends (RACE)-polymerase chain reaction (PCR) and three multiplex-PCR approaches, when applied to the same T cell sample. We found marked differences in accuracy and intra- and inter-method reproducibility for T cell receptor α (TRA) and T cell receptor β (TRB) TCR chains. Most methods showed a lower ability to capture TRA than TRB diversity. Low RNA input generated non-representative repertoires. Results from the 5' RACE-PCR methods were consistent among themselves but differed from the RNA-based multiplex-PCR results. Using an in silico meta-repertoire generated from 108 replicates, we found that one genomic DNA-based method and two non-unique molecular identifier (UMI) RNA-based methods were more sensitive than UMI methods in detecting rare clonotypes, despite the better clonotype quantification accuracy of the latter.

• MeSH
• dospělí MeSH
• Jurkat buňky MeSH
• lidé středního věku MeSH
• lidé MeSH
• počítačová simulace MeSH
• receptory antigenů T-buněk alfa-beta genetika   MeSH
• receptory antigenů T-buněk genetika   MeSH
• reprodukovatelnost výsledků MeSH
• vysoce účinné nukleotidové sekvenování metody   MeSH
• zkreslení výsledků (epidemiologie) MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Intramural MeSH

INTRODUCTION: Cutaneous T-cell lymphoid infiltrate can represent reactive lesion or a malignant T-cell lymphoma. However, clinical and histopathological appearance can overlap in both groups with a risk of misdiagnosis. Aberrant expression of T-cell markers is not always applicable and T-cell receptor (TCR) gene rearrangement is not always accessible and diagnosis in borderline cases can be challenging. AIMS: Several types of TCR antibodies are currently available with limited knowledge of their expression in different cutaneous lymphoid infiltrates. Aim of the study is a comparison of expression of TCR antibodies in benign and malignant lymphoid infiltrates and their utility in borderline cases. METHODS: Representative cases of reactive and malignant lymphoproliferations were collected. Separate group of lesions with borderline morphology was selected for comparison. Immunohistochemical expression of TCR-V-betaF1 (TCRBF1), TCR-C-beta1 (TCRJOVI.1), TCR gamma/delta (TCRGD) and TCR delta (TCRD) was performed in all cases. TCR gene rearrangement evaluation was performed in all cases using PCR BIOMED-2 assay. RESULTS: Benign lymphoid infiltrates were all negative in TCRD and TCRGD. Expression of TCRJOVI.1 was seen in 3/10 cases and TCRBF1 in one. T-cell lymphomas were positive for TCRBF1 and TCRGD in 60% and 30% of cases respectively. TCR gene rearrangement was confirmed in 90% of lymphoma cases. All benign lesions were polyclonal. Morphologically borderline lesions showed expression of TCRBF1 in 6/10 cases and TCR gene rearrangement in 4/10 cases. Re-evaluation of the cases and clinical correlation led to the change of the diagnosis and confirmation of T-cell lymphoma in 4/10 cases. CONCLUSIONS: Expression of TCRBF1 and TCR-gene rearrangement was significantly associated with malignant infiltrates. TCRBF1 positivity in borderline cutaneous lymphoproliferations can raise the suspicion of malignancy but confirmation by TCR gene rearrangement and careful clinical correlation is still advisable.

• MeSH
• antigeny CD7 analýza   MeSH
• diferenciální diagnóza MeSH
• dospělí MeSH
• genová přestavba T-lymfocytů * MeSH
• imunohistochemie * MeSH
• kožní T-buněčný lymfom genetika   imunologie   patologie   MeSH
• kůže imunologie   metabolismus   MeSH
• lidé středního věku MeSH
• lidé MeSH
• lymfoproliferativní nemoci genetika   imunologie   patologie   MeSH
• prediktivní hodnota testů MeSH
• protilátky imunologie   MeSH
• receptory antigenů T-buněk alfa-beta analýza   genetika   MeSH
• receptory antigenů T-buněk gama-delta analýza   genetika   MeSH
• senioři MeSH
• specificita protilátek MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• srovnávací studie MeSH

• MeSH
• buněčné linie MeSH
• finanční podpora výzkumu jako téma MeSH
• kultivované buňky MeSH
• lidé MeSH
• monoklonální protilátky imunologie   MeSH
• myši MeSH
• receptory antigenů T-buněk alfa-beta chemie   imunologie   MeSH
• receptory antigenů T-buněk chemie   imunologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH

OBJECTIVE: One of the current hypotheses to explain the proinflammatory immune response in IBD is a dysregulated T cell reaction to yet unknown intestinal antigens. As such, it may be possible to identify disease-associated T cell clonotypes by analysing the peripheral and intestinal T-cell receptor (TCR) repertoire of patients with IBD and controls. DESIGN: We performed bulk TCR repertoire profiling of both the TCR alpha and beta chains using high-throughput sequencing in peripheral blood samples of a total of 244 patients with IBD and healthy controls as well as from matched blood and intestinal tissue of 59 patients with IBD and disease controls. We further characterised specific T cell clonotypes via single-cell RNAseq. RESULTS: We identified a group of clonotypes, characterised by semi-invariant TCR alpha chains, to be significantly enriched in the blood of patients with Crohn's disease (CD) and particularly expanded in the CD8+ T cell population. Single-cell RNAseq data showed an innate-like phenotype of these cells, with a comparable gene expression to unconventional T cells such as mucosal associated invariant T and natural killer T (NKT) cells, but with distinct TCRs. CONCLUSIONS: We identified and characterised a subpopulation of unconventional Crohn-associated invariant T (CAIT) cells. Multiple evidence suggests these cells to be part of the NKT type II population. The potential implications of this population for CD or a subset thereof remain to be elucidated, and the immunophenotype and antigen reactivity of CAIT cells need further investigations in future studies.

• MeSH
• CD8-pozitivní T-lymfocyty MeSH
• Crohnova nemoc * genetika   MeSH
• lidé MeSH
• NKT buňky * MeSH
• receptory antigenů T-buněk alfa-beta genetika   MeSH
• receptory antigenů T-buněk metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Immunosuppressive therapy (IST), consisting of antithymocyte globulin and cyclosporine A, is effective in refractory cytopenia of childhood (RCC), suggesting that, similar to low-grade myelodysplastic syndromes in adult patients, T lymphocytes are involved in suppressing hematopoiesis in a subset of RCC patients. However, the potential role of a T-cell-mediated pathophysiology in RCC remains poorly explored. In a cohort of 92 RCC patients, we prospectively assessed the frequency of T-cell receptor (TCR) β-chain variable (Vβ) domain skewing in bone marrow and peripheral blood by heteroduplex PCR, and analyzed T-cell subsets in peripheral blood by flow cytometry. TCRVβ skewing was present in 40% of RCC patients. TCRVβ skewing did not correlate with bone marrow cellularity, karyotype, transfusion history, HLA-DR15 or the presence of a PNH clone. In 28 patients treated with IST, TCRVβ skewing was not clearly related with treatment response. However, TCRVβ skewing did correlate with a disturbed CD4(+)/CD8(+) T-cell ratio, a reduction in naive CD8(+) T cells, an expansion of effector CD8(+) T cells and an increase in activated CD8(+) T cells (defined as HLA-DR(+), CD57(+) or CD56(+)). These data suggest that T lymphocytes contribute to RCC pathogenesis in a proportion of patients, and provide a rationale for treatment with IST in selected patients with RCC.

• MeSH
• cytotoxické T-lymfocyty imunologie   MeSH
• dítě MeSH
• imunosupresivní léčba MeSH
• kojenec MeSH
• lidé MeSH
• mladiství MeSH
• myelodysplastické syndromy imunologie   patologie   MeSH
• pancytopenie imunologie   MeSH
• předškolní dítě MeSH
• prospektivní studie MeSH
• receptory antigenů T-buněk alfa-beta imunologie   MeSH
• studie případů a kontrol MeSH
• T-lymfocyty - podskupiny chemie   MeSH
• Check Tag
• dítě MeSH
• kojenec MeSH
• lidé MeSH
• mladiství MeSH
• mužské pohlaví MeSH
• předškolní dítě MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• multicentrická studie MeSH
• práce podpořená grantem MeSH

The organizational integrity of the adaptive immune system is determined by functionally discrete subsets of CD4+ T cells, but it has remained unclear to what extent lineage choice is influenced by clonotypically expressed T-cell receptors (TCRs). To address this issue, we used a high-throughput approach to profile the αβ TCR repertoires of human naive and effector/memory CD4+ T-cell subsets, irrespective of antigen specificity. Highly conserved physicochemical and recombinatorial features were encoded on a subset-specific basis in the effector/memory compartment. Clonal tracking further identified forbidden and permitted transition pathways, mapping effector/memory subsets related by interconversion or ontogeny. Public sequences were largely confined to particular effector/memory subsets, including regulatory T cells (Tregs), which also displayed hardwired repertoire features in the naive compartment. Accordingly, these cumulative repertoire portraits establish a link between clonotype fate decisions in the complex world of CD4+ T cells and the intrinsic properties of somatically rearranged TCRs.

• MeSH
• buněčný rodokmen imunologie   MeSH
• CD4-pozitivní T-lymfocyty imunologie   MeSH
• lidé MeSH
• receptory antigenů T-buněk alfa-beta imunologie   MeSH
• T-lymfocyty - podskupiny imunologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

αβT-cell-depleted allogeneic hematopoietic cell transplantation holds promise for the safe and accessible therapy of both malignant and non-malignant blood disorders. Here we employed molecular barcoding normalized T-cell receptor (TCR) profiling to quantitatively track T-cell immune reconstitution after TCRαβ-/CD19-depleted transplantation in children. We demonstrate that seemingly early reconstitution of αβT-cell counts 2 months after transplantation is based on only several hundred rapidly expanded clones originating from non-depleted graft cells. In further months, frequency of these hyperexpanded clones declines, and after 1 year the observed T-cell counts and TCRβ diversity are mostly provided by the newly produced T cells. We also demonstrate that high TCRβ diversity at day 60 observed for some of the patients is determined by recipient T cells and intrathymic progenitors that survived conditioning regimen. Our results indicate that further efforts on optimization of TCRαβ-/CD19-depleted transplantation protocols should be directed toward providing more efficient T-cell defense in the first months after transplantation.

• MeSH
• antigeny CD19 * MeSH
• časové faktory MeSH
• dítě MeSH
• kojenec MeSH
• krevní nemoci terapie   MeSH
• lidé MeSH
• lymfocytární deplece metody   MeSH
• mladiství MeSH
• mladý dospělý MeSH
• předškolní dítě MeSH
• přežívání štěpu * MeSH
• receptory antigenů T-buněk alfa-beta * MeSH
• T-lymfocyty imunologie   MeSH
• transplantace hematopoetických kmenových buněk metody   MeSH
• Check Tag
• dítě MeSH
• kojenec MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• předškolní dítě MeSH
• Publikační typ
• časopisecké články MeSH

BACKGROUND AND AIMS: Intestinal inflammation in inflammatory bowel diseases [IBD] is thought to be T cell mediated and therefore dependent on the interaction between the T cell receptor [TCR] and human leukocyte antigen [HLA] proteins expressed on antigen presenting cells. The collection of all TCRs in one individual, known as the TCR repertoire, is characterised by enormous diversity and inter-individual variability. It was shown that healthy monozygotic [MZ] twins are more similar in their TCR repertoire than unrelated individuals. Therefore MZ twins, concordant or discordant for IBD, may be useful to identify disease-related and non-genetic factors in the TCR repertoire which could potentially be used as disease biomarkers. METHODS: Employing unique molecular barcoding that can distinguish between polymerase chain reaction [PCR] artefacts and true sequence variation, we performed deep TCRα and TCRβ repertoire profiling of the peripheral blood of 28 MZ twin pairs from Denmark and Germany, 24 of whom were discordant and four concordant for IBD. RESULTS: We observed disease- and smoking-associated traits such as sharing, diversity and abundance of specific clonotypes in the TCR repertoire of IBD patients, and particularly in patients with active disease, compared with their healthy twins. CONCLUSIONS: Our findings identified TCR repertoire features specific for smokers and IBD patients, particularly when signs of disease activity were present. These findings are a first step towards the application of TCR repertoire analyses as a valuable tool to characterise inflammatory bowel diseases and to identify potential biomarkers and true disease causes.

• MeSH
• C-reaktivní protein analýza   MeSH
• Crohnova nemoc * diagnóza   imunologie   patofyziologie   MeSH
• dospělí MeSH
• dvojčata monozygotní MeSH
• feces MeSH
• geny TcR alfa * MeSH
• geny TcR beta * MeSH
• kouření imunologie   MeSH
• leukocytární L1-antigenní komplex analýza   MeSH
• lidé MeSH
• posouzení stavu pacienta MeSH
• receptory antigenů T-buněk alfa-beta krev   MeSH
• sekvenční analýza DNA MeSH
• ulcerózní kolitida * diagnóza   imunologie   patofyziologie   MeSH
• Check Tag
• dospělí MeSH
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• studie na dvojčatech MeSH
• Geografické názvy
• Dánsko MeSH
• Německo MeSH

A novel immature human T-ALL cell line, UHKT-42, was established from a 12 year old male patient with acute undifferentiated leukemia. The cell line expressed surface CD7, CD5 and cytoplasmic CD3 antigens. All other T-lymphocytic antigens were undetectable on the surface or in the cytoplasm of cultured cells. Expression of the T-cell receptor (TCR) beta, TCR delta, CD3 delta and CD3 epsilon genes was detected by Northern blotting in total cellular RNA extracts, however, the expression of TCR alpha and TCR gamma was undetectable. After stimulation by TPA for 3 days, only the appearance of CD25 (Tac antigen) was detected by immunofluorescence and flow cytometry. Secretion of interleukin-2 (IL-2) into the culture media was also detected after stimulation by PHA or TPA, but not in unstimulated cells. These results suggest that UHKT-42 cells are early precursors of T cells, with TCR beta/delta expression.

• MeSH
• akutní lymfatická leukemie imunologie   patologie   MeSH
• antigeny CD5 MeSH
• antigeny CD7 MeSH
• CD antigeny metabolismus   MeSH
• diferenciační antigeny T-lymfocytů metabolismus   MeSH
• dítě MeSH
• lidé MeSH
• nádorové buňky kultivované MeSH
• receptory antigenů T-buněk alfa-beta genetika   metabolismus   MeSH
• T-lymfocyty * metabolismus   patologie   MeSH
• Check Tag
• dítě MeSH
• lidé MeSH
• mužské pohlaví MeSH
• Publikační typ
• kazuistiky MeSH