scholarly journals Molecular Diagnostics on Microfabricated Electrophoretic Devices: From Slab Gel- to Capillary- to Microchip-based Assays for T- and B-Cell Lymphoproliferative Disorders

1999 ◽  
Vol 45 (11) ◽  
pp. 1906-1917 ◽  
Author(s):  
Nicole J Munro ◽  
Karen Snow ◽  
Jeffrey A Kant ◽  
James P Landers
Keyword(s):  
Capillary Electrophoresis ◽  
T Cell ◽  
Molecular Biology ◽  
B Cell ◽  
Dna Sequences ◽  
Gel Electrophoresis ◽  
Variable Region ◽  
Cell Receptor ◽  
Relevant Information ◽  
Chain Gene

Abstract Background: Current methods for molecular-based diagnosis of disease rely heavily on modern molecular biology techniques for interrogating the genome for aberrant DNA sequences. These techniques typically include amplification of the target DNA sequences followed by separation of the amplified fragments by slab gel electrophoresis. As a result of the labor-intensive, time-consuming nature of slab gel electrophoresis, alternative electrophoretic formats have been developed in the form of capillary electrophoresis and, more recently, multichannel microchip electrophoresis. Methods: Capillary electrophoresis was explored as an alternative to slab gel electrophoresis for the analysis of PCR-amplified products indicative of T- and B-cell malignancies as a means of defining the elements for silica microchip-based diagnosis. Capillary-based separations were replicated on electrophoretic microchips. Results: The microchip-based electrophoretic separation effectively resolved PCR-amplified fragments from the variable region of the T-cell receptor-γ gene (150–250 bp range) and the immunoglobulin heavy chain gene (80–140 bp range), yielding diagnostically relevant information regarding the presence of clonal DNA populations. Although hydroxyethylcellulose provided adequate separation power, the need for a coated microchannel for effective resolution necessitated additional preparative steps. In addition, preliminary data are shown indicating that polyvinylpyrrolidone may provide an adequate matrix without the need for microchannel coating. Conclusions: Separation of B- and T-cell gene rearrangement PCR products on microchips provides diagnostic information in dramatically reduced time (160 s vs 2.5 h) with no loss of diagnostic capacity when compared with current methodologies. As illustrated, this technology and methodology holds great potential for extrapolation to the abundance of similar molecular biology-based techniques.

T-cell receptor variable region of the β-chain gene use in peripheral blood and multiple synovial membranes during rheumatoid arthritis

1995 ◽  
Vol 42 (4) ◽  
pp. 331-339 ◽  
Author(s):  
Antoine Alam ◽  
Jacqueline Lulé ◽  
Héléne Coppin ◽  
Nathalie Lambert ◽  
Bernard Maziéres ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
T Cell ◽  
T Cell Receptor ◽  
Peripheral Blood ◽  
Variable Region ◽  
Cell Receptor ◽  
Chain Gene ◽  
Synovial Membranes ◽  
Β Chain

An (8;14)(q24;q11) translocation involving the T-cell receptor α-chain gene and theMYC oncogene 3′ region in a B-cell lymphoma

1989 ◽  
Vol 1 (1) ◽  
pp. 15-22 ◽  
Author(s):  
James K. Park ◽  
Timothy W. McKeithan ◽  
Michelle M. le Beau ◽  
Mitchell A. Bitter ◽  
Wilbur A. Franklin ◽  
...  
Keyword(s):  
T Cell ◽  
B Cell ◽  
T Cell Receptor ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Cell Receptor ◽  
Chain Gene ◽  
Α Chain

scholarly journals Germline PTEN mutations are associated with a skewed peripheral immune repertoire in humans and mice

2020 ◽  
Vol 29 (14) ◽  
pp. 2353-2364
Author(s):  
Ritika Jaini ◽  
Matthew G Loya ◽  
Alexander T King ◽  
Stetson Thacker ◽  
Nicholas B Sarn ◽  
...  
Keyword(s):  
T Cell ◽  
B Cell ◽  
Immune Tolerance ◽  
Stress Responses ◽  
Variable Region ◽  
Cell Receptor ◽  
Autism Spectrum ◽  
Gene Repertoire ◽  
Cell Reactivity ◽  
Diverse Range

Abstract Individuals with germline mutations in the gene encoding phosphatase and tensin homolog on chromosome ten (PTEN) are diagnosed with PTEN hamartoma tumor syndrome (PHTS) and are at high risk for developing breast, thyroid and other cancers and/or autoimmunity or neurodevelopmental issues including autism spectrum disorders. Although well recognized as a tumor suppressor, involvement of PTEN mutations in mediating such a diverse range of phenotypes indicates a more central involvement for PTEN in immunity than previously recognized. To address this, sequencing of the T-cell receptor variable-region β-chain was performed on peripheral blood from PHTS patients. Based on patient findings, we performed mechanistic studies in two Pten knock-in murine models, distinct from each other in cell compartment-specific predominance of Pten. We found that PTEN mutations in humans and mice are associated with a skewed T- and B-cell gene repertoire, characterized by increased prevalence of high-frequency clones. Immunological characterization showed that Pten mutants have increased B-cell proliferation and a proclivity towards increased T-cell reactivity upon Toll-like-receptor stimulation. Furthermore, decreases in nuclear but not cytoplasmic Pten levels associated with a reduction in expression of the autoimmune regulator (Aire), a critical mediator of central immune tolerance. Mechanistically, we show that nuclear PTEN most likely regulates Aire expression via its emerging role in splicing regulation. We conclude that germline disruption of PTEN, both in human and mouse, results in compromised central immune tolerance processes that may significantly impact individual stress responses and therefore predisposition to autoimmunity and cancer.

Constitutive Lymphoid Expression of the Nuclear Form of RNase H1 Is Associated with a Developmental Bottleneck at the Pro-B Cell Stage of B Cell Differentiation.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4702-4702
Author(s):  
Lina A. Gugliotti ◽  
Kiran B. Sakhuja ◽  
Hongsheng Wang ◽  
Julia Pinkhasov ◽  
Paul E. Love ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Dna Sequences ◽  
Conflicts Of Interest ◽  
Cell Receptor ◽  
Chain Gene ◽  
B Cell Differentiation ◽  
Loop Formation ◽  
Dna Breaks ◽  
Cell Stage

Abstract Abstract 4702 The development of B lymphocytes and the process of lineage determination are initiated by expression of a set of transcriptional regulators leading to V(D)J recombination events initiated by double-strand DNA breaks. Subsequently, these recombinations form DNAs that permit transcription of immunoglobulin genes and translation of the corresponding mRNAs, first by joining the V(D)J DNA sequences, then by recombination, that generates various isotypes of immunoglobulins by class-switch recombination (CSR). Formation of R-loops, regions containing RNA/DNA hybrid and a displaced single-stranded DNA, have been shown to lead to recombination in bacteria, yeast, HeLa and chick cells. Expression in each of these cases of excess ribonuclease H1 (RNase H1), a class of enzymes that degrade RNA in RNA/DNA hybrids, has ameliorated the deleterious effects and decreased recombinational events associated with R-loop formation. R-loops have been observed following transcription of the switch regions that occurs during CSR. The possibility that R-loops are important in V(D)J recombination has not been addressed, and whether ribonucleases H (RNases H) play a role in this process is still uncertain. Transgenic (TG) mice that overexpress RNase H1 in B and T cells (M27F7) were employed in this study. FACS analysis of hematopoietic cells from TG mice revealed a decrease in pre-B cells in the bone marrow. The data indicate a block at the pro-B to pre-B stage of B cell development, which may be the result of apoptosis due to the failure to generate a productive VH-D-JH rearrangement and expression of the pre-B cell receptor. A few B cells that successfully passed these checkpoints predominately differentiated into marginal zone and B1a cells in the peripheral lymphoid organs of the TG mice. These data suggest that R-loops are important in H chain gene rearrangement. This research is supported by the Intramural Research Program of the National Institutes of Health, the Eunice Kennedy Shriver National Institute of Child Health and Human Development and the National Institute of Allergy and Infectious Diseases. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Activation and Tolerance in CD4+ T Cells Reactive to an Immunoglobulin Variable Region

2004 ◽  
Vol 200 (1) ◽  
pp. 1-11 ◽  
Author(s):  
Christopher M. Snyder ◽  
Katja Aviszus ◽  
Ryan A. Heiser ◽  
Daniel R. Tonkin ◽  
Amanda M. Guth ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Cell Activation ◽  
Variable Region ◽  
Cell Receptor ◽  
B Cell Activation ◽  
Antibody Diversity ◽  
Helper Activity

Antibody diversity creates an immunoregulatory challenge for T cells that must cooperate with B cells, yet discriminate between self and nonself. To examine the consequences of T cell reactions to the B cell receptor (BCR), we generated a transgenic (Tg) line of mice expressing a T cell receptor (TCR) specific for a κ variable region peptide in monoclonal antibody (mAb) 36-71. The κ epitope was originally generated by a pair of somatic mutations that arose naturally during an immune response. By crossing this TCR Tg mouse with mice expressing the κ chain of mAb 36-71, we found that κ-specific T cells were centrally deleted in thymi of progeny that inherited the κTg. Maternally derived κTg antibody also induced central deletion. In marked contrast, adoptive transfer of TCR Tg T cells into κTg recipients resulted in T and B cell activation, lymphadenopathy, splenomegaly, and the production of IgG antichromatin antibodies by day 14. In most recipients, autoantibody levels increased with time, Tg T cells persisted for months, and a state of lupus nephritis developed. Despite this, Tg T cells appeared to be tolerant as assessed by severely diminished proliferative responses to the Vκ peptide. These results reveal the importance of attaining central and peripheral T cell tolerance to BCR V regions. They suggest that nondeletional forms of T tolerance in BCR-reactive T cells may be insufficient to preclude helper activity for chromatin-reactive B cells.

scholarly journals IGHV sequencing reveals acquired N-glycosylation sites as a clonal and stable event during follicular lymphoma evolution

Blood ◽  
2020 ◽  
Vol 135 (11) ◽  
pp. 834-844 ◽  
Author(s):  
Mariette Odabashian ◽  
Emanuela Carlotti ◽  
Shamzah Araf ◽  
Jessica Okosun ◽  
Filomena Spada ◽  
...  
Keyword(s):  
Follicular Lymphoma ◽  
B Cell ◽  
Dna Sequences ◽  
Somatic Hypermutation ◽  
Variable Region ◽  
Cell Receptor ◽  
Tumor Evolution ◽  
Growth And Survival ◽  
Calcium Dependent ◽  
Glycosylation Sites

Abstract Follicular lymphoma B cells undergo continuous somatic hypermutation (SHM) of their immunoglobulin variable region genes, generating a heterogeneous tumor population. SHM introduces DNA sequences encoding N-glycosylation sites asparagine-X-serine/threonine (N-gly sites) within the V-region that are rarely found in normal B-cell counterparts. Unique attached oligomannoses activate B-cell receptor signaling pathways after engagement with calcium-dependent lectins expressed by tissue macrophages. This novel interaction appears critical for tumor growth and survival. To elucidate the significance of N-gly site presence and loss during ongoing SHM, we tracked site behavior during tumor evolution and progression in a diverse group of patients through next-generation sequencing. A hierarchy of subclones was visualized through lineage trees based on SHM semblance between subclones and their discordance from the germline sequence. We observed conservation of N-gly sites in more than 96% of subclone populations within and across diagnostic, progression, and transformation events. Rare N-gly-negative subclones were lost or negligible from successive events, in contrast to N-gly-positive subclones, which could additionally migrate between anatomical sites. Ongoing SHM of the N-gly sites resulted in subclones with different amino acid compositions across disease events, yet the vast majority of resulting DNA sequences still encoded for an N-gly site. The selection and expansion of only N-gly-positive subclones is evidence of the tumor cells’ dependence on sites, despite the changing genomic complexity as the disease progresses. N-gly sites were gained in the earliest identified lymphoma cells, indicating they are an early and stable event of pathogenesis. Targeting the inferred mannose-lectin interaction holds therapeutic promise.

scholarly journals T Cell Receptor Beta (TRB) Germline Variability is Revealed by Inference From Repertoire Data

2021 ◽  
Author(s):  
Aviv Omer ◽  
Ayelet Peres ◽  
Oscar L Rodrigues ◽  
Corey T Watson ◽  
William Lees ◽  
...  
Keyword(s):  
T Cell ◽  
B Cell ◽  
T Cell Receptor ◽  
Clinical Importance ◽  
Variable Region ◽  
Cell Receptor ◽  
Genomic Variation ◽  
Variable Regions ◽  
T Cell Receptor Beta ◽  
Receptor Beta

T and B cell repertoires constitute the foundation of adaptive immunity. Adaptive immune receptor repertoire sequencing (AIRR-seq) is a common approach to study immune system dynamics. Understanding the genetic factors influencing the composition and dynamics of these repertoires is of major scientific and clinical importance. The chromosomal loci encoding for the variable regions of T and B cell receptors (TCRs and BCRs, respectively) are challenging to decipher due to repetitive elements and undocumented structural variants. To confront this challenge, AIRR-seq-based methods have been developed recently for B cells, enabling genotype and haplotype inference and discovery of undocumented alleles. Applying these methods to AIRR-seq data reveals a plethora of undocumented genomic variations. However, this approach relies on complete coverage of the receptors' variable regions, and most T cell studies sequence only a small fraction of the variable region. Here, we adapted BCR inference methods to full and partial TCR sequences, and identified 38 undocumented polymorphisms in TRBV, 15 of them were also observed in genomic data assemblies. Further, we identified 31 undocumented 5' UTR sequences. A subset of these inferences was also observed using independent genomic approaches. We found the two documented TRBD2 alleles to be equally abundant in the population, and show that the single nucleotide that differentiates them is strongly associated with dramatic changes in the expressed repertoire. Our findings expand the knowledge of genomic variation in the TRB (T Cell Receptor Beta) locus and provide a basis for annotation of TCR repertoires for future basic and clinical studies.

scholarly journals Pathogenesis of B cell lymphoma in a patient with AIDS

Blood ◽  
1986 ◽  
Vol 67 (3) ◽  
pp. 612-615 ◽  
Author(s):  
JE Groopman ◽  
JL Sullivan ◽  
C Mulder ◽  
D Ginsburg ◽  
SH Orkin ◽  
...  
Keyword(s):  
T Cell ◽  
B Cells ◽  
B Cell ◽  
T Cell Receptor ◽  
Dna Sequences ◽  
Receptor Gene ◽  
B Cell Lymphoma ◽  
Cell Receptor ◽  
Molecular Techniques ◽  
Barr Virus

Abstract Lymphoma occurs at increased frequency in patients with the acquired immunodeficiency syndrome (AIDS). We studied, using serologic and molecular techniques, one such lymphoma for (a) evidence of infection with human T lymphotropic virus, type III (HTLV-III), and Epstein-Barr virus (EBV), (b) monoclonal rearrangement of immunoglobulin and T cell receptor genes, and (c) rearrangement of the c-myc oncogene. Immunoglobulin and T cell receptor gene studies demonstrated that the tumor was of monoclonal B cell origin. Similar to cases of Burkitt's lymphoma unrelated to AIDS, there were DNA sequences in the lymphoma that hybridized to EBV-specific probes and demonstrated evidence of c- myc rearrangement. HTLV-III sequences were not detected in the malignant B cells. The pathogenesis of some B cell neoplasms in patients with the syndrome may involve transformation by EBV and deregulation of oncogene expression without direct infection of the malignant B cells by HTLV-III.

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