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British Journal of Haematology Mar 2020Rearrangements 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...
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.
Topics: DNA, Neoplasm; Female; Gene Rearrangement, alpha-Chain T-Cell Antigen Receptor; Hematologic Neoplasms; High-Throughput Nucleotide Sequencing; Humans; Lymphoproliferative Disorders; Male; Multiplex Polymerase Chain Reaction
PubMed: 31587259
DOI: 10.1111/bjh.16230 -
Immunology Apr 2021Within each individual, the adaptive immune system generates a repertoire of cells expressing receptors capable of recognizing diverse potential pathogens. The...
Within each individual, the adaptive immune system generates a repertoire of cells expressing receptors capable of recognizing diverse potential pathogens. The theoretical diversity of the T-cell receptor (TCR) repertoire exceeds the actual size of the T-cell population in an individual by several orders of magnitude - making the observation of identical TCRs in different individuals extremely improbable if all receptors were equally likely. Despite this disparity between the theoretical and the realized diversity of the repertoire, these 'public' receptor sequences have been identified in autoimmune, cancer and pathogen interaction contexts. Biased generation processes explain the presence of public TCRs in the naive repertoire, but do not adequately explain the different abundances of these public TCRs. We investigate and characterize the distribution of genomic TCR-β sequences of naive CD8+ T cells from three genetically identical mice, comparing non-productive (non-functional sequences) and productive sequences. We find public TCR-β sequences at higher abundances compared with unshared sequences in the productive, but not in the non-productive, repertoire. We show that neutral processes such as recombination biases, codon degeneracy and generation probability do not fully account for these differences, and conclude that thymic or peripheral selection plays an important role in increasing the abundances of public TCR-β sequences.
Topics: Animals; CD8-Positive T-Lymphocytes; Cells, Cultured; Clonal Selection, Antigen-Mediated; Codon Usage; Genes, T-Cell Receptor beta; Humans; Mice; Mice, Inbred C57BL; Receptors, Antigen, T-Cell, alpha-beta; Recombination, Genetic; Thymus Gland
PubMed: 33345304
DOI: 10.1111/imm.13299 -
Journal of Insect Science (Online) Jan 2017transformer (tra) is a switch gene of sex determination in many insects, particularly in Dipterans. However, the sex determination pathway in Bactrocera cucurbitae...
transformer (tra) is a switch gene of sex determination in many insects, particularly in Dipterans. However, the sex determination pathway in Bactrocera cucurbitae (Coquillett), a very destructive pest on earth, remains largely uncharacterized. In this study, we have isolated and characterized one female-specific and two male-specific transcripts of the tra gene (Bcutra) of B. cucurbitae. The genomic structure of Bcutra has been determined and the presence of multiple conserved Transformer (TRA)/TRA-2 binding sites in Bcutra has been found. BcuTRA is highly conservative with its homologues in other tephritid fruit flies. Gene expression analysis of Bcutra at different developmental stages demonstrates that the female transcript of Bcutra appears earlier than the male counterparts, indicating that the maternal TRA is inherited in eggs and might play a role in the regulation of TRA expression. The conservation of protein sequence and sex-specific splicing of Bcutra and its expression patterns during development suggest that Bcutra is probably the master gene of sex determination of B. cucurbitae. Isolation of Bcutra will facilitate the development of a genetic sexing strain for its biological control.
Topics: Amino Acid Sequence; Animals; DNA, Complementary; Female; Insect Proteins; Larva; Male; Phylogeny; Pupa; RNA, Messenger; Sequence Alignment; Tephritidae
PubMed: 28931159
DOI: 10.1093/jisesa/iex031 -
Frontiers in Immunology 2021The complete germline repertoires of the channel catfish, , T cell receptor (TR) loci, TRAD, TRB, and TRG were obtained by analyzing genomic data from PacBio sequencing.... (Comparative Study)
Comparative Study
The complete germline repertoires of the channel catfish, , T cell receptor (TR) loci, TRAD, TRB, and TRG were obtained by analyzing genomic data from PacBio sequencing. The catfish TRB locus spans 214 kb, and contains 112 TRBV genes, a single TRBD gene, 31 TRBJ genes and two TRBC genes. In contrast, the TRAD locus is very large, at 1,285 kb. It consists of four TRDD genes, one TRDJ gene followed by the exons for TRDC, 125 TRAJ genes and the exons encoding the TRAC. Downstream of the TRAC, are 140 TRADV genes, and all of them are in the opposite transcriptional orientation. The catfish TRGC locus spans 151 kb and consists of four diverse V-J-C cassettes. Altogether, this locus contains 15 TRGV genes and 10 TRGJ genes. To place our data into context, we also analyzed the zebrafish TR germline gene repertoires. Overall, our findings demonstrated that catfish possesses a more restricted repertoire compared to the zebrafish. For example, the 140 TRADV genes in catfish form eight subgroups based on members sharing 75% nucleotide identity. However, the 149 TRAD genes in zebrafish form 53 subgroups. This difference in subgroup numbers between catfish and zebrafish is best explained by expansions of catfish TRADV subgroups, which likely occurred through multiple, relatively recent gene duplications. Similarly, 112 catfish TRBV genes form 30 subgroups, while the 51 zebrafish TRBV genes are placed into 36 subgroups. Notably, several catfish and zebrafish TRB subgroups share ancestor nodes. In addition, the complete catfish TR gene annotation was used to compile a TR gene segment database, which was applied in clonotype analysis of an available gynogenetic channel catfish transcriptome. Combined, the TR annotation and clonotype analysis suggested that the expressed TRA, TRB, and TRD repertoires were generated by different mechanisms. The diversity of the TRB repertoire depends on the number of TRBV subgroups and TRBJ genes, while TRA diversity relies on the many different TRAJ genes, which appear to be only minimally trimmed. In contrast, TRD diversity relies on nucleotide additions and the utilization of up to four TRDD segments.
Topics: Animals; Evolution, Molecular; Fish Proteins; Genes, T-Cell Receptor; Genes, T-Cell Receptor alpha; Genes, T-Cell Receptor beta; Genes, T-Cell Receptor delta; Genes, T-Cell Receptor gamma; Genetic Loci; Ictaluridae; Phylogeny; Receptors, Antigen, T-Cell; Receptors, Antigen, T-Cell, alpha-beta; Receptors, Antigen, T-Cell, gamma-delta; Species Specificity; Zebrafish; Zebrafish Proteins
PubMed: 34899754
DOI: 10.3389/fimmu.2021.786402 -
BMC Genomics Sep 2022Cynomolgus macaque (Macaca fascicularis) is an attractive animal model for the study of human disease and is extensively used in biomedical research. Cynomolgus macaques...
BACKGROUND
Cynomolgus macaque (Macaca fascicularis) is an attractive animal model for the study of human disease and is extensively used in biomedical research. Cynomolgus macaques share behavioral, physiological, and genomic traits with humans and recapitulate human disease manifestations not observed in other animal species. To improve the use of the cynomolgus macaque model to investigate immune responses, we defined and characterized the T cell receptor (TCR) repertoire.
RESULT
We identified and analyzed the alpha (TRA), beta (TRB), gamma (TRG), and delta (TRD) TCR loci of the cynomolgus macaque. The expressed repertoire was determined using 22 unique lung samples from Mycobacterium tuberculosis infected cynomolgus macaques by single cell RNA sequencing. Expressed TCR alpha (TRAV) and beta (TRBV) variable region genes were enriched and identified using gene specific primers, which allowed their functional status to be determined. Analysis of the primers used for cynomolgus macaque TCR variable region gene enrichment showed they could also be used to amplify rhesus macaque (M. mulatta) variable region genes.
CONCLUSION
The genomic organization of the cynomolgus macaque has great similarity with the rhesus macaque and they shared > 90% sequence similarity with the human TCR repertoire. The identification of the TCR repertoire facilitates analysis of T cell immunity in cynomolgus macaques.
Topics: Animals; Genome; Genomics; Humans; Macaca fascicularis; Macaca mulatta; Mycobacterium tuberculosis
PubMed: 36096729
DOI: 10.1186/s12864-022-08867-0 -
Cancer Medicine Jul 2021Considering melanoma is the deadliest malignancy among dermatoma and presently lacks effective therapies, there is an urgent need to investigate the potential mechanisms...
BACKGROUND
Considering melanoma is the deadliest malignancy among dermatoma and presently lacks effective therapies, there is an urgent need to investigate the potential mechanisms underlying melanoma metastasis and determine prospective therapeutic targets for precise treatment of melanoma.
METHOD
Hub genes in melanoma metastasis were identified by analyzing RNA-seq data (mRNA, miRNA, and lncRNA) obtained from TCGA database. Then the identified hub genes were validated in human tissues with qRT-PCR, followed by survival analysis. Competing endogenous RNAs of the hub genes were defined to clarify potential molecular mechanism of melanoma progression. Then central gene-related signaling pathways were analyzed, followed by immune cell abundance analysis in tumor microenvironment with CYTERSORTx.
RESULT
A tetrad of IL2RA, IL2RG, IFNG, and IL7R genes were determined as hub genes and verified by qRT-PCR, which were significantly associated with unfavorable prognosis in melanoma. LINC02446, LINC01857, and LINC02384 may act as competing endogenous lncRNAs of IL2RA and IL7R through absorbing their shared miR.891a.5p and miR.203b.3p. JAK-STAT signaling pathway identified as the most relevant pathway in melanoma metastasis, as well as a wealthy of genes including TNFRSF 13B, TNFRSF17, TNFRSF9, TNFRSF8, TNFRSF13C, TNFRSF11B, LAG3, NRP1, ENTPD1, NT5E, CCL21, and CCR7, may induce tumor autoimmune suppression through enhancing regulatory T-cell abundance and performance in the tumor microenvironment. And regulatory T-cell proportion was indeed critically elevated in metastatic melanoma relative to primary melanoma, as well as in highly expressed IL2RA, IL2RG, IL7R, and IFNG group than their respective counterparts.
CONCLUSION
Elevated IL2RA, IL2RG, IL7R, and IFNG expression may play a central role in promoting melanoma metastasis through up regulation of intratumoral regulatory T-cell proportion mainly by activation of JAK-STAT signaling pathway. LINC02446, LINC01857, and LINC02384 may stimulate melanoma progression by reducing tumor-protecting miR.891a.5p and miR.203b.3p. A number of identified molecules including TNFRSF13B, LAG3, NRP1, ENTPD1, NT5E, CCL21, and CCR7 can serve as future therapeutic targets in melanoma treatment.
Topics: Disease Progression; Gene Expression Profiling; Humans; Immune Tolerance; Interferon-gamma; Interleukin-2 Receptor alpha Subunit; Interleukin-7 Receptor alpha Subunit; Melanoma; MicroRNAs; Molecular Targeted Therapy; Prognosis; RNA, Long Noncoding; RNA, Messenger; Skin Neoplasms; T-Lymphocytes, Regulatory; Tumor Microenvironment; Up-Regulation
PubMed: 34159752
DOI: 10.1002/cam4.3963 -
Blood Aug 2016Primary immunodeficiency diseases comprise a group of heterogeneous genetic defects that affect immune system development and/or function. Here we use in vitro...
Primary immunodeficiency diseases comprise a group of heterogeneous genetic defects that affect immune system development and/or function. Here we use in vitro differentiation of human induced pluripotent stem cells (iPSCs) generated from patients with different recombination-activating gene 1 (RAG1) mutations to assess T-cell development and T-cell receptor (TCR) V(D)J recombination. RAG1-mutants from severe combined immunodeficient (SCID) patient cells showed a failure to sustain progression beyond the CD3(--)CD4(-)CD8(-)CD7(+)CD5(+)CD38(-)CD31(-/lo)CD45RA(+) stage of T-cell development to reach the CD3(-/+)CD4(+)CD8(+)CD7(+)CD5(+)CD38(+)CD31(+)CD45RA(-) stage. Despite residual mutant RAG1 recombination activity from an Omenn syndrome (OS) patient, similar impaired T-cell differentiation was observed, due to increased single-strand DNA breaks that likely occur due to heterodimers consisting of both an N-terminal truncated and a catalytically dead RAG1. Furthermore, deep-sequencing analysis of TCR-β (TRB) and TCR-α (TRA) rearrangements of CD3(-)CD4(+)CD8(-) immature single-positive and CD3(+)CD4(+)CD8(+) double-positive cells showed severe restriction of repertoire diversity with preferential usage of few Variable, Diversity, and Joining genes, and skewed length distribution of the TRB and TRA complementary determining region 3 sequences from SCID and OS iPSC-derived cells, whereas control iPSCs yielded T-cell progenitors with a broadly diversified repertoire. Finally, no TRA/δ excision circles (TRECs), a marker of TRA/δ locus rearrangements, were detected in SCID and OS-derived T-lineage cells, consistent with a pre-TCR block in T-cell development. This study compares human T-cell development of SCID vs OS patients, and elucidates important differences that help to explain the wide range of immunologic phenotypes that result from different mutations within the same gene of various patients.
Topics: Cells, Cultured; DNA Breaks; Genes, RAG-1; Homeodomain Proteins; Humans; Induced Pluripotent Stem Cells; Infant; Mutation; Receptors, Antigen, T-Cell, alpha-beta; Severe Combined Immunodeficiency; T-Lymphocytes; V(D)J Recombination
PubMed: 27301863
DOI: 10.1182/blood-2015-10-676304 -
Frontiers in Immunology 2020Chimeric antigen receptor (CAR) T cells have emerged as a promising treatment for patients with advanced B-cell cancers. However, widespread application of the therapy... (Review)
Review
Chimeric antigen receptor (CAR) T cells have emerged as a promising treatment for patients with advanced B-cell cancers. However, widespread application of the therapy is currently limited by potentially life-threatening toxicities due to a lack of control of the highly potent transfused cells. Researchers have therefore developed several regulatory mechanisms in order to control CAR T cells . Clinical adoption of these control systems will depend on several factors, including the need for temporal and spatial control, the immunogenicity of the requisite components as well as whether the system allows reversible control or induces permanent elimination. Here we describe currently available and emerging control methods and review their function, advantages, and limitations.
Topics: Antigens, Neoplasm; CRISPR-Cas Systems; Cell Hypoxia; Cetuximab; Cytokine Release Syndrome; Cytokines; Genes, Transgenic, Suicide; Humans; Immunotherapy, Adoptive; Interleukin 1 Receptor Antagonist Protein; Lymphocyte Activation; Protease Inhibitors; Protein Binding; Protein Domains; Receptors, Antigen, T-Cell, alpha-beta; Receptors, Chimeric Antigen; Rituximab; T-Cell Antigen Receptor Specificity; T-Lymphocyte Subsets; Tetracycline; Transcription, Genetic; Transfection; Tumor Microenvironment
PubMed: 32194561
DOI: 10.3389/fimmu.2020.00326 -
Microbial Genomics May 2023Rising rates of multidrug-resistant infections necessitate a comprehensive understanding of the major strains and plasmids driving spread of resistance elements. Here,...
Rising rates of multidrug-resistant infections necessitate a comprehensive understanding of the major strains and plasmids driving spread of resistance elements. Here, we analysed 540 clinical, screen and environmental isolates recovered from across Wales between 2007 and 2020 using combined short- and long-read sequencing approaches. We identified resistant clones that have spread within and between hospitals including the high-risk strain sequence type (ST)307, which acquired the carbapenemase gene on a pOXA-48-like plasmid. We found evidence that this strain, which caused an acute outbreak largely centred on a single hospital in 2019, had been circulating undetected across South Wales for several years prior to the outbreak. In addition to clonal transmission, our analyses revealed evidence for substantial plasmid spread, mostly notably involving and (including ) carbapenemase genes that were found among many species and strain backgrounds. Two thirds (20/30) of the genes were carried on the Tn transposon and associated with IncF plasmids. These were mostly recovered from patients in North Wales, reflecting an outward expansion of the plasmid-driven outbreak of -producing in North-West England. A total of 92.1 % (105/114) of isolates with a carbapenemase carried the gene on a pOXA-48-like plasmid. While this plasmid family is highly conserved, our analyses revealed novel accessory variation including integrations of additional resistance genes. We also identified multiple independent deletions involving the gene cluster among pOXA-48-like plasmids in the ST307 outbreak lineage. These resulted in loss of conjugative ability and signal adaptation of the plasmids to carriage by the host strain. Altogether, our study provides, to our knowledge, the first high resolution view of the diversity, transmission and evolutionary dynamics of major resistant clones and plasmids of in Wales, and forms an important basis for ongoing surveillance efforts. This article contains data hosted by Microreact.
Topics: Klebsiella pneumoniae; Klebsiella; Wales; Plasmids; Genomics
PubMed: 37227259
DOI: 10.1099/mgen.0.001016 -
Microbiology (Reading, England) Dec 2013Expression of DNA transfer (tra) genes of F-type conjugative plasmids is required for the assembly of a functional type IV secretion machinery and subsequent plasmid DNA...
Expression of DNA transfer (tra) genes of F-type conjugative plasmids is required for the assembly of a functional type IV secretion machinery and subsequent plasmid DNA transfer from donor to recipient cells. Transcription of tra genes depends on the activation of a single promoter, designated PY, by the plasmid encoded TraJ protein. We here determine plasmid specificity of TraJ proteins from various subgroups of F-like plasmids and find that plasmid R1 conjugation and PY promoter activation can be achieved only by its cognate activator and by TraJ of the Salmonella plasmid pSLT and not by F or R100 TraJ proteins. In addition, we characterize the PY promoter of plasmid R1. We show that TraJ binds to PY DNA in vivo and that H-NS acts as a silencer of the PY promoter. In the natural plasmid context, H-NS silences transfer gene expression and horizontal plasmid DNA transfer. In contrast to what was found for the F plasmid, lack of H-NS did not abolish the requirement for ArcA and TraJ to reach full tra gene expression and DNA transfer activity. We propose that, besides a passive de-silencing activity, both ArcA and TraJ play a direct role in synergistically stimulating tra operon transcription and subsequent DNA transfer.
Topics: Bacterial Outer Membrane Proteins; Bacterial Proteins; Bacterial Secretion Systems; DNA-Binding Proteins; Drug Resistance, Bacterial; F Factor; Gene Silencing; Gene Transfer, Horizontal; Promoter Regions, Genetic; R Factors; Salmonella; Transcriptional Activation
PubMed: 24085835
DOI: 10.1099/mic.0.071738-0