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Nature Mar 2023T cell receptors (TCRs) enable T cells to specifically recognize mutations in cancer cells. Here we developed a clinical-grade approach based on CRISPR-Cas9...
T cell receptors (TCRs) enable T cells to specifically recognize mutations in cancer cells. Here we developed a clinical-grade approach based on CRISPR-Cas9 non-viral precision genome-editing to simultaneously knockout the two endogenous TCR genes TRAC (which encodes TCRα) and TRBC (which encodes TCRβ). We also inserted into the TRAC locus two chains of a neoantigen-specific TCR (neoTCR) isolated from circulating T cells of patients. The neoTCRs were isolated using a personalized library of soluble predicted neoantigen-HLA capture reagents. Sixteen patients with different refractory solid cancers received up to three distinct neoTCR transgenic cell products. Each product expressed a patient-specific neoTCR and was administered in a cell-dose-escalation, first-in-human phase I clinical trial ( NCT03970382 ). One patient had grade 1 cytokine release syndrome and one patient had grade 3 encephalitis. All participants had the expected side effects from the lymphodepleting chemotherapy. Five patients had stable disease and the other eleven had disease progression as the best response on the therapy. neoTCR transgenic T cells were detected in tumour biopsy samples after infusion at frequencies higher than the native TCRs before infusion. This study demonstrates the feasibility of isolating and cloning multiple TCRs that recognize mutational neoantigens. Moreover, simultaneous knockout of the endogenous TCR and knock-in of neoTCRs using single-step, non-viral precision genome-editing are achieved. The manufacture of neoTCR engineered T cells at clinical grade, the safety of infusing up to three gene-edited neoTCR T cell products and the ability of the transgenic T cells to traffic to the tumours of patients are also demonstrated.
Topics: Humans; Antigens, Neoplasm; Biopsy; Cell- and Tissue-Based Therapy; Cytokine Release Syndrome; Disease Progression; Encephalitis; Gene Editing; Gene Knock-In Techniques; Gene Knockout Techniques; Genes, T-Cell Receptor alpha; Genes, T-Cell Receptor beta; Mutation; Neoplasms; Patient Safety; Precision Medicine; Receptors, Antigen, T-Cell; T-Lymphocytes; Transgenes; HLA Antigens; CRISPR-Cas Systems
PubMed: 36356599
DOI: 10.1038/s41586-022-05531-1 -
Proceedings of the National Academy of... Dec 1989To design and direct at will the specificity of T cells in a non-major histocompatibility complex (MHC)-restricted manner, we have generated and expressed chimeric...
To design and direct at will the specificity of T cells in a non-major histocompatibility complex (MHC)-restricted manner, we have generated and expressed chimeric T-cell receptor (TcR) genes composed of the TcR constant (C) domains fused to the antibody's variable (V) domains. Genomic expression vectors have been constructed containing the rearranged gene segments coding for the V region domains of the heavy (VH) and light (VL) chains of an anti-2,4,6-trinitrophenyl (TNP) antibody (SP6) spliced to either one of the C-region gene segments of the alpha or beta TcR chains. Following transfection into a cytotoxic T-cell hybridoma, expression of a functional TcR was detected. The chimeric TcR exhibited the idiotope of the Sp6 anti-TNP antibody and endowed the T cells with a non-MHC-restricted response to the hapten TNP. The transfectants specifically killed and produced interleukin 2 in response to TNP-bearing target cells across strain and species barriers. Moreover, such transfectants responded to immobilized TNP-protein conjugates, bypassing the need for cellular processing and presentation. In the particular system employed, both the TNP-binding site and the Sp6 idiotope reside almost exclusively in the VH chain region. Hence, introduction into T cells of TcR genes containing only the VHSp6 fused to either the C alpha or C beta was sufficient for the expression of a functional surface receptor. Apparently, the VHC alpha or VHC beta chimeric chains can pair with the endogenous beta or alpha chains of the recipient T cell to form a functional alpha beta heterodimeric receptor. Thus, this chimeric receptor provides the T cell with an antibody-like specificity and is able to effectively transmit the signal for T-cell activation and execution of its effector function.
Topics: Animals; Antibody Specificity; Blotting, Northern; Cell Line; Chimera; Cloning, Molecular; Cytotoxicity, Immunologic; Embryo, Mammalian; Exons; Gene Library; Gene Rearrangement, T-Lymphocyte; Genes, Immunoglobulin; Immunoglobulin Heavy Chains; Immunoglobulin Light Chains; Immunoglobulin Variable Region; Interleukin-2; Kinetics; Major Histocompatibility Complex; Mice; Nucleic Acid Hybridization; Receptors, Antigen, T-Cell; Restriction Mapping; Transcription, Genetic; Transfection
PubMed: 2513569
DOI: 10.1073/pnas.86.24.10024 -
Frontiers in Immunology 2021In jawed vertebrates, two major T cell populations have been characterized. They are defined as α/β or γ/δ T cells, based on the expressed T cell receptor. Salmonids... (Comparative Study)
Comparative Study
In jawed vertebrates, two major T cell populations have been characterized. They are defined as α/β or γ/δ T cells, based on the expressed T cell receptor. Salmonids (family ) include two key teleost species for aquaculture, rainbow trout () and Atlantic salmon ( which constitute important models for fish immunology and important targets for vaccine development. The growing interest to decipher the dynamics of adaptive immune responses against pathogens or vaccines has resulted in recent efforts to sequence the immunoglobulin (IG) or antibodies and T cell receptor (TR) repertoire in these species. In this context, establishing a comprehensive and coherent locus annotation is the fundamental basis for the analysis of high-throughput repertoire sequencing data. We therefore decided to revisit the description and annotation of TRA/TRD locus in Atlantic salmon and two strains of rainbow trout (Swanson and Arlee) using the now available high-quality genome assemblies. Phylogenetic analysis of functional TRA/TRD V genes from these three genomes led to the definition of 25 subgroups shared by both species, some with particular feature. A total of 128 TRAJ genes were identified in , the majority with a close counterpart in . Analysis of expressed TRA repertoire indicates that most TRAV gene subgroups are expressed at mucosal and systemic level. The present work on TRA/TRD locus annotation along with the analysis of TRA repertoire sequencing data show the feasibility and advantages of a common salmonid TRA/TRD nomenclature that allows an accurate annotation and analysis of high-throughput sequencing results, across salmonid T cell subsets.
Topics: Amino Acid Sequence; Animals; Conserved Sequence; Gene Expression Profiling; Gene Library; Genes, T-Cell Receptor; Genome; Models, Molecular; Molecular Sequence Annotation; Oncorhynchus mykiss; Phylogeny; Protein Conformation; RNA, Messenger; Receptors, Antigen, T-Cell; Salmo salar; Sequence Alignment; Sequence Homology, Amino Acid; Species Specificity; Terminology as Topic
PubMed: 34733285
DOI: 10.3389/fimmu.2021.753960 -
Genes Dec 2020The adaptive immune response provides the vertebrate immune system with the ability to recognize and remember specific pathogens to generate immunity, and mount stronger... (Comparative Study)
Comparative Study
The adaptive immune response provides the vertebrate immune system with the ability to recognize and remember specific pathogens to generate immunity, and mount stronger attacks each time the pathogen is encountered. T cell receptors are the antigen receptors of the adaptive immune response expressed by T cells, which specifically recognize processed antigens, presented as peptides by the highly polymorphic major histocompatibility (MH) proteins. T cell receptors (TR) are divided into two groups, αβ and γδ, which express distinct TR containing either α and β, or γ and δ chains, respectively. The TRα locus (TRA) and TRδ locus (TRD) of bovine () and the sheep () have recently been described and annotated by IMGT biocurators. The aim of the present study is to present the results of the biocuration and to compare the genes of the TRA/TRD loci among these ruminant species based on the repertoire. The comparative analysis shows similarities but also differences, including the fact that these two species have a TRA/TRD locus about three times larger than that of humans and therefore have many more genes which may demonstrate duplications and/or deletions during evolution.
Topics: Adaptive Immunity; Animals; Cattle; Evolution, Molecular; Genes, T-Cell Receptor alpha; Genes, T-Cell Receptor delta; Genetic Loci; Molecular Sequence Annotation; Sheep, Domestic; Species Specificity
PubMed: 33379283
DOI: 10.3390/genes12010030 -
Immunity Mar 2023Human T cell receptors (TCRs) are critical for mediating immune responses to pathogens and tumors and regulating self-antigen recognition. Yet, variations in the genes...
Human T cell receptors (TCRs) are critical for mediating immune responses to pathogens and tumors and regulating self-antigen recognition. Yet, variations in the genes encoding TCRs remain insufficiently defined. Detailed analysis of expressed TCR alpha, beta, gamma, and delta genes in 45 donors from four human populations-African, East Asian, South Asian, and European-revealed 175 additional TCR variable and junctional alleles. Most of these contained coding changes and were present at widely differing frequencies in the populations, a finding confirmed using DNA samples from the 1000 Genomes Project. Importantly, we identified three Neanderthal-derived, introgressed TCR regions including a highly divergent TRGV4 variant, which mediated altered butyrophilin-like molecule 3 (BTNL3) ligand reactivity and was frequent in all modern Eurasian population groups. Our results demonstrate remarkable variation in TCR genes in both individuals and populations, providing a strong incentive for including allelic variation in studies of TCR function in human biology.
Topics: Humans; Receptors, Antigen, T-Cell; Antigens; Genes, T-Cell Receptor
PubMed: 36796364
DOI: 10.1016/j.immuni.2023.01.026 -
Microbiology Spectrum Jun 2023Bacterial conjugation is one of the most abundant horizontal gene transfer (HGT) mechanisms, playing a fundamental role in prokaryote evolution. A better understanding...
Bacterial conjugation is one of the most abundant horizontal gene transfer (HGT) mechanisms, playing a fundamental role in prokaryote evolution. A better understanding of bacterial conjugation and its cross talk with the environment is needed for a more complete understanding of HGT mechanisms and to fight the dissemination of malicious genes between bacteria. Here, we studied the effect of outer space, microgravity, and additional key environmental cues on transfer ( gene expression and conjugation efficiency, using the under studied broad-host range plasmid pN3, as a model. High resolution scanning electron microscopy revealed the morphology of the pN3 conjugative pili and mating pair formation during conjugation. Using a nanosatellite carrying a miniaturized lab, we studied pN3 conjugation in outer space, and used qRT-PCR, Western blotting and mating assays to determine the effect of ground physicochemical parameters on gene expression and conjugation. We showed for the first time that bacterial conjugation can occur in outer space and on the ground, under microgravity-simulated conditions. Furthermore, we demonstrated that microgravity, liquid media, elevated temperature, nutrient depletion, high osmolarity and low oxygen significantly reduce pN3 conjugation. Interestingly, under some of these conditions we observed an inverse correlation between gene transcription and conjugation frequency and found that induction of at least and can negatively affect pN3 conjugation frequency in a dose-dependent manner. Collectively, these results uncover pN3 regulation by various environmental cues and highlight the diversity of conjugation systems and the different ways in which they may be regulated in response to abiotic signals. Bacterial conjugation is a highly ubiquitous and promiscuous process, by which a donor bacterium transfers a large portion of genetic material to a recipient cell. This mechanism of horizontal gene transfer plays an important role in bacterial evolution and in the ability of bacteria to acquire resistance to antimicrobial drugs and disinfectants. Bacterial conjugation is a complex and energy-consuming process, that is tightly regulated and largely affected by various environmental signals sensed by the bacterial cell. Comprehensive knowledge about bacterial conjugation and the ways it is affected by environmental cues is required to better understand bacterial ecology and evolution and to find new effective ways to counteract the threating dissemination of antibiotic resistance genes between bacterial populations. Moreover, characterizing this process under stress or suboptimal growth conditions such as elevated temperatures, high salinity or in the outer space, may provide insights relevant to future habitat environmental conditions.
Topics: Conjugation, Genetic; Cues; Plasmids; Bacteria; Gene Transfer, Horizontal
PubMed: 36995224
DOI: 10.1128/spectrum.03688-22 -
BioRxiv : the Preprint Server For... Nov 2023Allelic variability in the adaptive immune receptor loci, which harbor the gene segments that encode B cell and T cell receptors (BCR/TCR), has been shown to be of...
Allelic variability in the adaptive immune receptor loci, which harbor the gene segments that encode B cell and T cell receptors (BCR/TCR), has been shown to be of critical importance for immune responses to pathogens and vaccines. In recent years, B cell and T cell receptor repertoire sequencing (Rep-Seq) has become widespread in immunology research making it the most readily available source of information about allelic diversity in immunoglobulin (IG) and T cell receptor (TR) loci in different populations. Here we present a novel algorithm for extra-sensitive and specific variable (V) and joining (J) gene allele inference and genotyping allowing reconstruction of individual high-quality gene segment libraries. The approach can be applied for inferring allelic variants from peripheral blood lymphocyte BCR and TCR repertoire sequencing data, including hypermutated isotype-switched BCR sequences, thus allowing high-throughput genotyping and novel allele discovery from a wide variety of existing datasets. The developed algorithm is a part of the MiXCR software ( https://mixcr.com ) and can be incorporated into any pipeline utilizing upstream processing with MiXCR. We demonstrate the accuracy of this approach using Rep-Seq paired with long-read genomic sequencing data, comparing it to a widely used algorithm, TIgGER. We applied the algorithm to a large set of IG heavy chain (IGH) Rep-Seq data from 450 donors of ancestrally diverse population groups, and to the largest reported full-length TCR alpha and beta chain (TRA; TRB) Rep-Seq dataset, representing 134 individuals. This allowed us to assess the genetic diversity of genes within the IGH, TRA and TRB loci in different populations and demonstrate the connection between antibody repertoire gene usage and the number of allelic variants present in the population. Finally we established a database of allelic variants of V and J genes inferred from Rep-Seq data and their population frequencies with free public access at https://vdj.online .
PubMed: 38014266
DOI: 10.1101/2023.10.10.561703 -
Genes Apr 2021The role of γδ T cells in vertebrate immunity is still an unsolved puzzle. Species such as humans and mice display a low percentage of these T lymphocytes (i.e., "γδ...
The role of γδ T cells in vertebrate immunity is still an unsolved puzzle. Species such as humans and mice display a low percentage of these T lymphocytes (i.e., "γδ low species") with a restricted diversity of γδ T cell receptors (TR). Conversely, artiodactyl species (i.e., "γδ high species") account for a high proportion of γδ T cells with large γ and δ chain repertoires. The genomic organisation of the TR γ (TRG) and δ (TRD) loci has been determined in sheep and cattle, noting that a wide number of germline genes that encode for γ and δ chains characterise their genomes. Taking advantage of the current improved version of the genome assembly, we have investigated the genomic structure and gene content of the dromedary TRD locus, which, as in the other mammalian species, is nested within the TR α (TRA) genes. The most remarkable finding was the identification of a very limited number of variable germline genes (TRDV) compared to sheep and cattle, which supports our previous expression analyses for which the somatic hypermutation mechanism is able to enlarge and diversify the primary repertoire of dromedary δ chains. Furthermore, the comparison between genomic and expressed sequences reveals that genes, up to four incorporated in a transcript, greatly contribute to the increased diversity of the dromedary δ chain antigen binding-site.
Topics: Animals; Camelus; Computational Biology; Genetic Loci; Genome; Phylogeny; Receptors, Antigen, T-Cell, alpha-beta; Receptors, Antigen, T-Cell, gamma-delta
PubMed: 33918850
DOI: 10.3390/genes12040544 -
Frontiers in Immunology 2021T-cell antigen receptors (TRs) in vertebrates can be divided into αβ or γδ, encoded by TRA/D, TRG, or TRB loci. TRs play a central role in mammal cellular immunity,...
T-cell antigen receptors (TRs) in vertebrates can be divided into αβ or γδ, encoded by TRA/D, TRG, or TRB loci. TRs play a central role in mammal cellular immunity, which occurs by rearrangement of V, D, J, and C genes in the loci. The bat is the only mammal with flying ability and is considered the main host of zoonotic viruses, an important public health concern. However, at present, little is known about the composition of bat TR genes. Based on the whole genome sequence of the greater horseshoe bat () and referring to the TR/IG annotation rules formulated by the international ImMunoGeneTics information system (IMGT), we present a complete annotation of TRA/D, TRG, and TRB loci of . A total of 128 V segments, three D segments, 85 J segments, and 6 C segments were annotated and compared with other known mammalian data. The characteristics of the TR locus and germline genes of are analyzed.
Topics: Animals; Biological Evolution; Cats; Cattle; Chiroptera; Computational Biology; Genetic Loci; Humans; Immunogenetics; Mice; Molecular Sequence Annotation; Phylogeny; Rabbits; Receptors, Antigen, T-Cell, alpha-beta; Receptors, Antigen, T-Cell, gamma-delta; Sequence Alignment; T-Lymphocytes; Whole Genome Sequencing
PubMed: 34659234
DOI: 10.3389/fimmu.2021.741408 -
Frontiers in Cellular and Infection... 2014Bacteria frequently acquire novel genes by horizontal gene transfer (HGT). HGT through the process of bacterial conjugation is highly efficient and depends on the... (Review)
Review
Bacteria frequently acquire novel genes by horizontal gene transfer (HGT). HGT through the process of bacterial conjugation is highly efficient and depends on the presence of conjugative plasmids (CPs) or integrated conjugative elements (ICEs) that provide the necessary genes for DNA transmission. This review focuses on recent advancements in our understanding of ssDNA transfer systems and regulatory networks ensuring timely and spatially controlled DNA transfer (tra) gene expression. As will become obvious by comparing different systems, by default, tra genes are shut off in cells in which conjugative elements are present. Only when conditions are optimal, donor cells-through epigenetic alleviation of negatively acting roadblocks and direct stimulation of DNA transfer genes-become transfer competent. These transfer competent cells have developmentally transformed into specialized cells capable of secreting ssDNA via a T4S (type IV secretion) complex directly into recipient cells. Intriguingly, even under optimal conditions, only a fraction of the population undergoes this transition, a finding that indicates specialization and cooperative, social behavior. Thereby, at the population level, the metabolic burden and other negative consequences of tra gene expression are greatly reduced without compromising the ability to horizontally transfer genes to novel bacterial hosts. This undoubtedly intelligent strategy may explain why conjugative elements-CPs and ICEs-have been successfully kept in and evolved with bacteria to constitute a major driving force of bacterial evolution.
Topics: Bacterial Physiological Phenomena; Biofilms; Biological Evolution; Conjugation, Genetic; DNA Transposable Elements; DNA, Bacterial; DNA, Single-Stranded; Gene Transfer, Horizontal; Gram-Negative Bacteria; Gram-Positive Bacteria; Host-Pathogen Interactions; Plasmids; Quorum Sensing
PubMed: 24809026
DOI: 10.3389/fcimb.2014.00054