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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 -
Immunogenetics Feb 2020The domestic ferret, Mustela putorius furo, is an important mammalian animal model to study human respiratory infection. However, insufficient genomic annotation hampers... (Review)
Review
The domestic ferret, Mustela putorius furo, is an important mammalian animal model to study human respiratory infection. However, insufficient genomic annotation hampers detailed studies of ferret T cell responses. In this study, we analyzed the published T cell receptor beta (TRB) locus and performed high-throughput sequencing (HTS) of peripheral blood of four healthy adult ferrets to identify expressed V, D, J, and C genes. The HTS data is used as a guide to manually curate the expressed V, D, J, and C genes. The ferret locus appears to be most similar to that of the dog. Like other mammalian TRB loci, the ferret TRB locus contains a library of variable genes located upstream of two D-J-C gene clusters, followed by a (in the ferret non-functional) V gene with an inverted transcriptional orientation. All TRB genes (expressed or not) reported here have been approved by the IMGT/WHO-IUIS nomenclature committee.
Topics: Animals; Ferrets; Gene Expression Regulation; Gene Rearrangement, beta-Chain T-Cell Antigen Receptor; High-Throughput Nucleotide Sequencing; Receptors, Antigen, T-Cell, alpha-beta
PubMed: 31797007
DOI: 10.1007/s00251-019-01142-9 -
Frontiers in Immunology 2018The α/β T cell receptor (TR) is a complex heterodimer that recognizes antigenic peptides and binds to major histocompatibility complex (MH) molecules. Both α and β...
The α/β T cell receptor (TR) is a complex heterodimer that recognizes antigenic peptides and binds to major histocompatibility complex (MH) molecules. Both α and β chains are encoded by different genes localized on two distinct chromosomal loci: TRA and TRB. The present study employed the recent release of the swine genome assembly to define the genomic organization of the TRB locus. According to the sequencing data, the pig TRB locus spans approximately 400 kb of genomic DNA and consists of 38 TRBV genes belonging to 24 subgroups located upstream of three in tandem TRBD-J-C clusters, which are followed by a TRBV gene in an inverted transcriptional orientation. Comparative analysis confirms that the general organization of the TRB locus is similar among mammalian species, but the number of germline TRBV genes varies greatly even between species belonging to the same order, determining the diversity and specificity of the immune response. However, sequence analysis of the TRB locus also suggests the presence of blocks of conserved homology in the genomic region across mammals. Furthermore, by analysing a public cDNA collection, we identified the usage pattern of the TRBV, TRBD, and TRBJ genes in the adult pig TRB repertoire, and we noted that the expressed TRBV repertoire seems to be broader and more diverse than the germline repertoire, in line with the presence of a high level of TRBV gene polymorphisms. Because the nucleotide differences seems to be principally concentrated in the CDR2 region, it is reasonable to presume that most T cell β-chain diversity can be related to polymorphisms in pig MH molecules. Domestic pigs represent a valuable animal model as they are even more anatomically, genetically and physiologically similar to humans than are mice. Therefore, present knowledge on the genomic organization of the pig TRB locus allows the collection of increased information on the basic aspects of the porcine immune system and contributes to filling the gaps left by rodent models.
Topics: Adaptive Immunity; Amino Acid Sequence; Animals; Genes, T-Cell Receptor alpha; Genes, T-Cell Receptor beta; Genome; Polymorphism, Single Nucleotide; Receptors, Antigen, T-Cell, alpha-beta; Sus scrofa; T-Lymphocytes; Whole Genome Sequencing
PubMed: 30455691
DOI: 10.3389/fimmu.2018.02526 -
Frontiers in Genetics 2019T cells can be separated into two major subsets based on the heterodimer that forms their T cell receptors. αβ T cells have receptors consisting of α and β chains,...
T cells can be separated into two major subsets based on the heterodimer that forms their T cell receptors. αβ T cells have receptors consisting of α and β chains, while γδ T cells are composed of γ and δ chains. αβ T cells play an essential role within the adaptive immune responses against pathogens. The recent genomic characterization of the T cell receptor β (TRB) locus has allowed us to infer the structure of this locus from the draft genome sequences of its wild and domestic Bactrian congeners, and . The general structural organization of the wild and domestic Bactrian TRB locus is similar to that of the dromedary, with a pool of TRBV genes positioned at the 5' end of D-J-C clusters, followed by a single TRBV gene located at the 3' end with an inverted transcriptional orientation. Despite the fragmented nature of the assemblies, comparative genomics reveals the existence of a perfect co-linearity between the three Old World camel TRB genomic sequences, which enables the transfer of information from one sequence to another and the filling of gaps in the genomic sequences. A virtual camelid TRB locus is hypothesized with the presence of 33 TRBV genes distributed in 26 subgroups. Likewise, in the artiodactyl species, three in-tandem D-J-C clusters, each composed of one TRBD gene, six or seven TRBJ genes, and one TRBC gene, are placed at the 3' end of the locus. As reported in the ruminant species, a group of four functional TRY genes at the 5' end and only one gene at the 3' end, complete the camelid TRB locus. Although the gene content is similar, differences are observed in the TRBV functional repertoire, and genes that are functional in one species are pseudogenes in the other species. Hence, variations in the functional repertoire between dromedary, wild and domestic Bactrian camels, rather than differences in the gene content, may represent the molecular basis explaining the disparity in the TRB repertoire between the species. Finally, our data contribute to the knowledge about the evolutionary history of Old World camelids.
PubMed: 31231418
DOI: 10.3389/fgene.2019.00482 -
Genes Apr 2021The bottlenose dolphin () belongs to the Cetartiodactyla and, similarly to other cetaceans, represents the most successful mammalian colonization of the aquatic... (Comparative Study)
Comparative Study
The bottlenose dolphin () belongs to the Cetartiodactyla and, similarly to other cetaceans, represents the most successful mammalian colonization of the aquatic environment. Here we report a genomic, evolutionary, and expression study of T cell receptor beta (TRB) genes. Although the organization of the dolphin TRB locus is similar to that of the other artiodactyl species, with three in tandem D-J-C clusters located at its 3' end, its uniqueness is given by the reduction of the total length due essentially to the absence of duplications and to the deletions that have drastically reduced the number of the germline TRBV genes. We have analyzed the relevant mature transcripts from two subjects. The simultaneous availability of rearranged T cell receptor α (TRA) and TRB cDNA from the peripheral blood of one of the two specimens, and the human/dolphin amino acids multi-sequence alignments, allowed us to calculate the most likely interactions at the protein interface between the alpha/beta heterodimer in complex with major histocompatibility class I (MH1) protein. Interacting amino acids located in the complementarity-determining region according to IMGT numbering (CDR-IMGT) of the dolphin variable V-alpha and beta domains were identified. According to comparative modelization, the atom pair contact sites analysis between the human MH1 grove (G) domains and the T cell receptor (TR) V domains confirms conservation of the structure of the dolphin TR/pMH.
Topics: Animals; Bottle-Nosed Dolphin; Chromosome Mapping; Female; Gene Rearrangement, alpha-Chain T-Cell Antigen Receptor; Gene Rearrangement, beta-Chain T-Cell Antigen Receptor; Histocompatibility Antigens Class I; Humans; Male; Receptors, Antigen, T-Cell, alpha-beta; Sequence Alignment; Sequence Analysis, DNA; Sequence Analysis, Protein; beta 2-Microglobulin
PubMed: 33919966
DOI: 10.3390/genes12040571 -
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 -
BMC Genomics Sep 2020Goats (Capra hircus), one of the first domesticated species, are economically important for milk and meat production, and their broad geographical distribution reflects...
BACKGROUND
Goats (Capra hircus), one of the first domesticated species, are economically important for milk and meat production, and their broad geographical distribution reflects their successful adaptation to diverse environmental conditions. Despite the relevance of this species, the genetic research on the goat traits is limited compared to other domestic species. Thanks to the latest goat reference genomic sequence (ARS1), which is considered to be one of the most continuous assemblies in livestock, we deduced the genomic structure of the T cell receptor beta (TRB) and gamma (TRG) loci in this ruminant species.
RESULTS
Our analyses revealed that although the organization of the goat TRB locus is broadly similar to that of the other artiodactyl species, with three in-tandem D-J-C clusters located at the 3' end, a complex and extensive series of duplications have occurred in the V genes at the 5' end, leading to a marked expansion in the number of the TRBV genes. This phenomenon appears to be a feature of the ruminant lineage since similar gene expansions have also occurred in sheep and cattle. Likewise, the general organization of the goat TRG genes is typical of ruminant species studied so far, with two paralogous TRG loci, TRG1 and TRG2, located in two distinct and distant positions on the same chromosome as result of a split in the ancestral locus. Each TRG locus consists of reiterated V-J-J-C cassettes, with the goat TRG2 containing an additional cassette relative to the corresponding sheep and cattle loci.
CONCLUSIONS
Taken together, these findings demonstrate that strong evolutionary pressures in the ruminant lineage have selected for the development of enlarged sets of TRB and TRG genes that contribute to a diverse T cell receptor repertoire. However, differences observed among the goat, sheep and cattle TRB and TRG genes indicate that distinct evolutionary histories, with independent expansions and/or contractions, have also affected each ruminant species.
Topics: Animals; Evolution, Molecular; Gene Dosage; Genes, T-Cell Receptor beta; Genes, T-Cell Receptor gamma; Goats; Phylogeny
PubMed: 32912163
DOI: 10.1186/s12864-020-07022-x -
Frontiers in Immunology 2023T-cell receptor (TR) diversity of the variable domains is generated by recombination of both the alpha (TRA) and beta (TRB) chains. The textbook process of TRB chain...
T-cell receptor (TR) diversity of the variable domains is generated by recombination of both the alpha (TRA) and beta (TRB) chains. The textbook process of TRB chain production starts with TRBD and TRBJ gene rearrangement, followed by the rearrangement of a TRBV gene to the partially rearranged D-J gene. Unsuccessful V-D-J TRB rearrangements lead to apoptosis of the cell. Here, we performed deep sequencing of the poorly explored pool of partial TRBD1-TRBD2 rearrangements in T-cell genomic DNA. We reconstructed full repertoires of human partial TRBD1-TRBD2 rearrangements using novel sequencing and validated them by detecting V-D-J recombination-specific byproducts: excision circles containing the recombination signal (RS) joint 5'D2-RS - 3'D1-RS. Identified rearrangements were in compliance with the classical 12/23 rule, common for humans, rats, and mice and contained typical V-D-J recombination footprints. Interestingly, we detected a bimodal distribution of D-D junctions indicating two active recombination sites producing long and short D-D rearrangements. Long TRB D-D rearrangements with two D-regions are coding joints D1-D2 remaining classically on the chromosome. The short TRB D-D rearrangements with no D-region are signal joints, the coding joint D1-D2 being excised from the chromosome. They both contribute to the TRB V-(D)-J combinatorial diversity. Indeed, short D-D rearrangements may be followed by direct V-J2 recombination. Long D-D rearrangements may recombine further with J2 and V genes forming partial D1-D2-J2 and then complete V-D1-D2-J2 rearrangement. Productive TRB V-D1-D2-J2 chains are present and expressed in thousands of clones of human antigen-experienced memory T cells proving their capacity for antigen recognition and actual participation in the immune response.
Topics: Animals; Humans; Mice; Rats; Apoptosis; Chromosome Aberrations; Clone Cells; Genes, T-Cell Receptor beta; Memory T Cells; V(D)J Recombination
PubMed: 37744336
DOI: 10.3389/fimmu.2023.1245175 -
BMC Genomics Apr 2009Diverse TR and IG repertoires are generated by V(D)J somatic recombination. Genomic studies have been pivotal in cataloguing the V, D, J and C genes present in the... (Comparative Study)
Comparative Study
BACKGROUND
Diverse TR and IG repertoires are generated by V(D)J somatic recombination. Genomic studies have been pivotal in cataloguing the V, D, J and C genes present in the various TR/IG loci and describing how duplication events have expanded the number of these genes. Such studies have also provided insights into the evolution of these loci and the complex mechanisms that regulate TR/IG expression. In this study we analyze the sequence of the third bovine genome assembly to characterize the germline repertoire of bovine TRB genes and compare the organization, evolution and regulatory structure of the bovine TRB locus with that of humans and mice.
RESULTS
The TRB locus in the third bovine genome assembly is distributed over 5 scaffolds, extending to approximately 730 Kb. The available sequence contains 134 TRBV genes, assigned to 24 subgroups, and 3 clusters of DJC genes, each comprising a single TRBD gene, 5-7 TRBJ genes and a single TRBC gene. Seventy-nine of the TRBV genes are predicted to be functional. Comparison with the human and murine TRB loci shows that the gene order, as well as the sequences of non-coding elements that regulate TRB expression, are highly conserved in the bovine. Dot-plot analyses demonstrate that expansion of the genomic TRBV repertoire has occurred via a complex and extensive series of duplications, predominantly involving DNA blocks containing multiple genes. These duplication events have resulted in massive expansion of several TRBV subgroups, most notably TRBV6, 9 and 21 which contain 40, 35 and 16 members respectively. Similarly, duplication has lead to the generation of a third DJC cluster. Analyses of cDNA data confirms the diversity of the TRBV genes and, in addition, identifies a substantial number of TRBV genes, predominantly from the larger subgroups, which are still absent from the genome assembly. The observed gene duplication within the bovine TRB locus has created a repertoire of phylogenetically diverse functional TRBV genes, which is substantially larger than that described for humans and mice.
CONCLUSION
The analyses completed in this study reveal that, although the gene content and organization of the bovine TRB locus are broadly similar to that of humans and mice, multiple duplication events have led to a marked expansion in the number of TRB genes. Similar expansions in other ruminant TR loci suggest strong evolutionary pressures in this lineage have selected for the development of enlarged sets of TR genes that can contribute to diverse TR repertoires.
Topics: Amino Acid Sequence; Animals; Base Sequence; Cattle; Conserved Sequence; DNA, Complementary; Enhancer Elements, Genetic; Evolution, Molecular; Gene Duplication; Gene Order; Genes, T-Cell Receptor beta; Genome; Mice; Molecular Sequence Data; Phylogeny; Sequence Alignment; Sequence Analysis, DNA; Synteny
PubMed: 19393068
DOI: 10.1186/1471-2164-10-192