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Michael S. Krangel - One of the best experts on this subject based on the ideXlab platform.
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specification of vδ and vα usage by tcra tcrd locus v Gene Segment promoters
Journal of Immunology, 2015Co-Authors: Abani Kanta Naik, Abbas Hawwari, Michael S. KrangelAbstract:The Tcra/Tcrd locus undergoes V-Dδ-Jδ rearrangement in CD4 − CD8 − thymocytes to form the TCRδ chain of the γδ TCR and V-Jα rearrangement in CD4 + CD8 + thymocytes to form the TCRα-chain of the αβ TCR. Most V Segments in the locus participate in V-Jα rearrangement, but only a small and partially overlapping subset participates in V-Dδ-Jδ rearrangement. What specifies any particular Tcra/Tcrd locus V Gene Segment as a Vδ, a Vα, or both is currently unknown. We tested the hypothesis that V Segment usage is specified by V Segment promoter-dependent chromatin accessibility in developing thymocytes. TRAV15/DV6 family V Gene Segments contribute to both the Tcrd and the Tcra repertoires, whereas TRAV12 family V Gene Segments contribute almost exclusively to the Tcra repertoire. To understand whether the TRAV15/DV6 promoter region specifies TRAV15/DV6 as a Vδ, we used Gene targeting to replace the promoter region of a TRAV12 family member with one from a TRAV15/DV6 family member. The TRAV15/DV6 promoter region conferred increased germline transcription and histone modifications to TRAV12 in double-negative thymocytes and caused a substantial increase in usage of TRAV12 in Tcrd recombination events. Our results demonstrate that usage of TRAV15/DV6 family V Gene Segments for Tcrd recombination in double-negative thymocytes is regulated, at least in part, by intrinsic features of TRAV15/DV6 promoters, and argue that Tcra/Tcrd locus Vδ Gene Segments are defined by their local chromatin accessibility in CD4 − CD8 − thymocytes.
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Specification of Vδ and Vα Usage by Tcra/Tcrd Locus V Gene Segment Promoters
Journal of immunology (Baltimore Md. : 1950), 2014Co-Authors: Abani Kanta Naik, Abbas Hawwari, Michael S. KrangelAbstract:The Tcra/Tcrd locus undergoes V-Dδ-Jδ rearrangement in CD4 − CD8 − thymocytes to form the TCRδ chain of the γδ TCR and V-Jα rearrangement in CD4 + CD8 + thymocytes to form the TCRα-chain of the αβ TCR. Most V Segments in the locus participate in V-Jα rearrangement, but only a small and partially overlapping subset participates in V-Dδ-Jδ rearrangement. What specifies any particular Tcra/Tcrd locus V Gene Segment as a Vδ, a Vα, or both is currently unknown. We tested the hypothesis that V Segment usage is specified by V Segment promoter-dependent chromatin accessibility in developing thymocytes. TRAV15/DV6 family V Gene Segments contribute to both the Tcrd and the Tcra repertoires, whereas TRAV12 family V Gene Segments contribute almost exclusively to the Tcra repertoire. To understand whether the TRAV15/DV6 promoter region specifies TRAV15/DV6 as a Vδ, we used Gene targeting to replace the promoter region of a TRAV12 family member with one from a TRAV15/DV6 family member. The TRAV15/DV6 promoter region conferred increased germline transcription and histone modifications to TRAV12 in double-negative thymocytes and caused a substantial increase in usage of TRAV12 in Tcrd recombination events. Our results demonstrate that usage of TRAV15/DV6 family V Gene Segments for Tcrd recombination in double-negative thymocytes is regulated, at least in part, by intrinsic features of TRAV15/DV6 promoters, and argue that Tcra/Tcrd locus Vδ Gene Segments are defined by their local chromatin accessibility in CD4 − CD8 − thymocytes.
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Specification Of Va And Vd Usage By Tcra/Tcrd Locus V Gene Segment Promoters
Qatar Foundation Annual Research Conference Proceedings Volume 2014 Issue 1, 2014Co-Authors: Abbas Hawwari, Abani Kanta Naik, Michael S. KrangelAbstract:The Tcra/Tcrd locus undergoes V-Dδ-Jδ rearrangement in CD4-CD8- thymocytes to form the TCRδ chain of the γδ TCR and V-Jα rearrangement in CD4+CD8+ thymocytes to form the TCRα chain of the αβ TCR. Most V Segments in the locus participate in V-Jα rearrangement, but only a small and partially overlapping subset participates in V-Dδ-Jδ rearrangement. What determines any particular Tcra/Tcrd locus V Gene Segment as a Vδ, a Vα, or both is currently unknown. We tested the hypothesis that V Segment usage is specified by V Segment promoter-dependent chromatin accessibility in developing thymocytes. TRAV15/DV6 family V Gene Segments contribute to both the Tcrd and Tcra repertoires, whereas TRAV12 family V Gene Segments contribute almost exclusively to the Tcra repertoire. To understand whether the TRAV15/DV6 promoter region specifies TRAV15/DV6 as a Vδ, we used Gene targeting to replace the promoter region of a TRAV12 family member with one from a TRAV15/DV6 family member. The TRAV15/DV6 promoter region conferred increased germline transcription and histone modifications to TRAV12 in DN thymocytes and caused a substantial increase in usage of TRAV12 in Tcrd recombination events. Our results demonstrate that usage of TRAV15/DV6 family V Gene Segments for Tcrd recombination in DN thymocytes is regulated at least in part by intrinsic features of TRAV15/DV6 promoters, and argue that Tcra/Tcrd locus Vδ Gene Segments are defined by their local chromatin accessibility in CD4-CD8- thymocytes.
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Regulation of TCRβ Allelic Exclusion by Gene Segment Proximity and Accessibility
Journal of immunology (Baltimore Md. : 1950), 2011Co-Authors: Hrisavgi D. Kondilis-mangum, Barry P. Sleckman, Han-yu Shih, Grace K. Mahowald, Michael S. KrangelAbstract:Ag receptor loci are regulated to promote allelic exclusion, but the mechanisms are not well understood. Assembly of a functional TCR β-chain Gene triggers feedback inhibition of Vβ-to-DJβ recombination in double-positive (DP) thymocytes, which correlates with reduced Vβ chromatin accessibility and a locus conformational change that separates Vβ from DJβ Gene Segments. We previously Generated a Tcrb allele that maintained Vβ accessibility but was still subject to feedback inhibition in DP thymocytes. We have now further analyzed the contributions of chromatin accessibility and locus conformation to feedback inhibition using two novel TCR alleles. We show that reduced Vβ accessibility and increased distance between Vβ and DJβ Gene Segments both enforce feedback inhibition in DP thymocytes.
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Long-Distance Regulation of Fetal Vδ Gene Segment TRDV4 by the Tcrd Enhancer
Journal of immunology (Baltimore Md. : 1950), 2011Co-Authors: Bingtao Hao, Michael S. KrangelAbstract:Murine Tcra and Tcrd Gene Segments are organized into a single Genetic locus ( Tcra/Tcrd locus) that undergoes V(D)J recombination in CD4 − CD8 − double-negative (DN) thymocytes to assemble Tcrd Genes and in CD4 + CD8 + double-positive thymocytes to assemble Tcra Genes. Recombination events are regulated by two developmental stage-specific enhancers, E δ and E α . Effects of E α on Trca/Tcrd locus chromatin have been well documented, but effects of E δ have not. In this regard, E α acts over long distances to activate many V α and J α Segments for recombination in double-positive thymocytes. However, in DN thymocytes, it is unclear whether E δ functions over long distances to regulate V δ Gene Segments or functions only locally to regulate D δ and J δ Gene Segments. In this study, we analyzed germline transcription, histone modifications, and recombination on wild-type and E δ -deficient alleles in adult and fetal thymocytes. We found that E δ functions as a local enhancer whose influence is limited to no more than ∼10 kb in either direction (including D δ , J δ , and TRDV5 Gene Segments) in adult DN thymocytes. However, we identified a unique long-distance role for E δ promoting accessibility and recombination of fetal V δ Gene Segment TRDV4, over a distance of 55 kb, in fetal thymocytes. TRDV4 recombination is specifically repressed in adult thymocytes. We found that this repression is enforced by a developmentally regulated loss of histone acetylation. Constitutively high levels of a suppressive modification, histone H3 lysine 9 dimethylation, may contribute to repression as well.
J D Capra - One of the best experts on this subject based on the ideXlab platform.
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Biased utilization of DHQ52 and JH4 Gene Segments in a human Ig transgenic minilocus is independent of antigenic selection
Journal of immunology (Baltimore Md. : 1950), 1994Co-Authors: N Tuaillon, Philip W Tucker, A B Miller, N Longberg, J D CapraAbstract:We have assessed the effect of antigenic selection on human Ig heavy chain D and JH Gene Segment utilization in mice that contain transGenes composed of 2 VH (psi VH3-105 and VH5-251), 10 D, 6 JH, C mu, and C gamma 1 human Gene Segments. Human heavy chains using the functional VH5-251 Gene Segment are expressed in the serum and on the surface of murine B cells. The second VH Gene Segment (psi VH3-105) is not expressed as a protein but is rearranged and transcribed into mRNA. We previously reported that the functional VH5-251 mu transcripts preferentially used the DHQ52 and JH4 Gene Segments similar to their use in the human repertoire. Here, we demonstrate that the nonfunctional (psi VH3-105) Gene Segment shows the same bias in D and JH Gene Segment utilization. Because transcripts using the pseudo-VH Gene Segment cannot be subjected to antigenic selection, we conclude that the restricted repertoire observed is Ag independent. Analysis of pseudo VH Gene Segment recombination products reveals no bias in D Gene Segment reading frame utilization. Finally, we demonstrate that in the human transGene coding sequence complementarities can affect the recombination site and that D inversion is common.
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Analysis of Ig H chain Gene Segment utilization in human fetal liver. Revisiting the "proximal utilization hypothesis".
Journal of immunology (Baltimore Md. : 1950), 1993Co-Authors: Virginia Pascual, L Verkruyse, M L Casey, J D CapraAbstract:The utilization of Ig H chain Gene Segments during ontogeny is postulated to result from an immature recombination machinery that preferentially rearranges Genes according to chromosomal position. We have tested the "proximal utilization hypothesis" using the two functional members of the VH5 family as a model. Nucleotide sequence analyses of transcripts involving these VH Gene Segments in 11- to 12-wk fetal liver samples revealed that they were utilized frequently, even though they are 500 kb apart. Although 50% of the H chain rearrangements in our study use the DQ52 Gene Segment, the most 3' end proximal human D Segment, the preference for rearranging D Gene Segments based on 3' end proximity does not apply to other D Segments because different members of the DLR family that are interspersed throughout the D locus are equally rearranged in our sample. The recurrent utilization of the DQ52 and JH4 Gene Segments and a propensity to preserve the two nucleotides flanking the 3' end of the VH Gene Segment to Generate the amino acid residue at the V-D junction appear to be the major biases in the Generation of an otherwise diverse fetal repertoire.
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Molecular characterization of a cross-reactive idiotope on human immunoglobulins utilizing the VH4-21 Gene Segment.
The Journal of experimental medicine, 1993Co-Authors: Kathleen N. Potter, Virginia Pascual, M B Spellerberg, Freda K. Stevenson, R. C. Williams, L. C. Byres, J D CapraAbstract:The anti-idiotypic (anti-Id) antibody (Ab) 9G4 binds a cross-reactive idiotope (CRI) present in a select group of human autoantibodies. This Id has been localized to the portion of immunoglobulin (Ig) heavy (H) chains encoded by the VH4-21 Gene Segment, a member of the human VH4 family. This Gene Segment is utilized by essentially all cold agglutinin (CA) Abs with I/i specificity isolated from patients with CA disease stemming from chronic lymphoproliferative disorders. In this study, mutational analysis of a CA has been used to determine the structural basis for 9G4 binding to Abs utilizing the VH4-21 Gene Segment. Recombinant CA H chain mutants were produced and their 9G4 reactivity determined. Mutants were Generated by exchanging VH4-21 sequences in the FR1, CDR1, and CDR2 with corresponding sequences from a closely related Gene Segment V71-2, a VH4 family member that is associated neither with Abs having CA activity nor with Abs that react with 9G4. The results indicate that the motif AVY at amino acid positions 23-25 in FR1 defines the 9G4 idiotope. Reaction of these recombinant Abs with a polyclonal rabbit anti-CA antiserum absorbed to render it specific for a CA CRI also maps predominantly to FR1. These findings indicate that the solvent-exposed FR1 plays an important role in eliciting an immune response to Igs.
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human immunoglobulin heavy chain minilocus recombination in transgenic mice Gene Segment use in mu and gamma transcripts
Proceedings of the National Academy of Sciences of the United States of America, 1993Co-Authors: N Tuaillon, L D Taylor, N Lonberg, Philip W Tucker, J D CapraAbstract:Abstract We (N.L. and L.D.T.) have introduced a human heavy-chain minilocus into mice transgenically. Constructs contain 2 heavy-chain variable (VH; psi VH3-105 and VH5-251), 10 diversity (D), 6 heavy-chain joining (JH), and either constant (C)mu or C mu and C gamma Gene Segments. Several founder lines were established and studied before immunization. Seventy heavy-chain transcripts were cloned and sequenced from murine splenic B lymphocytes, and Gene-Segment use was assessed before and after class-switching. In General, the repertoire was "fetal" in appearance with little evidence of somatic mutation in any Gene Segment. The two VH Gene Segments were found rearranged to mu- and gamma-chain C Segments, with a preference of VH5-251. We observed a preponderance of the most-J-proximal D Gene (DHQ52) Segments among the mu transcripts (44%). The JH Gene-Segment use mimics most patterns seen in human antibodies. Diversification in CDR3 was extensive and included clear examples of D inversions and D-D fusions. These data suggest that a human immunoglobulin minilocus can undergo recombinatorial processes in a manner analogous to that seen in the human fetal/preimmune repertoire. This model, in addition to providing a potential source of human monoclonal antibodies, is ideal for the study of further questions concerning immunoglobulin Gene-Segment recombination.
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VH restriction among human cold agglutinins. The VH4-21 Gene Segment is required to encode anti-I and anti-i specificities.
Journal of immunology (Baltimore Md. : 1950), 1992Co-Authors: Virginia Pascual, K Victor, M B Spellerberg, T J Hamblin, Freda K. Stevenson, J D CapraAbstract:We previously reported that human autoantibodies with cold agglutinin activity contained a single human VH Gene Segment (VH4-21) which was also responsible for the cross-idiotypic specificity characteristic of the cold agglutinin response. To confirm and extend this observation we have analyzed at the nucleotide level the H and L chains of six new cold agglutinin molecules derived from different patients. We found that regardless of whether the antibody recognizes the i or the I red cell Ag, restriction at the VH Gene Segment level is absolute. We also found that even in the absence of somatic mutation the VH4-21 Gene Segment can encode both anti-i and anti-I specificities. Finally, although the VH4-21 Gene Segment is essential for cold agglutinin activity, the other Genetic elements that contribute to the V region of the antibody molecules can be extremely diverse. The structural information provided in this report sharply restricts the requirement for encoding pathogenic cold agglutinin activity to one of the components of the H chain V region, specifically the VH Gene Segment. The implications of this apparently absolute requirement for a single VH Gene Segment, unprecedented in the human autoimmune response, are discussed.
Abbas Hawwari - One of the best experts on this subject based on the ideXlab platform.
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specification of vδ and vα usage by tcra tcrd locus v Gene Segment promoters
Journal of Immunology, 2015Co-Authors: Abani Kanta Naik, Abbas Hawwari, Michael S. KrangelAbstract:The Tcra/Tcrd locus undergoes V-Dδ-Jδ rearrangement in CD4 − CD8 − thymocytes to form the TCRδ chain of the γδ TCR and V-Jα rearrangement in CD4 + CD8 + thymocytes to form the TCRα-chain of the αβ TCR. Most V Segments in the locus participate in V-Jα rearrangement, but only a small and partially overlapping subset participates in V-Dδ-Jδ rearrangement. What specifies any particular Tcra/Tcrd locus V Gene Segment as a Vδ, a Vα, or both is currently unknown. We tested the hypothesis that V Segment usage is specified by V Segment promoter-dependent chromatin accessibility in developing thymocytes. TRAV15/DV6 family V Gene Segments contribute to both the Tcrd and the Tcra repertoires, whereas TRAV12 family V Gene Segments contribute almost exclusively to the Tcra repertoire. To understand whether the TRAV15/DV6 promoter region specifies TRAV15/DV6 as a Vδ, we used Gene targeting to replace the promoter region of a TRAV12 family member with one from a TRAV15/DV6 family member. The TRAV15/DV6 promoter region conferred increased germline transcription and histone modifications to TRAV12 in double-negative thymocytes and caused a substantial increase in usage of TRAV12 in Tcrd recombination events. Our results demonstrate that usage of TRAV15/DV6 family V Gene Segments for Tcrd recombination in double-negative thymocytes is regulated, at least in part, by intrinsic features of TRAV15/DV6 promoters, and argue that Tcra/Tcrd locus Vδ Gene Segments are defined by their local chromatin accessibility in CD4 − CD8 − thymocytes.
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Specification of Vδ and Vα Usage by Tcra/Tcrd Locus V Gene Segment Promoters
Journal of immunology (Baltimore Md. : 1950), 2014Co-Authors: Abani Kanta Naik, Abbas Hawwari, Michael S. KrangelAbstract:The Tcra/Tcrd locus undergoes V-Dδ-Jδ rearrangement in CD4 − CD8 − thymocytes to form the TCRδ chain of the γδ TCR and V-Jα rearrangement in CD4 + CD8 + thymocytes to form the TCRα-chain of the αβ TCR. Most V Segments in the locus participate in V-Jα rearrangement, but only a small and partially overlapping subset participates in V-Dδ-Jδ rearrangement. What specifies any particular Tcra/Tcrd locus V Gene Segment as a Vδ, a Vα, or both is currently unknown. We tested the hypothesis that V Segment usage is specified by V Segment promoter-dependent chromatin accessibility in developing thymocytes. TRAV15/DV6 family V Gene Segments contribute to both the Tcrd and the Tcra repertoires, whereas TRAV12 family V Gene Segments contribute almost exclusively to the Tcra repertoire. To understand whether the TRAV15/DV6 promoter region specifies TRAV15/DV6 as a Vδ, we used Gene targeting to replace the promoter region of a TRAV12 family member with one from a TRAV15/DV6 family member. The TRAV15/DV6 promoter region conferred increased germline transcription and histone modifications to TRAV12 in double-negative thymocytes and caused a substantial increase in usage of TRAV12 in Tcrd recombination events. Our results demonstrate that usage of TRAV15/DV6 family V Gene Segments for Tcrd recombination in double-negative thymocytes is regulated, at least in part, by intrinsic features of TRAV15/DV6 promoters, and argue that Tcra/Tcrd locus Vδ Gene Segments are defined by their local chromatin accessibility in CD4 − CD8 − thymocytes.
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Specification Of Va And Vd Usage By Tcra/Tcrd Locus V Gene Segment Promoters
Qatar Foundation Annual Research Conference Proceedings Volume 2014 Issue 1, 2014Co-Authors: Abbas Hawwari, Abani Kanta Naik, Michael S. KrangelAbstract:The Tcra/Tcrd locus undergoes V-Dδ-Jδ rearrangement in CD4-CD8- thymocytes to form the TCRδ chain of the γδ TCR and V-Jα rearrangement in CD4+CD8+ thymocytes to form the TCRα chain of the αβ TCR. Most V Segments in the locus participate in V-Jα rearrangement, but only a small and partially overlapping subset participates in V-Dδ-Jδ rearrangement. What determines any particular Tcra/Tcrd locus V Gene Segment as a Vδ, a Vα, or both is currently unknown. We tested the hypothesis that V Segment usage is specified by V Segment promoter-dependent chromatin accessibility in developing thymocytes. TRAV15/DV6 family V Gene Segments contribute to both the Tcrd and Tcra repertoires, whereas TRAV12 family V Gene Segments contribute almost exclusively to the Tcra repertoire. To understand whether the TRAV15/DV6 promoter region specifies TRAV15/DV6 as a Vδ, we used Gene targeting to replace the promoter region of a TRAV12 family member with one from a TRAV15/DV6 family member. The TRAV15/DV6 promoter region conferred increased germline transcription and histone modifications to TRAV12 in DN thymocytes and caused a substantial increase in usage of TRAV12 in Tcrd recombination events. Our results demonstrate that usage of TRAV15/DV6 family V Gene Segments for Tcrd recombination in DN thymocytes is regulated at least in part by intrinsic features of TRAV15/DV6 promoters, and argue that Tcra/Tcrd locus Vδ Gene Segments are defined by their local chromatin accessibility in CD4-CD8- thymocytes.
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Regulation of TCR δ and α repertoires by local and long-distance control of variable Gene Segment chromatin structure
The Journal of experimental medicine, 2005Co-Authors: Abbas Hawwari, Michael S. KrangelAbstract:Murine Tcrd and Tcra Gene Segments reside in a single Genetic locus and undergo recombination in CD4 − CD8 − (double negative [DN]) and CD4 + CD8 + (double positive [DP]) thymocytes, respectively. TcraTcrd locus variable Gene Segments are subject to complex regulation. Only a small subset of ∼100 variable Gene Segments contributes substantially to the adult TCR δ repertoire. Moreover, although most contribute to the TCR α repertoire, variable Gene Segments that are J α proximal are preferentially used during primary Tcra recombination. We investigate the role of local chromatin accessibility in determining the developmental pattern of TcraTcrd locus variable Gene Segment recombination. We find variable Gene Segments to be heteroGeneous with respect to acetylation of histones H3 and H4. Those that dominate the adult TCR δ repertoire are hyperacetylated in DN thymocytes, independent of their position in the locus. Moreover, proximal variable Gene Segments show dramatic increases in histone acetylation and germline transcription in DP thymocytes, a result of super long-distance regulation by the Tcra enhancer. Our results imply that differences in chromatin accessibility contribute to biases in TcraTcrd locus variable Gene Segment recombination in DN and DP thymocytes and extend the distance over which the Tcra enhancer can regulate chromatin structure to a remarkable 525 kb.
Abani Kanta Naik - One of the best experts on this subject based on the ideXlab platform.
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specification of vδ and vα usage by tcra tcrd locus v Gene Segment promoters
Journal of Immunology, 2015Co-Authors: Abani Kanta Naik, Abbas Hawwari, Michael S. KrangelAbstract:The Tcra/Tcrd locus undergoes V-Dδ-Jδ rearrangement in CD4 − CD8 − thymocytes to form the TCRδ chain of the γδ TCR and V-Jα rearrangement in CD4 + CD8 + thymocytes to form the TCRα-chain of the αβ TCR. Most V Segments in the locus participate in V-Jα rearrangement, but only a small and partially overlapping subset participates in V-Dδ-Jδ rearrangement. What specifies any particular Tcra/Tcrd locus V Gene Segment as a Vδ, a Vα, or both is currently unknown. We tested the hypothesis that V Segment usage is specified by V Segment promoter-dependent chromatin accessibility in developing thymocytes. TRAV15/DV6 family V Gene Segments contribute to both the Tcrd and the Tcra repertoires, whereas TRAV12 family V Gene Segments contribute almost exclusively to the Tcra repertoire. To understand whether the TRAV15/DV6 promoter region specifies TRAV15/DV6 as a Vδ, we used Gene targeting to replace the promoter region of a TRAV12 family member with one from a TRAV15/DV6 family member. The TRAV15/DV6 promoter region conferred increased germline transcription and histone modifications to TRAV12 in double-negative thymocytes and caused a substantial increase in usage of TRAV12 in Tcrd recombination events. Our results demonstrate that usage of TRAV15/DV6 family V Gene Segments for Tcrd recombination in double-negative thymocytes is regulated, at least in part, by intrinsic features of TRAV15/DV6 promoters, and argue that Tcra/Tcrd locus Vδ Gene Segments are defined by their local chromatin accessibility in CD4 − CD8 − thymocytes.
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Specification of Vδ and Vα Usage by Tcra/Tcrd Locus V Gene Segment Promoters
Journal of immunology (Baltimore Md. : 1950), 2014Co-Authors: Abani Kanta Naik, Abbas Hawwari, Michael S. KrangelAbstract:The Tcra/Tcrd locus undergoes V-Dδ-Jδ rearrangement in CD4 − CD8 − thymocytes to form the TCRδ chain of the γδ TCR and V-Jα rearrangement in CD4 + CD8 + thymocytes to form the TCRα-chain of the αβ TCR. Most V Segments in the locus participate in V-Jα rearrangement, but only a small and partially overlapping subset participates in V-Dδ-Jδ rearrangement. What specifies any particular Tcra/Tcrd locus V Gene Segment as a Vδ, a Vα, or both is currently unknown. We tested the hypothesis that V Segment usage is specified by V Segment promoter-dependent chromatin accessibility in developing thymocytes. TRAV15/DV6 family V Gene Segments contribute to both the Tcrd and the Tcra repertoires, whereas TRAV12 family V Gene Segments contribute almost exclusively to the Tcra repertoire. To understand whether the TRAV15/DV6 promoter region specifies TRAV15/DV6 as a Vδ, we used Gene targeting to replace the promoter region of a TRAV12 family member with one from a TRAV15/DV6 family member. The TRAV15/DV6 promoter region conferred increased germline transcription and histone modifications to TRAV12 in double-negative thymocytes and caused a substantial increase in usage of TRAV12 in Tcrd recombination events. Our results demonstrate that usage of TRAV15/DV6 family V Gene Segments for Tcrd recombination in double-negative thymocytes is regulated, at least in part, by intrinsic features of TRAV15/DV6 promoters, and argue that Tcra/Tcrd locus Vδ Gene Segments are defined by their local chromatin accessibility in CD4 − CD8 − thymocytes.
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Specification Of Va And Vd Usage By Tcra/Tcrd Locus V Gene Segment Promoters
Qatar Foundation Annual Research Conference Proceedings Volume 2014 Issue 1, 2014Co-Authors: Abbas Hawwari, Abani Kanta Naik, Michael S. KrangelAbstract:The Tcra/Tcrd locus undergoes V-Dδ-Jδ rearrangement in CD4-CD8- thymocytes to form the TCRδ chain of the γδ TCR and V-Jα rearrangement in CD4+CD8+ thymocytes to form the TCRα chain of the αβ TCR. Most V Segments in the locus participate in V-Jα rearrangement, but only a small and partially overlapping subset participates in V-Dδ-Jδ rearrangement. What determines any particular Tcra/Tcrd locus V Gene Segment as a Vδ, a Vα, or both is currently unknown. We tested the hypothesis that V Segment usage is specified by V Segment promoter-dependent chromatin accessibility in developing thymocytes. TRAV15/DV6 family V Gene Segments contribute to both the Tcrd and Tcra repertoires, whereas TRAV12 family V Gene Segments contribute almost exclusively to the Tcra repertoire. To understand whether the TRAV15/DV6 promoter region specifies TRAV15/DV6 as a Vδ, we used Gene targeting to replace the promoter region of a TRAV12 family member with one from a TRAV15/DV6 family member. The TRAV15/DV6 promoter region conferred increased germline transcription and histone modifications to TRAV12 in DN thymocytes and caused a substantial increase in usage of TRAV12 in Tcrd recombination events. Our results demonstrate that usage of TRAV15/DV6 family V Gene Segments for Tcrd recombination in DN thymocytes is regulated at least in part by intrinsic features of TRAV15/DV6 promoters, and argue that Tcra/Tcrd locus Vδ Gene Segments are defined by their local chromatin accessibility in CD4-CD8- thymocytes.
Thierry Hercend - One of the best experts on this subject based on the ideXlab platform.
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an experimentally validated panel of subfamily specific oligonucleotide primers vα1 w29 vβ1 w24 for the study of human t cell receptor variable v Gene Segment usage by polymerase chain reaction
European Journal of Immunology, 1992Co-Authors: Catherine Genevee, Janine Nierat, Anne Caignard, P Y Dietrich, Laurent Ferradini, Sergio Romanroman, Fredearic Triebel, Thierry HercendAbstract:We report here the characterization of a series of T cell receptor (TcR) Vα or Vβ subfamily-specific oligonucleotide primers. Criteria that have guided the design of each oligonucleotide include appropriate thermodynamic parameters as well as differential base-pairing scores with related and unrelated target sequences. The specificity of the oligonucleotides for each Vα or Vβ subfamily was tested by polymerase chain reaction (PCR) on both a series of TcR encoding plasmid DNA and clonal T cell populations. Unexpected cross-reactivities were observed with plasmid cDNA sequences corresponding to unrelated subfamily Gene Segments. This led to the synthesis of additional series of oligonucleotides to obtain a relevant panel. A series of Vα1-w29/Vβ1-w24 TcR subfamily-specific oligonucleotides was eventually selected which Generates little, if any, cross-reactivity. The use of Cα or Cβ primers for the amplification of internal positive control templates (i.e. Cβ for the Vα series and Cα for the Vβ series) has been tested in PCR performed with cDNA derived from peripheral blood lymphocytes; it was shown not to alter the amplification of the V subfamily-specific DNA fragments. This panel of oligonucleotides will be helpful in the study of TcR V Gene Segment usage and, thus, may lead to a better characterization of T cell responses in physiological and pathological situations.
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an experimentally validated panel of subfamily specific oligonucleotide primers vα1 w29 vβ1 w24 for the study of human t cell receptor variable v Gene Segment usage by polymerase chain reaction
European Journal of Immunology, 1992Co-Authors: Catherine Genevee, Janine Nierat, Anne Caignard, P Y Dietrich, Laurent Ferradini, Sergio Romanroman, Fredearic Triebel, Anita Diu, Thierry HercendAbstract:We report here the characterization of a series of T cell receptor (TcR) V alpha or V beta subfamily-specific oligonucleotide primers. Criteria that have guided the design of each oligonucleotide include appropriate thermodynamic parameters as well as differential base-pairing scores with related and unrelated target sequences. The specificity of the oligonucleotides for each V alpha or V beta subfamily was tested by polymerase chain reaction (PCR) on both a series of TcR encoding plasmid DNA and clonal T cell populations. Unexpected cross-reactivities were observed with plasmid cDNA sequences corresponding to unrelated subfamily Gene Segments. This led to the synthesis of additional series of oligonucleotides to obtain a relevant panel. A series of V alpha 1-w29/V beta 1-w24 TcR subfamily-specific oligonucleotides was eventually selected which Generates little, if any, cross-reactivity. The use of C alpha or C beta primers for the amplification of internal positive control templates (i.e. C beta for the V alpha series and C alpha for the V beta series) has been tested in PCR performed with cDNA derived from peripheral blood lymphocytes; it was shown not to alter the amplification of the V subfamily-specific DNA fragments. This panel of oligonucleotides will be helpful in the study of TcRV Gene Segment usage and, thus, may lead to a better characterization of T cell responses in physiological and pathological situations.