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Krangel, Michael S.

Overview:

The process of V(D)J recombination assembles T cell receptor (TCR) genes (α,β,γ,δ) from variable (V), diversity (D) and joining (J) gene segments during T cell development, and is essential for the formation of diverse antigen receptor repertoires on αβ and γδ T lymphocytes. We are interested in the molecular basis for developmentally regulated rearrangement and expression of murine TCR genes. One focus of our studies is the TCRα/δ locus, because it represents an intriguing model with two sets of gene segments that are differentially activated for recombination during T cell development. We are also studying the TCRβ locus, because this locus presents a model in which there is a developmental inactivation of V(D)J recombination associated with the process of allelic exclusion. V(D)J recombination depends on the ability of recombinase proteins RAG1 and RAG2 to recognize and generate double-strand breaks at recombination signal sequences that flank TCR gene segments. Our main focus has been on the role of chromatin structure in defining the portions of these loci that are accessible to the RAG recombinase and therefore active for V(D)J recombination, and on the mechanisms by which cis-regulatory elements within these loci (enhancers, promoters) function as developmental regulators of chromatin structure. Our primary approach has been to manipulate cis-acting elements within these loci by gene targeting, and to study the effects of these manipulations on locus chromatin structure and recombination events in developing thymocytes in vivo. An important outcome of this work has been our demonstration that enhancer- and promoter-directed transcription through recombination signal sequences can displace and covalently modify nucleosomes to provide accessibility for RAG binding and V(D)J recombination.

Recent work in our laboratory and elsewhere has highlighted additional properties of antigen receptor loci that likely to play important roles in developmental regulation. One area of interest is subnuclear positioning. We have used three-dimensional fluorescence in situ hybridization (3D-FISH) to show that TCRβ alleles interact stochastically and at high frequency with the nuclear lamina and with foci of pericentromeric heterochromatin, and that these interactions are inhibitory to V(D)J recombination. We suspect that these inhibitory interactions help to promote allelic exclusion by diminishing the likelihood of simultaneous V to DJ recombination on both alleles. Current work is aimed at developing a better understanding of how the TCRβ locus interacts with the nuclear lamina and the mechanism by which this interaction impacts recombination events.

A second area of interest is locus conformation. It is now appreciated that recombination events at antigen receptor loci depend on locus conformational changes that bring into proximity gene segments that may be widely separated in the linear DNA sequence. Conformational states can be defined using 3D-FISH or a chemical crosslinking approach called chromosome conformation capture (3C). Recent studies indicate that developmental changes in locus conformation contribute to allelic exclusion at the TCRβ locus and mediate a transition from TCRδ to TCRα rearrangement at the TCRα/δ locus. Current work aims to address at a molecular level how locus conformational states are maintained and modified during T cell development and how these changes impact long-distance transactions including enhancer-promoter communication and V(D)J recombination.

Positions:

Mary Bernheim Professor of Immunology

Immunology
School of Medicine

Professor of Immunology

Immunology
School of Medicine

Member of the Duke Cancer Institute

Duke Cancer Institute
School of Medicine

Chair, Department of Immunology

Immunology
School of Medicine

Education:

Ph.D. 1982

Ph.D. — Harvard University

Grants:

Control of TCR Delta and TCR Alpha Rearrangement

Administered By
Immunology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
August 01, 1989
End Date
July 31, 2020

Basic Immunology Training Program

Administered By
Immunology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
July 01, 2014
End Date
June 30, 2019

Training Program in Inflammatory and Immunological Diseases

Administered By
Medicine, Rheumatology and Immunology
AwardedBy
National Institutes of Health
Role
Preceptor
Start Date
September 30, 1980
End Date
August 31, 2017

Control of TCR V Beta Rearrangement & Allelic Exclusion

Administered By
Immunology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
July 01, 2001
End Date
May 31, 2017

The role of BATF in allergic inflammation and anti-helminth immunity

Administered By
Immunology
AwardedBy
National Institutes of Health
Role
Co Investigator
Start Date
July 01, 2015
End Date
June 30, 2016

Orphan Nuclear Receptor in Thymocyte Differentiation

Administered By
Immunology
AwardedBy
National Institutes of Health
Role
Consultant
Start Date
August 28, 2002
End Date
July 31, 2007

Structure And Function Of I-309 And Other Chemokines

Administered By
Immunology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
June 01, 1996
End Date
May 31, 2002

Control Of Tcr * And Trc * Gene Rearrangement

Administered By
Immunology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
August 01, 1994
End Date
July 31, 1998

Control Of Tcr Delta And Tcr Alpha Rearrangement

Administered By
Immunology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
August 01, 1989
End Date
July 31, 1998

Human Gamma Delta T Cell Receptor

Administered By
Immunology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
August 01, 1993
End Date
July 31, 1994

Human Ganna Delta T Cell Receptor

Administered By
Immunology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
August 01, 1990
End Date
July 01, 1994
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Awards:

AAAS Fellows. American Association for the Advancement of Science, The.

Type
National
Awarded By
American Association for the Advancement of Science, The
Date
January 01, 2012

Publications:

An Ectopic CTCF Binding Element Inhibits Tcrd Rearrangement by Limiting Contact between Vδ and Dδ Gene Segments.

Chromatin looping mediated by the CCCTC binding factor (CTCF) regulates V(D)J recombination at Ag receptor loci. CTCF-mediated looping can influence recombination signal sequence (RSS) accessibility by regulating enhancer activation of germline promoters. CTCF-mediated looping has also been shown to limit directional tracking of the RAG recombinase along chromatin, and to regulate long-distance interactions between RSSs, independent of the RAG recombinase. However, in all prior instances in which CTCF-mediated looping was shown to influence V(D)J recombination, it was not possible to fully resolve the relative contributions to the V(D)J recombination phenotype of changes in accessibility, RAG tracking, and RAG-independent long-distance interactions. In this study, to assess mechanisms by which CTCF-mediated looping can impact V(D)J recombination, we introduced an ectopic CTCF binding element (CBE) immediately downstream of Eδ in the murine Tcra-Tcrd locus. The ectopic CBE impaired inversional rearrangement of Trdv5 in the absence of measurable effects on Trdv5 transcription and chromatin accessibility. The ectopic CBE also limited directional RAG tracking from the Tcrd recombination center, demonstrating that a single CBE can impact the distribution of RAG proteins along chromatin. However, such tracking cannot account for Trdv5-to-Trdd2 inversional rearrangement. Rather, the defect in Trdv5 rearrangement could only be attributed to a reconfigured chromatin loop organization that limited RAG-independent contacts between the Trdv5 and Trdd2 RSSs. We conclude that CTCF can regulate V(D)J recombination by segregating RSSs into distinct loop domains and inhibiting RSS synapsis, independent of any effects on transcription, RSS accessibility, and RAG tracking.

Authors
Chen, L; Zhao, L; Alt, FW; Krangel, MS
MLA Citation
Chen, L, Zhao, L, Alt, FW, and Krangel, MS. "An Ectopic CTCF Binding Element Inhibits Tcrd Rearrangement by Limiting Contact between Vδ and Dδ Gene Segments." Journal of immunology (Baltimore, Md. : 1950) 197.8 (October 2016): 3188-3197.
PMID
27613698
Source
epmc
Published In
Journal of immunology (Baltimore, Md. : 1950)
Volume
197
Issue
8
Publish Date
2016
Start Page
3188
End Page
3197

Orientation-specific RAG activity in chromosomal loop domains contributes to Tcrd V(D)J recombination during T cell development.

T cell antigen receptor δ (Tcrd) variable region exons are assembled by RAG-initiated V(D)J recombination events in developing γδ thymocytes. Here, we use linear amplification-mediated high-throughput genome-wide translocation sequencing (LAM-HTGTS) to map hundreds of thousands of RAG-initiated Tcrd D segment (Trdd1 and Trdd2) rearrangements in CD4(-)CD8(-) double-negative thymocyte progenitors differentiated in vitro from bone marrow-derived hematopoietic stem cells. We find that Trdd2 joins directly to Trdv, Trdd1, and Trdj segments, whereas Trdd1 joining is ordered with joining to Trdd2, a prerequisite for further rearrangement. We also find frequent, previously unappreciated, Trdd1 and Trdd2 rearrangements that inactivate Tcrd, including sequential rearrangements from V(D)J recombination signal sequence fusions. Moreover, we find dozens of RAG off-target sequences that are generated via RAG tracking both upstream and downstream from the Trdd2 recombination center across the Tcrd loop domain that is bounded by the upstream INT1-2 and downstream TEA elements. Disruption of the upstream INT1-2 boundary of this loop domain allows spreading of RAG on- and off-target activity to the proximal Trdv domain and, correspondingly, shifts the Tcrd V(D)J recombination landscape by leading to predominant V(D)J joining to a proximal Trdv3 pseudogene that lies just upstream of the normal boundary.

Authors
Zhao, L; Frock, RL; Du, Z; Hu, J; Chen, L; Krangel, MS; Alt, FW
MLA Citation
Zhao, L, Frock, RL, Du, Z, Hu, J, Chen, L, Krangel, MS, and Alt, FW. "Orientation-specific RAG activity in chromosomal loop domains contributes to Tcrd V(D)J recombination during T cell development." The Journal of experimental medicine 213.9 (August 15, 2016): 1921-1936.
PMID
27526713
Source
epmc
Published In
The Journal of Experimental Medicine
Volume
213
Issue
9
Publish Date
2016
Start Page
1921
End Page
1936
DOI
10.1084/jem.20160670

The Ties that Bind (the Igh Locus).

Immunoglobulin heavy-chain locus V(D)J recombination requires a 3D chromatin organization which permits widely distributed variable (V) gene segments to contact distant diversity (D) and joining (J) gene segments. A recent study has identified key nodes in the locus interactome, paving the way for new molecular insights into how the locus is configured for recombination.

Authors
Krangel, MS
MLA Citation
Krangel, MS. "The Ties that Bind (the Igh Locus)." Trends in genetics : TIG 32.5 (May 2016): 253-255.
PMID
27012539
Source
epmc
Published In
Trends in Genetics
Volume
32
Issue
5
Publish Date
2016
Start Page
253
End Page
255
DOI
10.1016/j.tig.2016.03.003

Pillars Article: Identification of a Putative Second T-cell Receptor. Nature. 1986. 322: 145-149.

Authors
Brenner, MB; McLean, J; Dialynas, DP; Strominger, JL; Smith, JA; Owen, FL; Seidman, JG; Ip, S; Rosen, F; Krangel, MS
MLA Citation
Brenner, MB, McLean, J, Dialynas, DP, Strominger, JL, Smith, JA, Owen, FL, Seidman, JG, Ip, S, Rosen, F, and Krangel, MS. "Pillars Article: Identification of a Putative Second T-cell Receptor. Nature. 1986. 322: 145-149." Journal of immunology (Baltimore, Md. : 1950) 196.9 (May 2016): 3509-3513.
PMID
27183647
Source
epmc
Published In
Journal of immunology (Baltimore, Md. : 1950)
Volume
196
Issue
9
Publish Date
2016
Start Page
3509
End Page
3513

Yin Yang 1 Promotes Thymocyte Survival by Downregulating p53.

Yin Yang 1 (YY1) is a zinc finger protein that functions as a transcriptional activator or repressor and participates in multiple biological processes, including development and tumorigenesis. To investigate the role of YY1 in developing T cells, we used mouse models that depleted YY1 at two distinct stages of thymocyte development. When YY1 was depleted in CD4(-)CD8(-) double-negative thymocytes, development to the CD4(+)CD8(+) double-positive stage was impaired, due to increased apoptosis that prevented expansion of post-β-selection thymocytes. When YY1 was depleted in double-positive thymocytes, they underwent increased cell-autonomous apoptosis in vitro and displayed a shorter lifespan in vivo, as judged by their ability to undergo secondary Vα-to-Jα recombination. Mechanistically, we found that the increased apoptosis in YY1-deficient thymocytes was attributed to overexpression of p53, because concurrent loss of p53 completely rescued the developmental defects of YY1-deficient thymocytes. These results indicated that YY1 functions as a critical regulator of thymocyte survival and that it does so by suppressing the expression of p53.

Authors
Chen, L; Foreman, DP; Sant'Angelo, DB; Krangel, MS
MLA Citation
Chen, L, Foreman, DP, Sant'Angelo, DB, and Krangel, MS. "Yin Yang 1 Promotes Thymocyte Survival by Downregulating p53." Journal of immunology (Baltimore, Md. : 1950) 196.6 (March 2016): 2572-2582.
PMID
26843327
Source
epmc
Published In
Journal of immunology (Baltimore, Md. : 1950)
Volume
196
Issue
6
Publish Date
2016
Start Page
2572
End Page
2582
DOI
10.4049/jimmunol.1501916

Inactivation of nuclear GSK3β by Ser(389) phosphorylation promotes lymphocyte fitness during DNA double-strand break response.

Variable, diversity and joining (V(D)J) recombination and immunoglobulin class switch recombination (CSR) are key processes in adaptive immune responses that naturally generate DNA double-strand breaks (DSBs) and trigger a DNA repair response. It is unclear whether this response is associated with distinct survival signals that protect T and B cells. Glycogen synthase kinase 3β (GSK3β) is a constitutively active kinase known to promote cell death. Here we show that phosphorylation of GSK3β on Ser(389) by p38 MAPK (mitogen-activated protein kinase) is induced selectively by DSBs through ATM (ataxia telangiectasia mutated) as a unique mechanism to attenuate the activity of nuclear GSK3β and promote survival of cells undergoing DSBs. Inability to inactivate GSK3β through Ser(389) phosphorylation in Ser(389)Ala knockin mice causes a decrease in the fitness of cells undergoing V(D)J recombination and CSR. Preselection-Tcrβ repertoire is impaired and antigen-specific IgG antibody responses following immunization are blunted in Ser(389)GSK3β knockin mice. Thus, GSK3β emerges as an important modulator of the adaptive immune response.

Authors
Thornton, TM; Delgado, P; Chen, L; Salas, B; Krementsov, D; Fernandez, M; Vernia, S; Davis, RJ; Heimann, R; Teuscher, C; Krangel, MS; Ramiro, AR; Rincón, M
MLA Citation
Thornton, TM, Delgado, P, Chen, L, Salas, B, Krementsov, D, Fernandez, M, Vernia, S, Davis, RJ, Heimann, R, Teuscher, C, Krangel, MS, Ramiro, AR, and Rincón, M. "Inactivation of nuclear GSK3β by Ser(389) phosphorylation promotes lymphocyte fitness during DNA double-strand break response." Nature communications 7 (January 29, 2016): 10553-.
PMID
26822034
Source
epmc
Published In
Nature Communications
Volume
7
Publish Date
2016
Start Page
10553
DOI
10.1038/ncomms10553

Beyond Hypothesis: Direct Evidence That V(D)J Recombination Is Regulated by the Accessibility of Chromatin Substrates.

Authors
Krangel, MS
MLA Citation
Krangel, MS. "Beyond Hypothesis: Direct Evidence That V(D)J Recombination Is Regulated by the Accessibility of Chromatin Substrates." Journal of immunology (Baltimore, Md. : 1950) 195.11 (December 2015): 5103-5105.
PMID
26589747
Source
epmc
Published In
Journal of immunology (Baltimore, Md. : 1950)
Volume
195
Issue
11
Publish Date
2015
Start Page
5103
End Page
5105
DOI
10.4049/jimmunol.1502150

A discrete chromatin loop in the mouse Tcra-Tcrd locus shapes the TCRδ and TCRα repertoires.

The locus encoding the T cell antigen receptor (TCR) α-chain and δ-chain (Tcra-Tcrd) undergoes recombination of its variable-diversity-joining (V(D)J) segments in CD4(-)CD8(-) double-negative thymocytes and CD4(+)CD8(+) double-positive thymocytes to generate diverse TCRδ repertoires and TCRα repertoires, respectively. Here we identified a chromatin-interaction network in the Tcra-Tcrd locus in double-negative thymocytes that was formed by interactions between binding elements for the transcription factor CTCF. Disruption of a discrete chromatin loop encompassing the D, J and constant (C) segments of Tcrd allowed a single V segment to frequently contact and rearrange to D and J segments and dominate the adult TCRδ repertoire. Disruption of this loop also narrowed the TCRα repertoire, which, we believe, followed as a consequence of the restricted TCRδ repertoire. Hence, a single CTCF-mediated chromatin loop directly regulated TCRδ diversity and indirectly regulated TCRα diversity.

Authors
Chen, L; Carico, Z; Shih, H-Y; Krangel, MS
MLA Citation
Chen, L, Carico, Z, Shih, H-Y, and Krangel, MS. "A discrete chromatin loop in the mouse Tcra-Tcrd locus shapes the TCRδ and TCRα repertoires." Nature immunology 16.10 (October 2015): 1085-1093.
Website
http://hdl.handle.net/10161/12705
PMID
26258942
Source
epmc
Published In
Nature Immunology
Volume
16
Issue
10
Publish Date
2015
Start Page
1085
End Page
1093
DOI
10.1038/ni.3232

An anti-silencer- and SATB1-dependent chromatin hub regulates Rag1 and Rag2 gene expression during thymocyte development.

Rag1 and Rag2 gene expression in CD4(+)CD8(+) double-positive (DP) thymocytes depends on the activity of a distant anti-silencer element (ASE) that counteracts the activity of an intergenic silencer. However, the mechanistic basis for ASE activity is unknown. Here, we show that the ASE physically interacts with the distant Rag1 and Rag2 gene promoters in DP thymocytes, bringing the two promoters together to form an active chromatin hub. Moreover, we show that the ASE functions as a classical enhancer that can potently activate these promoters in the absence of the silencer or other locus elements. In thymocytes lacking the chromatin organizer SATB1, we identified a partial defect in Tcra gene rearrangement that was associated with reduced expression of Rag1 and Rag2 at the DP stage. SATB1 binds to the ASE and Rag promoters, facilitating inclusion of Rag2 in the chromatin hub and the loading of RNA polymerase II to both the Rag1 and Rag2 promoters. Our results provide a novel framework for understanding ASE function and demonstrate a novel role for SATB1 as a regulator of Rag locus organization and gene expression in DP thymocytes.

Authors
Hao, B; Naik, AK; Watanabe, A; Tanaka, H; Chen, L; Richards, HW; Kondo, M; Taniuchi, I; Kohwi, Y; Kohwi-Shigematsu, T; Krangel, MS
MLA Citation
Hao, B, Naik, AK, Watanabe, A, Tanaka, H, Chen, L, Richards, HW, Kondo, M, Taniuchi, I, Kohwi, Y, Kohwi-Shigematsu, T, and Krangel, MS. "An anti-silencer- and SATB1-dependent chromatin hub regulates Rag1 and Rag2 gene expression during thymocyte development." The Journal of experimental medicine 212.5 (May 2015): 809-824.
PMID
25847946
Source
epmc
Published In
The Journal of Experimental Medicine
Volume
212
Issue
5
Publish Date
2015
Start Page
809
End Page
824
DOI
10.1084/jem.20142207

T-cell receptor α enhancer is inactivated in αβ T lymphocytes.

The Tcra enhancer (Eα) is essential for Tcra locus germ-line transcription and primary Vα-to-Jα recombination during thymocyte development. We found that Eα is inhibited late during thymocyte differentiation and in αβ T lymphocytes, indicating that it is not required to drive transcription of rearranged Tcra genes. Eα inactivation resulted in the disruption of functional long-range enhancer-promoter interactions and was associated with loss of Eα-dependent histone modifications at promoter and enhancer regions, and reduced expression and recruitment of E2A to the Eα enhanceosome in T cells. Enhancer activity could not be recovered by T-cell activation, by forced expression of E2A or by the up-regulation of this and other transcription factors in the context of T helper differentiation. Our results argue that the major function of Eα is to coordinate the formation of a chromatin hub that drives Vα and Jα germ-line transcription and primary rearrangements in thymocytes and imply the existence of an Eα-independent mechanism to activate transcription of the rearranged Tcra locus in αβ T cells.

Authors
del Blanco, B; Angulo, Ú; Krangel, MS; Hernández-Munain, C
MLA Citation
del Blanco, B, Angulo, Ú, Krangel, MS, and Hernández-Munain, C. "T-cell receptor α enhancer is inactivated in αβ T lymphocytes." Proceedings of the National Academy of Sciences of the United States of America 112.14 (April 2015): E1744-E1753.
PMID
25831496
Source
epmc
Published In
Proceedings of the National Academy of Sciences of USA
Volume
112
Issue
14
Publish Date
2015
Start Page
E1744
End Page
E1753
DOI
10.1073/pnas.1406551112

IL-7 coordinates proliferation, differentiation and Tcra recombination during thymocyte β-selection

© 2015 Nature America, Inc. All rights reserved.Signaling via the pre-T cell antigen receptor (pre-TCR) and the receptor Notch1 induces transient self-renewal (β-selection) of TCRβ+ CD4-CD8- double-negative stage 3 (DN3) and DN4 progenitor cells that differentiate into CD4+CD8+ double-positive (DP) thymocytes, which then rearrange the locus encoding the TCR α-chain (Tcra). Interleukin 7 (IL-7) promotes the survival of TCRβ-DN thymocytes by inducing expression of the pro-survival molecule Bcl-2, but the functions of IL-7 during β-selection have remained unclear. Here we found that IL-7 signaled TCRβ+ DN3 and DN4 thymocytes to upregulate genes encoding molecules involved in cell growth and repressed the gene encoding the transcriptional repressor Bcl-6. Accordingly, IL-7-deficient DN4 cells lacked trophic receptors and did not proliferate but rearranged Tcra prematurely and differentiated rapidly. Deletion of Bcl6 partially restored the self-renewal of DN4 cells in the absence of IL-7, but overexpression of BCL2 did not. Thus, IL-7 critically acts cooperatively with signaling via the pre-TCR and Notch1 to coordinate proliferation, differentiation and Tcra recombination during β-selection.

Authors
Boudil, A; Matei, IR; Shih, HY; Bogdanoski, G; Yuan, JS; Chang, SG; Montpellier, B; Kowalski, PE; Voisin, V; Bashir, S; Bader, GD; Krangel, MS; Guidos, CJ
MLA Citation
Boudil, A, Matei, IR, Shih, HY, Bogdanoski, G, Yuan, JS, Chang, SG, Montpellier, B, Kowalski, PE, Voisin, V, Bashir, S, Bader, GD, Krangel, MS, and Guidos, CJ. "IL-7 coordinates proliferation, differentiation and Tcra recombination during thymocyte β-selection." Nature Immunology 16.4 (January 1, 2015): 397-405.
Source
scopus
Published In
Nature Immunology
Volume
16
Issue
4
Publish Date
2015
Start Page
397
End Page
405
DOI
10.1038/ni.3122

Specification of Vδ and Vα usage by Tcra/Tcrd locus V gene segment promoters.

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.

Authors
Naik, AK; Hawwari, A; Krangel, MS
MLA Citation
Naik, AK, Hawwari, A, and Krangel, MS. "Specification of Vδ and Vα usage by Tcra/Tcrd locus V gene segment promoters." Journal of immunology (Baltimore, Md. : 1950) 194.2 (January 2015): 790-794.
PMID
25472997
Source
epmc
Published In
Journal of immunology (Baltimore, Md. : 1950)
Volume
194
Issue
2
Publish Date
2015
Start Page
790
End Page
794
DOI
10.4049/jimmunol.1402423

Lineage-specific compaction of Tcrb requires a chromatin barrier to protect the function of a long-range tethering element.

Gene regulation relies on dynamic changes in three-dimensional chromatin conformation, which are shaped by composite regulatory and architectural elements. However, mechanisms that govern such conformational switches within chromosomal domains remain unknown. We identify a novel mechanism by which cis-elements promote long-range interactions, inducing conformational changes critical for diversification of the TCRβ antigen receptor locus (Tcrb). Association between distal Vβ gene segments and the highly expressed DβJβ clusters, termed the recombination center (RC), is independent of enhancer function and recruitment of V(D)J recombinase. Instead, we find that tissue-specific folding of Tcrb relies on two distinct architectural elements located upstream of the RC. The first, a CTCF-containing element, directly tethers distal portions of the Vβ array to the RC. The second element is a chromatin barrier that protects the tether from hyperactive RC chromatin. When the second element is removed, active RC chromatin spreads upstream, forcing the tether to serve as a new barrier. Acquisition of barrier function by the CTCF element disrupts contacts between distal Vβ gene segments and significantly alters Tcrb repertoires. Our findings reveal a separation of function for RC-flanking regions, in which anchors for long-range recombination must be cordoned off from hyperactive RC landscapes by chromatin barriers.

Authors
Majumder, K; Koues, OI; Chan, EAW; Kyle, KE; Horowitz, JE; Yang-Iott, K; Bassing, CH; Taniuchi, I; Krangel, MS; Oltz, EM
MLA Citation
Majumder, K, Koues, OI, Chan, EAW, Kyle, KE, Horowitz, JE, Yang-Iott, K, Bassing, CH, Taniuchi, I, Krangel, MS, and Oltz, EM. "Lineage-specific compaction of Tcrb requires a chromatin barrier to protect the function of a long-range tethering element." The Journal of experimental medicine 212.1 (January 2015): 107-120.
PMID
25512470
Source
epmc
Published In
The Journal of Experimental Medicine
Volume
212
Issue
1
Publish Date
2015
Start Page
107
End Page
120
DOI
10.1084/jem.20141479

Chromatin Dynamics and the Development of the TCRα and TCRδ Repertoires.

The adaptive immune system allows vertebrates to orchestrate highly specific responses to a virtually unlimited milieu of antigens. Effective adaptive immune responses depend on the capacity of T and B lymphocytes to generate diverse repertoires of antigen receptors through the recombination of variable (V), diversity (D), and joining (J) gene segments at antigen receptor loci. V(D)J recombination must be carefully regulated during the early stages of T and B lymphocyte development to ensure the proper development of lymphocyte subsets and to maximize antigen receptor combinatorial diversity. Among all T cell receptor (TCR) and immunoglobulin loci, the TCRα/δ (Tcra/Tcrd) locus is unique in its complexity since it undergoes recombination at two distinct stages of T cell development to create distinct TCR proteins that are used by different lineages of T cells. Here, we review the mechanisms that regulate V(D)J recombination at the Tcra/Tcrd locus, with a focus on the dynamic chromatin environment and how it instructs the assembly of the Tcra and Tcrd repertoires. We discuss the dynamics of Tcra and Tcrd repertoire formation in the context of T cell development, and we consider how the recombination program is directed by localized changes in chromatin structure that regulate the accessibility of Tcra and Tcrd gene segments to the V(D)J recombinase. We then move beyond local to address spatial relationships in the nucleus, emphasizing the three-dimensional organization of the Tcra/Tcrd locus as a critical player in understanding long-distance interactions between chromatin regulatory elements as well as long-distance interactions between recombination substrates.

Authors
Carico, Z; Krangel, MS
MLA Citation
Carico, Z, and Krangel, MS. "Chromatin Dynamics and the Development of the TCRα and TCRδ Repertoires." Advances in immunology 128 (January 2015): 307-361. (Review)
PMID
26477370
Source
epmc
Published In
Advances in immunology
Volume
128
Publish Date
2015
Start Page
307
End Page
361
DOI
10.1016/bs.ai.2015.07.005

KAP-1 promotes resection of broken DNA ends not protected by γ-H2AX and 53BP1 in G₁-phase lymphocytes.

The resection of broken DNA ends is required for DNA double-strand break (DSB) repair by homologous recombination (HR) but can inhibit normal repair by nonhomologous end joining (NHEJ), the main DSB repair pathway in G1-phase cells. Antigen receptor gene assembly proceeds through DNA DSB intermediates generated in G1-phase lymphocytes by the RAG endonuclease. These DSBs activate ATM, which phosphorylates H2AX, forming γ-H2AX in flanking chromatin. γ-H2AX prevents CtIP from initiating resection of RAG DSBs. Whether there are additional proteins required to promote resection of these DNA ends is not known. KRAB-associated protein 1 (KAP-1) (TRIM28) is a transcriptional repressor that modulates chromatin structure and has been implicated in the repair of DNA DSBs in heterochromatin. Here, we show that in murine G1-phase lymphocytes, KAP-1 promotes resection of DSBs that are not protected by H2AX and its downstream effector 53BP1. In these murine cells, KAP-1 activity in DNA end resection is attenuated by a single-amino-acid change that reflects a KAP-1 polymorphism between primates and other mammalian species. These findings establish KAP-1 as a component of the machinery that can resect DNA ends in G1-phase cells and suggest that there may be species-specific features to this activity.

Authors
Tubbs, AT; Dorsett, Y; Chan, E; Helmink, B; Lee, B-S; Hung, P; George, R; Bredemeyer, AL; Mittal, A; Pappu, RV; Chowdhury, D; Mosammaparast, N; Krangel, MS; Sleckman, BP
MLA Citation
Tubbs, AT, Dorsett, Y, Chan, E, Helmink, B, Lee, B-S, Hung, P, George, R, Bredemeyer, AL, Mittal, A, Pappu, RV, Chowdhury, D, Mosammaparast, N, Krangel, MS, and Sleckman, BP. "KAP-1 promotes resection of broken DNA ends not protected by γ-H2AX and 53BP1 in G₁-phase lymphocytes." Molecular and cellular biology 34.15 (August 2014): 2811-2821.
PMID
24842905
Source
epmc
Published In
Molecular and Cellular Biology
Volume
34
Issue
15
Publish Date
2014
Start Page
2811
End Page
2821
DOI
10.1128/mcb.00441-14

Peripheral subnuclear positioning suppresses Tcrb recombination and segregates Tcrb alleles from RAG2.

Allelic exclusion requires that the two alleles at antigen-receptor loci attempt to recombine variable (V), diversity (D), and joining (J) gene segments [V(D)J recombination] asynchronously in nuclei of developing lymphocytes. It previously was shown that T-cell receptor β (Tcrb) alleles frequently and stochastically associate with the nuclear lamina and pericentromeric heterochromatin in CD4(-)CD8(-) thymocytes. Moreover, rearranged alleles were underrepresented at these locations. Here we used 3D immunofluorescence in situ hybridization to identify recently rearranged Tcrb alleles based on the accumulation of the DNA-repair protein 53BP1. We found that Tcrb alleles recombine asynchronously in double-negative thymocytes and that V(D)J recombination is suppressed on peripheral as compared with central Tcrb alleles. Moreover, the recombination events that did take place at the nuclear periphery preferentially occurred on Tcrb alleles that were partially dissociated from the nuclear lamina. To understand better the mechanism by which V(D)J recombination is suppressed at the nuclear periphery, we evaluated the subnuclear distribution of recombination-activating gene 2 (RAG2) protein. We found that RAG2 abundance was reduced at the nuclear periphery. Moreover, RAG2 was distributed differently from RNA polymerase II and histone H3K4 trimethylation. Our data suggest that the nuclear periphery suppresses V(D)J recombination, at least in part, by segregating Tcrb alleles from RAG proteins.

Authors
Chan, EAW; Teng, G; Corbett, E; Choudhury, KR; Bassing, CH; Schatz, DG; Krangel, MS
MLA Citation
Chan, EAW, Teng, G, Corbett, E, Choudhury, KR, Bassing, CH, Schatz, DG, and Krangel, MS. "Peripheral subnuclear positioning suppresses Tcrb recombination and segregates Tcrb alleles from RAG2." Proc Natl Acad Sci U S A 110.48 (November 26, 2013): E4628-E4637.
PMID
24218622
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
110
Issue
48
Publish Date
2013
Start Page
E4628
End Page
E4637
DOI
10.1073/pnas.1310846110

Chromatin architecture, CCCTC-binding factor, and V(D)J recombination: managing long-distance relationships at antigen receptor loci.

The rearrangement of T and B lymphocyte Ag receptor loci occurs within a highly complex chromosomal environment and is orchestrated through complex mechanisms. During the past decade, a large body of literature has highlighted the significance of chromatin architecture at Ag receptor loci in supporting the genomic assembly process: in preparation for recombination, these loci tend to contract and form multiple loops that shorten the distances between gene segments and facilitate recombination events. CCCTC-binding factor, CTCF, has received much attention in this regard since it has emerged as an important regulator of chromatin organization and transcription. In this review, we summarize recent work outlining conformational dynamics at Ag receptor loci during lymphocyte development and we discuss the role of CTCF in Ag receptor locus conformation and repertoire development.

Authors
Shih, H-Y; Krangel, MS
MLA Citation
Shih, H-Y, and Krangel, MS. "Chromatin architecture, CCCTC-binding factor, and V(D)J recombination: managing long-distance relationships at antigen receptor loci." J Immunol 190.10 (May 15, 2013): 4915-4921. (Review)
PMID
23645930
Source
pubmed
Published In
Journal of immunology (Baltimore, Md. : 1950)
Volume
190
Issue
10
Publish Date
2013
Start Page
4915
End Page
4921
DOI
10.4049/jimmunol.1300218

The DNA damage- and transcription-associated protein paxip1 controls thymocyte development and emigration.

Histone 3 lysine 4 trimethylation (H3K4me3) is associated with promoters of active genes and found at hot spots for DNA recombination. Here we have shown that PAXIP1 (also known as PTIP), a protein associated with MLL3 and MLL4 methyltransferase and the DNA damage response, regulates RAG-mediated cleavage and repair during V(D)J recombination in CD4(+) CD8(+) DP thymocytes. Loss of PAXIP1 in developing thymocytes diminished Jα H3K4me3 and germline transcription, suppressed double strand break formation at 3' Jα segments, but resulted in accumulation of unresolved T cell receptor α-chain gene (Tcra) breaks. Moreover, PAXIP1 was essential for release of mature single positive (SP) αβ T cells from the thymus through transcriptional activation of sphingosine-1-phosphate receptor S1pr1 as well as for natural killer T cell development. Thus, in addition to maintaining genome integrity during Tcra rearrangements, PAXIP1 controls distinct transcriptional programs during DP differentiation necessary for Tcra locus accessibility, licensing mature thymocytes for trafficking and natural killer T cell development.

Authors
Callen, E; Faryabi, RB; Luckey, M; Hao, B; Daniel, JA; Yang, W; Sun, H-W; Dressler, G; Peng, W; Chi, H; Ge, K; Krangel, MS; Park, J-H; Nussenzweig, A
MLA Citation
Callen, E, Faryabi, RB, Luckey, M, Hao, B, Daniel, JA, Yang, W, Sun, H-W, Dressler, G, Peng, W, Chi, H, Ge, K, Krangel, MS, Park, J-H, and Nussenzweig, A. "The DNA damage- and transcription-associated protein paxip1 controls thymocyte development and emigration." Immunity 37.6 (December 14, 2012): 971-985.
PMID
23159437
Source
pubmed
Published In
Immunity
Volume
37
Issue
6
Publish Date
2012
Start Page
971
End Page
985
DOI
10.1016/j.immuni.2012.10.007

Tcra gene recombination is supported by a Tcra enhancer- and CTCF-dependent chromatin hub.

Antigen receptor locus V(D)J recombination requires interactions between widely separated variable (V), diversity (D), and joining (J) gene segments, but the mechanisms that generate these interactions are not well understood. Here we assessed mechanisms that direct developmental stage-specific long-distance interactions at the Tcra/Tcrd locus. The Tcra/Tcrd locus recombines Tcrd gene segments in CD4(-)CD8(-) double-negative thymocytes and Tcra gene segments in CD4(+)CD8(+) double-positive thymocytes. Initial V(α)-to-J(α) recombination occurs within a chromosomal domain that displays a contracted conformation in both thymocyte subsets. We used chromosome conformation capture to demonstrate that the Tcra enhancer (E(α)) interacts directly with V(α) and J(α) gene segments distributed across this domain, specifically in double-positive thymocytes. Moreover, E(α) promotes interactions between these V(α) and J(α) segments that should facilitate their synapsis. We found that the CCCTC-binding factor (CTCF) binds to E(α) and to many locus promoters, biases E(α) to interact with these promoters, and is required for efficient V(α)-J(α) recombination. Our data indicate that E(α) and CTCF cooperate to create a developmentally regulated chromatin hub that supports V(α)-J(α) synapsis and recombination.

Authors
Shih, H-Y; Verma-Gaur, J; Torkamani, A; Feeney, AJ; Galjart, N; Krangel, MS
MLA Citation
Shih, H-Y, Verma-Gaur, J, Torkamani, A, Feeney, AJ, Galjart, N, and Krangel, MS. "Tcra gene recombination is supported by a Tcra enhancer- and CTCF-dependent chromatin hub." Proc Natl Acad Sci U S A 109.50 (December 11, 2012): E3493-E3502.
PMID
23169622
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
109
Issue
50
Publish Date
2012
Start Page
E3493
End Page
E3502
DOI
10.1073/pnas.1214131109

Cohesin, CTCF and lymphocyte antigen receptor locus rearrangement.

The somatic recombination of lymphocyte antigen receptor loci is integral to lymphocyte differentiation and adaptive immunity. Here we review the relation of this highly choreographed process with the zinc finger protein CTCF and with cohesin, a protein complex best known for its essential functions in post-replicative DNA repair and chromosome segregation during the cell cycle. At lymphocyte antigen receptor loci, CTCF and cohesin shape long-range interactions and contribute to V(D)J recombination by facilitating lineage- and developmental-stage-specific transcription and accessibility.

Authors
Seitan, VC; Krangel, MS; Merkenschlager, M
MLA Citation
Seitan, VC, Krangel, MS, and Merkenschlager, M. "Cohesin, CTCF and lymphocyte antigen receptor locus rearrangement." Trends Immunol 33.4 (April 2012): 153-159. (Review)
PMID
22440186
Source
pubmed
Published In
Trends in Immunology
Volume
33
Issue
4
Publish Date
2012
Start Page
153
End Page
159
DOI
10.1016/j.it.2012.02.004

Regulation of TCRβ allelic exclusion by gene segment proximity and accessibility.

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 V(β)-to-DJ(β) recombination in double-positive (DP) thymocytes, which correlates with reduced V(β) chromatin accessibility and a locus conformational change that separates V(β) from DJ(β) gene segments. We previously generated a Tcrb allele that maintained V(β) 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 V(β) accessibility and increased distance between V(β) and DJ(β) gene segments both enforce feedback inhibition in DP thymocytes.

Authors
Kondilis-Mangum, HD; Shih, H-Y; Mahowald, G; Sleckman, BP; Krangel, MS
MLA Citation
Kondilis-Mangum, HD, Shih, H-Y, Mahowald, G, Sleckman, BP, and Krangel, MS. "Regulation of TCRβ allelic exclusion by gene segment proximity and accessibility." J Immunol 187.12 (December 15, 2011): 6374-6381.
PMID
22079986
Source
pubmed
Published In
Journal of immunology (Baltimore, Md. : 1950)
Volume
187
Issue
12
Publish Date
2011
Start Page
6374
End Page
6381
DOI
10.4049/jimmunol.1102611

Long-distance regulation of fetal V(δ) gene segment TRDV4 by the Tcrd enhancer.

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.

Authors
Hao, B; Krangel, MS
MLA Citation
Hao, B, and Krangel, MS. "Long-distance regulation of fetal V(δ) gene segment TRDV4 by the Tcrd enhancer." J Immunol 187.5 (September 1, 2011): 2484-2491.
PMID
21784972
Source
pubmed
Published In
Journal of immunology (Baltimore, Md. : 1950)
Volume
187
Issue
5
Publish Date
2011
Start Page
2484
End Page
2491
DOI
10.4049/jimmunol.1100468

A role for cohesin in T-cell-receptor rearrangement and thymocyte differentiation.

Cohesin enables post-replicative DNA repair and chromosome segregation by holding sister chromatids together from the time of DNA replication in S phase until mitosis. There is growing evidence that cohesin also forms long-range chromosomal cis-interactions and may regulate gene expression in association with CTCF, mediator or tissue-specific transcription factors. Human cohesinopathies such as Cornelia de Lange syndrome are thought to result from impaired non-canonical cohesin functions, but a clear distinction between the cell-division-related and cell-division-independent functions of cohesion--as exemplified in Drosophila--has not been demonstrated in vertebrate systems. To address this, here we deleted the cohesin locus Rad21 in mouse thymocytes at a time in development when these cells stop cycling and rearrange their T-cell receptor (TCR) α locus (Tcra). Rad21-deficient thymocytes had a normal lifespan and retained the ability to differentiate, albeit with reduced efficiency. Loss of Rad21 led to defective chromatin architecture at the Tcra locus, where cohesion-binding sites flank the TEA promoter and the Eα enhancer, and demarcate Tcra from interspersed Tcrd elements and neighbouring housekeeping genes. Cohesin was required for long-range promoter-enhancer interactions, Tcra transcription, H3K4me3 histone modifications that recruit the recombination machinery and Tcra rearrangement. Provision of pre-rearranged TCR transgenes largely rescued thymocyte differentiation, demonstrating that among thousands of potential target genes across the genome, defective Tcra rearrangement was limiting for the differentiation of cohesin-deficient thymocytes. These findings firmly establish a cell-division-independent role for cohesin in Tcra locus rearrangement and provide a comprehensive account of the mechanisms by which cohesin enables cellular differentiation in a well-characterized mammalian system.

Authors
Seitan, VC; Hao, B; Tachibana-Konwalski, K; Lavagnolli, T; Mira-Bontenbal, H; Brown, KE; Teng, G; Carroll, T; Terry, A; Horan, K; Marks, H; Adams, DJ; Schatz, DG; Aragon, L; Fisher, AG; Krangel, MS; Nasmyth, K; Merkenschlager, M
MLA Citation
Seitan, VC, Hao, B, Tachibana-Konwalski, K, Lavagnolli, T, Mira-Bontenbal, H, Brown, KE, Teng, G, Carroll, T, Terry, A, Horan, K, Marks, H, Adams, DJ, Schatz, DG, Aragon, L, Fisher, AG, Krangel, MS, Nasmyth, K, and Merkenschlager, M. "A role for cohesin in T-cell-receptor rearrangement and thymocyte differentiation. (Published online)" Nature 476.7361 (August 10, 2011): 467-471.
PMID
21832993
Source
pubmed
Published In
Nature
Volume
476
Issue
7361
Publish Date
2011
Start Page
467
End Page
471
DOI
10.1038/nature10312

Immunology at Duke: 2011.

Authors
Krangel, MS
MLA Citation
Krangel, MS. "Immunology at Duke: 2011." Immunol Res 49.1-3 (April 2011): 1-2.
PMID
21116870
Source
pubmed
Published In
Immunologic Research
Volume
49
Issue
1-3
Publish Date
2011
Start Page
1
End Page
2
DOI
10.1007/s12026-010-8198-2

Orchestrating T-cell receptor α gene assembly through changes in chromatin structure and organization.

V(D)J recombination is regulated through changes in chromatin structure that allow recombinase proteins access to recombination signal sequences and through changes in three-dimensional chromatin organization that bring pairs of distant recombination signal sequences into proximity. The Tcra/Tcrd locus is complex and undergoes distinct recombination programs in double negative and double positive thymocytes that lead to the assembly of Tcrd and Tcra genes, respectively. Our studies provide insights into how locus chromatin structure is regulated and how changes in locus chromatin structure can target and then retarget the recombinase to create developmental progressions of recombination events. Our studies also reveal distinct locus conformations in double negative and double positive thymocytes and suggest how these conformations may support the distinct recombination programs in the two compartments.

Authors
Shih, H-Y; Hao, B; Krangel, MS
MLA Citation
Shih, H-Y, Hao, B, and Krangel, MS. "Orchestrating T-cell receptor α gene assembly through changes in chromatin structure and organization." Immunol Res 49.1-3 (April 2011): 192-201.
PMID
21128009
Source
pubmed
Published In
Immunologic Research
Volume
49
Issue
1-3
Publish Date
2011
Start Page
192
End Page
201
DOI
10.1007/s12026-010-8181-y

A barrier-type insulator forms a boundary between active and inactive chromatin at the murine TCRβ locus.

In CD4(-)CD8(-) double-negative thymocytes, the murine Tcrb locus is composed of alternating blocks of active and inactive chromatin containing Tcrb gene segments and trypsinogen genes, respectively. Although chromatin structure is appreciated to be critical for regulated recombination and expression of Tcrb gene segments, the molecular mechanisms that maintain the integrity of these differentially regulated Tcrb locus chromatin domains are not understood. We localized a boundary between active and inactive chromatin by mapping chromatin modifications across the interval extending from Prss2 (the most 3' trypsinogen gene) to D(β)1. This boundary, located 6 kb upstream of D(β)1, is characterized by a transition from repressive (histone H3 lysine 9 dimethylation [H3K9me2]) to active (histone H3 acetylation [H3ac]) chromatin and is marked by a peak of histone H3 lysine 4 dimethylation (H3K4me2) that colocalizes with a retroviral long terminal repeat (LTR). Histone H3 lysine 4 dimethylation is retained and histone H3 lysine 9 dimethylation fails to spread past the LTR even on alleles lacking the Tcrb enhancer (E(β)) suggesting that these features may be determined by the local DNA sequence. Notably, we found that LTR-containing DNA functions as a barrier-type insulator that can protect a transgene from negative chromosomal position effects. We propose that, in vivo, the LTR blocks the spread of heterochromatin, and thereby helps to maintain the integrity of the E(β)-regulated chromatin domain. We also identified low-abundance, E(β)-dependent transcripts that initiate at the border of the LTR and an adjacent long interspersed element. We speculate that this transcription, which extends across D(β), J(β) and C(β) gene segments, may play an additional role promoting initial opening of the E(β)-regulated chromatin domain.

Authors
Carabana, J; Watanabe, A; Hao, B; Krangel, MS
MLA Citation
Carabana, J, Watanabe, A, Hao, B, and Krangel, MS. "A barrier-type insulator forms a boundary between active and inactive chromatin at the murine TCRβ locus." J Immunol 186.6 (March 15, 2011): 3556-3562.
PMID
21317385
Source
pubmed
Published In
Journal of immunology (Baltimore, Md. : 1950)
Volume
186
Issue
6
Publish Date
2011
Start Page
3556
End Page
3562
DOI
10.4049/jimmunol.1003164

Promoters, enhancers, and transcription target RAG1 binding during V(D)J recombination.

V(D)J recombination assembles antigen receptor genes in a well-defined order during lymphocyte development. This sequential process has long been understood in the context of the accessibility model, which states that V(D)J recombination is regulated by controlling the ability of the recombination machinery to gain access to its chromosomal substrates. Indeed, many features of "open" chromatin correlate with V(D)J recombination, and promoters and enhancers have been strongly implicated in creating a recombinase-accessible configuration in neighboring chromatin. An important prediction of the accessibility model is that cis-elements and transcription control binding of the recombination-activating gene 1 (RAG1) and RAG2 proteins to their DNA targets. However, this prediction has not been tested directly. In this study, we use mutant Tcra and Tcrb alleles to demonstrate that enhancers control RAG1 binding globally at Jα or Dβ/Jβ gene segments, that promoters and transcription direct RAG1 binding locally, and that RAG1 binding can be targeted in the absence of RAG2. These findings reveal important features of the genetic mechanisms that regulate RAG binding and provide a direct confirmation of the accessibility model.

Authors
Ji, Y; Little, AJ; Banerjee, JK; Hao, B; Oltz, EM; Krangel, MS; Schatz, DG
MLA Citation
Ji, Y, Little, AJ, Banerjee, JK, Hao, B, Oltz, EM, Krangel, MS, and Schatz, DG. "Promoters, enhancers, and transcription target RAG1 binding during V(D)J recombination." J Exp Med 207.13 (December 20, 2010): 2809-2816.
PMID
21115692
Source
pubmed
Published In
The Journal of Experimental Medicine
Volume
207
Issue
13
Publish Date
2010
Start Page
2809
End Page
2816
DOI
10.1084/jem.20101136

Distinct contracted conformations of the Tcra/Tcrd locus during Tcra and Tcrd recombination.

Studies have suggested that antigen receptor loci adopt contracted conformations to promote long-distance interactions between gene segments during V(D)J recombination. The Tcra/Tcrd locus is unique because it undergoes highly divergent Tcrd and Tcra recombination programs in CD4(-)CD8(-) double negative (DN) and CD4(+)CD8(+) double positive (DP) thymocytes, respectively. Using three-dimensional fluorescence in situ hybridization, we asked whether these divergent recombination programs are supported by distinct conformational states of the Tcra/Tcrd locus. We found that the 3' portion of the locus is contracted in DN and DP thymocytes but not in B cells. Remarkably, the 5' portion of the locus is contracted in DN thymocytes but is decontracted in DP thymocytes. We propose that the fully contracted conformation in DN thymocytes allows Tcrd rearrangements involving V(delta) gene segments distributed over 1 Mb, whereas the unique 3'-contracted, 5'-decontracted conformation in DP thymocytes biases initial Tcra rearrangements to the most 3' of the available V(alpha) gene segments. This would maintain a large pool of distal 5' V(alpha) gene segments for subsequent rounds of recombination. Thus, distinct contracted conformations of the Tcra/Tcrd locus may facilitate a transition from a Tcrd to a Tcra mode of recombination during thymocyte development.

Authors
Shih, H-Y; Krangel, MS
MLA Citation
Shih, H-Y, and Krangel, MS. "Distinct contracted conformations of the Tcra/Tcrd locus during Tcra and Tcrd recombination." J Exp Med 207.9 (August 30, 2010): 1835-1841.
PMID
20696701
Source
pubmed
Published In
The Journal of Experimental Medicine
Volume
207
Issue
9
Publish Date
2010
Start Page
1835
End Page
1841
DOI
10.1084/jem.20100772

Transcription-dependent mobilization of nucleosomes at accessible TCR gene segments in vivo.

Accessibility of chromosomal recombination signal sequences to the RAG protein complex is known to be essential for V(D)J recombination at Ag receptor loci in vivo. Previous studies have addressed the roles of cis-acting regulatory elements and germline transcription in the covalent modification of nucleosomes at Ag receptor loci. However, a detailed picture of nucleosome organization at accessible and inaccessible recombination signal sequences has been lacking. In this study, we have analyzed the nucleosome organization of accessible and inaccessible Tcrb and Tcra alleles in primary murine thymocytes in vivo. We identified highly positioned arrays of nucleosomes at Dbeta, Jbeta, and Jalpha segments and obtained evidence indicating that positioning is established at least in part by the regional DNA sequence. However, we found no consistent positioning of nucleosomes with respect to recombination signal sequences, which could be nucleosomal or internucleosomal even in their inaccessible configurations. Enhancer- and promoter-dependent accessibility was characterized by diminished abundance of certain nucleosomes and repositioning of others. Moreover, some changes in nucleosome positioning and abundance at Jalpha61 were shown to be a direct consequence of germline transcription. We suggest that enhancer- and promoter-dependent transcription generates optimal recombinase substrates in which some nucleosomes are missing and others are covalently modified.

Authors
Kondilis-Mangum, HD; Cobb, RM; Osipovich, O; Srivatsan, S; Oltz, EM; Krangel, MS
MLA Citation
Kondilis-Mangum, HD, Cobb, RM, Osipovich, O, Srivatsan, S, Oltz, EM, and Krangel, MS. "Transcription-dependent mobilization of nucleosomes at accessible TCR gene segments in vivo." J Immunol 184.12 (June 15, 2010): 6970-6977.
PMID
20483751
Source
pubmed
Published In
Journal of immunology (Baltimore, Md. : 1950)
Volume
184
Issue
12
Publish Date
2010
Start Page
6970
End Page
6977
DOI
10.4049/jimmunol.0903923

CD69 gene is differentially regulated in T and B cells by evolutionarily conserved promoter-distal elements.

CD69 is a type II C-type lectin involved in lymphocyte migration and cytokine secretion. CD69 expression represents one of the earliest available indicators of leukocyte activation and its rapid induction occurs through transcriptional activation. In this study we examined the molecular mechanism underlying mouse CD69 gene transcription in vivo in T and B cells. Analysis of the 45-kb region upstream of the CD69 gene revealed evolutionary conservation at the promoter and at four noncoding sequences (CNS) that were called CNS1, CNS2, CNS3, and CNS4. These regions were found to be hypersensitive sites in DNase I digestion experiments, and chromatin immunoprecipitation assays showed specific epigenetic modifications. CNS2 and CNS4 displayed constitutive and inducible enhancer activity in transient transfection assays in T cells. Using a transgenic approach to test CNS function, we found that the CD69 promoter conferred developmentally regulated expression during positive selection of thymocytes but could not support regulated expression in mature lymphocytes. Inclusion of CNS1 and CNS2 caused suppression of CD69 expression, whereas further addition of CNS3 and CNS4 supported developmental-stage and lineage-specific regulation in T cells but not in B cells. We concluded CNS1-4 are important cis-regulatory elements that interact both positively and negatively with the CD69 promoter and that differentially contribute to CD69 expression in T and B cells.

Authors
Vazquez, BN; Laguna, T; Carabana, J; Krangel, MS; Lauzurica, P
MLA Citation
Vazquez, BN, Laguna, T, Carabana, J, Krangel, MS, and Lauzurica, P. "CD69 gene is differentially regulated in T and B cells by evolutionarily conserved promoter-distal elements." J Immunol 183.10 (November 15, 2009): 6513-6521.
PMID
19841192
Source
pubmed
Published In
Journal of immunology (Baltimore, Md. : 1950)
Volume
183
Issue
10
Publish Date
2009
Start Page
6513
End Page
6521
DOI
10.4049/jimmunol.0900839

Mechanics of T cell receptor gene rearrangement.

The four T cell receptor genes (Tcra, Tcrb, Tcrg, Tcrd) are assembled by V(D)J recombination according to distinct programs during intrathymic T cell development. These programs depend on genetic factors, including gene segment order and recombination signal sequences. They also depend on epigenetic factors. Regulated changes in chromatin structure, directed by enhancers and promoter, can modify the availability of recombination signal sequences to the RAG recombinase. Regulated changes in locus conformation may control the synapsis of distant recombination signal sequences, and regulated changes in subnuclear positioning may influence locus recombination events by unknown mechanisms. Together these influences may explain the ordered activation and inactivation of T cell receptor locus recombination events and the phenomenon of Tcrb allelic exclusion.

Authors
Krangel, MS
MLA Citation
Krangel, MS. "Mechanics of T cell receptor gene rearrangement." Curr Opin Immunol 21.2 (April 2009): 133-139. (Review)
PMID
19362456
Source
pubmed
Published In
Current Opinion in Immunology
Volume
21
Issue
2
Publish Date
2009
Start Page
133
End Page
139
DOI
10.1016/j.coi.2009.03.009

Germline transcription: a key regulator of accessibility and recombination.

The developmental control of V(D)J recombination is imposed at the level of chromatin accessibility of recombination signal sequences (RSSs) to the recombinase machinery. Cis-acting transcriptional regulatory elements such as promoters and enhancers play a central role in the control of accessibility in vivo. However, the molecular mechanisms by which these elements influence accessibility are still under investigation. Although accessibility for V(D)J recombination is usually accompanied by germline transcription at antigen receptor loci, the functional significance of this transcription in directing RSS accessibility has been elusive. In this chapter, we review past studies outlining the complex relationship between V(D)J recombination and transcription as well as our current understanding on how chromatin structure is regulated during gene expression. We then summarize recent work that directly addresses the functional role of transcription in V(D)J recombination.

Authors
Abarrategui, I; Krangel, MS
MLA Citation
Abarrategui, I, and Krangel, MS. "Germline transcription: a key regulator of accessibility and recombination." Adv Exp Med Biol 650 (2009): 93-102. (Review)
PMID
19731804
Source
pubmed
Published In
Advances in experimental medicine and biology
Volume
650
Publish Date
2009
Start Page
93
End Page
102

Initiation of allelic exclusion by stochastic interaction of Tcrb alleles with repressive nuclear compartments.

Studies of antigen-receptor loci have linked directed monoallelic association with pericentromeric heterochromatin to the initiation or maintenance of allelic exclusion. Here we provide evidence for a fundamentally different basis for T cell antigen receptor-beta (Tcrb) allelic exclusion. Using three-dimensional immunofluorescence in situ hybridization, we found that germline Tcrb alleles associated stochastically and at high frequency with the nuclear lamina or with pericentromeric heterochromatin in developing thymocytes and that such interactions inhibited variable-to-diversity-joining (V(beta)-to-D(beta)J(beta)) recombination before beta-selection. The introduction of an ectopic enhancer into Tcrb resulted in fewer such interactions and impaired allelic exclusion. We propose that initial V(beta)-to-D(beta)J(beta) recombination events are generally monoallelic in developing thymocytes because of frequent stochastic, rather than directed, interactions of Tcrb alleles with repressive nuclear compartments. Such interactions may be essential for Tcrb allelic exclusion.

Authors
Schlimgen, RJ; Reddy, KL; Singh, H; Krangel, MS
MLA Citation
Schlimgen, RJ, Reddy, KL, Singh, H, and Krangel, MS. "Initiation of allelic exclusion by stochastic interaction of Tcrb alleles with repressive nuclear compartments." Nat Immunol 9.7 (July 2008): 802-809.
PMID
18536719
Source
pubmed
Published In
Nature Immunology
Volume
9
Issue
7
Publish Date
2008
Start Page
802
End Page
809
DOI
10.1038/ni.1624

Noncoding transcription controls downstream promoters to regulate T-cell receptor alpha recombination.

The T early alpha (TEA) promoter in the murine Tcra locus generates noncoding transcripts that extend across the 65 kb Jalpha array. Here, we have analyzed the significance of TEA transcription for Tcra locus regulation through the targeted introduction of a transcription terminator downstream of the TEA promoter. We demonstrate that noncoding transcription driven by this single promoter can instruct both positively and negatively the activity of downstream Jalpha promoters, and can similarly instruct alterations in Jalpha chromatin structure and Jalpha recombination. TEA transcription activates promoters associated with relatively proximal Jalpha segments and stimulates histone acetylation, histone methylation and chromatin accessibility in this region. In contrast, at more distal locations, TEA transcription inhibits promoter activity through transcriptional interference and suppresses chromatin accessibility. In combination, these effects target initial Valpha-to-Jalpha recombination to TEA-proximal Jalpha segments and promote the ordered usage of the Jalpha array. The ability of TEA transcription to coordinate the activity of multiple downstream promoters maximizes the biological potential of the Jalpha array and diversifies the Tcra repertoire.

Authors
Abarrategui, I; Krangel, MS
MLA Citation
Abarrategui, I, and Krangel, MS. "Noncoding transcription controls downstream promoters to regulate T-cell receptor alpha recombination." EMBO J 26.20 (October 17, 2007): 4380-4390.
PMID
17882258
Source
pubmed
Published In
EMBO Journal
Volume
26
Issue
20
Publish Date
2007
Start Page
4380
End Page
4390
DOI
10.1038/sj.emboj.7601866

Multiple constraints at the level of TCRalpha rearrangement impact Valpha14i NKT cell development.

CD1d-restricted NKT cells that express an invariant Valpha14 TCR represent a subset of T cells implicated in the regulation of several immune responses, including autoimmunity, infectious disease, and cancer. Proper rearrangement of Valpha14 with the Jalpha18 gene segment in immature thymocytes is a prerequisite to the production of a TCR that can be subsequently positively selected by CD1d/self-ligand complexes in the thymus and gives rise to the NKT cell population. We show here that Valpha14 to Jalpha rearrangements are temporally regulated during ontogeny providing a molecular explanation to their late appearance in the thymus. Using mice deficient for the transcription factor RORgamma and the germline promoters T early-alpha and Jalpha49, we show that developmental constraints on both Valpha and Jalpha usage impact NKT cell development. Finally, we demonstrate that rearrangements using Valpha14 and Jalpha18 occur normally in the absence of FynT, arguing that the effect of FynT on NKT cell development occurs subsequent to alpha-chain rearrangement. Altogether, this study provides evidence that there is no directed rearrangement of Valpha14 to Jalpha18 segments and supports the instructive selection model for NKT cell selection.

Authors
Hager, E; Hawwari, A; Matsuda, JL; Krangel, MS; Gapin, L
MLA Citation
Hager, E, Hawwari, A, Matsuda, JL, Krangel, MS, and Gapin, L. "Multiple constraints at the level of TCRalpha rearrangement impact Valpha14i NKT cell development." J Immunol 179.4 (August 15, 2007): 2228-2234.
PMID
17675483
Source
pubmed
Published In
Journal of immunology (Baltimore, Md. : 1950)
Volume
179
Issue
4
Publish Date
2007
Start Page
2228
End Page
2234

T cell development: better living through chromatin.

T lymphocyte development is directed by a gene-expression program that occurs in the complex nucleoprotein environment of chromatin. This review examines basic principles of chromatin regulation and evaluates ongoing progress toward understanding how the chromatin template is manipulated to control gene expression and gene recombination in developing thymocytes. Special attention is devoted to the loci encoding T cell receptors alpha and beta, T cell coreceptors CD4 and CD8, and the enzyme terminal deoxynucleotidyl transferase. The properties of SATB1, a notable organizer of thymocyte chromatin, are also addressed.

Authors
Krangel, MS
MLA Citation
Krangel, MS. "T cell development: better living through chromatin." Nat Immunol 8.7 (July 2007): 687-694. (Review)
PMID
17579647
Source
pubmed
Published In
Nature Immunology
Volume
8
Issue
7
Publish Date
2007
Start Page
687
End Page
694
DOI
10.1038/ni1484

T-B+NK+ severe combined immunodeficiency caused by complete deficiency of the CD3zeta subunit of the T-cell antigen receptor complex.

CD3zeta is a subunit of the T-cell antigen receptor (TCR) complex required for its assembly and surface expression that also plays an important role in TCR-mediated signal transduction. We report here a patient with T(-)B(+)NK(+) severe combined immunodeficiency (SCID) who was homozygous for a single C insertion following nucleotide 411 in exon 7 of the CD3zeta gene. The few T cells present contained no detectable CD3zeta protein, expressed low levels of cell surface CD3epsilon, and were nonfunctional. CD4(+)CD8(-)CD3epsilon(low), CD4(-)CD8(+)CD3epsilon(low), and CD4(-)CD8(-)CD3epsilon(low) cells were detected in the periphery, and the patient also exhibited an unusual population of CD56(-)CD16(+) NK cells with diminished cytolytic activity. Additional studies demonstrated that retrovirally transduced patient mutant CD3zeta cDNA failed to rescue assembly of nascent complete TCR complexes or surface TCR expression in CD3zeta-deficient MA5.8 murine T-cell hybridoma cells. Nascent transduced mutant CD3zeta protein was also not detected in metabolically labeled MA5.8 cells, suggesting that it was unstable and rapidly degraded. Taken together, these findings provide the first demonstration that complete CD3zeta deficiency in humans can cause SCID by preventing normal TCR assembly and surface expression.

Authors
Roberts, JL; Lauritsen, JPH; Cooney, M; Parrott, RE; Sajaroff, EO; Win, CM; Keller, MD; Carpenter, JH; Carabana, J; Krangel, MS; Sarzotti, M; Zhong, X-P; Wiest, DL; Buckley, RH
MLA Citation
Roberts, JL, Lauritsen, JPH, Cooney, M, Parrott, RE, Sajaroff, EO, Win, CM, Keller, MD, Carpenter, JH, Carabana, J, Krangel, MS, Sarzotti, M, Zhong, X-P, Wiest, DL, and Buckley, RH. "T-B+NK+ severe combined immunodeficiency caused by complete deficiency of the CD3zeta subunit of the T-cell antigen receptor complex." Blood 109.8 (April 15, 2007): 3198-3206.
PMID
17170122
Source
pubmed
Published In
Blood
Volume
109
Issue
8
Publish Date
2007
Start Page
3198
End Page
3206
DOI
10.1182/blood-2006-08-043166

Role for rearranged variable gene segments in directing secondary T cell receptor alpha recombination.

During the recombination of variable (V) and joining (J) gene segments at the T cell receptor alpha locus, a ValphaJalpha joint resulting from primary rearrangement can be replaced by subsequent rounds of secondary rearrangement that use progressively more 5' Valpha segments and progressively more 3' Jalpha segments. To understand the mechanisms that target secondary T cell receptor alpha recombination, we studied the behavior of a T cell receptor alpha allele (HYalpha) engineered to mimic a natural primary rearrangement of TRAV17 to Jalpha57. The introduced ValphaJalpha segment was shown to provide chromatin accessibility to Jalpha segments situated within several kilobases downstream and to suppress germ-line Jalpha promoter activity and accessibility at greater distances. As a consequence, the ValphaJalpha segment directed secondary recombination events to a subset of Jalpha segments immediately downstream from the primary rearrangement. The data provide the mechanistic basis for a model of primary and secondary T cell receptor alpha recombination in which recombination events progress in multiple small steps down the Jalpha array.

Authors
Hawwari, A; Krangel, MS
MLA Citation
Hawwari, A, and Krangel, MS. "Role for rearranged variable gene segments in directing secondary T cell receptor alpha recombination." Proc Natl Acad Sci U S A 104.3 (January 16, 2007): 903-907.
PMID
17210914
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
104
Issue
3
Publish Date
2007
Start Page
903
End Page
907
DOI
10.1073/pnas.0608248104

Regulation of T cell receptor-alpha gene recombination by transcription.

Despite the longstanding correlation between transcription and variable-(diversity)-joining (V(D)J) recombination, it is unknown whether transcription itself can direct recombinase targeting. Here we show that blockade of transcriptional elongation through the mouse T cell receptor-alpha (Tcra) locus suppressed V(alpha)-to-J(alpha) recombination and chromatin remodeling of J(alpha) segments. Transcriptional blockade also derepressed cryptic J(alpha) promoters. Our results demonstrate two key functions for transcription in Tcra locus regulation. Transcription increases the recombination of J(alpha) segments located within several kilobases of a promoter and prevents the activation of downstream promoters through transcriptional interference. These influences promote an ordered progression of Tcra locus recombination events and selection of a robust Tcra repertoire.

Authors
Abarrategui, I; Krangel, MS
MLA Citation
Abarrategui, I, and Krangel, MS. "Regulation of T cell receptor-alpha gene recombination by transcription." Nat Immunol 7.10 (October 2006): 1109-1115.
PMID
16936730
Source
pubmed
Published In
Nature Immunology
Volume
7
Issue
10
Publish Date
2006
Start Page
1109
End Page
1115
DOI
10.1038/ni1379

A role for MAPK in feedback inhibition of Tcrb recombination.

The Tcrb locus is subject to a host of regulatory mechanisms that impart a strict cell and developmental stage-specific order to variable (V), diversity (D), and joining (J) gene segment recombination. The Tcrb locus is also regulated by allelic exclusion mechanisms, which restrict functional rearrangements to a single allele. The production of a functional rearrangement in CD4-CD8- double-negative (DN) thymocytes leads to the assembly of a pre-TCR and initiates signaling cascades that allow for DN to CD4+CD8+ double-positive (DP) differentiation, proliferation, and feedback inhibition of further Vbeta to DJbeta rearrangement. Feedback inhibition is believed to be controlled, in part, by the loss of Vbeta gene segment accessibility during the DN to DP transition. However, the pre-TCR signaling pathways that lead to the inactivation of Vbeta chromatin have not been determined. Because activation of the MAPK pathway is documented to promote DP differentiation in the absence of allelic exclusion, we characterized the properties of Vbeta chromatin within DP thymocytes generated by a constitutively active Raf1 (Raf-CAAX) transgene. Consistent with previous reports, we show that the Raf-CAAX transgene does not inhibit Tcrb recombination in DN thymocytes. Nevertheless, DP thymocytes generated by Raf-CAAX signals display normal down-regulation of Vbeta segment accessibility and normal feedback inhibition of the Vbeta to DJbeta rearrangement. Therefore, our results emphasize the distinct requirements for feedback inhibition in the DN and DP compartments. Although MAPK activation cannot impose feedback in DN thymocytes, it contributes to feedback inhibition through developmental changes that are tightly linked to DN to DP differentiation.

Authors
Jackson, AM; Krangel, MS
MLA Citation
Jackson, AM, and Krangel, MS. "A role for MAPK in feedback inhibition of Tcrb recombination." J Immunol 176.11 (June 1, 2006): 6824-6830.
PMID
16709842
Source
pubmed
Published In
Journal of immunology (Baltimore, Md. : 1950)
Volume
176
Issue
11
Publish Date
2006
Start Page
6824
End Page
6830

Some nuts are tougher to crack than others.

In this issue of Immunity, Oestreich et al. (2006) show that, during V(D)J recombination, RSSs may have distinct accessibility requirements. Some rely on an enhancer-intrinsic, general chromatin opening function, whereas others require enhancer-promoter interactions that direct local chromatin remodeling.

Authors
Krangel, MS; Carabana, J; Abarrategui, I
MLA Citation
Krangel, MS, Carabana, J, and Abarrategui, I. "Some nuts are tougher to crack than others." Immunity 24.4 (April 2006): 361-363. (Review)
PMID
16618593
Source
pubmed
Published In
Immunity
Volume
24
Issue
4
Publish Date
2006
Start Page
361
End Page
363
DOI
10.1016/j.immuni.2006.04.002

Turning T-cell receptor beta recombination on and off: more questions than answers.

Successful V(D)J recombination at the T-cell receptor beta (Tcrb) locus is critical for early thymocyte development. The locus is subject to a host of regulatory mechanisms that impart a strict developmental order to Tcrb recombination events and that insure that Tcrb recombination occurs in an allelically excluded fashion. Progress has been made in the understanding of the cis-acting control of Tcrb locus chromatin structure and the extent to which such accessibility control can account for the developmental regulation of Tcrb recombination. However, recent studies in our laboratory and elsewhere have made it abundantly clear that accessibility control is only part of the story, and multiple additional mechanisms impact both the developmental activation and inactivation of locus recombination events. Here we evaluate our current understanding of developmental regulation at the Tcrb locus. We highlight the many unresolved issues and we discuss how recent concepts emerging from studies of other antigen receptor loci may (or may not) help to resolve these issues.

Authors
Jackson, AM; Krangel, MS
MLA Citation
Jackson, AM, and Krangel, MS. "Turning T-cell receptor beta recombination on and off: more questions than answers." Immunol Rev 209 (February 2006): 129-141. (Review)
PMID
16448539
Source
pubmed
Published In
Immunological Reviews
Volume
209
Publish Date
2006
Start Page
129
End Page
141
DOI
10.1111/j.0105-2896.2006.00342.x

Allele-specific regulation of TCR beta variable gene segment chromatin structure.

Allelic exclusion of the murine Tcrb locus is imposed at the level of recombination and restricts each cell to produce one functional VDJbeta rearrangement. Allelic exclusion is achieved through asynchronous Vbeta to DJbeta recombination as well as feedback inhibition that terminates recombination once a functional rearrangement has occurred. Because the accessibility of Vbeta gene segment chromatin is diminished as thymocytes undergo allelic exclusion at the CD4(-)CD8(-) (double-negative) to CD4(+)CD8(+) (double-positive) transition, chromatin regulation was thought to be an important component of the feedback inhibition process. However, previous studies of chromatin regulation addressed the status of Tcrb alleles using genetic models in which both alleles remained in a germline configuration. Under physiological conditions, developing thymocytes would undergo Vbeta to DJbeta recombination on one or both alleles before the enforcement of feedback. On rearranged alleles, Vbeta gene segments that in germline configuration are regulated independently of the Tcrb enhancer are now brought into its proximity. We show in this study that in contrast to Vbeta segments on a nonrearranged allele, those situated upstream of a functionally rearranged Vbeta segment are contained in active chromatin as judged by histone H3 acetylation, histone H3 lysine 4 (K4) methylation, and germline transcription. Nevertheless, these Vbeta gene segments remain refractory to recombination in double-positive thymocytes. These results suggest that a unique feedback mechanism may operate independent of chromatin structure to inhibit Vbeta to DJbeta recombination after the double-negative stage of thymocyte development.

Authors
Jackson, AM; Krangel, MS
MLA Citation
Jackson, AM, and Krangel, MS. "Allele-specific regulation of TCR beta variable gene segment chromatin structure." J Immunol 175.8 (October 15, 2005): 5186-5191.
PMID
16210623
Source
pubmed
Published In
Journal of immunology (Baltimore, Md. : 1950)
Volume
175
Issue
8
Publish Date
2005
Start Page
5186
End Page
5191

Regulation of TCR delta and alpha repertoires by local and long-distance control of variable gene segment chromatin structure.

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 approximately 100 variable gene segments contributes substantially to the adult TCRdelta repertoire. Moreover, although most contribute to the TCRalpha repertoire, variable gene segments that are Jalpha 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 TCRdelta 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.

Authors
Hawwari, A; Krangel, MS
MLA Citation
Hawwari, A, and Krangel, MS. "Regulation of TCR delta and alpha repertoires by local and long-distance control of variable gene segment chromatin structure." J Exp Med 202.4 (August 15, 2005): 467-472.
PMID
16087716
Source
pubmed
Published In
The Journal of Experimental Medicine
Volume
202
Issue
4
Publish Date
2005
Start Page
467
End Page
472
DOI
10.1084/jem.20050680

Regulation of T cell receptor alpha gene assembly by a complex hierarchy of germline Jalpha promoters.

Assembly of the gene encoding T cell receptor alpha (Tcra) is characterized by an orderly progression of primary and secondary V(alpha)-to-J(alpha) recombination events across the J(alpha) array, but the targeting mechanisms responsible for this progression are mostly unknown. Studies have shown that the T early-alpha promoter is important in targeting primary Tcra rearrangements. We found that T early-alpha and a previously unknown promoter associated with J(alpha)49 targeted primary recombination to discrete sets of constant alpha region (C(alpha))-distal J(alpha) segments and together directed nearly all normal primary recombination events. Furthermore, deletion of the T early-alpha promoter activated previously suppressed downstream promoters and stimulated primary rearrangement to centrally located J(alpha) segments. Central promoter derepression also occurred after primary rearrangement, thereby providing a mechanism to target secondary recombination events.

Authors
Hawwari, A; Bock, C; Krangel, MS
MLA Citation
Hawwari, A, Bock, C, and Krangel, MS. "Regulation of T cell receptor alpha gene assembly by a complex hierarchy of germline Jalpha promoters." Nat Immunol 6.5 (May 2005): 481-489.
PMID
15806105
Source
pubmed
Published In
Nature Immunology
Volume
6
Issue
5
Publish Date
2005
Start Page
481
End Page
489
DOI
10.1038/ni1189

Regulation of the murine Ddelta2 promoter by upstream stimulatory factor 1, Runx1, and c-Myb.

Accessibility control of V(D)J recombination at Ag receptor loci depends on the coordinate activities of transcriptional enhancers and germline promoters. Recombination of murine Tcrd gene segments is known to be regulated, at least in part, by the Tcrd enhancer (Edelta) situated in the Jdelta2-Cdelta intron. However, there has been little characterization of promoters and other cis-acting elements that are activated by or collaborate with Edelta and that might function to regulate Tcrd gene recombination events. We now describe a strong promoter that is tightly associated with the murine Ddelta2 gene segment. EMSAs reveal that upstream stimulatory factor 1, Runx1, c-Myb, lymphoid enhancer binding factor 1, NF1, and E47 all interact with this promoter in vitro. Of these, upstream stimulatory factor 1, Runx1, and c-Myb appear necessary for full promoter activity in transiently transfected cells. Moreover, the same three factors were found to interact with the promoter in vivo by chromatin immunoprecipitation. We suggest that these factors play important roles as Edelta-dependent regulators of Ddelta2 accessibility in vivo. Consistent with the established roles of c-Myb and Runx factors in Edelta function, we detected low level, enhancer-independent activity of the Ddelta2 promoter in transient transfection experiments. We speculate that the Ddelta2 promoter may play a role as a weak, enhancer-independent regulator in vivo, and might contribute to residual Tcrd rearrangement in Edelta(-/-) mice.

Authors
Carabana, J; Ortigoza, E; Krangel, MS
MLA Citation
Carabana, J, Ortigoza, E, and Krangel, MS. "Regulation of the murine Ddelta2 promoter by upstream stimulatory factor 1, Runx1, and c-Myb." J Immunol 174.7 (April 1, 2005): 4144-4152.
PMID
15778374
Source
pubmed
Published In
Journal of immunology (Baltimore, Md. : 1950)
Volume
174
Issue
7
Publish Date
2005
Start Page
4144
End Page
4152

Regulation of T cell receptor beta allelic exclusion at a level beyond accessibility.

Allelic exclusion of V(beta)-to-DJ(beta) recombination depends on asynchronous rearrangement of alleles of the gene encoding T cell receptor beta in double-negative thymocytes and feedback inhibition that is maintained in double-positive thymocytes. Feedback is thought to be enforced through downregulation of V(beta) accessibility. In an attempt to override this negative regulation, we introduced the enhancer of the gene encoding T cell receptor alpha into the V(beta) gene cluster downstream of V(beta)12. In double-negative thymocytes, the introduced enhancer had no measurable effect on accessibility, but V(beta)12 rearrangement was stimulated and V(beta)12 allelic exclusion was partially subverted. In contrast, double-positive thymocytes showed increased V(beta) transcription and accessibility, but feedback inhibition of V(beta)-to-DJ(beta) recombination remained intact. Our results indicate additional regulatory constraints on V(beta)-to-DJ(beta) recombination that operate beyond the accessibility barrier.

Authors
Jackson, A; Kondilis, HD; Khor, B; Sleckman, BP; Krangel, MS
MLA Citation
Jackson, A, Kondilis, HD, Khor, B, Sleckman, BP, and Krangel, MS. "Regulation of T cell receptor beta allelic exclusion at a level beyond accessibility." Nat Immunol 6.2 (February 2005): 189-197.
PMID
15640803
Source
pubmed
Published In
Nature Immunology
Volume
6
Issue
2
Publish Date
2005
Start Page
189
End Page
197
DOI
10.1038/ni1157

Developmental activation of the TCR alpha enhancer requires functional collaboration among proteins bound inside and outside the core enhancer.

The TCR delta enhancer (Edelta) and TCR alpha enhancer (Ealpha) play critical roles in the temporal and lineage-specific control of V(D)J recombination and transcription at the TCR alphadelta locus, working as a developmental switch controlling a transition from TCR delta to TCR alpha activity during thymocyte development. Previous experiments using a transgenic reporter substrate revealed that substitution of the 116-bp minimal Ealpha, denoted Talpha1-Talpha2, for the entire 1.4-kb Ealpha led to a premature activation of V(D)J recombination. This suggested that binding sites outside of Talpha1-Talpha2 are critical for the strict developmental regulation of TCR alpha rearrangement. We have further analyzed Ealpha to better understand the mechanisms responsible for appropriate developmental regulation in vivo. We found that a 275-bp Ealpha fragment, denoted Talpha1-Talpha4, contains all binding sites required for proper developmental regulation in vivo. This suggests that developmentally appropriate enhancer activation results from a functional interaction between factors bound to Talpha1-Talpha2 and Talpha3-Talpha4. In support of this, EMSAs reveal the formation of a large enhanceosome complex that reflects the cooperative assembly of proteins bound to both Talpha1-Talpha2 and Talpha3-Talpha4. Our data suggest that enhanceosome assembly is critical for developmentally appropriate activation of Ealpha in vivo, and that transcription factors, Sp1 and pCREB, may play unique roles in this process.

Authors
Balmelle, N; Zamarreño, N; Krangel, MS; Hernández-Munain, C
MLA Citation
Balmelle, N, Zamarreño, N, Krangel, MS, and Hernández-Munain, C. "Developmental activation of the TCR alpha enhancer requires functional collaboration among proteins bound inside and outside the core enhancer." J Immunol 173.8 (October 15, 2004): 5054-5063.
PMID
15470049
Source
pubmed
Published In
Journal of immunology (Baltimore, Md. : 1950)
Volume
173
Issue
8
Publish Date
2004
Start Page
5054
End Page
5063

Enforcing order within a complex locus: current perspectives on the control of V(D)J recombination at the murine T-cell receptor alpha/delta locus.

V(D)J recombination proceeds according to defined developmental programs at T-cell receptor (TCR) and immunoglobulin loci as a function of cell lineage and stage of differentiation. Although the molecular details are still lacking, such regulation is thought to occur at the level of accessibility of chromosomal recombination signal sequences to the recombinase. The unique and complex organization of the TCRalpha/delta locus poses intriguing regulatory challenges in this regard: embedded TCRalpha and TCRdelta gene segments rearrange at distinct stages of thymocyte development, there is a highly regulated progression of primary followed by secondary rearrangements involving Jalpha segments, and there are important developmental constraints on V gene segment usage. The locus therefore provides a fascinating laboratory in which to explore the basic mechanisms underlying developmental control. We provide here a current view of cis-acting mechanisms that enforce the TCRalpha/delta locus developmental program, and we emphasize the unresolved issues that command the attention of our and other laboratories.

Authors
Krangel, MS; Carabana, J; Abbarategui, I; Schlimgen, R; Hawwari, A
MLA Citation
Krangel, MS, Carabana, J, Abbarategui, I, Schlimgen, R, and Hawwari, A. "Enforcing order within a complex locus: current perspectives on the control of V(D)J recombination at the murine T-cell receptor alpha/delta locus." Immunol Rev 200 (August 2004): 224-232. (Review)
PMID
15242408
Source
pubmed
Published In
Immunological Reviews
Volume
200
Publish Date
2004
Start Page
224
End Page
232
DOI
10.1111/j.0105-2896.2004.00155.x

Targeted inhibition of V(D)J recombination by a histone methyltransferase.

The tissue- and stage-specific assembly of antigen receptor genes by V(D)J recombination is regulated by changes in the chromatin accessibility of target gene segments. This dynamic remodeling process is coordinated by cis-acting promoters and enhancers, which function as accessibility control elements. The basic epigenetic mechanisms that activate or repress chromatin accessibility to V(D)J recombinase remain unclear. We now demonstrate that a histone methyltransferase overrides accessibility control element function and cripples V(D)J recombination of chromosomal gene segments. The recruited histone methyltransferase induces extensive revisions in the local chromatin environment, including altered histone modifications and de novo methylation of DNA. These findings indicate a key function for histone methyltransferases in the tissue- and stage-specific suppression of antigen receptor gene assembly during lymphocyte development.

Authors
Osipovich, O; Milley, R; Meade, A; Tachibana, M; Shinkai, Y; Krangel, MS; Oltz, EM
MLA Citation
Osipovich, O, Milley, R, Meade, A, Tachibana, M, Shinkai, Y, Krangel, MS, and Oltz, EM. "Targeted inhibition of V(D)J recombination by a histone methyltransferase." Nat Immunol 5.3 (March 2004): 309-316.
PMID
14985714
Source
pubmed
Published In
Nature Immunology
Volume
5
Issue
3
Publish Date
2004
Start Page
309
End Page
316
DOI
10.1038/ni1042

Gene segment selection in V(D)J recombination: accessibility and beyond.

V(D)J recombination assembles genes encoding antigen receptors according to defined developmental programs in immature B and T lymphocytes. The 'accessibility hypothesis' was initially invoked to explain how a single recombinase complex could control the locus and allele specificity of V(D)J recombination. It has been since shown that recombination signal sequences themselves influence recombination efficiency and specificity in ways that had not been previously appreciated. Recent developments have increased our understanding of how the chromatin barrier to V(D)J recombination is regulated, and how chromatin control and the properties of the underlying recombination signal sequences may cooperate to create diverse, lineage-restricted and allelically excluded repertoires of antigen receptors.

Authors
Krangel, MS
MLA Citation
Krangel, MS. "Gene segment selection in V(D)J recombination: accessibility and beyond." Nat Immunol 4.7 (July 2003): 624-630. (Review)
PMID
12830137
Source
pubmed
Published In
Nature Immunology
Volume
4
Issue
7
Publish Date
2003
Start Page
624
End Page
630
DOI
10.1038/ni0703-624

Allelic Exclusion, Isotypic Exclusion, and the Developmental Regulation of V(D)J Recombination

© 2003 Elsevier Ltd. All rights reserved.This chapter focuses on the various mechanisms that impart developmental control to V(D)J recombination and that yield allelically and isotypically excluded antigen receptor repertoires. The lymphoid specificity of V(D)J recombination reflects the regulated expression of recombinase proteins RAG1 and RAG2. Variation in RAG gene expression and protein stability accounts for the two waves of V(D)J recombination events in developing B and T lymphocytes However, neither RAG gene expression nor the RSS constraints can account for the locus-specific developmental control of V(D)J recombination. Rather, developmental control is thought to occur largely through regulation of RAG protein access to RSSs within chromatin. Transcriptional enhancers appear to function, at least in part, by activating germline promoters. These promoters then appear to influence V(D)J recombination in a relatively localized fashion. TCRβ, like IgH, is subject to stringent allelic exclusion, with the product of a functional rearrangement providing a potent feedback signal that blocks further rearrangement at the V-to-DJ step. Co-expression of two functional TCRβ proteins has been detected, but occurs rarely. Available evidence suggests that TCRβ allelic exclusion is associated with changes in Vβ but not DβJβCβ chromatin. TCRα, the TCRγ, and TCRδ genes rearrange without evidence of allelic exclusion. B cells are isotypically excluded in that they usually express κ or λ light chains, but not both. The isotypic exclusion reflects a defined developmental sequence of light chain rearrangement, with κ preceding λ, or with a much higher probability of κ rearrangement.

Authors
Krangel, MS; Schlissel, MS
MLA Citation
Krangel, MS, and Schlissel, MS. "Allelic Exclusion, Isotypic Exclusion, and the Developmental Regulation of V(D)J Recombination." Molecular Biology of B Cells. February 18, 2003. 127-140.
Source
scopus
Publish Date
2003
Start Page
127
End Page
140
DOI
10.1016/B978-012053641-2/50009-5

Distinct roles for c-Myb and core binding factor/polyoma enhancer-binding protein 2 in the assembly and function of a multiprotein complex on the TCR delta enhancer in vivo.

Enhancers and promoters within TCR loci functionally collaborate to modify chromatin structure and to confer accessibility to the transcription and V(D)J recombination machineries during T cell development in the thymus. Two enhancers at the TCRalphadelta locus, the TCR alpha enhancer and the TCR delta enhancer (Edelta), are responsible for orchestrating the distinct developmental programs for V(D)J recombination and transcription of the TCR alpha and delta genes, respectively. Edelta function depends critically on transcription factors core binding factor (CBF)/polyoma enhancer-binding protein 2 (PEBP2) and c-Myb as measured by transcriptional activation of transiently transfected substrates in Jurkat cells, and by activation of V(D)J recombination within chromatin-integrated substrates in transgenic mice. To understand the molecular mechanisms for synergy between these transcription factors in the context of chromatin, we used in vivo footprinting to study the requirements for protein binding to Edelta within wild-type and mutant versions of a human TCR delta minilocus in stably transfected Jurkat cells. Our data indicate that CBF/PEBP2 plays primarily a structural role as it induces a conformational change in the enhanceosome that is associated with augmented binding of c-Myb. In contrast, c-Myb has no apparent affect on CBF/PEBP2 binding, but is critical for transcriptional activation. Thus, our data reveal distinct functions for c-Myb and CBF/PEBP2 in the assembly and function of an Edelta enhanceosome in the context of chromatin in vivo.

Authors
Hernández-Munain, C; Krangel, MS
MLA Citation
Hernández-Munain, C, and Krangel, MS. "Distinct roles for c-Myb and core binding factor/polyoma enhancer-binding protein 2 in the assembly and function of a multiprotein complex on the TCR delta enhancer in vivo." J Immunol 169.8 (October 15, 2002): 4362-4369.
PMID
12370369
Source
pubmed
Published In
Journal of immunology (Baltimore, Md. : 1950)
Volume
169
Issue
8
Publish Date
2002
Start Page
4362
End Page
4369

Regulation of V(D)J recombination: a dominant role for promoter positioning in gene segment accessibility.

Antigen receptor gene assembly is regulated by transcriptional promoters and enhancers, which control the accessibility of gene segments to a lymphocyte-specific V(D)J recombinase. However, it remained unclear whether accessibility depends on the process of transcription itself or chromatin modifications that accompany transcription. By using T cell receptor beta substrates that integrate stably into nuclear chromatin, we show that promoter location, rather than germ-line transcription or histone acetylation, is a primary determinant of recombination efficiency. These spatial constraints on promoter positioning may reflect an RNA polymerase-independent mechanism to target adjacent gene segments for chromatin remodeling events that facilitate rearrangement.

Authors
Sikes, ML; Meade, A; Tripathi, R; Krangel, MS; Oltz, EM
MLA Citation
Sikes, ML, Meade, A, Tripathi, R, Krangel, MS, and Oltz, EM. "Regulation of V(D)J recombination: a dominant role for promoter positioning in gene segment accessibility." Proc Natl Acad Sci U S A 99.19 (September 17, 2002): 12309-12314.
PMID
12196630
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
99
Issue
19
Publish Date
2002
Start Page
12309
End Page
12314
DOI
10.1073/pnas.182166699

Regulation of the TCRalpha repertoire by the survival window of CD4(+)CD8(+) thymocytes.

T cell receptor (TCR) alpha alleles undergo primary and secondary rearrangement in double-positive (DP) thymocytes. By analyzing TCRalpha rearrangement in orphan nuclear receptor RORgamma-deficient mice, in which the DP lifespan is shorter, and in Bcl-x(L)-transgenic mice, in which the DP lifespan is extended, we show that the progression of secondary V(alpha) to J(alpha) rearrangements is controlled by DP thymocyte survival. In addition, because Bcl-x(L) induces a bias towards 3' J(alpha) usage in peripheral T cells, we conclude that the programmed cell death of DP thymocytes is not simply a consequence of failed positive selection. Rather, it limits the progression of rearrangement along the J(alpha) locus and the opportunities for positive selection, thereby regulating the TCRalpha repertoire.

Authors
Guo, J; Hawwari, A; Li, H; Sun, Z; Mahanta, SK; Littman, DR; Krangel, MS; He, Y-W
MLA Citation
Guo, J, Hawwari, A, Li, H, Sun, Z, Mahanta, SK, Littman, DR, Krangel, MS, and He, Y-W. "Regulation of the TCRalpha repertoire by the survival window of CD4(+)CD8(+) thymocytes." Nat Immunol 3.5 (May 2002): 469-476.
PMID
11967541
Source
pubmed
Published In
Nature Immunology
Volume
3
Issue
5
Publish Date
2002
Start Page
469
End Page
476
DOI
10.1038/ni791

A change in the structure of Vbeta chromatin associated with TCR beta allelic exclusion.

To investigate chromatin control of TCR beta rearrangement and allelic exclusion, we analyzed TCR beta chromatin structure in double negative (DN) thymocytes, which are permissive for TCR beta recombination, and in double positive (DP) thymocytes, which are postallelic exclusion and nonpermissive for Vbeta to DbetaJbeta recombination. Histone acetylation mapping and DNase I sensitivity studies indicate Vbeta and DbetaJbeta segments to be hyperacetylated and accessible in DN thymocytes. However, they are separated from each other by hypoacetylated and inaccessible trypsinogen chromatin. The transition from DN to DP is accompanied by selective down-regulation of Vbeta acetylation and accessibility. The level of DP acetylation and accessibility is minimal for five of six Vbeta segments studied but remains substantial for one. Hence, the observed changes in Vbeta chromatin structure appear sufficient to account for allelic exclusion of many Vbeta segments. They may contribute to, but not by themselves fully account for, allelic exclusion of others.

Authors
Tripathi, R; Jackson, A; Krangel, MS
MLA Citation
Tripathi, R, Jackson, A, and Krangel, MS. "A change in the structure of Vbeta chromatin associated with TCR beta allelic exclusion." J Immunol 168.5 (March 1, 2002): 2316-2324.
PMID
11859121
Source
pubmed
Published In
Journal of immunology (Baltimore, Md. : 1950)
Volume
168
Issue
5
Publish Date
2002
Start Page
2316
End Page
2324

Assessing a role for enhancer-blocking activity in gene regulation within the murine T-cell receptor alpha/delta locus.

Although situated close together within the T-cell receptor (TCR) alpha/delta locus, TCR delta and TCR alpha gene segments are controlled by two developmental stage-specific enhancers and are activated according to distinct developmental programmes. We previously used a stable transfection colony assay to identify an enhancer-blocking element, blocking element alpha/delta-1 (BEAD-1), between the TCR delta and alpha gene segments of the human TCR alpha/delta locus. We hypothesized that enhancer-blocking by BEAD-1 might be required to prevent the TCR delta enhancer from activating TCR alpha gene segment transcription and rearrangement at the double negative stage of thymocyte development. Here, we used a transfection approach to define partial enhancer-blocking activity in an analogous position of the murine TCR alpha/delta locus. To test the functional significance of this activity in vivo, we used gene targeting to delete the region from the endogenous locus. We found no perturbation of TCR delta and TCR alpha gene expression and rearrangement on targeted alleles, indicating that enhancer-blocking activity in this region is not required to maintain the developmentally distinct activation profiles of the two genes. We suggest that appropriate regulation may be achieved as a result of intrinsic biases in enhancer-promoter interactions or a developmental stage specificity to promoter function that is distinct from any additional specificity imposed by the enhancers themselves.

Authors
Sleckman, BP; Carabana, J; Zhong, X; Krangel, MS
MLA Citation
Sleckman, BP, Carabana, J, Zhong, X, and Krangel, MS. "Assessing a role for enhancer-blocking activity in gene regulation within the murine T-cell receptor alpha/delta locus." Immunology 104.1 (September 2001): 11-18.
PMID
11576215
Source
pubmed
Published In
Immunology
Volume
104
Issue
1
Publish Date
2001
Start Page
11
End Page
18

V(D)J recombination becomes accessible.

Authors
Krangel, MS
MLA Citation
Krangel, MS. "V(D)J recombination becomes accessible." J Exp Med 193.7 (April 2, 2001): F27-F30.
PMID
11283161
Source
pubmed
Published In
The Journal of Experimental Medicine
Volume
193
Issue
7
Publish Date
2001
Start Page
F27
End Page
F30

Chemokines have diverse abilities to form solid phase gradients.

Chemokines play critical roles in leukocyte recruitment into sites of inflammation such as rheumatoid arthritis (RA). While chemokines immobilized on endothelium (solid-phase), but not soluble chemokines, direct rolling leukocytes to firmly adhere to endothelium, soluble and solid-phase chemokine gradients may play important roles in leukocyte extravasation into the tissue. In this study, we have sought to determine (1) if chemokines can be immobilized on structures in the extravascular space, (2) the mechanisms by which chemokines may be immobilized, and (3) if different chemokines have similar potentials to form solid-phase gradients. While secreted alkaline phosphatase (SEAP)-tagged chemokines SLC (CCL21), TARC (CCL17), and RANTES (CCL5) bound to mast cells and the extracellular matrix (ECM) in RA synovium under physiologic salt conditions, MCP1 (CCL2), MIP1 alpha (CCL3), MIP1 beta (CCL4), and fractalkine (FKN, CX3CL1) fusion proteins did not detectably bind. Chemokine binding to ECM and mast cells in situ and to immobilized heparin was inhibited by high salt and glycosaminoglycans (GAGs) heparin, heparan sulfate, chondroitin sulfate, and dermatan sulfate, but not by dextran or hyaluronan, indicating that the chemokines bind to highly sulfated GAGs. Chemokine binding to synovial structures correlated strongly with avidity of chemokine binding to heparin (SLC > TARC > RANTES > MIP1 beta > MCP1 > MIP1 alpha > FKN). A RANTES mutant with decreased avidity for heparin was not able to bind to ECM or mast cells. Thus, these data indicate that chemokines can bind to ECM and mast cell granule constituents in situ via interactions with GAGs. Further, only a subset of chemokines were able to bind efficiently to structures in the extravascular space, indicating that chemokines may form different types of gradients based on their GAG binding ability and that chemotactic gradients in tissues may be quite complex.

Authors
Patel, DD; Koopmann, W; Imai, T; Whichard, LP; Yoshie, O; Krangel, MS
MLA Citation
Patel, DD, Koopmann, W, Imai, T, Whichard, LP, Yoshie, O, and Krangel, MS. "Chemokines have diverse abilities to form solid phase gradients." Clin Immunol 99.1 (April 2001): 43-52.
PMID
11286540
Source
pubmed
Published In
Clinical Immunology
Volume
99
Issue
1
Publish Date
2001
Start Page
43
End Page
52
DOI
10.1006/clim.2000.4997

Chemokine production by G protein-coupled receptor activation in a human mast cell line: roles of extracellular signal-regulated kinase and NFAT.

Chemoattractants are thought to be the first mediators generated at sites of bacterial infection. We hypothesized that signaling through G protein-coupled chemoattractant receptors may stimulate cytokine production. To test this hypothesis, a human mast cell line (HMC-1) that normally expresses receptors for complement components C3a and C5a at low levels was stably transfected to express physiologic levels of fMLP receptors. We found that fMLP, but not C3a or C5a, induced macrophage inflammatory protein (MIP)-1ss (CCL4) and monocyte chemoattractant protein-1 (CCL2) mRNA and protein. Although fMLP stimulated both sustained Ca(2+) mobilization and phosphorylation of extracellular signal-regulated kinase (ERK), these responses to C3a or C5a were transient. However, transient expression of C3a receptors in HMC-1 cells rendered the cells responsive to C3a for sustained Ca(2+) mobilization and MIP-1ss production. The fMLP-induced chemokine production was blocked by pertussis toxin, PD98059, and cyclosporin A, which respectively inhibit G(i)alpha activation, mitgen-activated protein kinase kinase-mediated ERK phosphorylation, and calcineurin-mediated activation of NFAT. Furthermore, fMLP, but not C5a, stimulated NFAT activation in HMC-1 cells. These data indicate that chemoattractant receptors induce chemokine production in HMC-1 cells with a selectivity that depends on the level of receptor expression, the length of their signaling time, and the synergistic interaction of multiple signaling pathways, including extracellular signal-regulated kinase phosphorylation, sustained Ca(2+) mobilization and NFAT activation.

Authors
Ali, H; Ahamed, J; Hernandez-Munain, C; Baron, JL; Krangel, MS; Patel, DD
MLA Citation
Ali, H, Ahamed, J, Hernandez-Munain, C, Baron, JL, Krangel, MS, and Patel, DD. "Chemokine production by G protein-coupled receptor activation in a human mast cell line: roles of extracellular signal-regulated kinase and NFAT." J Immunol 165.12 (December 15, 2000): 7215-7223.
PMID
11120854
Source
pubmed
Published In
Journal of immunology (Baltimore, Md. : 1950)
Volume
165
Issue
12
Publish Date
2000
Start Page
7215
End Page
7223

T cell receptor-dependent activation of human lymphocytes through cell surface ganglioside GT1b: implications for innate immunity.

Gangliosides form a component of the glycosphingolipid-rich membrane microdomains recently shown to play an important role in receptor signal transduction. Specific gangliosides also serve as receptors for binding and internalization of bacterial toxins. In the course of characterizing the basis of the native tetanus toxin (TTx) reactivity of a human gamma delta T cell clone, we observed that transfer of the TCR was required to impart TTx reactivity on a TCR-negative recipient T cell. However, the reconstitution of toxin reactivity could be achieved regardless of the antigen specificity of the TCR chains. Further analysis showed that the T cell recognition of native TTx was dependent on the presence of its ganglioside receptor, GT1b, on the T cell surface. Incorporation of exogenous GT1b into plasma membrane conferred TTx reactivity on otherwise non-reactive T cells provided these cells expressed the TCR. Finally, reconstitution of TCR-negative Jurkat T cells with a CD8-CD3zeta chain chimera demonstrated that the cytoplasmic region of the CD3zeta chain was sufficient to couple ganglioside-mediated TTx binding to T cell activation. These data reveal a novel mode of TCR-dependent reactivity to a bacterial toxin that could mobilize a large subset of T cells, thus representing a form of innate immunity. Given the possibility that endogenous ligands may bind to cell surface gangliosides, regulation of their levels and topology on the cell surface may constitute an immunoregulatory mechanism.

Authors
Bukowski, JF; Roncarolo, MG; Spits, H; Krangel, MS; Morita, CT; Brenner, MB; Band, H
MLA Citation
Bukowski, JF, Roncarolo, MG, Spits, H, Krangel, MS, Morita, CT, Brenner, MB, and Band, H. "T cell receptor-dependent activation of human lymphocytes through cell surface ganglioside GT1b: implications for innate immunity." Eur J Immunol 30.11 (November 2000): 3199-3206.
PMID
11093135
Source
pubmed
Published In
European Journal of Immunology
Volume
30
Issue
11
Publish Date
2000
Start Page
3199
End Page
3206
DOI
10.1002/1521-4141(200011)30:11<3199::AID-IMMU3199>3.0.CO;2-Y

A role for histone acetylation in the developmental regulation of VDJ recombination.

VDJ recombination is developmentally regulated in vivo by enhancer-dependent changes in the accessibility of chromosomal recombination signal sequences to the recombinase, but the molecular nature of these changes is unknown. Here histone H3 acetylation was measured along versions of a transgenic VDJ recombination reporter and the endogenous T cell receptor alpha/delta locus. Enhancer activity was shown to impart long-range, developmentally regulated changes in H3 acetylation, and H3 acetylation status was tightly linked to VDJ recombination. H3 hyperacetylation is proposed as a molecular mechanism coupling enhancer activity to accessibility for VDJ recombination.

Authors
McMurry, MT; Krangel, MS
MLA Citation
McMurry, MT, and Krangel, MS. "A role for histone acetylation in the developmental regulation of VDJ recombination." Science 287.5452 (January 21, 2000): 495-498.
PMID
10642553
Source
pubmed
Published In
Science
Volume
287
Issue
5452
Publish Date
2000
Start Page
495
End Page
498

Accessibility control of T cell receptor gene rearrangement in developing thymocytes. The TCR alpha/delta locus.

The joining of T cell receptor (TCR) and immunoglobulin (Ig) gene segments through the process of V(D)J recombination occurs in a lineage-specific and developmental-stage-specific way during the early stages of lymphocyte development. Such developmental regulation is thought to be mediated through the control of gene segment accessibility to the recombinase. We have studied the regulation of V(D)J recombination at the TCR alpha/delta locus, because this locus provides a fascinating model in which distinct sets of gene segments are activated at different stages of T cell development. The transcriptional enhancers Edelta and Ealpha have been implicated as critical regulators that, in conjunction with other cis-acting elements, confer region-specific and developmental-stage-specific changes in gene segment accessibility within TCR alpha/delta locus chromatin. Current work suggests that they may do so by functioning as regional modulators of histone acetylation.

Authors
Krangel, MS; McMurry, MT; Hernandez-Munain, C; Zhong, XP; Carabana, J
MLA Citation
Krangel, MS, McMurry, MT, Hernandez-Munain, C, Zhong, XP, and Carabana, J. "Accessibility control of T cell receptor gene rearrangement in developing thymocytes. The TCR alpha/delta locus." Immunol Res 22.2-3 (2000): 127-135. (Review)
PMID
11339350
Source
pubmed
Published In
Immunologic Research
Volume
22
Issue
2-3
Publish Date
2000
Start Page
127
End Page
135
DOI
10.1385/IR:22:2-3:127

Flanking nuclear matrix attachment regions synergize with the T cell receptor delta enhancer to promote V(D)J recombination.

Previous studies have identified nuclear matrix attachment regions (MARs) that are closely associated with transcriptional enhancers in the IgH, Igkappa, and T cell receptor (TCR) beta loci, but have yielded conflicting information regarding their functional significance. In this report, a combination of in vitro and in situ mapping approaches was used to localize three MARs associated with the human TCR delta gene. Two of these are located within the Jdelta3-Cdelta intron, flanking the core TCR delta enhancer (Edelta) both 5' and 3' in a fashion reminiscent of the Ig heavy chain intronic enhancer-associated MARs. The third is located about 20 kb upstream, tightly linked to Ddelta1 and Ddelta2. We have previously used a transgenic minilocus V(D)J recombination reporter to establish that Edelta functions as a developmental regulator of V(D)J recombination, and that it does so by modulating substrate accessibility to the V(D)J recombinase. We show here that the Edelta-associated MARs function synergistically with the core Edelta to promote V(D)J recombination in this system, as they are required for enhancer-dependent transgene rearrangement in single-copy transgene integrants.

Authors
Zhong, XP; Carabaña, J; Krangel, MS
MLA Citation
Zhong, XP, Carabaña, J, and Krangel, MS. "Flanking nuclear matrix attachment regions synergize with the T cell receptor delta enhancer to promote V(D)J recombination." Proc Natl Acad Sci U S A 96.21 (October 12, 1999): 11970-11975.
PMID
10518560
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
96
Issue
21
Publish Date
1999
Start Page
11970
End Page
11975

Structure and function of the glycosaminoglycan binding site of chemokine macrophage-inflammatory protein-1 beta.

The ability of chemokines to bind to glycosaminoglycans (GAGs) on cell surfaces and in the extracellular matrix is thought to play a crucial role in chemokine function. We investigated the structural basis for chemokine binding to GAGs by using in vitro mutagenesis to identify amino acids of chemokine macrophage-inflammatory protein-1 beta (MIP-1 beta) that contribute to its interaction with the model GAG heparin. Among six basic residues that are organized into a single basic domain in the folded MIP-1 beta monomer, three (R18, K45, and R46) were found to contribute significantly to heparin binding. Of these, R46 was found to play a dominant role, and proved essential for the interaction of MIP-1 beta with both heparin and heparan sulfate in physiological salt. The results of this mutational analysis have implications for the structure of the MIP-1 beta-heparin complex, and a comparison of these results with those obtained by mutational analysis of the MIP-1 alpha-heparin interaction suggests a possible structural difference between the MIP-1 beta-heparin and MIP-1 alpha-heparin complexes. To determine whether GAG binding plays an important role in receptor binding and cellular activation by MIP-1 beta, the activities of wild-type MIP-1 beta and R46-substituted MIP-1 beta were compared in assays of T lymphocyte chemotaxis. The two proteins proved equipotent in this assay, arguing that interaction of MIP-1 beta with GAGs is not intrinsically required for functional interaction of MIP-1 beta with its receptor.

Authors
Koopmann, W; Ediriwickrema, C; Krangel, MS
MLA Citation
Koopmann, W, Ediriwickrema, C, and Krangel, MS. "Structure and function of the glycosaminoglycan binding site of chemokine macrophage-inflammatory protein-1 beta." J Immunol 163.4 (August 15, 1999): 2120-2127.
PMID
10438952
Source
pubmed
Published In
Journal of immunology (Baltimore, Md. : 1950)
Volume
163
Issue
4
Publish Date
1999
Start Page
2120
End Page
2127

Enhancer-blocking activity within the DNase I hypersensitive site 2 to 6 region between the TCR alpha and Dad1 genes.

Although tightly linked, the TCR alpha and delta genes are expressed specifically in T lymphocytes, whereas the Dad1 gene is ubiquitously expressed. Between TCR alpha and Dad1 are eight DNase I hypersensitive sites (HS). HS1 colocalizes with the TCR alpha enhancer (Ealpha) and is T cell-specific; HS2, -3, -4, -5, and -6 map downstream of HS1 and are tissue-nonspecific. The region spanning HS2-6 was reported to display chromatin-opening activity and to confer copy number-dependent and integration site-independent transgene expression in transgenic mice. Here, we demonstrate that HS2-6 also displays enhancer-blocking activity, as it can block an enhancer from activating a promoter when located between the two in a chromatin-integrated context, and can do so without repressing either the enhancer or the promoter. Multiple enhancer-blocking elements are arrayed across HS2-6. We show that HS2-6 by itself does not activate transcription in chromatin context, but can synergize with an enhancer when located upstream of an enhancer and promoter. We propose that HS2-6 primarily functions as an insulator or boundary element that may be critical for the autonomous regulation of the TCR alpha and Dad1 genes.

Authors
Zhong, XP; Krangel, MS
MLA Citation
Zhong, XP, and Krangel, MS. "Enhancer-blocking activity within the DNase I hypersensitive site 2 to 6 region between the TCR alpha and Dad1 genes." J Immunol 163.1 (July 1, 1999): 295-300.
PMID
10384128
Source
pubmed
Published In
Journal of immunology (Baltimore, Md. : 1950)
Volume
163
Issue
1
Publish Date
1999
Start Page
295
End Page
300

A developmental switch from TCR delta enhancer to TCR alpha enhancer function during thymocyte maturation.

V(D)J recombination and transcription within the TCR alpha/delta locus are regulated by three characterized cis-acting elements: the TCR delta enhancer (Edelta), TCR alpha enhancer (Ealpha), and T early alpha (TEA) promoter. Analysis of enhancer and promoter occupancy and function in developing thymocytes in vivo indicates Edelta and Ealpha to be developmental-stage-specific enhancers, with Edelta "on" and Ealpha "off" in double-negative III thymocytes and Edelta "off" and Ealpha "on" in double-positive thymocytes. Edelta downregulation reflects a loss of occupancy. Surprisingly, Ealpha and TEA are extensively occupied even prior to activation. TCR delta downregulation in double-positive thymocytes depends on two events, Edelta inactivation and removal of TCR delta from the influence of Ealpha by chromosomal excision.

Authors
Hernández-Munain, C; Sleckman, BP; Krangel, MS
MLA Citation
Hernández-Munain, C, Sleckman, BP, and Krangel, MS. "A developmental switch from TCR delta enhancer to TCR alpha enhancer function during thymocyte maturation." Immunity 10.6 (June 1999): 723-733.
PMID
10403647
Source
pubmed
Published In
Immunity
Volume
10
Issue
6
Publish Date
1999
Start Page
723
End Page
733

Regulation of chromatin accessibility for V(D)J recombination.

Authors
Hernandez-Munain, C; McMurry, MT; Krangel, MS
MLA Citation
Hernandez-Munain, C, McMurry, MT, and Krangel, MS. "Regulation of chromatin accessibility for V(D)J recombination." Cold Spring Harb Symp Quant Biol 64 (1999): 183-189. (Review)
PMID
11232283
Source
pubmed
Published In
Cold Spring Harbor Laboratory: Symposia on Quantitative Biology
Volume
64
Publish Date
1999
Start Page
183
End Page
189

Developmental regulation of V(D)J recombination at the TCR alpha/delta locus.

The T-cell receptor (TCR) alpha/delta locus includes a large number of V, D, J and C gene segments that are used to produce functional TCR delta and TCR alpha chains expressed by distinct subsets of T lymphocytes. V(D)J recombination events within the locus are regulated as a function of developmental stage and cell lineage during T-lymphocyte differentiation in the thymus. The process of V(D)J recombination is regulated by cis-acting elements that modulate the accessibility of chromosomal substrates to the recombinase. Here we evaluate how the assembly of transcription factor complexes onto enhancers, promoters and other regulatory elements within the TCR alpha/delta locus imparts developmental control to VDJ delta and VJ alpha rearrangement events. Furthermore, we develop the notion that within a complex locus such as the TCR alpha/delta locus, highly localized and region-specific control is likely to require an interplay between positive regulatory elements and blocking or boundary elements that restrict the influence of the positive elements to defined regions of the locus.

Authors
Krangel, MS; Hernandez-Munain, C; Lauzurica, P; McMurry, M; Roberts, JL; Zhong, XP
MLA Citation
Krangel, MS, Hernandez-Munain, C, Lauzurica, P, McMurry, M, Roberts, JL, and Zhong, XP. "Developmental regulation of V(D)J recombination at the TCR alpha/delta locus." Immunol Rev 165 (October 1998): 131-147. (Review)
PMID
9850858
Source
pubmed
Published In
Immunological Reviews
Volume
165
Publish Date
1998
Start Page
131
End Page
147

Cooperation among multiple transcription factors is required for access to minimal T-cell receptor alpha-enhancer chromatin in vivo.

To understand the molecular basis for the dramatic functional synergy between transcription factors that bind to the minimal T-cell receptor alpha enhancer (Ealpha), we analyzed enhancer occupancy in thymocytes of transgenic mice in vivo by genomic footprinting. We found that the formation of a multiprotein complex on this enhancer in vivo results from the occupancy of previously identified sites for CREB/ATF, TCF/LEF, CBF/PEBP2, and Ets factors as well as from the occupancy of two new sites 5' of the CRE site, GC-I (which binds Sp1 in vitro) and GC-II. Significantly, although all sites are occupied on a wild-type Ealpha, all sites are unoccupied on versions of Ealpha with mutations in the TCF/LEF or Ets sites. Previous in vitro experiments demonstrated hierarchical enhancer occupancy with independent binding of LEF-1 and CREB. Our data indicate that the formation of a multiprotein complex on the enhancer in vivo is highly cooperative and that no single Ealpha binding factor can access chromatin in vivo to play a unique initiating role in its assembly. Rather, the simultaneous availability of multiple enhancer binding proteins is required for chromatin disruption and stable binding site occupancy as well as the activation of transcription and V(D)J recombination.

Authors
Hernandez-Munain, C; Roberts, JL; Krangel, MS
MLA Citation
Hernandez-Munain, C, Roberts, JL, and Krangel, MS. "Cooperation among multiple transcription factors is required for access to minimal T-cell receptor alpha-enhancer chromatin in vivo." Mol Cell Biol 18.6 (June 1998): 3223-3233.
PMID
9584163
Source
pubmed
Published In
Molecular and Cellular Biology
Volume
18
Issue
6
Publish Date
1998
Start Page
3223
End Page
3233

Enhancer control of local accessibility to V(D)J recombinase.

We have studied the role of transcriptional enhancers in providing recombination signal sequence (RSS) accessibility to V(D)J recombinase by examining mice carrying a transgenic human T-cell receptor (TCR) delta gene minilocus. This transgene is composed of unrearranged variable (Vdelta and Vdelta2), diversity (Ddelta3), joining (Jdelta1 and Jdelta3), and constant (Cdelta) gene segments. Previous data indicated that with the TCR delta enhancer (Edelta) present in the Jdelta3-Cdelta intron, V(D)J recombination proceeds stepwise, first V to D and then VD to J. With the enhancer deleted or mutated, V-to-D rearrangement is intact, but VD-to-J rearrangement is inhibited. We proposed that Edelta is necessary for J segment but not D segment accessibility and that J segment inaccessibility in the enhancerless minilocus resulted in the observed V(D)J recombination phenotype. In this study, we tested this notion by using ligation-mediated PCR to assess the formation of recombination-activating gene (RAG)-dependent double-strand breaks (DSBs) at RSSs 3' of Ddelta3 and 5' of Jdelta1. In five lines of mice carrying multicopy integrants of constructs that either lacked Edelta or carried an inactivated Edelta, the frequency of DSBs 5' of Jdelta1 was dramatically reduced relative to that in the wild type, whereas the frequency of DSBs 3' of Ddelta3 was unaffected. We interpret these results to indicate that Edelta is required for Jdelta1 but not Ddelta3 accessibility within the minilocus, and we conclude that enhancers regulate V(D)J recombination by providing local accessibility to the recombinase. cis-acting elements other than Edelta must maintain Ddelta3 in an accessible state in the absence of Edelta. The analysis of DSB formation in a single-copy minilocus integrant indicates that efficient DSB formation at the accessible RSS 3' of Ddelta3 requires an accessible partner RSS, arguing that RSS synapsis is required for DSB formation in chromosomal substrates in vivo.

Authors
McMurry, MT; Hernandez-Munain, C; Lauzurica, P; Krangel, MS
MLA Citation
McMurry, MT, Hernandez-Munain, C, Lauzurica, P, and Krangel, MS. "Enhancer control of local accessibility to V(D)J recombinase." Mol Cell Biol 17.8 (August 1997): 4553-4561.
PMID
9234713
Source
pubmed
Published In
Molecular and Cellular Biology
Volume
17
Issue
8
Publish Date
1997
Start Page
4553
End Page
4561

An enhancer-blocking element between alpha and delta gene segments within the human T cell receptor alpha/delta locus.

T cell receptor (TCR) alpha and delta gene segments are organized within a single genetic locus but are differentially regulated during T cell development. An enhancer-blocking element (BEAD-1, for blocking element alpha/delta 1) was localized to a 2.0-kb region 3' of TCR delta gene segments and 5' of TCR alpha joining gene segments within this locus. BEAD-1 blocked the ability of the TCR delta enhancer (Edelta) to activate a promoter when located between the two in a chromatin-integrated construct. We propose that BEAD-1 functions as a boundary that separates the TCR alpha/delta locus into distinct regulatory domains controlled by Edelta and the TCR alpha enhancer, and that it prevents Edelta from opening the chromatin of the TCR alpha joining gene segments for VDJ recombination at an early stage of T cell development.

Authors
Zhong, XP; Krangel, MS
MLA Citation
Zhong, XP, and Krangel, MS. "An enhancer-blocking element between alpha and delta gene segments within the human T cell receptor alpha/delta locus." Proc Natl Acad Sci U S A 94.10 (May 13, 1997): 5219-5224.
PMID
9144218
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
94
Issue
10
Publish Date
1997
Start Page
5219
End Page
5224

Identification of a glycosaminoglycan-binding site in chemokine macrophage inflammatory protein-1alpha.

Chemokines bind to receptors of the seven-transmembrane type on target cells and also bind to glycosaminoglycans (GAGs), including heparin. In this study, we have sought to identify structural motifs mediating binding of the beta-chemokine macrophage inflammatory protein-1alpha (MIP-1alpha) to GAGs. Alignment of beta-chemokine amino acid sequences revealed the presence of several highly conserved basic amino acids, and molecular modeling predicted that the side chains of three of the basic amino acids fold closely together in MIP-1alpha. Site-directed mutagenesis was used to change the conserved basic residues in MIP-1alpha to alanines, and both wild-type and mutant proteins were produced in a transient COS cell expression system. Wild-type MIP-1alpha bound to heparin-Sepharose, while three of the mutants, R18A, R46A, and R48A, failed to bind. Mutant K45A eluted from heparin-Sepharose at lower NaCl concentrations than wild type, while the binding of K61A, with a mutation in the C-terminal alpha-helix, was indistinguishable from that of the wild-type protein. To determine whether GAG-binding capacity is required for receptor binding and cell activation, we performed competition radioligand binding and calcium mobilization experiments using one of the non-heparin-binding mutants, R46A. R46A bound as efficiently as wild-type MIP-1alpha to CCR1 and was equally active in eliciting increases in intracellular free calcium concentrations. Our data define a GAG binding site in MIP-1alpha consisting of three noncontiguous basic amino acids and show that the capacity to bind to GAGs is not a prerequisite for receptor binding or signaling in vitro.

Authors
Koopmann, W; Krangel, MS
MLA Citation
Koopmann, W, and Krangel, MS. "Identification of a glycosaminoglycan-binding site in chemokine macrophage inflammatory protein-1alpha." J Biol Chem 272.15 (April 11, 1997): 10103-10109.
PMID
9092555
Source
pubmed
Published In
The Journal of biological chemistry
Volume
272
Issue
15
Publish Date
1997
Start Page
10103
End Page
10109

Regulation of T cell receptor delta gene rearrangement by CBF/PEBP2.

We have analyzed transgenic mice carrying versions of a human T cell receptor (TCR)-delta gene minilocus to study the developmental control of VDJ (variable/diversity/joining) recombination. Previous data indicated that a 1.4-kb DNA fragment carrying the TCR-delta enhancer (E(delta)) efficiently activates minilocus VDJ recombination in vivo. We tested whether the transcription factor CBF/PEBP2 plays an important role in the ability of E(delta) to activate VDJ recombination by analyzing VDJ recombination in mice carrying a minilocus in which the deltaE3 element of E(delta) includes a mutated CBF/PEBP2 binding site. The enhancer-dependent VD to J step of minilocus rearrangement was dramatically inhibited in three of four transgenic lines, arguing that the binding of CBF/PEBP2 plays a role in modulating local accessibility to the VDJ recombinase in vivo. Because mutation of the deltaE3 binding site for the transcription factor c-Myb had previously established a similar role for c-Myb, and because a 60-bp fragment of E(delta) carrying deltaE3 and deltaE4 binding sites for CBF/PEBP2, c-Myb, and GATA-3 displays significant enhancer activity in transient transfection experiments, we tested whether this fragment of E(delta) is sufficient to activate VDJ recombination in vivo. This fragment failed to efficiently activate the enhancer-dependent VD to J step of minilocus rearrangement in all three transgenic lines examined, indicating that the binding of CBF/PEBP2 and c-Myb to their cognate sites within E(delta), although necessary, is not sufficient for the activation of VDJ recombination by E(delta). These results imply that CBF/PEBP2 and c-Myb collaborate with additional factors that bind elsewhere within E(delta) to modulate local accessibility to the VDJ recombinase in vivo.

Authors
Lauzurica, P; Zhong, XP; Krangel, MS; Roberts, JL
MLA Citation
Lauzurica, P, Zhong, XP, Krangel, MS, and Roberts, JL. "Regulation of T cell receptor delta gene rearrangement by CBF/PEBP2." J Exp Med 185.7 (April 7, 1997): 1193-1201.
PMID
9104806
Source
pubmed
Published In
The Journal of Experimental Medicine
Volume
185
Issue
7
Publish Date
1997
Start Page
1193
End Page
1201

Regulation of I-309 gene expression in human monocytes by endogenous interleukin-1.

Activated human monocytes are a source of numerous beta-chemokines. The present study was conducted to determine whether these cells produce the human beta-chemokine I-309 and to compare the induction requirements of I-309 to those of other beta-chemokines. We demonstrate that appropriately stimulated adherence-purified human peripheral blood monocytes express I-309 transcripts and secreted I-309 protein. Two stimuli, immobilized IgG and lipopolysaccharide (LPS), synergize strongly to induce I-309 gene expression. We further demonstrate that the production of endogenous interleukin (IL)-1alpha plays a crucial role in I-309 induction. Thus, neutralization of endogenous IL-1alpha using an anti-IL-1alpha antiserum inhibits the induction of I-309 transcripts in response to stimulation with immobilized IgG and LPS, and exogenous IL-1alpha or IL-1beta induces I-309 transcripts in monocytes stimulated with immobilized IgG. Immobilized IgG and LPS have the opposite effect on monocyte chemoattractant protein-1 (MCP-1) gene expression, in that the induction observed with either stimulus alone is diminished using the two stimuli in combination. Furthermore, endogenous and exogenous IL-1 can be either stimulatory or inhibitory for MCP-1 gene expression depending on other signals delivered to the monocytes. Immobilized IgG and LPS synergize to induce macrophage inflammatory protein-1alpha transcripts, but endogenous IL-1 does not play a significant role. Thus, each of these beta-chemokine genes is under distinct regulatory control in human monocytes.

Authors
Selvan, RS; Zhou, LJ; Krangel, MS
MLA Citation
Selvan, RS, Zhou, LJ, and Krangel, MS. "Regulation of I-309 gene expression in human monocytes by endogenous interleukin-1." Eur J Immunol 27.3 (March 1997): 687-694.
PMID
9079810
Source
pubmed
Published In
European Journal of Immunology
Volume
27
Issue
3
Publish Date
1997
Start Page
687
End Page
694
DOI
10.1002/eji.1830270317

Developmental regulation of VDJ recombination by the core fragment of the T cell receptor alpha enhancer.

The role of T cell receptor alpha enhancer (E alpha) cis-acting elements in the developmental regulation of VDJ recombination at the TCR alpha/delta locus was examined in transgenic mice containing variants of a minilocus VDJ recombination substrate. We demonstrate that the 116-bp T alpha 1,2 core enhancer fragment of the 1.4-kb E alpha is sufficient to activate the enhancer-dependent step of minilocus rearrangement, and that within T alpha 1,2, intact binding sites for TCF/LEF and Ets family transcription factors are essential. Although minilocus rearrangement under the control of the 1.4-kb E alpha initiates at fetal day 16.5 and is strictly limited to alpha beta T cells, we find that rearrangement under the control of T alpha 1,2 initiates slightly earlier during ontogeny and occurs in both gamma delta and alpha beta T cells. We conclude that the core fragment of E alpha can establish accessibility to the recombinase in developing thymocytes in vivo in a fashion that is dependent on the binding of TCF/LEF and Ets family transcription factors, but that these and other factors that bind to the E alpha core cannot account for the precise developmental onset of accessibility that is provided by the intact E alpha. Rather, our data suggests a critical role for factors that bind E alpha outside of the core T alpha 1,2 region in establishing the precise developmental onset of TCR alpha rearrangement in vivo.

Authors
Roberts, JL; Lauzurica, P; Krangel, MS
MLA Citation
Roberts, JL, Lauzurica, P, and Krangel, MS. "Developmental regulation of VDJ recombination by the core fragment of the T cell receptor alpha enhancer." J Exp Med 185.1 (January 6, 1997): 131-140.
PMID
8996249
Source
pubmed
Published In
The Journal of Experimental Medicine
Volume
185
Issue
1
Publish Date
1997
Start Page
131
End Page
140

Regulation of T cell receptor delta gene rearrangement by c-Myb.

Developmental activation of VDJ recombination at the T cell receptor (TCR) delta locus is controlled by an intronic transcriptional enhancer (E delta). Transcriptional activation by E delta is dependent on c-Myb. To determine whether c-Myb plays a role in the activation of TCR-delta gene rearrangement, we compared VDJ recombination in transgenic mice carrying two versions of a human TCR-delta gene minilocus recombination substrate. One includes a wild-type E delta, whereas the other carries an E delta with a mutation that abrogates c-Myb binding. We demonstrate that an intact Myb binding site is necessary for efficient rearrangement of the minilocus substrate, suggesting that c-Myb plays a crucial role in activating VDJ recombination at the endogenous TCR-delta locus.

Authors
Hernández-Munain, C; Lauzurica, P; Krangel, MS
MLA Citation
Hernández-Munain, C, Lauzurica, P, and Krangel, MS. "Regulation of T cell receptor delta gene rearrangement by c-Myb." J Exp Med 183.1 (January 1, 1996): 289-293.
PMID
8551234
Source
pubmed
Published In
The Journal of Experimental Medicine
Volume
183
Issue
1
Publish Date
1996
Start Page
289
End Page
293

Transcriptional regulation of the human T cell receptor delta gene.

T cell receptor delta gene expression is regulated by a T cell-specific transcriptional enhancer located within the J delta 3-C delta intron. An essential element of the enhancer was localized to a small 30 bp segment denoted delta E3. Two specific factors, CBF/PEBP2 and c-Myb, bind to adjacent sites within delta E3 and cooperate functionally to mediate transcriptional activation. These factors are likely to play essential roles in the developmental activation of the TCR delta gene in vivo.

Authors
Redondo, JM; Hernandez-Munain, C; Krangel, MS
MLA Citation
Redondo, JM, Hernandez-Munain, C, and Krangel, MS. "Transcriptional regulation of the human T cell receptor delta gene." Immunobiology 193.2-4 (July 1995): 288-292. (Review)
PMID
8530157
Source
pubmed
Published In
Immunobiology
Volume
193
Issue
2-4
Publish Date
1995
Start Page
288
End Page
292

c-Myb and core-binding factor/PEBP2 display functional synergy but bind independently to adjacent sites in the T-cell receptor delta enhancer.

A T-cell-specific transcriptional enhancer lies within the J delta 3-C delta intron of the human T-cell receptor delta gene. We have previously shown that a 30-bp element, denoted delta E3, acts as the minimal TCR delta enhancer and that within delta E3, adjacent and precisely spaced binding sites for core-binding factor (CBF/PEBP2) and c-Myb are essential for transcriptional activity. These data suggested that CBF/PEBP2 and c-Myb synergize to mediate transcriptional activity but did not establish the molecular basis for synergy. In this study, we have examined in detail the binding of CBF/PEBP2 and c-Myb to delta E3. We found that CBF/PEBP2 and c-Myb could simultaneously occupy the core site and one of two overlapping Myb sites within delta E3. However, equilibrium binding and kinetic dissociation experiments suggest that the two factors bind to delta E3 independently, rather than cooperatively. This was found to be true by using isoforms of these factors present in extracts of transfected COS-7 cells, as well as the natural factors present in nuclear extracts of the Jurkat T-cell line. We further showed that CBF/PEBP2 and c-Myb provide unique transactivation functions, since the core-Myb combination cannot be substituted by dimerized core or Myb sites. We propose that spatially precise synergy between CBF/PEBP2 and c-Myb may result from the ability of the two factors to form a composite surface that makes unique and stereospecific contacts with one or more additional components of the transcriptional machinery.

Authors
Hernandez-Munain, C; Krangel, MS
MLA Citation
Hernandez-Munain, C, and Krangel, MS. "c-Myb and core-binding factor/PEBP2 display functional synergy but bind independently to adjacent sites in the T-cell receptor delta enhancer." Mol Cell Biol 15.6 (June 1995): 3090-3099.
PMID
7760805
Source
pubmed
Published In
Molecular and Cellular Biology
Volume
15
Issue
6
Publish Date
1995
Start Page
3090
End Page
3099

Erratum: c-Myb and core-binding factor/PEBP2 display functional synergy but bind independently to adjacent sites in the T-cell receptor δ enhancer (Molecular and Cellular Biology (1995) 15:6 (3095))

Authors
Hernandez-Munain, C; Krangel, MS
MLA Citation
Hernandez-Munain, C, and Krangel, MS. "Erratum: c-Myb and core-binding factor/PEBP2 display functional synergy but bind independently to adjacent sites in the T-cell receptor δ enhancer (Molecular and Cellular Biology (1995) 15:6 (3095))." Molecular and Cellular Biology 15.8 (1995): 4659--.
Source
scival
Published In
Molecular and Cellular Biology
Volume
15
Issue
8
Publish Date
1995
Start Page
4659-

The chemokines IL-8, monocyte chemoattractant protein-1, and I-309 are monomers at physiologically relevant concentrations.

The chemokines are a family of immune mediators involved in a wide range of inflammatory processes, most importantly as chemoattractants of monocytes, neutrophils, lymphocytes, and fibroblasts to sites of inflammation. Nuclear magnetic resonance and x-ray crystallographic studies have shown that IL-8 and macrophage-inflammatory protein-1 beta (MIP-1 beta) form noncovalent dimers and that platelet factor-4 (PF-4) forms noncovalent dimers and tetramers, leading to the assumption that, as a family, the chemokines would form multimeric structures. In this study, we analyze the association states of the chemokines IL-8, monocyte chemoattractant protein-1 (MCP-1), and I-309, by using a combination of size exclusion HPLC, sedimentation equilibrium ultracentrifugation, and chemical cross-linking. We find that the association states of MCP-1 and IL-8 are characterized by an equilibrium between monomers and dimers: although dimers predominate at concentrations above 100 microM, these chemokines are almost exclusively monomeric at the nanomolar concentrations at which they display maximal chemotactic activity. I-309, by contrast, remains a monomer at all concentrations tested. I-309 contains two additional cysteine residues (C26 and C68) that are not found in any other members of the chemokine family. We used cyanogen bromide and trypsin digestion strategies to demonstrate that these two residues are linked in a unique intramolecular disulfide bond. Furthermore, by using site-directed mutagenesis, we show that the integrity of this bond is crucial for protein secretion.

Authors
Paolini, JF; Willard, D; Consler, T; Luther, M; Krangel, MS
MLA Citation
Paolini, JF, Willard, D, Consler, T, Luther, M, and Krangel, MS. "The chemokines IL-8, monocyte chemoattractant protein-1, and I-309 are monomers at physiologically relevant concentrations." J Immunol 153.6 (September 15, 1994): 2704-2717.
PMID
8077676
Source
pubmed
Published In
Journal of immunology (Baltimore, Md. : 1950)
Volume
153
Issue
6
Publish Date
1994
Start Page
2704
End Page
2717

Immunochemical and molecular analysis of antigen binding to lipid anchored and soluble forms of an MHC independent human alpha/beta T cell receptor.

We have constructed antigen-specific chimeric human T cell receptor (TCR) molecules deleted of the transmembrane domain and containing the signal sequence for the biosynthesis of the phosphatidyl inositol glycan (GPI) linkage. These membrane-anchored forms of the TCR alpha and beta chains have been expressed in non-T cells, and they are recognized by alpha or beta TCR specific monoclonal antibodies. We have utilized both immunochemical methods and flow cytometry to prove that the enzyme phosphatidylinositol phospholipase C (PI/PLC) is able to cleave the GPI anchored TCR as a heterodimer from the CHO cell surface. We have demonstrated that the alpha/beta TCR heterodimer on the surface of CHO cells will recognize and bind polymers containing fluorescein (FL-polymer), and the binding activity is completely eliminated by the enzyme, PI/PLC. Moreover, soluble forms of the alpha/beta heterodimer will bind tightly to FL substituted sepharose, which demonstrates the retention of biological activity by the TCR after solubilization. Molecular modelling of the putative antigen binding site of the alpha FL beta FL TCR was derived from the known atomic coordinates of eight different hapten or peptide specific antibodies. Mutagenesis of several residues predicted from the model to be important in FL binding gave results consistent with involvement of Ig equivalent CDR2 and CDR3 domains in the antigen binding pocket. Therefore, using a model hapten system in studying recognition of the TCR independent of MHC interactions, we conclude that amino acid residues located in similar positions within CDR domains as compared to the case of MHC restricted TCR recognition are used in the binding of either hapten or peptide antigens.

Authors
Buchwalder, A; Krangel, MS; Hao, P; Diamond, DJ
MLA Citation
Buchwalder, A, Krangel, MS, Hao, P, and Diamond, DJ. "Immunochemical and molecular analysis of antigen binding to lipid anchored and soluble forms of an MHC independent human alpha/beta T cell receptor." Mol Immunol 31.11 (August 1994): 857-872.
PMID
8047075
Source
pubmed
Published In
Molecular Immunology
Volume
31
Issue
11
Publish Date
1994
Start Page
857
End Page
872

Temporal and lineage-specific control of T cell receptor alpha/delta gene rearrangement by T cell receptor alpha and delta enhancers.

To analyze the regulation of gene rearrangement at the T cell receptor (TCR) alpha/delta locus during T cell development, we generated transgenic mice carrying a human TCR delta gene minilocus. We previously showed that the presence of the TCR delta enhancer (E delta) within the J delta 3-C delta intron was required to activate a specific step (V-D to J) of transgene rearrangement, and that rearrangement was activated equivalently in the precursors of alpha beta and gamma delta T cells. To further explore the role of transcriptional enhancers in establishing the developmental pattern of gene rearrangement at the TCR alpha/delta locus, we substituted the TCR alpha enhancer (E alpha) in place of E delta within the transgenic minilocus. We found that V-D-J rearrangement of the E alpha+ minilocus was restricted to the alpha beta T cell subset. Further, we found that although V-D-J rearrangement of the E delta+ minilocus was initiated in the fetal thymus by day 14.5, V-D-J rearrangement of the E alpha+ minilocus did not occur until fetal day 16.5. Finally, whereas V-D-J rearrangement of the E delta+ minilocus is essentially completed within the triple negative population of postnatal thymocytes, V-D-J rearrangement of the E alpha+ minilocus is only initiated late within this population. Since the properties of minilocus rearrangement under the control of E delta and E alpha parallel the properties of V delta-D delta-J delta and V alpha-J alpha rearrangement at the endogenous TCR alpha/delta locus, we conclude that these enhancers play an important role in orchestrating the developmental program of rearrangements at this locus.

Authors
Lauzurica, P; Krangel, MS
MLA Citation
Lauzurica, P, and Krangel, MS. "Temporal and lineage-specific control of T cell receptor alpha/delta gene rearrangement by T cell receptor alpha and delta enhancers." J Exp Med 179.6 (June 1, 1994): 1913-1921.
PMID
8195717
Source
pubmed
Published In
The Journal of Experimental Medicine
Volume
179
Issue
6
Publish Date
1994
Start Page
1913
End Page
1921

Expression of multiple chemokine genes by a human mast cell leukemia.

The chemokines are a large group of cytokines that are recognized to be important mediators of inflammation. In this study we show that the human mast cell leukemia line HMC-1 is a source of multiple chemokines, including I-309, monocyte chemoattractant protein 1, macrophage inflammatory protein-1 alpha, macrophage inflammatory protein-1 beta, RANTES, and interleukin-8. I-309 and MCP-1 transcripts are expressed at low levels in unstimulated HMC-1. However, phorbol ester treatment up-regulates these and other chemokine transcript levels and also up-regulates chemokine protein synthesis and secretion. Induction of chemokine transcripts in HMC-1 requires de novo protein synthesis. We compared the effects of anti-inflammatory glucocorticoids on the expression of chemokine genes in HMC-1 to their effects in activated T-cells. We find that methyl-prednisolone reduces MCP-1 but not other chemokine transcripts in HMC-1, even though there are distinct and more general effects on chemokine transcripts in activated T-cells. These effects are attributed to inhibition of transcription rather than transcript stability. Our results suggest that human mast cells may be a source of multiple chemokines, that glucocorticoids may inhibit the expression of only a subset of these chemokines, and that mast cells and T-cell chemokine expression may occur via distinct regulatory pathways.

Authors
Selvan, RS; Butterfield, JH; Krangel, MS
MLA Citation
Selvan, RS, Butterfield, JH, and Krangel, MS. "Expression of multiple chemokine genes by a human mast cell leukemia." J Biol Chem 269.19 (May 13, 1994): 13893-13898.
PMID
8188667
Source
pubmed
Published In
The Journal of biological chemistry
Volume
269
Issue
19
Publish Date
1994
Start Page
13893
End Page
13898

Enhancer-dependent and -independent steps in the rearrangement of a human T cell receptor delta transgene.

The rearrangement and expression of T cell receptor (TCR) gene segments occurs in a highly ordered fashion during thymic ontogeny of T lymphocytes. To study the regulation of gene rearrangement within the TCR alpha/delta locus, we generated transgenic mice that carry a germline human TCR delta minilocus that includes V delta 1, V delta 2, D delta 3, J delta 1, J delta 3, and C delta segments, and either contains or lacks the TCR delta enhancer. We found that the enhancer-positive construct rearranges stepwise, first V to D, and then V-D to J. Construct V-D rearrangement mimics a unique property of the endogenous TCR delta locus. V-D-J rearrangement is T cell specific, but is equivalent in alpha/beta and gamma/delta T lymphocytes. Thus, either there is no commitment to the alpha/beta and gamma/delta T cell lineages before TCR delta gene rearrangement, or if precommitment occurs, it does not operate directly on TCR delta gene cis-acting regulatory elements to control TCR delta gene rearrangement. Enhancer-negative mice display normal V to D rearrangement, but not V-D to J rearrangement. Thus, the V-D to J step is controlled by the enhancer, but the V to D step is controlled by separate elements. The enhancer apparently controls access to J delta 1 but not D delta 3, suggesting that a boundary between two independently regulated domains of the minilocus lies between these elements. Within the endogenous TCR alpha/delta locus, this boundary may represent the 5' end of a chromatin regulatory domain that is opened by the TCR delta enhancer during T cell development. The position of this boundary may explain the unique propensity of the TCR delta locus to undergo early V to D rearrangement. Our results indicate that the TCR delta enhancer performs a crucial targeting function to regulate TCR delta gene rearrangement during T cell development.

Authors
Lauzurica, P; Krangel, MS
MLA Citation
Lauzurica, P, and Krangel, MS. "Enhancer-dependent and -independent steps in the rearrangement of a human T cell receptor delta transgene." J Exp Med 179.1 (January 1, 1994): 43-55.
PMID
8270882
Source
pubmed
Published In
The Journal of Experimental Medicine
Volume
179
Issue
1
Publish Date
1994
Start Page
43
End Page
55

Regulation of the T-cell receptor delta enhancer by functional cooperation between c-Myb and core-binding factors.

A T-cell-specific transcriptional enhancer lies within the J delta 3-C delta intron of the human T-cell receptor (TCR) delta gene. The 30-bp minimal enhancer element denoted delta E3 carries a core sequence (TGTGGTTT) that binds a T-cell-specific factor, and that is necessary but not sufficient for transcriptional activation. Here we demonstrate that the transcription factor c-Myb regulates TCR delta enhancer activity through a binding site in delta E3 that is adjacent to the core site. Both v-Myb and c-Myb bind specifically to delta E3. The Myb site is necessary for enhancer activity, because a mutation that eliminates Myb binding abolishes transcriptional activation by the delta E3 element and by the 370-bp TCR delta enhancer. Transfection of cells with a c-Myb expression construct upregulates delta E3 enhancer activity, whereas treatment of cells with an antisense c-myb oligonucleotide inhibits delta E3 enhancer activity. Since intact Myb and core sites are both required for delta E3 function, our data argue that c-Myb and core binding factors must cooperate to mediate transcriptional activation through delta E3. Efficient cooperation depends on the relative positioning of the Myb and core sites, since only one of two overlapping Myb sites within delta E3 is functional and alterations of the distance between this site and the core site disrupt enhancer activity. Cooperative regulation by c-Myb and core-binding factors is likely to play an important role in the control of gene expression during T-cell development.

Authors
Hernandez-Munain, C; Krangel, MS
MLA Citation
Hernandez-Munain, C, and Krangel, MS. "Regulation of the T-cell receptor delta enhancer by functional cooperation between c-Myb and core-binding factors." Mol Cell Biol 14.1 (January 1994): 473-483.
PMID
8264615
Source
pubmed
Published In
Molecular and Cellular Biology
Volume
14
Issue
1
Publish Date
1994
Start Page
473
End Page
483

Indistinguishable nuclear factor binding to functional core sites of the T-cell receptor delta and murine leukemia virus enhancers.

We have previously shown that the delta E3 site is an essential element for transcriptional activation by the human T-cell receptor (TCR) delta enhancer and identified two factors, NF-delta E3A and NF-delta E3C, that bound to overlapping core (TGTGGTTT) and E-box motifs within delta E3. In this study, we show that protein binding to the core motif is necessary but not sufficient for transcriptional activation by the delta E3 element. In contrast, protein binding to the E-box motif does not contribute significantly to enhancer activity. A similar core motif present within the enhancers of T-cell-tropic murine retroviruses has been shown to contribute to transcriptional activity of the viral long terminal repeat in T lymphocytes and to viral T-cell tropism. We therefore determined the relationship between the nuclear factors that bind to the TCR delta and Moloney murine leukemia virus core motifs. On the basis of electrophoretic mobility shift binding and competition studies, biochemical analysis of affinity-labeled DNA-binding proteins, and the binding of a purified core binding factor, the proteins that bound to the TCR delta core site were indistinguishable from those that bound to the murine leukemia virus core site. These data argue that DNA-binding proteins that interact with the core site of murine leukemia virus long terminal repeats and contribute to viral T-cell tropism also play an essential role in the T-cell-specific expression of cellular genes.

Authors
Redondo, JM; Pfohl, JL; Hernandez-Munain, C; Wang, S; Speck, NA; Krangel, MS
MLA Citation
Redondo, JM, Pfohl, JL, Hernandez-Munain, C, Wang, S, Speck, NA, and Krangel, MS. "Indistinguishable nuclear factor binding to functional core sites of the T-cell receptor delta and murine leukemia virus enhancers." Mol Cell Biol 12.11 (November 1992): 4817-4823.
PMID
1328863
Source
pubmed
Published In
Molecular and Cellular Biology
Volume
12
Issue
11
Publish Date
1992
Start Page
4817
End Page
4823

The human cytokine I-309 is a monocyte chemoattractant.

The human cytokine I-309 is a small glycoprotein secreted by activated T lymphocytes and structurally related to a number of inflammatory cytokines. To investigate the biological activities of I-309 protein, we produced a stable Chinese hamster ovary cell transfectant, CDI.10, which constitutively secretes I-309 protein into culture supernatant. Affinity chromatography on a heparin-Sepharose matrix followed by reverse-phase HPLC was used to purify to homogeneity a glycoprotein doublet of 15-16 kDa from culture supernatant. Biochemical analysis showed the purified recombinant I-309 glycoprotein to be indistinguishable from the natural I-309 glycoprotein constitutively secreted by the T-cell line IDP2. Purified recombinant I-309 stimulated migration of human monocytes but not neutrophils when tested by in vitro chemotaxis assay. Furthermore, the purified protein transiently increased cytoplasmic free calcium concentration in human peripheral blood monocytes but did not do so in lymphocytes or neutrophils. These results demonstrate that the I-309 gene encodes an inflammatory mediator that specifically stimulates human monocytes.

Authors
Miller, MD; Krangel, MS
MLA Citation
Miller, MD, and Krangel, MS. "The human cytokine I-309 is a monocyte chemoattractant." Proc Natl Acad Sci U S A 89.7 (April 1, 1992): 2950-2954.
PMID
1557400
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
89
Issue
7
Publish Date
1992
Start Page
2950
End Page
2954

Biology and biochemistry of the chemokines: a family of chemotactic and inflammatory cytokines.

Studies conducted in many laboratories over the past several years have resulted in the identification and initial characterization of a large superfamily of structurally and functionally related inflammatory cytokines. This superfamily currently includes 14 distinct members: platelet factor 4, beta-thromboglobulin, neutrophil activating peptide-1/interleukin-8, gro, IP-10, mig, ENA-78, macrophage inflammatory proteins-1 alpha and -1 beta, monocyte chemoattractant protein-1/JE, RANTES, HC-14, C-10, and I-309. Although numerous biological activities have been assigned to these molecules, a common theme is their ability to stimulate the chemotactic migration of distinct sets of cells, including neutrophils, monocytes, lymphocytes, and fibroblasts. Accumulating evidence indicates that these molecules play important roles in mediating cell recruitment and activation necessary for inflammation and the repair of tissue damage.

Authors
Miller, MD; Krangel, MS
MLA Citation
Miller, MD, and Krangel, MS. "Biology and biochemistry of the chemokines: a family of chemotactic and inflammatory cytokines." Crit Rev Immunol 12.1-2 (1992): 17-46. (Review)
PMID
1418604
Source
pubmed
Published In
Critical Reviews in Immunology
Volume
12
Issue
1-2
Publish Date
1992
Start Page
17
End Page
46

Identification of an essential site for transcriptional activation within the human T-cell receptor delta enhancer.

A T-cell-specific transcriptional enhancer was previously identified within the J delta 3-C delta intron of the human T-cell receptor (TCR) delta gene, and seven distinct binding sites for nuclear factors (delta E1 to delta E7) were defined by DNase I footprinting. In this study, we conducted a detailed functional analysis of the various cis-acting DNA sequence elements of the enhancer and show that a 60-bp fragment encompassing delta E3 and delta E4 displays potent enhancer activity, as judged by its ability to activate transcription from the V delta 1 promoter. We show that the interaction of nuclear factors with the delta E3 site is essential for enhancer activity. This element displays significant activity in the absence of additional segments of the enhancer. Further, methylation interference and in vitro mutagenesis identify a site within delta E3 that mediates the binding of two nuclear factors (NF-delta E3A and NF-delta E3C) and that is required for significant transcriptional activation by the enhancer. NF-delta E3C is ubiquitous and may be identical to a previously characterized microE3-binding factor. NF-delta E3A is preferentially expressed in T lymphocytes, and we suggest that this factor may play the dominant role in transcriptional activation through the delta E3 site. This factor interacts with the sequence TGTGGTTT, a motif that is also found within the enhancers of additional TCR and CD3 genes. Nuclear factor binding to delta E4 is also analyzed. One of three specific complexes formed with a delta E4 probe appears to be T-cell specific.

Authors
Redondo, JM; Pfohl, JL; Krangel, MS
MLA Citation
Redondo, JM, Pfohl, JL, and Krangel, MS. "Identification of an essential site for transcriptional activation within the human T-cell receptor delta enhancer." Mol Cell Biol 11.11 (November 1991): 5671-5680.
PMID
1833636
Source
pubmed
Published In
Molecular and Cellular Biology
Volume
11
Issue
11
Publish Date
1991
Start Page
5671
End Page
5680

Evidence for controlled gene rearrangements and cytokine production during development of human TCRγδ+ lymphocytes

Authors
Spits, H; Yssel, H; Brockelhurst, C; Krangel, M
MLA Citation
Spits, H, Yssel, H, Brockelhurst, C, and Krangel, M. "Evidence for controlled gene rearrangements and cytokine production during development of human TCRγδ+ lymphocytes." Current Topics in Microbiology and Immunology 173 (1991): 47-55.
PMID
1833159
Source
scival
Published In
Current topics in microbiology and immunology
Volume
173
Publish Date
1991
Start Page
47
End Page
55

Sequence and chromosomal location of the I-309 gene. Relationship to genes encoding a family of inflammatory cytokines.

We previously reported the isolation and characterization of a cDNA clone, I-309, that encodes a small secreted protein produced by activated human T lymphocytes. This protein is structurally related to a large number of recently identified proteins that are secreted upon cellular activation. In this report we describe the isolation and characterization of the gene encoding I-309. The genomic organization is essentially identical to that found in the genes encoding the structurally similar proteins TCA-3, hJE/MCP-1, and mJE, strengthening the hypothesis that these genes are evolutionarily related. The region of the I-309 gene 5' of the mRNA cap site exhibits extensive nucleotide sequence homology with the same region of the murine gene TCA-3, providing additional evidence that I-309 and TCA-3 are likely to be homologs. Finally, panels of rodent-human somatic cell hybrids were used to map the I-309 gene to human chromosome 17. In conjunction with recent mapping data from other laboratories, this result suggests the presence of a cluster of related genes on this chromosome.

Authors
Miller, MD; Wilson, SD; Dorf, ME; Seuanez, HN; O'Brien, SJ; Krangel, MS
MLA Citation
Miller, MD, Wilson, SD, Dorf, ME, Seuanez, HN, O'Brien, SJ, and Krangel, MS. "Sequence and chromosomal location of the I-309 gene. Relationship to genes encoding a family of inflammatory cytokines." J Immunol 145.8 (October 15, 1990): 2737-2744.
PMID
2212659
Source
pubmed
Published In
Journal of immunology (Baltimore, Md. : 1950)
Volume
145
Issue
8
Publish Date
1990
Start Page
2737
End Page
2744

A distinct wave of human T cell receptor gamma/delta lymphocytes in the early fetal thymus: evidence for controlled gene rearrangement and cytokine production.

The rearrangement and expression of human T cell receptor (TCR)-gamma and -delta gene segments in clonal and polyclonal populations of early fetal and postnatal human TCR-gamma/delta thymocytes were examined. The data suggest that the TCR-gamma and -delta loci rearrange in an ordered and coordinated fashion. Initial rearrangements at the TCR-delta locus join V delta 2 to D delta 3, and initial rearrangements at the TCR-gamma locus join downstream V gamma gene segments (V gamma 1.8 and V gamma 2) to upstream J gamma gene segments associated with C gamma 1. These rearrangements are characterized by minimal junctional diversity. At later times there is a switch at the TCR-delta locus such that V delta 1 is joined to upstream D delta gene segments, and a switch at the TCR-gamma locus such that upstream V gamma gene segments are joined to downstream J gamma gene segments associated with C gamma 2. These rearrangements are characterized by extensive junctional diversity. Programmed rearrangement explains in part the origin of discrete subpopulations of peripheral blood TCR-gamma/delta lymphocytes that have been defined in previous studies. In addition, cytokine production by early fetal and postnatal TCR-gamma/delta thymocyte clones was examined. Fetal thymocyte clones produced significant levels of IL-4 and IL-5 following stimulation, whereas postnatal thymocyte clones did not produce these cytokines. Thus, these cell populations may represent functionally distinct subsets as well.

Authors
Krangel, MS; Yssel, H; Brocklehurst, C; Spits, H
MLA Citation
Krangel, MS, Yssel, H, Brocklehurst, C, and Spits, H. "A distinct wave of human T cell receptor gamma/delta lymphocytes in the early fetal thymus: evidence for controlled gene rearrangement and cytokine production." J Exp Med 172.3 (September 1, 1990): 847-859.
PMID
2167345
Source
pubmed
Published In
The Journal of Experimental Medicine
Volume
172
Issue
3
Publish Date
1990
Start Page
847
End Page
859

A T cell-specific transcriptional enhancer within the human T cell receptor delta locus.

The T cell antigen receptor (TCR) delta gene is located within the TCR alpha locus. A T cell-specific transcriptional enhancer, distinct from the TCR alpha enhancer, has been identified within the J delta 3-C delta intron of the human T cell receptor delta gene. This enhancer activates transcription from the V delta 1 and V delta 3 promoters as well as from heterologous promoters. Enhancer activity has been localized to a 250-bp region that contains multiple binding sites for nuclear proteins. Thus, transcriptional control of the TCR delta and TCR alpha genes is mediated by distinct regulatory elements.

Authors
Redondo, JM; Hata, S; Brocklehurst, C; Krangel, MS
MLA Citation
Redondo, JM, Hata, S, Brocklehurst, C, and Krangel, MS. "A T cell-specific transcriptional enhancer within the human T cell receptor delta locus." Science 247.4947 (March 9, 1990): 1225-1229.
PMID
2156339
Source
pubmed
Published In
Science
Volume
247
Issue
4947
Publish Date
1990
Start Page
1225
End Page
1229

Human T cell receptor-gamma and -delta chain pairing analyzed by transfection of a T cell receptor-delta negative mutant cell line.

Specific TCR V gamma and V delta segments are found to be coordinately used on subpopulations of gamma delta T lymphocytes. The reasons for this phenomenon are unknown, but may include the inability of particular chains expressing unique V delta and V gamma segments to physically associate. V delta 2 is typically used together with V gamma 2 on human peripheral blood gamma delta T lymphocytes. To examine whether V delta 2 can be used in conjunction with distinct V gamma segments, a TCR- mutant of the human gamma delta T cell line MOLT-13, which expresses parental TCR gamma (V gamma 1.3) but not TCR-delta protein, was transfected with plasmids containing full-length TCR-delta cDNA using either V delta 2 or V delta 3. TCR reconstitution was successful in both transfectants and resulted in TCR protein and RNA levels similar to that of the parental MOLT-13 cell line. These cell lines could be activated through their receptors as assessed by increases in cytoplasmic free calcium. These studies imply that physical constraints cannot explain the observed chain pairing preferences. Other possible explanations are discussed.

Authors
Solomon, KR; Krangel, MS; McLean, J; Brenner, MB; Band, H
MLA Citation
Solomon, KR, Krangel, MS, McLean, J, Brenner, MB, and Band, H. "Human T cell receptor-gamma and -delta chain pairing analyzed by transfection of a T cell receptor-delta negative mutant cell line." J Immunol 144.3 (February 1, 1990): 1120-1126.
PMID
2136897
Source
pubmed
Published In
Journal of immunology (Baltimore, Md. : 1950)
Volume
144
Issue
3
Publish Date
1990
Start Page
1120
End Page
1126

A novel polypeptide secreted by activated human T lymphocytes.

We have identified two cDNA clones, I-309 and G-26, which define genes expressed abundantly in activated human PBMC, but at low or undetectable levels in resting PBMC. Based upon nucleotide sequence analysis, both clones are predicted to encode small, structurally related polypeptides, each containing a hydrophobic leader sequence characteristic of secreted proteins and a motif of four conserved cysteine residues. Further, I-309 and G-26 are structurally related to a growing family of genes that apparently encode small polypeptides whose secretion is induced upon cell activation. I-309 represents a previously undescribed human gene. We have generated an anti-peptide antiserum to the I-309 gene product which recognizes proteins in culture supernatants of an activated T cell clone and of COS cells transfected with the I-309 cDNA, supporting the idea that I-309 encodes a secreted protein. Because I-309 encodes a small protein secreted by activated T cells that displays structural features similar to other cytokines, we believe that it defines a novel cytokine with as yet unknown function.

Authors
Miller, MD; Hata, S; De Waal Malefyt, R; Krangel, MS
MLA Citation
Miller, MD, Hata, S, De Waal Malefyt, R, and Krangel, MS. "A novel polypeptide secreted by activated human T lymphocytes." J Immunol 143.9 (November 1, 1989): 2907-2916.
PMID
2809212
Source
pubmed
Published In
Journal of immunology (Baltimore, Md. : 1950)
Volume
143
Issue
9
Publish Date
1989
Start Page
2907
End Page
2916

Expression of human T cell receptor-gamma delta structural forms.

The human TCR-gamma delta occurs in three biochemically distinct forms (forms 1, 2bc, and 2abc). A 40-kDa TCR gamma-chain is disulfide-linked to the TCR delta-chain in form 1, whereas 40-kDa or 55-kDa TCR-gamma polypeptides are noncovalently associated with the TCR delta-chain in forms 2bc and 2abc, respectively. Sequence analysis of TCR-gamma cDNA clones indicates that form 1 utilizes the C gamma 1 gene segment, whereas forms 2bc and 2abc appear to use allelic C gamma 2 gene segments containing either two copies (b and c) or three copies (a, b, and c) of the CII exon, respectively. We transfected TCR-gamma cDNA encoding form 1 or form 2abc into the MOLT-13 cell line that expresses form 2bc. The transfected TCR gamma-chains associate with the resident MOLT-13 TCR-delta, normally part of form 2bc, to yield CD3-associated TCR-gamma delta heterodimers identical to those seen on the donor cell lines (form 1 or 2abc). These transfection experiments show directly that, 1) when a single TCR-delta subunit is available, the presence or absence of disulfide linkage between TCR gamma- and TCR delta-chains is controlled by the TCR gamma-chain, and 2) the difference in the amount of N-linked carbohydrate attached to the transfected TCR-gamma proteins of form 2bc vs form 2abc is influenced by the presence or absence of CII exon copy "a" which appears to alter the secondary and/or tertiary structure of these TCR gamma-chain constant regions, thereby affecting the attachment of N-linked glycans. In contrast to the similar structure and usage of C beta 1 and C beta 2, TCR-gamma delta forms show striking differences in structure and are not equally represented in peripheral blood. Although the role of each form is unknown, it is possible that variable or joining-gene segment selection events or functional differences account for their unequal usage.

Authors
Band, H; Hochstenbach, F; Parker, CM; McLean, J; Krangel, MS; Brenner, MB
MLA Citation
Band, H, Hochstenbach, F, Parker, CM, McLean, J, Krangel, MS, and Brenner, MB. "Expression of human T cell receptor-gamma delta structural forms." J Immunol 142.10 (May 15, 1989): 3627-3633.
PMID
2785559
Source
pubmed
Published In
Journal of immunology (Baltimore, Md. : 1950)
Volume
142
Issue
10
Publish Date
1989
Start Page
3627
End Page
3633

Diversity and organization of human T cell receptor delta variable gene segments.

Previous studies of the human TCR-delta gene identified a single commonly used V delta segment, denoted V delta 1. To better understand the extent of the human TCR-delta V gene repertoire, TCR-delta transcripts and gene rearrangements were examined in a new panel of cloned human TCR-gamma/delta lymphocytes. Through this analysis we identified and determined the structures of two new V delta segments, denoted V delta 2 and V delta 3. These V delta segments are different from previously characterized V alpha segments, supporting the notion that the human V delta and V alpha repertoires are distinct. Examination of V gamma gene segment usage in these cells reveals that the V delta 2 gene segment is used in conjunction with the V gamma 2 gene segment. Blot hybridization indicates that the V delta 2 gene segment lies between V delta 1 and D delta-J delta-C delta, and within 100 kb of the latter. Analysis of genomic clones indicates that the V delta 3 gene segment lies in an inverted orientation, approximately 2 kb 3' of C delta. This implies that rearrangement of V delta 3 to D delta-J delta-C delta occurs by inversion. Together with previous mapping studies, these results indicate that human V delta segments are dispersed, rather than clustered, within the TCR-alpha/delta locus. The analysis of rearrangements in polyclonal thymocyte DNA suggests that there may be a limited number of additional V delta gene segments yet to be characterized.

Authors
Hata, S; Clabby, M; Devlin, P; Spits, H; De Vries, JE; Krangel, MS
MLA Citation
Hata, S, Clabby, M, Devlin, P, Spits, H, De Vries, JE, and Krangel, MS. "Diversity and organization of human T cell receptor delta variable gene segments." J Exp Med 169.1 (January 1, 1989): 41-57.
PMID
2521243
Source
pubmed
Published In
The Journal of Experimental Medicine
Volume
169
Issue
1
Publish Date
1989
Start Page
41
End Page
57

Human T cell receptor gamma delta structure.

Authors
Brenner, MB; Hochstenbach, F; Band, H; Parker, C; McLean, J; Hata, S; Krangel, M
MLA Citation
Brenner, MB, Hochstenbach, F, Band, H, Parker, C, McLean, J, Hata, S, and Krangel, M. "Human T cell receptor gamma delta structure." Advances in Experimental Medicine and Biology 254 (1989): 17-19.
PMID
2530852
Source
scival
Published In
Advances in Experimental Medicine and Biology
Volume
254
Publish Date
1989
Start Page
17
End Page
19

Genomic organization of the human T-cell antigen-receptor alpha/delta locus.

Two clusters of overlapping cosmid clones comprising about 100 kilobases (kb) at the human T-cell antigen-receptor alpha/delta locus were isolated from a genomic library. The structure of the germ-line V delta 1 variable gene segment was determined. V delta 1 is located 8.5 kb downstream of the V alpha 13.1 gene segment, and both V segments are arranged in the same transcriptional orientation. The V alpha 17.1 segment is located between V delta 1 and the D delta, J delta, C delta region (containing the diversity, joining, and constant gene segments). Thus, V delta and V alpha segments are interspersed along the chromosome. The germ-line organization of the D delta 2, J delta 1, and J delta 2 segments was determined. Linkage of C delta to the J alpha region was established by identification of J alpha segments within 20 kb downstream of C delta. The organization of the locus was also analyzed by field-inversion gel electrophoresis. The unrearranged V delta 1 and D delta, J delta, C delta regions are quite distant from each other, apparently separated by a minimum of 175-180 kb.

Authors
Satyanarayana, K; Hata, S; Devlin, P; Roncarolo, MG; De Vries, JE; Spits, H; Strominger, JL; Krangel, MS
MLA Citation
Satyanarayana, K, Hata, S, Devlin, P, Roncarolo, MG, De Vries, JE, Spits, H, Strominger, JL, and Krangel, MS. "Genomic organization of the human T-cell antigen-receptor alpha/delta locus." Proc Natl Acad Sci U S A 85.21 (November 1988): 8166-8170.
PMID
3186718
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
85
Issue
21
Publish Date
1988
Start Page
8166
End Page
8170

Extensive junctional diversity of rearranged human T cell receptor delta genes.

The human T cell receptor delta (TCR delta) gene encodes one component of the TCR gamma delta-CD3 complex found on subsets of peripheral blood and thymic T cells. Human TCR delta diversity was estimated by characterizing rearrangements in TCR gamma delta cell lines and determining the structures of complementary DNA clones representing functional and nonfunctional transcripts in these cell lines. One V delta segment and one J delta segment were identified in all functional transcripts, although a distinct J delta segment was identified in a truncated transcript. Further, one D delta element was identified, and evidence for the use of an additional D delta element was obtained. Thus human TCR delta genes appear to use a limited number of germline elements. However, the apparent use of two D delta elements in tandem coupled with imprecise joining and extensive incorporation of N nucleotides generates unprecedented variability in the junctional region.

Authors
Hata, S; Satyanarayana, K; Devlin, P; Band, H; McLean, J; Strominger, JL; Brenner, MB; Krangel, MS
MLA Citation
Hata, S, Satyanarayana, K, Devlin, P, Band, H, McLean, J, Strominger, JL, Brenner, MB, and Krangel, MS. "Extensive junctional diversity of rearranged human T cell receptor delta genes." Science 240.4858 (June 10, 1988): 1541-1544.
PMID
3259726
Source
pubmed
Published In
Science
Volume
240
Issue
4858
Publish Date
1988
Start Page
1541
End Page
1544

The gamma delta T cell receptor.

Authors
Brenner, MB; Strominger, JL; Krangel, MS
MLA Citation
Brenner, MB, Strominger, JL, and Krangel, MS. "The gamma delta T cell receptor." Adv Immunol 43 (1988): 133-192. (Review)
PMID
3055853
Source
pubmed
Published In
Advances in immunology
Volume
43
Publish Date
1988
Start Page
133
End Page
192

The γδ T cell receptor

Authors
Brenner, MB; Strominger, JL; Krangel, MS
MLA Citation
Brenner, MB, Strominger, JL, and Krangel, MS. "The γδ T cell receptor." Advances in Immunology 43 (1988): 133-192.
Source
scival
Published In
Advances in Immunology
Volume
43
Publish Date
1988
Start Page
133
End Page
192

Identification of putative human T cell receptor delta complementary DNA clones.

A novel T cell receptor (TCR) subunit termed TCR delta, associated with TCR gamma and CD3 polypeptides, was recently found on a subpopulation of human T lymphocytes. T cell-specific complementary DNA clones present in a human TCR gamma delta T cell complementary DNA library were obtained and characterized in order to identify candidate clones encoding TCR delta. One cross-hybridizing group of clones detected transcripts that are expressed in lymphocytes bearing TCR gamma delta but not in other T lymphocytes and are encoded by genes that are rearranged in TCR gamma delta lymphocytes but deleted in other T lymphocytes. Their sequences indicate homology to the variable, joining, and constant elements of other TCR and immunoglobulin genes. These characteristics, as well as the immunochemical data presented in a companion paper, are strong evidence that the complementary DNA clones encode TCR delta.

Authors
Hata, S; Brenner, MB; Krangel, MS
MLA Citation
Hata, S, Brenner, MB, and Krangel, MS. "Identification of putative human T cell receptor delta complementary DNA clones." Science 238.4827 (October 30, 1987): 678-682.
PMID
3499667
Source
pubmed
Published In
Science
Volume
238
Issue
4827
Publish Date
1987
Start Page
678
End Page
682

Immunochemical proof that a novel rearranging gene encodes the T cell receptor delta subunit.

The T cell receptor (TCR) delta protein is expressed as part of a heterodimer with TCR gamma, in association with the CD3 polypeptides on a subset of functional peripheral blood T lymphocytes, thymocytes, and certain leukemic T cell lines. A monoclonal antibody directed against TCR delta was produced that binds specifically to the surface of several TCR gamma delta cell lines and immunoprecipitates the TCR gamma delta as a heterodimer from Triton X-100 detergent lysates and also immunoprecipitates the TCR delta subunit alone after chain separation. A candidate human TCR delta complementary DNA clone (IDP2 O-240/38), reported in a companion paper, was isolated by the subtractive library approach from a TCR gamma delta cell line. This complementary DNA clone was used to direct the synthesis of a polypeptide that is specifically recognized by the monoclonal antibody to TCR delta. This complementary DNA clone thus corresponds to the gene that encodes the TCR delta subunit.

Authors
Band, H; Hochstenbach, F; McLean, J; Hata, S; Krangel, MS; Brenner, MB
MLA Citation
Band, H, Hochstenbach, F, McLean, J, Hata, S, Krangel, MS, and Brenner, MB. "Immunochemical proof that a novel rearranging gene encodes the T cell receptor delta subunit." Science 238.4827 (October 30, 1987): 682-684.
PMID
3672118
Source
pubmed
Published In
Science
Volume
238
Issue
4827
Publish Date
1987
Start Page
682
End Page
684

T-cell receptors of human suppressor cells.

Cells which can suppress the immune response to an antigen (TS cells) appear to be essential for regulation of the immune system. But the characterization of the TS lineage has not been extensive and many are sceptical of studies using uncloned or hybrid T-cell lines. The nature of the antigen receptor on these cells is unclear. T cells of the helper or cytotoxic lineages appear to recognize their targets using the T-cell receptor (TCR) alpha beta-CD3 complex. TCR beta-gene rearrangements are also found in some murine and human suppressor cell lines but others have been shown not to rearrange or express the beta-chain or alpha-chain genes. We previously established TS clones derived from lepromatous leprosy patients which carry the CD8 antigen and recognize antigen in the context of the major histocompatibility complex (MHC) class II molecules in vitro. We here report the characterization of additional MHC-restricted TS clones which rearrange TCR beta genes, express messenger RNA for the alpha and beta chains of the TCR and express clonally unique CD3-associated TCR alpha beta structures on their cell surface but do not express the gamma chain of the gamma delta TCR on the cell surface. We conclude that antigen recognition by at least some human CD8+ suppressor cells is likely to be mediated by TCR alpha beta heterodimers.

Authors
Modlin, RL; Brenner, MB; Krangel, MS; Duby, AD; Bloom, BR
MLA Citation
Modlin, RL, Brenner, MB, Krangel, MS, Duby, AD, and Bloom, BR. "T-cell receptors of human suppressor cells." Nature 329.6139 (October 8, 1987): 541-545.
PMID
2958712
Source
pubmed
Published In
Nature
Volume
329
Issue
6139
Publish Date
1987
Start Page
541
End Page
545
DOI
10.1038/329541a0

Two forms of HLA class I molecules in human plasma.

Soluble HLA-A,-B antigens have previously been detected in human plasma. More recently, these molecules have been demonstrated to be secreted in water soluble form by cell lines and peripheral blood lymphocytes in vitro due to RNA splicing events which delete exon five from a fraction of class I antigen transcripts. In order to determine whether plasma HLA-A,-B molecules arise by this mechanism, their biochemical properties are analyzed. Based upon molecular weight, detergent binding, and lipid binding properties, it was demonstrated that two forms of HLA-A,-B molecules are present in the plasma. A 43 kd form that binds both lipids and detergents is presumed to be a shed version of the membrane form. However, a 39 kd form that fails to bind lipids and detergents has properties identical to those molecules secreted in vitro. It is suggested that these molecules arise by alternative splicing events in vivo that are identical to those characterized in vitro. Although all tested individuals display shed and/or secreted plasma class I molecules, it is striking that HLA-A24 occurs in both a shed and a secreted form at levels higher than all other alleles examined.

Authors
Krangel, MS
MLA Citation
Krangel, MS. "Two forms of HLA class I molecules in human plasma." Hum Immunol 20.2 (October 1987): 155-165.
PMID
3679904
Source
pubmed
Published In
Human Immunology
Volume
20
Issue
2
Publish Date
1987
Start Page
155
End Page
165

Structurally divergent human T cell receptor gamma proteins encoded by distinct C gamma genes.

The human T cell receptor (TCR) gamma polypeptide occurs in structurally distinct forms on certain peripheral blood T lymphocytes. Complementary DNA clones representing the transcripts of functionally rearranged TCR gamma genes in these cells have been analyzed. The expression of a disulfide-linked and a nondisulfide-linked form of TCR gamma correlates with the use of the C gamma 1 and C gamma 2 constant-region gene segments, respectively. Variability in TCR gamma polypeptide size and disulfide linkage is determined by the number of copies and the sequence of a repeated segment of the constant region. Thus C gamma 1 and C gamma 2 are used to generate structurally distinct, yet functional, T3-associated receptor complexes on peripheral blood lymphocytes. Tryptic peptide mapping suggests that the T3-associated TCR gamma and delta peptides in the nondisulfide-linked form are distinct.

Authors
Krangel, MS; Band, H; Hata, S; McLean, J; Brenner, MB
MLA Citation
Krangel, MS, Band, H, Hata, S, McLean, J, and Brenner, MB. "Structurally divergent human T cell receptor gamma proteins encoded by distinct C gamma genes." Science 237.4810 (July 3, 1987): 64-67.
PMID
2955517
Source
pubmed
Published In
Science
Volume
237
Issue
4810
Publish Date
1987
Start Page
64
End Page
67

T3 glycoprotein is functional although structurally distinct on human T-cell receptor gamma T lymphocytes.

The T-cell receptor (TCR) gamma gene product occurs in association with T3 (CD3) polypeptides on the surface of human T lymphocytes. TCR gamma lymphocytes express arrays of T3 polypeptides distinct from those typically observed on TCR alpha beta lymphocytes. This report demonstrates that identical T3 gamma, delta, and epsilon polypeptides are synthesized by TCR gamma lymphocytes and TCR alpha beta lymphocytes. However, the processing of T3 delta oligosaccharides is distinct in the two cell types. This observation may suggest distinct quaternary structures of these receptor complexes. Despite these structural differences, the T3 molecule on TCR gamma lymphocytes is functional. It is associated with and comodulates with TCR gamma and it serves as a substrate for protein kinase C-mediated phosphorylation. Anti-T3 monoclonal antibodies induce a rapid increase in cytoplasmic free calcium, indicating that the receptor complex is involved in signal transduction and triggering of TCR gamma lymphocytes.

Authors
Krangel, MS; Bierer, BE; Devlin, P; Clabby, M; Strominger, JL; McLean, J; Brenner, MB
MLA Citation
Krangel, MS, Bierer, BE, Devlin, P, Clabby, M, Strominger, JL, McLean, J, and Brenner, MB. "T3 glycoprotein is functional although structurally distinct on human T-cell receptor gamma T lymphocytes." Proc Natl Acad Sci U S A 84.11 (June 1987): 3817-3821.
PMID
3108880
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
84
Issue
11
Publish Date
1987
Start Page
3817
End Page
3821

Endocytosis and recycling of the T3-T cell receptor complex. The role of T3 phosphorylation.

An assay has been developed to assess the dynamics of cell surface glycoproteins, in which neuraminidase digestion of intact cells is used to determine the fate of cell surface molecules initially labelled via lactoperoxidase-catalyzed iodination. This approach has been used to demonstrate the constitutive endocytosis and recycling of the T3-T cell receptor (T CR) complex on the human T leukemic cell line HPB-MLT. Stable populations of both phosphorylated and nonphosphorylated forms of the T3 gamma peptide have been identified in these cells. Whereas the former are constitutively endocytosed, the latter appear to be excluded from this pathway. The results presented indicate that T3 gamma phosphorylation may control the endocytosis and recycling of the T3-TCR complex on this cell line.

Authors
Krangel, MS
MLA Citation
Krangel, MS. "Endocytosis and recycling of the T3-T cell receptor complex. The role of T3 phosphorylation." J Exp Med 165.4 (April 1, 1987): 1141-1159.
PMID
3104527
Source
pubmed
Published In
The Journal of Experimental Medicine
Volume
165
Issue
4
Publish Date
1987
Start Page
1141
End Page
1159

Two forms of the T-cell receptor gamma protein found on peripheral blood cytotoxic T lymphocytes.

The T-cell receptor (TCR) gamma polypeptide is expressed associated with CD3 (T3) on the surface of normal human peripheral blood lymphocytes. These cells function as non-MHC-restricted cytotoxic T lymphocytes (CTL)and thus may play an important role in host immune defence. The TCR gamma polypeptide occurs as a dimer in at least two molecular forms based on the absence or presence of disulphide linkage. These forms use TCR gamma polypeptides with strikingly different peptide backbone sizes.

Authors
Brenner, MB; McLean, J; Scheft, H; Riberdy, J; Ang, SL; Seidman, JG; Devlin, P; Krangel, MS
MLA Citation
Brenner, MB, McLean, J, Scheft, H, Riberdy, J, Ang, SL, Seidman, JG, Devlin, P, and Krangel, MS. "Two forms of the T-cell receptor gamma protein found on peripheral blood cytotoxic T lymphocytes." Nature 325.6106 (February 19, 1987): 689-694.
PMID
3102967
Source
pubmed
Published In
Nature
Volume
325
Issue
6106
Publish Date
1987
Start Page
689
End Page
694
DOI
10.1038/325689a0

Identification of a putative second T-cell receptor.

Framework monoclonal antibodies have identified a population of human lymphocytes that express the T3 glycoprotein but not the T-cell receptor (TCR) alpha- and beta-subunits. Chemical crosslinking experiments reveal that these lymphocytes express novel T3-associated polypeptides, one of which appears to be the product of the T gamma gene. The other polypeptide may represent a fourth TCR subunit, designated T delta.

Authors
Brenner, MB; McLean, J; Dialynas, DP; Strominger, JL; Smith, JA; Owen, FL; Seidman, JG; Ip, S; Rosen, F; Krangel, MS
MLA Citation
Brenner, MB, McLean, J, Dialynas, DP, Strominger, JL, Smith, JA, Owen, FL, Seidman, JG, Ip, S, Rosen, F, and Krangel, MS. "Identification of a putative second T-cell receptor." Nature 322.6075 (July 10, 1986): 145-149.
PMID
3755221
Source
pubmed
Published In
Nature
Volume
322
Issue
6075
Publish Date
1986
Start Page
145
End Page
149
DOI
10.1038/322145a0

Secretion of HLA-A and -B antigens via an alternative RNA splicing pathway.

Human class I major histocompatibility antigens (HLA-A, -B and -C) are integral membrane protein heterodimers, which are anchored in the membrane via a stretch of hydrophobic amino acids near the carboxyl terminus of the heavy chain. It has previously been shown that a mutagenized cell line secretes a water soluble form of the HLA-A2 antigen, due to a pattern of RNA splicing that removes exon 5 (encoding the transmembrane hydrophobic amino acids) from mature, HLA-A2--encoding transcripts. The present study was undertaken to assess whether a similar process might be operative in nonmutagenized cells. It is shown that water soluble class I molecules (primarily HLA-A24) are secreted by the T leukemic cell line HPB-ALL, and that alternative splicing removes exon 5 from a fraction of HLA-A24--encoding transcripts. It is further shown that class I molecules are secreted, possibly in an allele-specific fashion, from a variety of tumor cells and normal cells. The possible relationship between these findings and previous reports of HLA-A and -B antigens in human serum is discussed.

Authors
Krangel, MS
MLA Citation
Krangel, MS. "Secretion of HLA-A and -B antigens via an alternative RNA splicing pathway." J Exp Med 163.5 (May 1, 1986): 1173-1190.
PMID
3701253
Source
pubmed
Published In
The Journal of Experimental Medicine
Volume
163
Issue
5
Publish Date
1986
Start Page
1173
End Page
1190

Structural analysis of the functional sites of class I HLA antigens.

Considerable knowledge of the molecular organization of class I HLA antigens has been attained through extensive structural analysis of these proteins and their genes. Particularly, the nature and location of the polymorphic regions has been established, as well as the basic patterns of structural variability. This work has not unveiled the functionally relevant sites of the HLA molecules but has provided the basis to develop new strategies to do so. The molecular analysis of the determinants recognized by specific antibodies and cytolytic T lymphocytes is being approached through the biochemical characterization of mutants induced in vitro and population variants that are selected by their loss of specific serological or CTL allodeterminants. Other approaches include the immunological analysis of sera raised against synthetic peptides whose structure mimics highly variable segments of class I HLA molecules. These studies have already revealed the participation of several regions in specific allorecognition by antibodies or CTLs and their potential is becoming increasingly evident. A new and possibly powerful approach is currently being used for the dissection of functional sites. It makes use of the structural information derived from sequence analysis and involves expression of cloned HLA genes in transfected mouse or human cells in conjunction with site-directed mutagenesis techniques. Although some difficulties still lie ahead in developing a system suitable for functional assays, the possibility of tailoring HLA mutants and studying the modulation of their recognition determinants by predetermined structural alterations open new pathways to the molecular analysis of HLA function.

Authors
Lopez de Castro, JA; Barbosa, JA; Krangel, MS; Biro, PA; Strominger, JL
MLA Citation
Lopez de Castro, JA, Barbosa, JA, Krangel, MS, Biro, PA, and Strominger, JL. "Structural analysis of the functional sites of class I HLA antigens." Immunol Rev 85 (July 1985): 149-168. (Review)
PMID
2412949
Source
pubmed
Published In
Immunological Reviews
Volume
85
Publish Date
1985
Start Page
149
End Page
168

Unusual RNA splicing generates a secreted form of HLA-A2 in a mutagenized B lymphoblastoid cell line.

Human class I major histocompatibility antigens (HLA-A, -B and -C) are integral membrane glycoprotein heterodimers. A mutagenized B lymphoblastoid cell line has been previously shown to synthesize two forms of the HLA-A2 antigen; a minor form which remains cell-associated at all times, and an abundant form, which is secreted. The present study reports the isolation of cDNA clones for both the wild-type HLA-A2 molecule synthesized by the parent cell line and the secreted molecule synthesized by the mutant cell line. A comparison of their structures indicates that transcripts encoding the mutant HLA-A2 molecule lack the 117 nucleotides encoded by exon five of the HLA-A2 gene. This exon encodes the hydrophobic amino acids which are thought to anchor the polypeptide in the plasma membrane. This result supports an alternative splicing model to explain the phenotype of the mutant cell line. Further, it implies that information encoded in exon five is essential for anchoring class I antigens in the plasma membrane. The potential for a similar splicing mechanism to generate soluble forms of class I antigens in vivo is discussed.

Authors
Krangel, MS
MLA Citation
Krangel, MS. "Unusual RNA splicing generates a secreted form of HLA-A2 in a mutagenized B lymphoblastoid cell line." EMBO J 4.5 (May 1985): 1205-1210.
PMID
3874058
Source
pubmed
Published In
EMBO Journal
Volume
4
Issue
5
Publish Date
1985
Start Page
1205
End Page
1210

Structural analysis of an HLA-B7 antigen variant detected by cytotoxic T lymphocytes.

It has been demonstrated previously that lymphocytes of donor CF (HLA-A29,w33; B7,14) are not recognized by the HLA-B7-specific CTL clone HG-31. This report presents a structural comparison of the HLA-B7 antigen of donor CF with a "normal" HLA-B7 antigen, derived from the cell line JY. Isoelectric focusing showed that CF HLA-B7 heavy chains were more acidic than JY HLA-B7 heavy chains by the equivalent of a single charge. High pressure liquid chromatography and ion exchange chromatography comparisons of double-labeled tryptic peptides revealed a single detectable difference, which corresponded to the tryptic peptide spanning residues 112 to 121 on the HLA-B7 heavy chain. Although the complete amino acid sequence of this peptide was not obtained, the partial sequence indicates a substitution of an unidentified amino acid for tyrosine at position 116 of the heavy chain. This residue is found to vary among HLA specificities and to be altered in many H-2Kb mutants.

Authors
Taketani, S; Krangel, MS; Spits, H; de Vries, J; Strominger, JL
MLA Citation
Taketani, S, Krangel, MS, Spits, H, de Vries, J, and Strominger, JL. "Structural analysis of an HLA-B7 antigen variant detected by cytotoxic T lymphocytes." J Immunol 133.2 (August 1984): 816-821.
PMID
6429245
Source
pubmed
Published In
Journal of immunology (Baltimore, Md. : 1950)
Volume
133
Issue
2
Publish Date
1984
Start Page
816
End Page
821

Cysteines in the transmembrane region of major histocompatibility complex antigens are fatty acylated via thioester bonds.

Exogenous radioactive palmitic acid is incorporated post-translationally into the HLA-B and -DR heavy chains, but not HLA-A heavy chains or -DR light chains of the human B lymphoblastoid cells JY and T51 . Protease digestions localize the label to the transmembrane region of the B7 heavy chain. Both B7 and DR heavy chains have a cysteine in the transmembrane hydrophobic region, while the A2 heavy chain and DR light chains have none. Palmitic acid is covalently linked to these transmembrane cysteines via a thioester bond since: 1) the label is not removed by organic extraction or boiling in sodium dodecyl sulfate and dithiothreitol, but is released at room temperature by methanolic KOH as methyl palmitate, and by hydroxylamine as palmitohydroxamate . 2) The pH sensitivity and kinetics of release by hydroxylamine and Tris are similar to those of palmitoyl-CoA (thioester linkage) and unlike those of methyl palmitate and palmitoyllysophosphatidylcholine ( hydroxyester linkages). 3) Neutral hydroxylamine treatment (but not neutral Tris treatment) generates sites that can be reduced and alkylated in the transmembrane region of B7 heavy chain and to a lesser extent in DR heavy chain. 4) Organic extraction of pronase digests of labeled B7 yields peptides containing palmitate and cysteine (but not serine or threonine) which co-migrate by thin layer chromatography. A population of beta 2-microglobulin molecules not associated with heavy chains is palmitylated , but not via a thioester linkage.

Authors
Kaufman, JF; Krangel, MS; Strominger, JL
MLA Citation
Kaufman, JF, Krangel, MS, and Strominger, JL. "Cysteines in the transmembrane region of major histocompatibility complex antigens are fatty acylated via thioester bonds." J Biol Chem 259.11 (June 10, 1984): 7230-7238.
PMID
6373770
Source
pubmed
Published In
The Journal of biological chemistry
Volume
259
Issue
11
Publish Date
1984
Start Page
7230
End Page
7238

Characterization of a B lymphoblastoid cell line mutant that secretes HLA-A2.

HLA-A2 antigen mutants were obtained previously from the B lymphoblastoid cell line T5-1 by mutagenesis followed by immunoselection. Here we present biochemical studies of one particular mutant, clone 8.14.1. These cells synthesize two forms of HLA-A2: a minor form, which remains cell-associated at all times, and an abundant form, which is secreted. The former appears by SDS-PAGE to be slightly larger than T5-1 HLA-A2, whereas the latter appears to be 4000 to 5000 daltons smaller. In vitro translation and in vivo pulse-chase studies suggest that these species are not related to each other by post-translational processing. Proteolytic digestion studies localize the resulting structural alteration in the mobility difference between wild-type and secreted HLA-A2 to a region near the carboxy terminus of the HLA-A2 heavy chain; however, their extreme carboxy termini appear similar, if not identical. We suggest that the secreted form may result from a pattern of RNA splicing in which the exon encoding the hydrophobic, membrane-spanning region is frequently deleted.

Authors
Krangel, MS; Pious, D; Strominger, JL
MLA Citation
Krangel, MS, Pious, D, and Strominger, JL. "Characterization of a B lymphoblastoid cell line mutant that secretes HLA-A2." J Immunol 132.6 (June 1984): 2984-2991.
PMID
6609965
Source
pubmed
Published In
Journal of immunology (Baltimore, Md. : 1950)
Volume
132
Issue
6
Publish Date
1984
Start Page
2984
End Page
2991

Structural analysis of HLA-A2 antigen from immunoselected mutant 8.6.1: further definition of an HLA-A2-specific serological determinant.

The HLA-A2 mutant cell line 8.6.1 was isolated previously from the lymphoblastoid B cell line T5-1 (HLA-A1, -A2, -B8, and -B27) by immunoselection with the mouse HLA-A2-specific monoclonal antibody BB7.2 and complement. The HLA-A2 molecules synthesized by 8.6.1 do not react with either the selecting antibody or with a second HLA-A2-specific monoclonal antibody, PA2.1. In this study, HLA-A2 heavy chains derived from 8.6.1 and those from the parent T5-1 cells have been analyzed by double-labeled tryptic peptide mapping by using reverse-phase HPLC, cation exchange chromatography, and microsequence analysis. We detect only a single difference between these molecules: 8.6.1 HLA-A2 differs from T5-1 HLA-A2 by the substitution of lysine for glutamic acid at position 161. This result is consistent with data derived from other immunoselected mutants, which implicate the second heavy chain domain (alpha 2) in the expression of the PA2.1 and BB7.2 epitopes, and suggests a crucial role for glutamic acid at position 161 in the formation of an HLA-A2-specific determinant.

Authors
Taketani, S; Krangel, MS; Pious, D; Strominger, JL
MLA Citation
Taketani, S, Krangel, MS, Pious, D, and Strominger, JL. "Structural analysis of HLA-A2 antigen from immunoselected mutant 8.6.1: further definition of an HLA-A2-specific serological determinant." J Immunol 131.6 (December 1983): 2935-2938.
PMID
6196407
Source
pubmed
Published In
Journal of immunology (Baltimore, Md. : 1950)
Volume
131
Issue
6
Publish Date
1983
Start Page
2935
End Page
2938

Recognition of HLA-A2 mutant and variant target cells by an HLA-A2 allospecific human cytotoxic T lymphocyte line.

HLA-A2 specific human cytotoxic T lymphocytes (CTL) cell lines have been developed using T cell growth factor and coculture of peripheral blood lymphocytes with selected allogeneic target cell lines. The CTL-8 line showed specificity for human leukocyte antigens (HLA)-A2 bearing target cells after 5 weeks in culture when tested against a panel of 14 lymphoblastoid cell lines in a 51Chromium (51Cr) release assay. Purified anti-human leukocyte antigens (HLA) monoclonal antibodies W6/32 and PA2.1 inhibited cytolysis by 85% and 60%, respectively. The CTL-8 line lysed non-HLA-A2 target cells in the presence of lectins concanavalin A (Con A) or phytohemagglutinin-P lectin (PHA-P) indicating the specificity of cytolysis was not due to nonspecific resistance of target cells to the CTL-lytic mechanism. The T5-1 HLA-A2 mutant cell series were tested as targets for the CTL-8 line. Cell clones 8.18.1, 8.21.1 and 8.6.1, which express altered HLA-A2 molecules as determined by their decreased reactivity with allospecific monoclonal antibodies, were lysed by the CTL-8 line as efficiently as the T5-1 wild type. These cell lines also acted as efficient cold target competitors for a normal HLA-A2 target cell. The 8.14.1 cell clone expressed a lower amount of HLA-A2 alloantigen and showed a corresponding decreased reactivity with CTL-8 in direct cytolytic and cold target competitive inhibition assays. In contrast, the M7 and DK1 HLA-A2 variant cell lines, which express normal HLA-A2 serological determinants, were inefficiently lysed by CTL-8 and did not act as competitive inhibitors of normal HLA-A2 target cells. These results support the concept that the alloantigenic determinant(s) recognized by T cells and antibodies occur at separate regions on the HLA-A2 molecule.

Authors
Ware, CF; Krangel, MS; Pious, D; Burakoff, SJ; Strominger, JL
MLA Citation
Ware, CF, Krangel, MS, Pious, D, Burakoff, SJ, and Strominger, JL. "Recognition of HLA-A2 mutant and variant target cells by an HLA-A2 allospecific human cytotoxic T lymphocyte line." J Immunol 131.3 (September 1983): 1312-1317.
PMID
6193184
Source
pubmed
Published In
Journal of immunology (Baltimore, Md. : 1950)
Volume
131
Issue
3
Publish Date
1983
Start Page
1312
End Page
1317

Comparative structural analysis of HLA-A2 antigens distinguishable by cytotoxic T lymphocytes. II. Variant DK1: evidence for a discrete CTL recognition region.

Multiple amino acid sequence differences distinguish individual HLA antigens. Those residues important in immune recognition events have not been defined. Recent studies have identified HLA-A2 structural variants that, although serologically indistinguishable from other HLA-A2 antigens, are recognized poorly, if at all, by HLA-A2-restricted, influenza virus-immune, or HLA-A2-specific alloimmune CTL. In this study we utilize double-label tryptic peptide comparisons performed by both reverse-phase HPLC and cation exchange chromatography, in conjunction with conventional and microsequence analysis, to characterize the HLA-A2 heavy chains derived from variant DK1. We detect a single tryptic peptide that distinguishes DK1 HLA-A2 from the predominant HLA-A2 heavy chain species. This peptide spans residues 147 to 157 in the second heavy chain domain, and carries substitutions at positions 149, 152, and 156. Residues in this segment of the polypeptide are also altered in another HLA-A2 variant, as well as one H-2Kb mutant. Thus, this segment appears to be critical in forming determinants important in CTL recognition of class I antigens in general. On the basis of these and other results, we suggest that in contrast to recognition by alloantibodies, a discrete region of class I antigens may be crucial for CTL recognition.

Authors
Krangel, MS; Biddison, WE; Strominger, JL
MLA Citation
Krangel, MS, Biddison, WE, and Strominger, JL. "Comparative structural analysis of HLA-A2 antigens distinguishable by cytotoxic T lymphocytes. II. Variant DK1: evidence for a discrete CTL recognition region." J Immunol 130.4 (April 1983): 1856-1862.
PMID
6601143
Source
pubmed
Published In
Journal of immunology (Baltimore, Md. : 1950)
Volume
130
Issue
4
Publish Date
1983
Start Page
1856
End Page
1862

HLA-A2 mutants immunoselected in vitro. Definition of residues contributing to an HLA-A2-specific serological determinant.

The HLA-A2-specific mouse monoclonal antibody BB7.2 plus complement has been used to immunoselect variant clones of the lymphoblastoid cell line T5-1 (HLA-A1, -A2, -B8, and -B27). Members of one class of variant clones appear to express cell surface HLA-A2 molecules that display reduced reactivity with the selecting antibody, but normal or near normal reactivities with some other HLA-A2-specific monoclonal antibodies and human alloantisera. The HLA-A2 heavy chains derived from two of these variant clones were characterized by comparative double-label tryptic peptide mapping in conjunction with microsequence analysis. These heavy chains were found to carry distinct mutations in the same peptide in the molecule. We conclude that residues within this short segment of the polypeptide contribute to an HLA-A2-specific serological determinant.

Authors
Krangel, MS; Taketani, S; Pious, D; Strominger, JL
MLA Citation
Krangel, MS, Taketani, S, Pious, D, and Strominger, JL. "HLA-A2 mutants immunoselected in vitro. Definition of residues contributing to an HLA-A2-specific serological determinant." J Exp Med 157.1 (January 1, 1983): 324-336.
PMID
6184441
Source
pubmed
Published In
The Journal of Experimental Medicine
Volume
157
Issue
1
Publish Date
1983
Start Page
324
End Page
336

HLA antigen structural gene mutants selected with an allospecific monoclonal antibody.

The HLA-A2 antigen-specific monoclonal antibody BB7.2 and complement were used to immunoselect mutants from an ethyl methanesulfonate-mutagenized human B lymphoid cell line, T5-1. Surviving colonies were screened by radioimmune binding with BB7.2 and with a monospecific HLA-A2 alloantiserum, Stewart, and HLA antigens of selected clones were immunoprecipitated and studied by isoelectric focusing. Several classes of mutants could be distinguished: mutants that expressed no HLA-A2 heavy chain; mutants that expressed an HLA-A2 heavy chain that was unable to associate with beta 2-microglobulin (beta 2m) and was not expressed at the cell surface; mutants with reduced HLA-A2 heavy chain-beta 2m association and cell surface expression of HLA-A2 dimer with or without heavy chain charge alterations; mutants with normal HLA-A2 heavy chain-beta 2m association and normal quantitative cell surface HLA-A2 expression but with HLA-A2 heavy chain charge alterations; and mutants with as yet incompletely defined lesions. Mutants with altered cell surface HLA antigens were not found in previous selections with alloantisera and should be useful for epitope mapping and structure-function studies of HLA molecules.

Authors
Pious, D; Krangel, MS; Dixon, LL; Parham, P; Strominger, JL
MLA Citation
Pious, D, Krangel, MS, Dixon, LL, Parham, P, and Strominger, JL. "HLA antigen structural gene mutants selected with an allospecific monoclonal antibody." Proc Natl Acad Sci U S A 79.24 (December 1982): 7832-7836.
PMID
6961455
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
79
Issue
24
Publish Date
1982
Start Page
7832
End Page
7836

Comparative structural analysis of HLA-A2 antigens distinguishable by cytotoxic T lymphocytes: variants M7 and DR1.

Comparative primary structural analyses have begun to elucidate polymorphic residues and segments of the class I antigens of the major histocompatibility complex, at least some of which presumably contribute to determinants important in immune recognition events. HLA-A2 structural variants have been described which are serologically indistinguishable from other HLA-A2 antigens, yet which can be recognized neither by HLA-A2 specific alloimmune nor by HLA-A2 restricted, virus immune cytotoxic T lymphocytes. This study utilizes double-label tryptic peptide comparisons in combination with both conventional and microsequence analyses to investigate the structure of two such variants, M7 and DR1. We find that these variants are identical with each other and differ from the predominant HLA-A2 heavy chain species by a glutamine to arginine substitution at residue 43, by an unidentified substitution in the tryptic peptide spanning residues 147-157, and by an as yet poorly defined alteration in glycosylation. Structural information from these and other variants should be useful in precisely defining functionally important determinants on the molecule.

Authors
Krangel, MS; Taketani, S; Biddison, WE; Strong, DM; Strominger, JL
MLA Citation
Krangel, MS, Taketani, S, Biddison, WE, Strong, DM, and Strominger, JL. "Comparative structural analysis of HLA-A2 antigens distinguishable by cytotoxic T lymphocytes: variants M7 and DR1." Biochemistry 21.24 (November 23, 1982): 6313-6321.
PMID
6983890
Source
pubmed
Published In
Biochemistry
Volume
21
Issue
24
Publish Date
1982
Start Page
6313
End Page
6321

Delineation of immunologically and biochemically distinct HLA-A2 antigens.

Cytotoxic T cell (CTL) recognition of influenza virus in conjunction with HLA-A2 was examined in a population study. Virus-infected target cells from three unrelated A2-positive donors were not lysed by virus-immune CTL from any donor matched only for A2. The A2 antigens of these three donors were indistinguishable from the A2 antigens of other A2-positive donors as assessed by extensive serologic analyses; however, isoelectric focusing (IEF) of A2 molecules from these three donors demonstrated that their A2 heavy polypeptide chains are structurally distinct from those of "normal" A2-positive donors. To date 11% of all A2-positive donors tested exhibited a "variant" A2-associated CTL restriction antigen, and IEF of A2 heavy chains from all "variant" A2-positive cells revealed structural differences in each of these polypeptides. These results suggest there may be considerably greater polymorphism of HLA-A gene products than has been revealed by current serologic techniques.

Authors
Biddison, WE; Kostyu, DD; Strominger, JL; Krangel, MS
MLA Citation
Biddison, WE, Kostyu, DD, Strominger, JL, and Krangel, MS. "Delineation of immunologically and biochemically distinct HLA-A2 antigens." J Immunol 129.2 (August 1982): 730-734.
PMID
6177780
Source
pubmed
Published In
Journal of immunology (Baltimore, Md. : 1950)
Volume
129
Issue
2
Publish Date
1982
Start Page
730
End Page
734

Human histocompatibility antigen mutants immunoselected in vitro. Biochemical analysis of a mutant which synthesizes an altered HLA-A2 heavy chain.

Immunoselection with HLA-A2 or HLA-A1 specific alloantisera has been utilized to isolate spontaneously arising and mutagen-induced variants from the B lymphoblastoid cell line T5-1 (HLA haplotypes DR3, B8, A1 and DR1, B27, Cw1, A2). Such variants are characterized by reduced reactivity with alloantisera of the selecting specificity, but normal reactivity with alloantisera directed to the other HLA specificities of T5-1. Biochemical analysis reveals two classes of variants. In all HLA-A1 and some HLA-A2 variants, the heavy chain in question cannot be detected; however, in other HLA-A2 variants, a structurally altered HLA-A2 heavy chain is found. In the HLA-A2 variant 6.6.5, this heavy chain is glycosylated and thus has presumably been inserted into the rough endoplasmic reticulum membrane in vivo. However, unlike all other HLA heavy chains, the 6.6.5 HLA-A2 heavy chain does not associate with beta 2-microglobulin, does not undergo processing of its high mannose oligosaccharide, and does not migrate to the cell surface, although it is relatively stably expressed within the cell. We suggest that the primary defect in these cells is the failure of the 6.6.5 HLA-A2 heavy chain to associate stably with beta 2-microglobulin. It is likely that the observed structural alteration in this heavy chain reflects a change in amino acid sequence, and thus, a mutation in the structural gene encoding HLA-A2.

Authors
Krangel, MS; Pious, D; Strominger, JL
MLA Citation
Krangel, MS, Pious, D, and Strominger, JL. "Human histocompatibility antigen mutants immunoselected in vitro. Biochemical analysis of a mutant which synthesizes an altered HLA-A2 heavy chain." J Biol Chem 257.9 (May 10, 1982): 5296-5305.
PMID
6978338
Source
pubmed
Published In
The Journal of biological chemistry
Volume
257
Issue
9
Publish Date
1982
Start Page
5296
End Page
5305

HLA antigen structural gene mutants selected with an allospecific monoclonal antibody

Authors
Pious, D; Krangel, MS; Dixon, LL; Parham, P; Strominger, JL
MLA Citation
Pious, D, Krangel, MS, Dixon, LL, Parham, P, and Strominger, JL. "HLA antigen structural gene mutants selected with an allospecific monoclonal antibody." Proceedings of the National Academy of Sciences of the United States of America 79.24 I (1982): 7832-7836.
Source
scival
Published In
Proceedings of the National Academy of Sciences of USA
Volume
79
Issue
24 I
Publish Date
1982
Start Page
7832
End Page
7836
DOI
10.1073/pnas.79.24.7832

Virus-immune cytotoxic T cells recognize structural differences between serologically indistinguishable HLA-A2 molecules.

The self-specificity of human influenza virus-immune cytotoxic T cells has been analyzed in order to identify the relationship between the self-determinants which they recognize and the serologically defined HLA-A and -B antigenic determinants. Virus-immune T cells were generated in vitro by culture of normal adult peripheral blood lymphocytes with A/HK influenza virus. Virus-immune effectors from HLA-A2 positive donors were tested on panels of virus-infected target cells from donors who were either HLA-mismatched or matched only for the HLA-A2 specificity. Virus-immune T cells from 11/11 A2-positive donors lysed all A2-matched virus-infected target cells (and no HLA-mismatched targets), except that each of these effector cell populations consistently failed to lyse the virus-infected target cells from one A2-positive donor (designated M7). Although the A2 antigen of donor M7 could also be distinguished from the A2 antigen of other donors by alloimmune cytotoxic T cells, no differences in the A2 antigen of donor M7 could be defined by extensive serological analyses. Results of isoelectric focusing of A2 molecules from three individuals plus M7 demonstrated that the M7 A2 heavy-polypeptide chain is structurally distinct. These results indicate that: 1) there is a strong but incomplete association between a self antigen recognized by virus-immune T cells and the serologically defined HLA-A2 specificity; and 2) there may be at least two structurally and functionally distinct epitopes on the same A2 molecule: one is the serologically defined HLA-A2 antigenic determinant; the other is the self determinant recognized by T cells on HLA-A2 molecules.

Authors
Biddison, WE; Krangel, MS; Strominger, JL; Ward, FE; Shearer, GM; Shaw, S
MLA Citation
Biddison, WE, Krangel, MS, Strominger, JL, Ward, FE, Shearer, GM, and Shaw, S. "Virus-immune cytotoxic T cells recognize structural differences between serologically indistinguishable HLA-A2 molecules." Hum Immunol 1.3 (October 1980): 225-232.
PMID
6167549
Source
pubmed
Published In
Human Immunology
Volume
1
Issue
3
Publish Date
1980
Start Page
225
End Page
232

Structure, function, and biosynthesis of the major human histocompatibility antigens (HLA-A and HLA-B).

Authors
Krangel, MS; Orr, HT; Strominger, JL
MLA Citation
Krangel, MS, Orr, HT, and Strominger, JL. "Structure, function, and biosynthesis of the major human histocompatibility antigens (HLA-A and HLA-B)." Scand J Immunol 11.6 (1980): 561-571.
PMID
7444379
Source
pubmed
Published In
Scandinavian Journal of Immunology
Volume
11
Issue
6
Publish Date
1980
Start Page
561
End Page
571

Assembly and maturation of HLA-A and HLA-B antigens in vivo.

HLA-A and HLA-B antigens are integral membrane glycoproteins which consist of a glycosylated heavy chain embedded in the membrane in noncovalent association with beta 2-microglobulin, a water-soluble polypeptide. The assembly and maturation of these antigens has been studied in vivo in the human B lymphoblastoid cell line T5-1 (HLA-A1, -A2, -B8, -B27). Two antigenically distinct populations of HLA-A and -B heavy chains can be detected by antisera which recognize determinants sensitive to the conformation of the heavy chain. One heavy chain population is associated with beta 2-microglobulin, whereas the other population is not. These populations can be further distinguished by their oligosaccharide structure and their localization within the cell. Pulse-chase experiments demonstrate a precursor-product relationship between these heavy chain populations and suggest the following pathway for the assembly and maturation of HLA-A and -B antigens. The completed heavy chains initially carry high mannose oligosaccharides and are largely or wholly associated with beta 2-microglobulin. During the next 10-15 min, association with beta 2-microglobulin occurs and the heavy chain conformation is altered. Beginning at about 30 min after synthesis, the oligosaccharides are converted from the high mannose form to the complex form, and between 60 and 80 min after synthesis, the mature antigens appear at the cell surface. These observations are discussed in relation to in vivo and in vitro studies on the biosynthesis of a variety of secreted proteins and membrane proteins.

Authors
Krangel, MS; Orr, HT; Strominger, JL
MLA Citation
Krangel, MS, Orr, HT, and Strominger, JL. "Assembly and maturation of HLA-A and HLA-B antigens in vivo." Cell 18.4 (December 1979): 979-991.
PMID
93026
Source
pubmed
Published In
Cell
Volume
18
Issue
4
Publish Date
1979
Start Page
979
End Page
991
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Research Areas:

  • Academic Medical Centers
  • Acetylation
  • Alanine
  • Alleles
  • Amino Acid Sequence
  • Amino Acids
  • Animals
  • Antibodies, Monoclonal
  • Antibody Diversity
  • Antibody Specificity
  • Antigen-Antibody Reactions
  • Antigens, CD3
  • Antigens, CD4
  • Antigens, CD8
  • Antigens, Surface
  • Arthritis, Rheumatoid
  • B-Lymphocytes
  • Base Sequence
  • Binding Sites
  • Binding, Competitive
  • Biological Factors
  • Biopolymers
  • Blotting, Northern
  • Blotting, Southern
  • CD4-Positive T-Lymphocytes
  • CD8-Positive T-Lymphocytes
  • CHO Cells
  • COS Cells
  • Calcium
  • Carrier Proteins
  • Cell Adhesion
  • Cell Cycle Proteins
  • Cell Differentiation
  • Cell Division
  • Cell Line
  • Cell Line, Transformed
  • Cell Lineage
  • Cell Membrane
  • Cell Movement
  • Cell Nucleus
  • Cell Survival
  • Cells, Cultured
  • Chemical Phenomena
  • Chemical Precipitation
  • Chemistry
  • Chemokine CCL1
  • Chemokine CCL2
  • Chemokine CCL3
  • Chemokine CCL4
  • Chemokines
  • Chemokines, CC
  • Chemotactic Factors
  • Chemotaxis, Leukocyte
  • Child
  • Chromatin
  • Chromatin Assembly and Disassembly
  • Chromatin Immunoprecipitation
  • Chromosomal Proteins, Non-Histone
  • Clone Cells
  • Cloning, Molecular
  • Cyanogen Bromide
  • Cytokines
  • Cytotoxicity, Immunologic
  • DNA
  • DNA Damage
  • DNA Footprinting
  • DNA Methylation
  • DNA Nucleotidyltransferases
  • DNA Repair
  • DNA-Binding Proteins
  • Deoxyribonuclease I
  • Down-Regulation
  • Drosophila
  • Electrophoresis
  • Endocytosis
  • Enhancer Elements, Genetic
  • Epitopes
  • Exons
  • Extracellular Space
  • Feedback, Physiological
  • Female
  • Fetus
  • Fibroblasts
  • Fluorescent Antibody Technique
  • Gangliosides
  • Gene Expression
  • Gene Expression Regulation
  • Gene Frequency
  • Gene Rearrangement
  • Gene Rearrangement, B-Lymphocyte
  • Gene Rearrangement, T-Lymphocyte
  • Gene Rearrangement, alpha-Chain T-Cell Antigen Receptor
  • Gene Rearrangement, beta-Chain T-Cell Antigen Receptor
  • Gene Rearrangement, delta-Chain T-Cell Antigen Receptor
  • Gene Targeting
  • Genes
  • Genes, Immunoglobulin
  • Genes, RAG-1
  • Genes, Switch
  • Genes, T-Cell Receptor
  • Genes, T-Cell Receptor alpha
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  • Genetic Linkage
  • Genetic Loci
  • Genetic Variation
  • Genotype
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  • Glycoproteins
  • Glycosaminoglycans
  • Glycosides
  • Glycosylphosphatidylinositols
  • HLA Antigens
  • HMGB1 Protein
  • Heparin
  • Histone-Lysine N-Methyltransferase
  • Histones
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  • Humans
  • Hydrogen-Ion Concentration
  • Hydroxylamines
  • Image Processing, Computer-Assisted
  • Immune Sera
  • Immune System Phenomena
  • Immunoglobulin Fragments
  • Immunoglobulin G
  • Immunoglobulin Joining Region
  • Immunoglobulin Variable Region
  • Immunologic Deficiency Syndromes
  • In Situ Hybridization, Fluorescence
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  • Isoelectric Point
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  • Microscopy, Confocal
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  • Molecular Sequence Data
  • Molecular Weight
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  • Nuclear Proteins
  • Nucleic Acid Conformation
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  • Trypsinogen
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  • V(D)J Recombination
  • VDJ Exons
  • VDJ Recombinases