of May 26, 2018.This information is current as

Update and ReappraisalAntigen Receptor Allelic Exclusion: An

Brenna L. Brady, Natalie C. Steinel and Craig H. Bassing

http://www.jimmunol.org/content/185/7/3801doi: 10.4049/jimmunol.1001158

2010; 185:3801-3808; ;J Immunol 

Referenceshttp://www.jimmunol.org/content/185/7/3801.full#ref-list-1

, 32 of which you can access for free at: cites 66 articlesThis article

        average*  

4 weeks from acceptance to publicationFast Publication! •    

Every submission reviewed by practicing scientistsNo Triage! •    

from submission to initial decisionRapid Reviews! 30 days* •    

Submit online. ?The JIWhy

Subscriptionhttp://jimmunol.org/subscription

is online at: The Journal of ImmunologyInformation about subscribing to

Permissionshttp://www.aai.org/About/Publications/JI/copyright.htmlSubmit copyright permission requests at:

Email Alertshttp://jimmunol.org/alertsReceive free email-alerts when new articles cite this article. Sign up at:

Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists, Inc. All rights reserved.Copyright © 2010 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

is published twice each month byThe Journal of Immunology

by guest on May 26, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

by guest on May 26, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

Antigen Receptor Allelic Exclusion: An Update andReappraisalBrenna L. Brady,1 Natalie C. Steinel,1 and Craig H. Bassing

Most lymphocytes express cell surface Ag receptorchains from single alleles of distinct Ig or TCR loci.Since the identification of Ag receptor allelic exclusion,the importance of this process and the precise molec-ular mechanisms by which it is achieved have remainedenigmatic. This brief review summarizes current knowl-edge of the extent to which Ig and TCR loci are subjectto allelic exclusion. Recent progress in studying and de-fining mechanistic steps and molecules that may controlthe monoallelic initiation and subsequent inhibition ofV-to-(D)-J recombination is outlined using the mouseTCRb locus as a model with frequent comparisons tothe mouse IgH and Igk loci. Potential consequences ofdefects in mechanisms that control Ag receptor allelicexclusion and a reappraisal of the physiologic relevanceof this immunologic process also are discussed. TheJournal of Immunology, 2010, 185: 3801–3808.

Antigen receptor allelic exclusion is defined as thesurface expression of Ig or TCR chains from a singleallelic copy of corresponding genetic loci. Pernis et al.

(1) identified this phenomenon in the 1960s while studying Igexpression on rabbit lymphocytes, providing evidence for the“one lymphocyte–one antigen receptor” concept of Burnett’sclonal selection theory. Analyses of Ig rearrangements in theearly 1980s suggested that the assembly and expression of anAg receptor chain from one allele inhibit further V-to-(D)-Jrecombination on the other allele (2). Evidence for such feed-back regulation was provided over the next decade by dem-onstrations that preassembled Ig or TCR transgenes enforceallelic exclusion through inhibiting V-to-(D)-J rearrange-ments (2). These observations helped establish the currentdogma that allelic exclusion is maintained by feedback regu-lation to ensure virtually every lymphocyte exhibits monospe-cific Ag recognition.Although Ag receptor allelic exclusion has been investigated

for almost 50 y, the importance of this process and the precisemechanisms by which it is achieved remain largely unknown.

Monoallelic gene expression is a general phenomenon criticalfor normal biology. This regulation is pervasive during geneticimprinting and X chromosome inactivation, enforcing genesilencing in all cell types (3). Defects in X chromosome in-activation and imprinting were found to cause human dis-orders, which defined the relevance of these processes andfacilitated their investigation by studying clear phenotypes. Incontrast, defects in olfactory or Ag receptor allelic exclusion,which silence tissue-specific genes in distinct lineages, havenot been linked unequivocally to any symptoms in humans(3). However, dysfunction of these tissue-specific processesmay result in subtle phenotypes or may be compensated byadditional mechanisms, either of which would mask theirsignificance and provide obstacles for investigation.

Allelic exclusion, self tolerance, and autoimmunity

A long-standing tenent of adaptive immunity is that virtuallyall lymphocytes express surface TCRor Ig chains fromone alleleto ensure monospecific Ag recognition and suppress autoim-munity by facilitating central tolerance to self-reactive lympho-cytes. However, this notion is not supported by current know-ledge. Flow cytometry reveals IgH allelic inclusion in only0.01% of mouse B cells (4), but allelic inclusion of Igk andTCR loci in at least 1–10% of mouse lymphocytes (Table I)(5–9). Thus, a significant fraction of normal mouse (and whereassayed human) lymphocytes express surface Igk or TCRchains from both alleles, refuting the “one lymphocyte–oneantigen receptor” concept. In addition, recognition of multipledistinct ligands is known now to be a general and inherentproperty of T and B cell Ag receptors (10), meaning that farmore than the 1–10% of allelically included lymphocytesexhibits poly-specific Ag recognition. Because primary TCRand BCR repertoires include receptors capable of bind-ing self-Ags, organisms with adaptive immune systems mustpossess central ability to tolerate the generation of autoreactivelymphocytes and thereby prevent autoimmunity. Central tol-erance mechanisms include deletion, stalled maturation, an-ergy, or receptor editing (9). In the 1990s, analyses of TCRand Ig transgenic mice demonstrated that dual expression ofself-reactive and non–self-reactive receptors enables developing

Immunology Graduate Group, Division of Cancer Pathobiology, Department of Pa-thology and Laboratory Medicine, Center for Childhood Cancer Research, Children’sHospital of Philadelphia, University of Pennsylvania School of Medicine, AbramsonFamily Cancer Research Institute, Philadelphia, PA 19104

1B.L.B. and N.C.S. contributed equally to this review.

Received for publication June 23, 2010. Accepted for publication August 5, 2010.

This work was supported by Training Grant TG GM-07229 of the University ofPennsylvania (to B.L.B.); the Department of Pathology and Laboratory Medicine andthe Center for Childhood Cancer Research of the Children’s Hospital of Philadelphia;the Abramson Family Cancer Research Institute of the University of Pennsylvania

School of Medicine; and National Institutes of Health Grant R01 CA125195 (toC.H.B.).

Address correspondence and reprint requests to Dr. Craig H. Bassing, Children’s Hos-pital of Philadelphia, 4054 Colket Translational Research Building, 3501 Civic CenterBoulevard, Philadelphia, PA 19104. E-mail address: bassing@mail.med.upenn.edu

Abbreviations used in this paper: ATM, ataxia telangiectasia mutated; DN, doublenegative; DP, double positive; Eb, TCRb enhancer; pDb1, Db1 promoter.

Copyright� 2010 by TheAmerican Association of Immunologists, Inc. 0022-1767/10/$16.00

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1001158

by guest on May 26, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

T and B cells to escape deletion and differentiate into maturelymphocytes that possess autoreactive potential in vitro, yetgenerally fail to cause autoimmunity in vivo (11–13). Theseadditional tolerance mechanisms restrain the systemic activa-tion of lymphocytes expressing both self-reactive and non–self-reactive receptors. A separate line of investigation demon-strated that the destructive potential of autoreactive lympho-cytes that escape central tolerance is restrained throughdominant peripheral tolerance mechanisms, such as those con-trolled by regulatory T and B cells (14, 15). In this context,allelic exclusion might function as an early cell-autonomoustolerance mechanism to reduce the frequency of developinglymphocytes with expression of two or more poly-specific Agreceptors and thereby facilitate central tolerance. If defects inallelic exclusion overwhelmed central tolerance mechanisms,peripheral tolerance checkpoints would be expected to functionas an additional barrier to restrain the destructive potential ofallelically included lymphocytes expressing autoreactive Agreceptors. Consistent with this notion, environmental factorssuch as viral infections can break down peripheral tolerancemechanisms and trigger autoimmunity driven by peripheralT cells expressing dual ab TCRs (16). However, allelic inclu-sion also can be beneficial because T lineage cells expressing dualabTCRs protect against infection by increasing the diversity ofreceptors that recognize foreign Ags (17).

Multiple mechanisms affect Ag receptor allelic exclusion

Although feedback inhibition is a major component by whichallelic exclusion is achieved, the sequence analyses of assembledTCR and Ig genes have revealed that additional mechanismscontribute to affect Ag receptor allelic exclusion. Because 1/3of V-(D)-J rearrangements occur in-frame, only 1/9 of de-veloping lymphocytes can assemble and express a particular Agreceptor gene from both alleles in the absence of feedbackregulation. Of the remaining developing cells, 4/9 would as-semble one in-frame and one out-of-frame gene and 4/9 wouldassemble out-of-frame genes on both alleles. Because the expres-sion of an Ag receptor chain is required for continued lympho-cyte differentiation, the 4/9 cells that fail to assemble aproductive gene on either allele are eliminated by apoptosis.Consequently, 1/5 is the theoretical maximum of maturelymphocytes that can express a particular Ag receptor gene fromboth alleles and exhibit allelic inclusion in the absence offeedback regulation and selection for or against such cells.Single-cell sequence analysis revealed that, of the 2–4% ofsplenic B cells containing two in-frame IgH genes, only onewas functional (Table I) (18). In contrast, sequencing demon-strated that ∼30% of ab T cells contain two in-frame TCRagenes (Table I) (9); yet, TCRa/TCRb pairing constraints andregulated TCRa-chain turnover reduce the frequency of

TCRa allelic inclusion to ∼10% (Table I) (9, 19). Similarsequence analyses of Igk, TCRb, TCRg, and TCRd rearrange-ments revealed two in-frame genes in 2–35% of cells depend-ing upon the locus (Table I) (5, 7, 20–22). These percentagesare higher than the corresponding allelic inclusion frequencies,suggesting that pairing restrictions or other mechanisms con-tribute to Igk, TCRb, TCRg, and TCRd allelic exclusion.Consistent with this notion, silencing of in-frame V-D-J-Cbgenes at the transcriptional and posttranscriptional levels con-tributes to TCRb allelic exclusion mouse ab T cells (20, 23).These data indicate thatmultiplemechanisms function in a suc-cessive manner to limit the frequency of cells with surfaceexpression of Ig or TCR chains from both allelic copies ofcorresponding loci. In this context, defects in mechanisms thatcontrol feedback inhibition could be countered by pairingrestrictions, transcriptional silencing, and posttranscriptionalsilencing to limit allelic inclusion and facilitate central toler-ance. Yet, dysfunction of these downstream allelic exclusionmechanisms, for example as an organism ages, could increasethe frequencies of allelically included mature lymphocytesexpressing autoreactive Ag receptors that need to be restrainedby peripheral tolerance checkpoints.

Developmental stage-specific control of allelic exclusion throughfeedback regulation

Most reviews of mechanisms that control Ag receptor allelicexclusion involve IgH and Igk loci, probably because studyingB lineage cells led to the discoveries of allelic exclusion, V(D)Jrecombination, and feedback regulation. In this work, themouse TCRb locus (Fig. 1) will serve as a paradigm for discus-sion of the developmental stage-specific control of allelic ex-clusion through feedback regulation because many recentadvances in this area have come from studying V-to-D-Jbrecombination. Frequent comparisons to the IgH and Igk loci(Fig. 1) also are provided.

Recombinational accessibility control of V-to-(D)-J recombination

Correlations between transcription, rearrangement, and nu-clease sensitivity of Ig gene segments led to the hypothesis thatmodulation of chromatin accessibility regulates V(D)J re-combination within the contexts of allelic exclusion (24).Consequently, the majority of studies investigating mecha-nisms that control Ag receptor allelic exclusion have focusedon the potential differential regulation of RAG accessibilitybetween loci on homologous chromosomes. For many years,recombinational (RAG) accessibility has been measured in-directly by germline transcription, general nuclease accessi-bility, and histone modifications associated with transcription(24). RAG accessibility also has been quantified by the ex-pression of reporter genes inserted into endogenous Ag re-ceptor loci (25–28); however, this indirect approach requires

Table I. Allelic inclusion of mouse Ag receptor loci

Locus Allelic Inclusion (%) Experimental ApproachBiallelic In-Frame

V(D)J (%) References

IgH 0.01 Natural allotypic differences 2–5 4, 18Igk 1–7 Hemizygous human Ck knock-in 11 5, 6TCRa 10 Anti-Va combinations 30 9, 19TCRb 1–3 Anti-Vb combinations 2–10 8, 20, 22TCRd 3 Anti-Vd combinations 35 7, 21TCRg 1 Anti-Vg combinations 10 7

3802 BRIEF REVIEWS: Ag RECEPTOR ALLELIC EXCLUSION

by guest on May 26, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

knowledge of local transcriptional regulation for unequivocalconclusions (27, 28). In addition to RAG accessibility, theV-to-(D)-J recombination step requires physical juxtapositionof RAG-accessible gene segments, RAG binding to a recom-bination signal sequence (RSS) flanking at least one of thesesegments, and capture of the other RSS to form productivesynaptic cleavage complexes. Recent advances in techniquesand reagents have enabled methods to investigate higher-orderstructural conformations of Ag receptor loci (29), RAG bind-ing to chromatin over genomic locations (30), RAG activity ata particular gene segment (31), and RAG cleavage withinindividual Ag receptor loci (32). Unfortunately, these assayshave inherent limitations that temper conclusions and canmeasure only a single mechanistic step required for V-to-(D)-J rearrangement. Such difficulties are substantial obstaclesfor elucidating the precise mechanisms that control V-to-(D)-J recombination within the contexts of feedback regulationand allelic exclusion.Ag receptor locus transcriptional enhancers and promoters

are required for V-to-(D)-J recombination; however, the pre-cise mechanisms by which these cis elements direct V rear-rangements and their potential role in allelic exclusion remainunknown. The IgH and Igk loci each contain two enhancers,whereas the TCRb locus contains only one known enhancer(Fig. 1). Promoters are known to reside upstream of each Vsegment within these loci, as well as upstream of the DQ52,Jk1, Db1, and Db2 segments (Fig. 1). The molecular mech-anisms by which enhancers and promoters direct V-to-(D)-Jrearrangements are understood most for the TCRb locus. TheTCRb enhancer (Eb) and the Db1 promoter (pDb1) forma holoenzyme complex that directs D-to-Jb1 and V-to-D-Jb1rearrangements, as well as germline transcription and chro-matin accessibility of Db1 and Jb1 segments (33). NeitherEb nor pDb1 controls Vb germline transcription or chroma-tin accessibility (34), which appear regulated at least in part byVb promoters (35). Collectively, these data suggest that Eband pDb1 may direct Vb rearrangements only through pro-moting accessibility of D–Jb complexes so the RAG proteinscan bind 59Db RSSs and capture Vb RSSs. The IgH and Igkenhancers and promoters most likely direct V-to-(D)-J re-combination through similar regulation of D–JH and Jk seg-ments. Consistent with this notion, experiments using acleavage-incompetent Rag1 protein have demonstrated thatRAG binding is detectable over germline D and J, but notV, segments within TCRb, IgH, and Igk loci (30). Yet, defin-

itive conclusions require similar analyses with TCRb and IgHalleles containing preassembled D–J complexes and incorpo-ration of assays that measure RAG/RSS interactions andjuxtaposition and synapsis of V and D–J segments. Becauseenhancers and promoters function together to direct V-to-(D)-J recombination, it is logical to assume that the enforce-ment of allelic exclusion could involve mechanisms that mod-ulate the activities of these cis elements. Support for thisnotion is provided by the observation that IgH allelic exclu-sion depends upon the IgH intronic enhancer maintaininghigh expression of V-D-J-CH genes during the pre-B to im-mature B cell transition (36).

Initiation of allelic exclusion

The V-to-(D)-J recombination step of Ag receptor loci subjectto allelic exclusion is thought to occur on one allele at a time,with the assembly and expression of a functional TCR or Iggene from the first allele inhibiting V-to-(D)-J rearrangementwithin the corresponding locus on the second allele (37). Toenforce allelic exclusion by feedback inhibition, only one al-lele can initiate V-to-(D)-J recombination during the timewindow required for feedback signals to exert their cellulareffects. Monoallelic initiation of V rearrangement could occurat any of the mechanistic steps required for V-to-(D)-J recombination.The correlation between transcription and rearrangement

led to studies suggesting that the differential positioning ofTCRb, IgH, and Igk loci at nuclear regions known to represstranscription might affect monoallelic initiation of V-to-(D)-Jrecombination (Fig. 2) (29). For example, TCRb loci associ-ate with inner nuclear membrane lamina or pericentromericheterochromatin at a higher frequency in CD42/CD82

(double-negative [DN]) thymocytes than in embryonic stemcells, B lineage cells, or CD4+/CD8+ (double-positive [DP])thymocytes (38, 39). TCRb alleles with an ectopic enhancerthat promotes TCRb allelic inclusion are localized less fre-quently at nuclear membrane lamina and pericentromericheterochromatin (39), providing indirect evidence that asso-ciation of Ag receptor loci with these transcriptional repressivenuclear regions may suppress V-to-(D)-J rearrangement. De-spite frequent association of TCRb loci with nuclear mem-brane lamina and pericentromeric heterochromatin, D-to-Jbrearrangements, germline Vb transcription, and Vb RAGaccessibility each occur on both TCRb alleles in developingthymocytes (31, 37). In addition, the association of IgH loci

FIGURE 1. Genomic configuration of

mouse TCRb, IgH, and Igk loci. Sche-

matic diagrams of germline TCRb, IgH,

and Igk loci depicting the relative loca-

tions of V segments (blue rectangles), D

segments (yellow rectangles), J segments

(green rectangles), C regions (gray rec-

tangles), enhancers (red ovals), and pro-

moters (blue circles). The relative

location of the Igk Sis element (blue

square) also is shown. The loci are not

drawn to scale, particularly with regard to

the genomic distances between V and D–

J segments.

The Journal of Immunology 3803

by guest on May 26, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

with pericentromeric heterochromatin does not inhibit tran-scription of germline or rearranged alleles (32, 40). Collec-tively, these data suggest that the positioning of TCR and Igalleles at inner nuclear membrane lamina or pericentromericheterochromatin may suppress V-to-(D)-J rearrangements byinhibiting the juxtaposition of V and D–J segments, ratherthan through suppressing transcription or RAG accessibility(Fig. 2). Consistent with this notion, germline and D–Jb-rearranged loci positioned at pericentromeric heterochromatindo not exhibit contraction by looping between Vb and D/Jbsegments as do unrearranged TCRb loci residing away fromthese nuclear regions (38). Identification of the cis elements andtrans factors that control the association of TCR and Ig lociwith inner nuclear membrane lamina and pericentromeric het-erochromatin is required to elucidate the potential function ofnuclear positioning in regulating V-to-(D)-J rearrangementsand allelic exclusion. Logical candidates have been providedby the discoveries of cis elements between V and J segmentswithin the TCRb, IgH, and Igk loci (41–43), and the demon-stration that this Igk Sis element binds the Ikaros transcrip-tional repressor, targets Igk transgenes to centromeric hetero-chromatin, and inhibits V-to-Jk rearrangement (42).Monoallelic initiation of V-to-(D)-J recombination also

could be affected by developmentally regulated conformationchanges of Ag receptor loci that control the juxtaposition ofRAG-accessible V and D–J segments (Fig. 2). Because germ-line TCRb, IgH, and Igk loci span large chromosomal dis-tances, the positioning of V and D–J segments in proximityby locus contraction most likely facilitates or is required for

primary V-to-(D)-J rearrangements within these loci. Germ-line and/or D–J-rearranged TCRb, IgH, and Igk loci exhibitmonoallelic contraction by chromosome looping between Vand D/J segments at a higher frequency in lymphocytes of thelineage and stage where these loci rearrange as compared within other cells (29, 38, 39). Data revealing that rearrangementsof Vb and VH segments inserted just upstream of Db or DH

segments cause allelic inclusion and/or are not subject tonormal feedback inhibition provide indirect evidence thatlocus contraction/decontraction may regulate the V-to-D-Jrecombination step within TCRb and IgH loci (23, 44).These Ag receptor locus conformational changes may be reg-ulated by mechanisms that direct association of TCR and Igloci with repressive nuclear regions and/or by distinct ciselements and trans factors such as enhancer/promoter inter-actions (45) and CCCTC-binding factor/cohesin proteins(46). However, factors in addition to locus decontractionmust control the juxtaposition of accessible V and D–J seg-ments within TCRb and Igk loci to regulate secondary Vrearrangements across short distances and recombination ofthe Vb14 segment, which resides in close proximity to D–Jbsegments.In addition to RAG accessibility and juxtaposition, intrinsic

properties of RSSs may contribute to affect allelic exclusion.The inherent inefficiency of Vb and VH RSSs could restrainthe overall rate at which accessible and juxtaposed V and D–Jsegments bind RAG and/or form productive synaptic com-plexes such that the frequency of synchronous V-to-D-J rear-rangements between TCRb or IgH alleles is rare (47). Vbs

FIGURE 2. Multiple redundant and successive mechanisms most likely cooperate to control Ag receptor allelic exclusion. Monoallelic initiation of V-to-(D)-J

rearrangement, feedback signals, and maintenance of feedback inhibition most likely function together to achieve allelic exclusion of TCRb, IgH, and Igk loci.

Monoallelic initiation of V-to-(D)-J rearrangement may be regulated by asynchronous replication, localization, conformations, transcription, and/or histone

modifications between TCRb, IgH, and Igk loci on homologous chromosomes. Feedback inhibition appears to involve signals that directly prevent V-to-(D)-J

rearrangement by down-regulating accessibility, juxtaposition, or RAG binding to Ag receptor loci, and may involve signals that indirectly prevent V-to-(D)-J

rearrangement by inactivating RAG activity or silencing germline V segments. Maintenance of feedback inhibition most likely is achieved through decontraction

and repositioning of loci, silencing of germline V segments, and developmental stage-specific expression of factors that promote or inhibit secondary V-to-(D)-J

rearrangements.

3804 BRIEF REVIEWS: Ag RECEPTOR ALLELIC EXCLUSION

by guest on May 26, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

rearrange to D–Jb complexes with little or no Vb-to-Jbjoining due to beyond 12/23 compatibility RSS joiningrestrictions (37). These RSS restrictions limit the number ofpotential Vb rearrangements on each allele to 70 (35 Vbs3 2Dbs), as compared with the 840 (35 Vbs 3 2 Dbs 3 12 Jbs)potential Vb rearrangements allowed by the 12/23 rule. To-gether, joining restrictions and inefficiencies of TCRb RSSsmay lower the probability that both alleles can initiate Vbrearrangement in the time window required for feedback in-hibition. Consistent with this notion, replacement of theVb14 RSS with the 10-fold more efficient 39Db1 RSS, whichrearranges to 59Db or Jb RSSs, short-circuits TCRb feedbacksignals, and increases the frequency of TCRb allelic inclusion(C.H. Bassing, unpublished observations). However, these dataalso support the notion that TCRb feedback signals activate59Db RSS-binding trans factors to prevent RAG accessibilityand V-to-D–Jb rearrangements (48, 49), analogous to inhibi-tion of V-to-Db rearrangements by c-fos binding 39Db RSSs(37). Considering that TCRb and IgH, but not TCRd genesare assembled through D–J intermediates, such inhibition ofRAG access to 59D RSSs could account for why feedbackregulation inhibits the assembly of V-D-Jb and V-D-JHexons more stringently than V-D-Jd exons (Table I) (7, 18,20–22).

Feedback inhibition

The assembly and expression of a functional TCRb or IgHgene in DN thymocytes or pro-B cells, respectively, activateintracellular pathways that signal feedback inhibition, cessa-tion of RAG expression, cellular proliferation/expansion, anddifferentiation into DP thymocytes or pre-B cells (Fig. 2).Although the pathways and mechanisms through whichTCRb and IgH chains signal these processes remain largelyunknown, experiments have revealed that DN thymocytesemploy distinct pathway(s) to signal feedback inhibitionthrough mechanisms involving the E47 and Ets-1 transcrip-tion factors (49–51). TCRb-signaled downregulation of E47appears to inhibit V-to-D-Jb rearrangements in DN cellsthrough rendering Vb segment RAG inaccessible (50).Ets-1 binds to and represses Eb (52), which is not requiredfor the expression of assembled V-D-J-Cb genes in DN cellsor TCRb-mediated DN-to-DP expansion and differentia-tion (53). Thus, TCRb feedback signals could modulateEts-1 activity to inhibit Eb in DN cells and thereby down-regulate RAG accessibility of 59Db RSSs on the D–Jb-rearranged allele while enabling continued TCRb expressionfrom the V-D-Jb–rearranged allele. This hypothetical mech-anism also would explain how TCRb feedback signals sup-press Vb14-to-D-Jb rearrangements without downregulatingRAG accessibility of Vb14 segments (31). The use of alleleswith preassembledD–J complexes or V-D-J-C genes alongwithassays of RAG binding, juxtaposition, and synapsis should fa-cilitate elucidation of the mechanisms through which TCRband IgH feedback signals inhibit V-to-D-J rearrangements inDN thymocytes and pro-B cells.In addition to the activation of mechanisms that directly

inhibit V-to-D-J rearrangements in DN or pro-B cells, TCRband IgH signals may indirectly promote feedback inhibitionthrough cessation of RAG expression, cellular proliferation/expansion, and differentiation into DP thymocytes or pre-B cells. For example, TCRb and IgH signals that drive DN

thymocytes or pro-B cells from G0/G1 and into S phase maycontribute to affect allelic exclusion by shortening the timewindow for additional V-to-D-J rearrangements (Fig. 2). Inaddition, considering that TCRb-driven cellular proliferationis required for progressive silencing of TCRg transcriptionduring the DN-to-DP transition (54), similar TCRb- andIgH-dependent epigenetic mechanisms may silence germlineV segment transcription to help suppress V-to-D-J rearrange-ments in DP thymocytes and pre-B cells. Finally, TCRb andIgH signals that inhibit RAG expression also could contributeto allelic exclusion by preventing DN or pro-B cells fromcontinuing V-to-D-J recombination (49).

Maintenance of allelic exclusion

To maintain TCRb and IgH allelic exclusion, V-to-D-J rear-rangements must remain suppressed on D–J-rearranged allelesfollowing RAG re-expression in DP thymocytes and pre-B cells. Evidence suggests that developmental stage-specificinhibition of RAG access to germline V segments and juxta-position of V and D–J segments most likely cooperate tomaintain feedback inhibition of V-to-D-J recombinationon D–J-rearranged alleles (Fig. 2) (37–39, 55, 56). Studiesinvestigating mechanisms that maintain TCRb feedback in-hibition have been conducted using DP cells of RAG-deficient mice either treated with anti-CD3 Abs or expressinga TCRb transgene to drive DN-to-DP differentiation. Onefinding often ignored in these experiments is that germlineVb transcripts and Vb chromatin marks associated with ac-tive transcription are present at higher levels in DP cells ofRAG-deficient mice treated with anti-CD3 Abs as comparedwith DP cells from RAG-deficient mice expressing a TCRbtransgene (41, 57). Germline Vb14 transcripts exhibit thelargest difference. These data indicate that anti-CD3 treat-ment and TCRb transgenes do not equally activate pre-TCR signaling pathways or thresholds required for Vb silenc-ing; determining which represents the more physiologic con-dition would be important. Germline transcription of D–Jbsegments in DP thymocytes has been interpreted that D–Jbcomplexes remain RAG accessible (41, 57); however, analysisof RAG binding to 59Db RSSs on alleles with preassembledD–Jb complexes is required for definitive conclusions.Maintenance of TCRb and IgH allelic exclusion in DP

thymocytes and pro-B cells also must involve the suppressionof secondary V rearrangements on alleles that have assembledout-of-frame V-D-J-C genes in DN or pro-B cells (Fig. 2). The12/23 rule and beyond 12/23 restrictions inhibit, but do notblock V-to-JH and V-to-Jb rearrangements in pro-B cells andDN thymocytes (48, 58). Such RSS joining restrictions mightcontribute to suppress V rearrangements over out-of-frameV-D-J-C genes in pre-B or DP cells (Fig. 2), particularly ifthe downstream J segments remain RAG accessible. BecauseVb rearrangements to Db2–Jb2 complexes are not restrictedby RSS joining constraints, additional factors must suppresssuch recombination events on alleles that have assembled out-of-frame V-D-J-Cb1 genes in DN thymocytes (Fig. 2).Germline Vb segments located immediately upstream of V-D-J-Cb genes remain transcribed in DP thymocytes (37, 59),yet Vb rearrangements to Db2–Jb2 complexes are largelysuppressed (37). This data indicate that V-to-D-Jb rearrange-ments must be controlled by developmental stage-specific fac-tor(s) that controls RAG binding to RSSs, juxtaposition, and/

The Journal of Immunology 3805

by guest on May 26, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

or synapsis in either DN or DP thymocytes (37). One suchcandidate is the E47 transcription factor because forcedexpression of this factor promotes Vb rearrangements to Db2–Jb2 complexes in DP cells (50). Notably, V-to-Db2 recombi-nation intermediates are detectable in DP thymocytes of wild-type mice, and intermediates involving Vb14, which residesclose to Db–Jb segments, are observed in DP cells of TCRbtransgenic mice (55, 60). Although such V(D)J recombinationintermediates could arise from contaminating DN cells, theseobservations suggest that Vb rearrangements to Db2–Jb2complexes on alleles with V-D-J-Cb1 genes are not com-pletely blocked in DP thymocytes. Such secondary Vb rear-rangements on alleles with out-of-frame V-D-J-Cb1 genescould lead to TCRb allelic inclusion either before or afterpositive selection of ab TCRs containing TCRb-chains fromthe other allele. In addition, secondary Vb rearrangements onalleles with in-frameV-D-J-Cb1 genes during positive selectioncould lead to the loss of an ab TCR or the replacement ofa selected ab TCR with an autoreactive receptor. The abilityof DNA sequences to form boundaries between active andinactive Vb chromatin domains upstream of assembled V-D-J-Cb1 genesmay have evolved to suppress the frequency of suchdeleterious Vb rearrangements in DP thymocytes (59).Interaction of self-Ags with autoreactive immature B cells or

naive ab T lymphocytes sustains or reinduces RAG expres-sion, respectively, to promote V rearrangements that replacein-frame V-Jk or V-D-Jb exons. To maintain allelic exclusionof TCRb, Igk, and possibly IgH loci during ab TCR revisionand BCR editing, V rearrangements need to be restricted onalleles with in-frame V-(D)-J-C genes. Studies of mechanismsthat potentially suppress V-to-(D)-J rearrangements on alleleslacking or containing out-of-frame V-(D)-J-C genes to main-tain allelic exclusion during BCR editing and ab TCR revisionare lacking. Yet, one study indicates that V-to-Jk rearrange-ments occur at equal frequency on both alleles during BCRediting, leading to allelic inclusion in ∼10% of cells (5). Con-sidering that the frequency of ab T cells exhibiting TCRballelic inclusion progressively increases as mice age (8), V-to-D-Jb rearrangements also might occur with equal probabilityon both alleles during TCRb revision. Because BCR editingand ab TCR revision will regenerate self-reactive receptorsthat could be edited/revised or restrained by peripheral toler-ance checkpoints, the immunologic consequences of allelicinclusion during these processes may not exert physiologicpressure to restrict V rearrangements on alleles with in-frameV-(D)-J-C genes.

Potential physiologic consequences of defects in allelic exclusion

Defects in mechanisms that control Ag receptor allelic exclusioncould have deleterious consequences for host organisms inaddition to causing autoimmunity. TCRb and IgH gene re-arrangements proceed through the programmed induction ofRAG DNA double-strand breaks (DSBs) in G1 phase DNthymocytes or pro-B cells with expression from in-frameV-D-J-C genes driving cells into S phase and through multiplecell cycles. The ataxia telangiectasia mutated (ATM) and p53tumor suppressor proteins inhibit the persistence of RAG DSBsthroughout the cell cycle (61); however, some fraction of RAGDSBs generated during V-to-D-Jb rearrangements normallyevades the G1/S checkpoint and induces apoptosis of DNthymocytes at the G2/M checkpoint (62). These RAG DSBs

could arise because the mechanisms that control monoallelicinitiation and feedback inhibition of Vb rearrangements areonly effective in 90–99% of DN thymocytes, as evidenced byTCRb allelic inclusion and biallelic in-frame V-D-J-Cbrearrangements in 1–10% of ab T ;cells (20). Failure to en-force monoallelic initiation or feedback inhibition of V-to-D-Jrearrangements at normal levels could result in the generationof RAG DSBs, whereas TCRb or IgH signals are driving de-veloping lymphocytes from G0/G1 through G1 and into Sphase. This would lead to increased elimination of DN orpro-B cells that express functional TCRb or IgH chains andcompromise host immunity by shrinking the repertoire of Agreceptors expressed on mature lymphocytes. Because RAGDSBs that persist into S phase can result in genomic instability,deficiencies in these mechanisms controlling monoallelic initi-ation and feedback inhibition of V-to-D-J rearrangements alsomight cause an increased predisposition to lymphomas drivenby TCRb or IgH translocations. Accordingly, monoallelic ini-tiation and feedback inhibition of V-to-D-J rearrangement mayhave evolved through pressure to generate broad Ag receptorrepertoires and restrain oncogenic translocations by enforcingregulation upon a random process. In this context, the morestringent allelic exclusion observed at the IgH locus as com-pared with the TCRb, TCRg, and TCRd loci (Table I) mayhave evolved in response to the greater oncogenic potential ofRAG DSBs introduced at the IgH locus than at these other loci(63). In the B cell lineage, similar pressure combined withgreater cellular proliferation upon the rearrangement and ex-pression of IgH genes than IgL genes may have led to morestrict enforcement of allelic exclusion at the IgH locus as com-pared with the Igk and Igl loci.

Evidence for lateral inhibition of V(D)J recombination

Since the discovery that allelic exclusion is regulated byfeedback inhibition, many experiments have been conductedto evaluate whether the monoallelic initiation of V rear-rangements occurs through deterministic versus stochasticmechanisms. TCRb, IgH, and Igk loci each replicate asyn-chronously in lymphocytes, with the order between allelesrandomly determined and clonally maintained (64). The early

FIGURE 3. Potential lateral inhibition of V(D)J recombination may

contribute to enforce allelic exclusion. RAG cleavage of Ag receptor loci in

developing lymphocytes activates ATM-dependent signals that may transiently

inhibit additional V(D)J recombination events. This regulation may enable

time for DNA repair, transcription, translation, surface expression, and signal-

ing required for feedback inhibition. These putative ATM signals could di-

rectly prevent V-to-(D)-J rearrangements or indirectly, such as through

downregulation of RAG activity.

3806 BRIEF REVIEWS: Ag RECEPTOR ALLELIC EXCLUSION

by guest on May 26, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

replicating Igk allele is preferentially demethylated, renderedRAG accessible, and selected for V-to-Jk rearrangement inpre-B cells (64), yet no connection has been reported betweenasynchronous TCRb or IgH locus replication and determina-tion of monoallele V-to-D-J rearrangement. In contrast, avail-able data suggest that stochastic association of TCRb alleleswith inner nuclear membrane lamina and pericentromericheterochromatin reduces the probability that biallelic V-to-D-Jb rearrangements occur before feedback inhibition (37).These Igk deterministic and TCRb stochastic models eachassume that a static time window exists between monoallelicinitiation and feedback inhibition of the V-to-(D)-J recom-bination step.One neglected proposal of the original feedback inhibition

model is that V(D)J recombination events on one allele couldactivate signals that transiently inhibit rearrangements on thesecond allele (65). In response to RAG DSBs, the ATM kinasephosphorylates numerous proteins and activates a geneticprogram that includes genes encoding proteins that regulatetranscription and chromatin accessibility (66). ATM-deficientlymphocytes exhibit increased RAG cleavage of IgH and Igkloci in G1 phase cells and biallelic Igk chromosome breaks andtranslocations upon re-entry into the cell cycle (32). These dataare consistent with the notion that RAG cleavage on one alleleactivates ATM-mediated lateral inhibition signals to suppressrecombination events on the other allele (Fig. 3) until DNArepair and termination of the DNA damage response, althoughother interpretations are possible. Although no obvious effect ofATM deficiency was observed upon IgH allelic exclusion inbone marrow cells of young mice (32), the frequency of TCRballelic inclusion in ATM-deficient thymocytes and peripheralab T cells is greater than in wild-type cells (N.C. Steinel andC.H. Bassing, unpublished observations). This difference couldreflect the relative dependence of TCRb and IgH loci uponATM for ensuring monoallelic RAG cleavage and/or sup-pressing aberrant rearrangement events that prevent the assem-bly of functional in-frame coding joins. However, confirmationand identification of these potential ATM-activated signalingpathways and the elucidation of their potential function incontrolling V-to-(D)-J rearrangements and contributing tothe initiation of allelic exclusion are required for unequivocalconclusions.

ConclusionsIn the past few years, studies incorporating advances in tech-niques and reagents have revealed that Ag receptor allelic ex-clusion most likely is controlled by multiple redundant andsuccessive mechanisms (Fig. 2). The next step within the field isto identify the DNA elements, protein factors, and potentialRNA molecules that may control each of these individualmechanisms. These efforts should be guided by the more ad-vanced knowledge of the related X chromosome inactivationand imprinting processes in mammals, as well as the generalcellular mechanisms known to silence developmentally regu-lated site-specific DNA recombination events in Schizosacchar-omyces pombe and Tetrahymena thermophila. Subsequent stepswill be to inactivate/mutate these candidate cis elements andtrans factors to confirm their potential role in regulating allelicexclusion and determine the precise mechanisms throughwhich they function in this capacity. To reach unequivocalconclusions, the design and interpretation of these experiments

will need to consider potential regulation at all mechanisticsteps, not just the one being assayed. In the long-term, experi-ments that manipulate all of the redundant and successivemechanisms that regulate Ag receptor allelic exclusion will berequired to reveal the biological relevance of this phenomenonthat has mystified immunologists for half a century.

DisclosuresThe authors have no financial conflicts of interest.

References1. Pernis, B. G., G. Chiappino, A. S. Kelus, and P. G. H. Gell. 1965. Cellular lo-

calization of immunoglobulins with different allotypic specificities in rabbit lym-phoid tissues. J. Exp. Med. 122: 853–876.

2. Mostoslavsky, R., F. W. Alt, and K. Rajewsky. 2004. The lingering enigma of theallelic exclusion mechanism. Cell 118: 539–544.

3. Zakharova, I. S., A. I. Shevchenko, and S. M. Zakian. 2009. Monoallelic geneexpression in mammals. Chromosoma 118: 279–290.

4. Barreto, V., and A. Cumano. 2000. Frequency and characterization of phenotypicIg heavy chain allelically included IgM-expressing B cells in mice. J. Immunol. 164:893–899.

5. Casellas, R., Q. Zhang, N. Y. Zheng, M. D. Mathias, K. Smith, and P. C. Wilson.2007. Igkappa allelic inclusion is a consequence of receptor editing. J. Exp. Med.204: 153–160.

6. Velez, M. G., M. Kane, S. Liu, S. B. Gauld, J. C. Cambier, R. M. Torres, andR. Pelanda. 2007. Ig allotypic inclusion does not prevent B cell development orresponse. J. Immunol. 179: 1049–1057.

7. Boucontet, L., N. Sepulveda, J. Carneiro, and P. Pereira. 2005. Mechanisms con-trolling termination of V-J recombination at the TCRgamma locus: implications forallelic and isotypic exclusion of TCRgamma chains. J. Immunol. 174: 3912–3919.

8. Balomenos, D., R. S. Balderas, K. P. Mulvany, J. Kaye, D. H. Kono, andA. N. Theofilopoulos. 1995. Incomplete T cell receptor V beta allelic exclusion anddual V beta-expressing cells. J. Immunol. 155: 3308–3312.

9. von Boehmer, H., and F. Melchers. 2010. Checkpoints in lymphocyte developmentand autoimmune disease. Nat. Immunol. 11: 14–20.

10. Wucherpfennig, K. W., P. M. Allen, F. Celada, I. R. Cohen, R. De Boer,K. C. Garcia, B. Goldstein, R. Greenspan, D. Hafler, P. Hodgkin, et al. 2007. Pol-yspecificity of T cell and B cell receptor recognition. Semin. Immunol. 19: 216–224.

11. Iliev, A., L. Spatz, S. Ray, and B. Diamond. 1994. Lack of allelic exclusion permitsautoreactive B cells to escape deletion. J. Immunol. 153: 3551–3556.

12. Zal, T., S. Weiss, A. Mellor, and B. Stockinger. 1996. Expression of a second re-ceptor rescues self-specific T cells from thymic deletion and allows activation ofautoreactive effector function. Proc. Natl. Acad. Sci. USA 93: 9102–9107.

13. Sarukhan, A., C. Garcia, A. Lanoue, and H. von Boehmer. 1998. Allelic inclusion ofT cell receptor alpha genes poses an autoimmune hazard due to low-level expressionof autospecific receptors. Immunity 8: 563–570.

14. Wing, K., and S. Sakaguchi. 2010. Regulatory T cells exert checks and balances onself tolerance and autoimmunity. Nat. Immunol. 11: 7–13.

15. Mizoguchi, A., and A. K. Bhan. 2006. A case for regulatory B cells. J. Immunol. 176:705–710.

16. Ji, Q., A. Perchellet, and J. M. Goverman. 2010. Viral infection triggers centralnervous system autoimmunity via activation of CD8+ T cells expressing dual TCRs.Nat. Immunol. 11: 628–634.

17. He, X., C. A. Janeway Jr., M. Levine, E. Robinson, P. Preston-Hurlburt, C. Viret,and K. Bottomly. 2002. Dual receptor T cells extend the immune repertoire forforeign antigens. Nat. Immunol. 3: 127–134.

18. ten Boekel, E., F. Melchers, and A. G. Rolink. 1998. Precursor B cells showing Hchain allelic inclusion display allelic exclusion at the level of pre-B cell receptorsurface expression. Immunity 8: 199–207.

19. Niederberger, N., K. Holmberg, S. M. Alam, W. Sakati, M. Naramura, H. Gu, andN. R. Gascoigne. 2003. Allelic exclusion of the TCR alpha-chain is an active processrequiring TCR-mediated signaling and c-Cbl. J. Immunol. 170: 4557–4563.

20. Steinel, N. C., B. L. Brady, A. C. Carpenter, K. S. Yang-Iott, and C. H. Bassing.2010. Posttranscriptional silencing of VbetaDJbetaCbeta genes contributes toTCRbeta allelic exclusion in mammalian lymphocytes. J. Immunol. 185: 1055–1062.

21. Sleckman, B. P., B. Khor, R. Monroe, and F. W. Alt. 1998. Assembly of productiveT cell receptor delta variable region genes exhibits allelic inclusion. J. Exp. Med. 188:1465–1471.

22. Aifantis, I., J. Buer, H. von Boehmer, and O. Azogui. 1997. Essential role of thepre-T cell receptor in allelic exclusion of the T cell receptor beta locus. [Publishederratum appears in 1997 Immunity 7: 895.] Immunity 7: 601–607.

23. Sieh, P., and J. Chen. 2001. Distinct control of the frequency and allelic ex-clusion of the V beta gene rearrangement at the TCR beta locus. J. Immunol. 167:2121–2129.

24. Bassing, C. H., W. Swat, and F. W. Alt. 2002. The mechanism and regulation ofchromosomal V(D)J recombination. Cell 109(Suppl.): S45–S55.

25. Jia, J., M. Kondo, and Y. Zhuang. 2007. Germline transcription from T-cell re-ceptor Vbeta gene is uncoupled from allelic exclusion. EMBO J. 26: 2387–2399.

26. Liang, H. E., L. Y. Hsu, D. Cado, and M. S. Schlissel. 2004. Variegated tran-scriptional activation of the immunoglobulin kappa locus in pre-B cells contributesto the allelic exclusion of light-chain expression. Cell 118: 19–29.

The Journal of Immunology 3807

by guest on May 26, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

27. Taylor, B., B. S. Cobb, L. Bruno, Z. Webster, A. G. Fisher, andM. Merkenschlager. 2009. A reappraisal of evidence for probabilistic models ofallelic exclusion. Proc. Natl. Acad. Sci. USA 106: 516–521.

28. Amin, R. H., D. Cado, H. Nolla, D. Huang, S. A. Shinton, Y. Zhou, R. R. Hardy,and M. S. Schlissel. 2009. Biallelic, ubiquitous transcription from the distalgermline Igkappa locus promoter during B cell development. Proc. Natl. Acad. Sci.USA 106: 522–527.

29. Jhunjhunwala, S., M. C. van Zelm, M. M. Peak, and C. Murre. 2009. Chro-matin architecture and the generation of antigen receptor diversity. Cell 138:435–448.

30. Ji, Y., W. Resch, E. Corbett, A. Yamane, R. Casellas, and D. G. Schatz. 2010. Thein vivo pattern of binding of RAG1 and RAG2 to antigen receptor loci. Cell 141:419–431.

31. Yang-Iott, K. S., A. C. Carpenter, M. A. Rowh, N. Steinel, B. L. Brady,K. Hochedlinger, R. Jaenisch, and C. H. Bassing. 2010. TCR beta feedbacksignals inhibit the coupling of recombinationally accessible V beta 14 segmentswith DJ beta complexes. J. Immunol. 184: 1369–1378.

32. Hewitt, S. L., B. Yin, Y. Ji, J. Chaumeil, K. Marszalek, J. Tenthorey, G. Salvagiotto,N. Steinel, L. B. Ramsey, J. Ghysdael, et al. 2009. RAG-1 and ATM coordinatemonoallelic recombination and nuclear positioning of immunoglobulin loci. Nat.Immunol. 10: 655–664.

33. Oestreich, K. J., R. M. Cobb, S. Pierce, J. Chen, P. Ferrier, and E. M. Oltz. 2006.Regulation of TCRbeta gene assembly by a promoter/enhancer holocomplex. Im-munity 24: 381–391.

34. Mathieu, N., W. M. Hempel, S. Spicuglia, C. Verthuy, and P. Ferrier. 2000.Chromatin remodeling by the T cell receptor (TCR)-beta gene enhancer duringearly T cell development: implications for the control of TCR-beta locus recombi-nation. J. Exp. Med. 192: 625–636.

35. Ryu, C. J., B. B. Haines, H. R. Lee, Y. H. Kang, D. D. Draganov, M. Lee,C. E. Whitehurst, H. J. Hong, and J. Chen. 2004. The T-cell receptor beta variablegene promoter is required for efficient V beta rearrangement but notallelic exclusion. Mol. Cell. Biol. 24: 7015–7023.

36. Li, F., and L. A. Eckhardt. 2009. A role for the IgH intronic enhancer E mu inenforcing allelic exclusion. J. Exp. Med. 206: 153–167.

37. Krangel, M. S. 2009. Mechanics of T cell receptor gene rearrangement. Curr. Opin.Immunol. 21: 133–139.

38. Skok, J. A., R. Gisler, M. Novatchkova, D. Farmer, W. de Laat, and M. Busslinger.2007. Reversible contraction by looping of the Tcra and Tcrb loci in rearrangingthymocytes. Nat. Immunol. 8: 378–387.

39. Schlimgen, R. J., K. L. Reddy, H. Singh, and M. S. Krangel. 2008. Initiation ofallelic exclusion by stochastic interaction of Tcrb alleles with repressive nuclearcompartments. Nat. Immunol. 9: 802–809.

40. Daly, J., S. Licence, A. Nanou, G. Morgan, and I. L. Martensson. 2007. Tran-scription of productive and nonproductive VDJ-recombined alleles after IgH allelicexclusion. EMBO J. 26: 4273–4282.

41. Chattopadhyay, S., C. E. Whitehurst, F. Schwenk, and J. Chen. 1998. Biochemicaland functional analyses of chromatin changes at the TCR-beta gene locus duringCD42CD82 to CD4+CD8+ thymocyte differentiation. J. Immunol. 160: 1256–1267.

42. Liu, Z., P. Widlak, Y. Zou, F. Xiao, M. Oh, S. Li, M. Y. Chang, J. W. Shay, andW. T. Garrard. 2006. A recombination silencer that specifies heterochromatinpositioning and ikaros association in the immunoglobulin kappa locus. Immunity24: 405–415.

43. Featherstone, K., A. L. Wood, A. J. Bowen, and A. E. Corcoran. 2010. The mouseimmunoglobulin heavy chain V-D intergenic sequence contains insulators that mayregulate ordered V(D)J recombination. J. Biol. Chem. 285: 9327–9338.

44. Bates, J. G., D. Cado, H. Nolla, and M. S. Schlissel. 2007. Chromosomal positionof a VH gene segment determines its activation and inactivation as a substrate for V(D)J recombination. J. Exp. Med. 204: 3247–3256.

45. Liu, Z., and W. T. Garrard. 2005. Long-range interactions between three transcrip-tional enhancers, active Vkappa gene promoters, and a 39 boundary sequence span-ning 46 kilobases. Mol. Cell. Biol. 25: 3220–3231.

46. Degner, S. C., T. P. Wong, G. Jankevicius, and A. J. Feeney. 2009. Cutting edge:developmental stage-specific recruitment of cohesin to CTCF sites throughout im-munoglobulin loci during B lymphocyte development. J. Immunol. 182: 44–48.

47. Liang, H. E., L. Y. Hsu, D. Cado, L. G. Cowell, G. Kelsoe, and M. S. Schlissel.2002. The “dispensable” portion of RAG2 is necessary for efficient V-to-DJ rear-rangement during B and T cell development. Immunity 17: 639–651.

48. Bassing, C. H., F. W. Alt, M. M. Hughes, M. D’Auteuil, T. D. Wehrly,B. B. Woodman, F. Gartner, J. M. White, L. Davidson, and B. P. Sleckman. 2000.Recombination signal sequences restrict chromosomal V(D)J recombination beyondthe 12/23 rule. Nature 405: 583–586.

49. Gartner, F., F. W. Alt, R. Monroe, M. Chu, B. P. Sleckman, L. Davidson, andW. Swat. 1999. Immature thymocytes employ distinct signaling pathways for allelicexclusion versus differentiation and expansion. Immunity 10: 537–546.

50. Agata, Y., N. Tamaki, S. Sakamoto, T. Ikawa, K. Masuda, H. Kawamoto, andC. Murre. 2007. Regulation of T cell receptor beta gene rearrangements and allelicexclusion by the helix-loop-helix protein, E47. Immunity 27: 871–884.

51. Eyquem, S., K. Chemin, M. Fasseu, and J. C. Bories. 2004. The Ets-1 transcriptionfactor is required for complete pre-T cell receptor function and allelic exclusion atthe T cell receptor beta locus. Proc. Natl. Acad. Sci. USA 101: 15712–15717.

52. Prosser, H. M., D. Wotton, A. Gegonne, J. Ghysdael, S. Wang, N. A. Speck, andM. J. Owen. 1992. A phorbol ester response element within the human T-cellreceptor beta-chain enhancer. Proc. Natl. Acad. Sci. USA 89: 9934–9938.

53. Busse, C. E., A. Krotkova, and K. Eichmann. 2005. The TCRbeta enhancer isdispensable for the expression of rearranged TCRbeta genes in thymic DN2/DN3populations but not at later stages. J. Immunol. 175: 3067–3074.

54. Ferrero, I., S. J. Mancini, F. Grosjean, A. Wilson, L. Otten, and H. R. MacDonald.2006. TCRgamma silencing during alphabeta T cell development depends uponpre-TCR-induced proliferation. J. Immunol. 177: 6038–6043.

55. Jackson, A., H. D. Kondilis, B. Khor, B. P. Sleckman, and M. S. Krangel. 2005.Regulation of T cell receptor beta allelic exclusion at a level beyond accessibility.Nat. Immunol. 6: 189–197.

56. Tripathi, R., A. Jackson, and M. S. Krangel. 2002. A change in the structure ofVbeta chromatin associated with TCR beta allelic exclusion. J. Immunol. 168:2316–2324.

57. Wang, L., M. Senoo, and S. Habu. 2002. Differential regulation between geneexpression and histone H3 acetylation in the variable regions of the TCRbeta locus.Biochem. Biophys. Res. Commun. 298: 420–426.

58. Koralov, S. B., T. I. Novobrantseva, J. Konigsmann, A. Ehlich, and K. Rajewsky.2006. Antibody repertoires generated by VH replacement and direct VH to JHjoining. Immunity 25: 43–53.

59. Brady, B. L., M. A. Oropallo, K. S. Yang-Iott, T. Serwold, K. Hochedlinger,R. Jaenisch, I. L. Weissman, and C. H. Bassing. 2010. Position-dependent silencingof germline Vb segments on TCRb alleles containing preassembled VbDJb1Cb1genes. J. Immunol. 185: 3564–3573.

60. Mathieu, N., S. Spicuglia, S. Gorbatch, O. Cabaud, C. Fernex, C. Verthuy,W. M. Hempel, A.-O. Hueber, and P. Ferrier. 2003. Assessing the role of the T cellreceptor beta gene enhancer in regulating coding joint formation during V(D)Jrecombination. J. Biol. Chem. 278: 18101–18109.

61. Dujka, M. E., N. Puebla-Osorio, O. Tavana, M. Sang, and C. Zhu. 2010. ATMand p53 are essential in the cell-cycle containment of DNA breaks during V(D)Jrecombination in vivo. Oncogene 29: 957–965.

62. Pedraza-Alva, G., M. Koulnis, C. Charland, T. Thornton, J. L. Clements,M. S. Schlissel, and M. Rincon. 2006. Activation of p38 MAP kinase by DNAdouble-strand breaks in V(D)J recombination induces a G2/M cell cycle check-point. EMBO J. 25: 763–773.

63. Rooney, S., J. Sekiguchi, S. Whitlow, M. Eckersdorff, J. P. Manis, C. Lee,D. O. Ferguson, and F. W. Alt. 2004. Artemis and p53 cooperate to suppressoncogenic N-myc amplification in progenitor B cells. Proc. Natl. Acad. Sci. USA101: 2410–2415.

64. Mostoslavsky, R., N. Singh, T. Tenzen, M. Goldmit, C. Gabay, S. Elizur, P. Qi,B. E. Reubinoff, A. Chess, H. Cedar, and Y. Bergman. 2001. Asynchronous rep-lication and allelic exclusion in the immune system. Nature 414: 221–225.

65. Alt, F. W., V. Enea, A. L. M. Bothwell, and D. Baltimore. 1980. Activity ofmultiple light chain genes in murine myeloma cells producing a single, functionallight chain. Cell 21: 1–12.

66. Bredemeyer, A. L., B. A. Helmink, C. L. Innes, B. Calderon, L. M. McGinnis,G. K. Mahowald, E. J. Gapud, L. M. Walker, J. B. Collins, B. K. Weaver, et al.2008. DNA double-strand breaks activate a multi-functional genetic program indeveloping lymphocytes. Nature 456: 819–823.

3808 BRIEF REVIEWS: Ag RECEPTOR ALLELIC EXCLUSION

by guest on May 26, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from