Antigen Receptor Allelic Exclusion: an Update and Reappraisal Brenna L

Antigen Receptor Allelic Exclusion: An Update and Reappraisal Brenna L. Brady, Natalie C. Steinel and Craig H. Bassing This information is current as J Immunol 2010; 185:3801-3808; ; of October 2, 2021. doi: 10.4049/jimmunol.1001158 http://www.jimmunol.org/content/185/7/3801

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2010 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Antigen Receptor Allelic Exclusion: An Update and Reappraisal Brenna L. Brady,1 Natalie C. Steinel,1 and Craig H. Bassing Most lymphocytes express cell surface Ag receptor Monoallelic gene expression is a general phenomenon critical chains from single alleles of distinct Ig or TCR loci. for normal biology. This regulation is pervasive during genetic Since the identiﬁcation of Ag receptor allelic exclusion, imprinting and X chromosome inactivation, enforcing gene the importance of this process and the precise molec- silencing in all cell types (3). Defects in X chromosome in- ular mechanisms by which it is achieved have remained activation and imprinting were found to cause human dis- enigmatic. This brief review summarizes current knowl- orders, which deﬁned the relevance of these processes and edge of the extent to which Ig and TCR loci are subject facilitated their investigation by studying clear phenotypes. In contrast, defects in olfactory or Ag receptor allelic exclusion,

to allelic exclusion. Recent progress in studying and de- Downloaded from fining mechanistic steps and molecules that may control which silence tissue-specific genes in distinct lineages, have the monoallelic initiation and subsequent inhibition of not been linked unequivocally to any symptoms in humans V-to-(D)-J recombination is outlined using the mouse (3). However, dysfunction of these tissue-specific processes may result in subtle phenotypes or may be compensated by TCRb locus as a model with frequent comparisons to additional mechanisms, either of which would mask their k the mouse IgH and Ig loci. Potential consequences of significance and provide obstacles for investigation. defects in mechanisms that control Ag receptor allelic http://www.jimmunol.org/ exclusion and a reappraisal of the physiologic relevance Allelic exclusion, self tolerance, and autoimmunity of this immunologic process also are discussed. The A long-standing tenent of adaptive immunity is that virtually Journal of Immunology, 2010, 185: 3801–3808. all lymphocytes express surface TCR or Ig chains from one allele to ensure monospecific Ag recognition and suppress autoimmunity by facilitating central tolerance to self-reactive lympho- ntigen receptor allelic exclusion is defined as the cytes. However, this notion is not supported by current know- surface expression of Ig or TCR chains from a single ledge. Flow cytometry reveals IgH allelic inclusion in only A allelic copy of corresponding genetic loci. Pernis et al. 0.01% of mouse B cells (4), but allelic inclusion of Igk and by guest on October 2, 2021 (1) identified this phenomenon in the 1960s while studying Ig TCR loci in at least 1–10% of mouse lymphocytes (Table I) expression on rabbit lymphocytes, providing evidence for the (5–9). Thus, a significant fraction of normal mouse (and where “one lymphocyte–one antigen receptor” concept of Burnett’s assayed human) lymphocytes express surface Igk or TCR clonal selection theory. Analyses of Ig rearrangements in the chains from both alleles, refuting the “one lymphocyte–one early 1980s suggested that the assembly and expression of an antigen receptor” concept. In addition, recognition of multiple Ag receptor chain from one allele inhibit further V-to-(D)-J distinct ligands is known now to be a general and inherent recombination on the other allele (2). Evidence for such feed- property of T and B cell Ag receptors (10), meaning that far back regulation was provided over the next decade by dem- more than the 1–10% of allelically included lymphocytes onstrations that preassembled Ig or TCR transgenes enforce exhibits poly-specific Ag recognition. Because primary TCR allelic exclusion through inhibiting V-to-(D)-J rearrange- and BCR repertoires include receptors capable of bind- ments (2). These observations helped establish the current ing self-Ags, organisms with adaptive immune systems must dogma that allelic exclusion is maintained by feedback regu- possess central ability to tolerate the generation of autoreactive lation to ensure virtually every lymphocyte exhibits monospe- lymphocytes and thereby prevent autoimmunity. Central tol- cific Ag recognition. erance mechanisms include deletion, stalled maturation, an- Although Ag receptor allelic exclusion has been investigated ergy, or receptor editing (9). In the 1990s, analyses of TCR for almost 50 y, the importance of this process and the precise and Ig transgenic mice demonstrated that dual expression of mechanisms by which it is achieved remain largely unknown. self-reactive and non–self-reactive receptors enables developing

Immunology Graduate Group, Division of Cancer Pathobiology, Department of Pa- School of Medicine; and National Institutes of Health Grant R01 CA125195 (to thology and Laboratory Medicine, Center for Childhood Cancer Research, Children’s C.H.B.). Hospital of Philadelphia, University of Pennsylvania School of Medicine, Abramson Address correspondence and reprint requests to Dr. Craig H. Bassing, Children’s Hos- Family Cancer Research Institute, Philadelphia, PA 19104 pital of Philadelphia, 4054 Colket Translational Research Building, 3501 Civic Center 1B.L.B. and N.C.S. contributed equally to this review. Boulevard, Philadelphia, PA 19104. E-mail address: [email protected] Received for publication June 23, 2010. Accepted for publication August 5, 2010. Abbreviations used in this paper: ATM, ataxia telangiectasia mutated; DN, double negative; DP, double positive; Eb, TCRb enhancer; pDb1, Db1 promoter. This work was supported by Training Grant TG GM-07229 of the University of Pennsylvania (to B.L.B.); the Department of Pathology and Laboratory Medicine and Ó the Center for Childhood Cancer Research of the Children’s Hospital of Philadelphia; Copyright 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 the Abramson Family Cancer Research Institute of the University of Pennsylvania www.jimmunol.org/cgi/doi/10.4049/jimmunol.1001158 3802 BRIEF REVIEWS: Ag RECEPTOR ALLELIC EXCLUSION

Table I. Allelic inclusion of mouse Ag receptor loci

Biallelic In-Frame Locus Allelic Inclusion (%) Experimental Approach V(D)J (%) References IgH 0.01 Natural allotypic differences 2–5 4, 18 Igk 1–7 Hemizygous human Ck knock-in 11 5, 6 TCRa 10 Anti-Va combinations 30 9, 19 TCRb 1–3 Anti-Vb combinations 2–10 8, 20, 22 TCRd 3 Anti-Vd combinations 35 7, 21 TCRg 1 Anti-Vg combinations 10 7

T and B cells to escape deletion and differentiate into mature TCRa allelic inclusion to ∼10% (Table I) (9, 19). Similar lymphocytes that possess autoreactive potential in vitro, yet sequence analyses of Igk, TCRb,TCRg, and TCRd rearrange- generally fail to cause autoimmunity in vivo (11–13). These ments revealed two in-frame genes in 2–35% of cells depend- additional tolerance mechanisms restrain the systemic activa- ing upon the locus (Table I) (5, 7, 20–22). These percentages tion of lymphocytes expressing both self-reactive and non–self- are higher than the corresponding allelic inclusion frequencies, reactive receptors. A separate line of investigation demon- suggesting that pairing restrictions or other mechanisms con- strated that the destructive potential of autoreactive lympho- tribute to Igk,TCRb, TCRg, and TCRd allelic exclusion. cytes that escape central tolerance is restrained through Consistent with this notion, silencing of in-frame V-D-J-Cb Downloaded from dominant peripheral tolerance mechanisms, such as those con- genes at the transcriptional and posttranscriptional levels controlled by regulatory T and B cells (14, 15). In this context, tributes to TCRb allelic exclusion mouse ab T cells (20, 23). allelic exclusion might function as an early cell-autonomous These data indicate that multiple mechanisms function in a suc- tolerance mechanism to reduce the frequency of developing cessive manner to limit the frequency of cells with surface lymphocytes with expression of two or more poly-specific Ag expression of Ig or TCR chains from both allelic copies of receptors and thereby facilitate central tolerance. If defects in corresponding loci. In this context, defects in mechanisms that http://www.jimmunol.org/ allelic exclusion overwhelmed central tolerance mechanisms, control feedback inhibition could be countered by pairing peripheral tolerance checkpoints would be expected to function restrictions, transcriptional silencing, and posttranscriptional as an additional barrier to restrain the destructive potential of silencing to limit allelic inclusion and facilitate central toler- allelically included lymphocytes expressing autoreactive Ag ance. Yet, dysfunction of these downstream allelic exclusion receptors. Consistent with this notion, environmental factors mechanisms, for example as an organism ages, could increase such as viral infections can break down peripheral tolerance the frequencies of allelically included mature lymphocytes mechanisms and trigger autoimmunity driven by peripheral expressing autoreactive Ag receptors that need to be restrained ab T cells expressing dual TCRs (16). However, allelic inclu- by peripheral tolerance checkpoints. by guest on October 2, 2021 sion also can be beneficial because T lineage cells expressing dual Developmental stage-specific control of allelic exclusion through ab TCRs protect against infection by increasing the diversity of feedback regulation receptors that recognize foreign Ags (17). Most reviews of mechanisms that control Ag receptor allelic exclusion involve IgH and Igk loci, probably because studying Multiple mechanisms affect Ag receptor allelic exclusion B lineage cells led to the discoveries of allelic exclusion, V(D)J Although feedback inhibition is a major component by which recombination, and feedback regulation. In this work, the allelic exclusion is achieved, the sequence analyses of assembled mouse TCRb locus (Fig. 1) will serve as a paradigm for discus- TCR and Ig genes have revealed that additional mechanisms sion of the developmental stage-specific control of allelic ex- contribute to affect Ag receptor allelic exclusion. Because 1/3 clusion through feedback regulation because many recent of V-(D)-J rearrangements occur in-frame, only 1/9 of de- advances in this area have come from studying V-to-D-Jb veloping lymphocytes can assemble and express a particular Ag recombination. Frequent comparisons to the IgH and Igk loci receptor gene from both alleles in the absence of feedback (Fig. 1) also are provided. regulation. Of the remaining developing cells, 4/9 would assemble one in-frame and one out-of-frame gene and 4/9 would Recombinational accessibility control of V-to-(D)-J recombination assemble out-of-frame genes on both alleles. Because the expres- Correlations between transcription, rearrangement, and nu- sion of an Ag receptor chain is required for continued lympho- clease sensitivity of Ig gene segments led to the hypothesis that cyte differentiation, the 4/9 cells that fail to assemble a modulation of chromatin accessibility regulates V(D)J re- productive gene on either allele are eliminated by apoptosis. combination within the contexts of allelic exclusion (24). Consequently, 1/5 is the theoretical maximum of mature Consequently, the majority of studies investigating mecha- lymphocytes that can express a particular Ag receptor gene from nisms that control Ag receptor allelic exclusion have focused both alleles and exhibit allelic inclusion in the absence of on the potential differential regulation of RAG accessibility feedback regulation and selection for or against such cells. between loci on homologous chromosomes. For many years, Single-cell sequence analysis revealed that, of the 2–4% of recombinational (RAG) accessibility has been measured in- splenic B cells containing two in-frame IgH genes, only one directly by germline transcription, general nuclease accessi- was functional (Table I) (18). In contrast, sequencing demon- bility, and histone modifications associated with transcription strated that ∼30% of ab T cells contain two in-frame TCRa (24). RAG accessibility also has been quantified by the ex- genes (Table I) (9); yet, TCRa/TCRb pairing constraints and pression of reporter genes inserted into endogenous Ag re- regulated TCRa-chain turnover reduce the frequency of ceptor loci (25–28); however, this indirect approach requires The Journal of Immunology 3803

FIGURE 1. Genomic conﬁguration of mouse TCRb, IgH, and Igk loci. Sche- matic diagrams of germline TCRb, IgH, and Igk loci depicting the relative locations of V segments (blue rectangles), D segments (yellow rectangles), J segments (green rectangles), C regions (gray rectangles), enhancers (red ovals), and promoters (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.

knowledge of local transcriptional regulation for unequivocal itive conclusions require similar analyses with TCRb and IgH conclusions (27, 28). In addition to RAG accessibility, the alleles containing preassembled D–J complexes and incorpo- Downloaded from V-to-(D)-J recombination step requires physical juxtaposition ration of assays that measure RAG/RSS interactions and of RAG-accessible gene segments, RAG binding to a recom- juxtaposition and synapsis of V and D–J segments. Because bination signal sequence (RSS) flanking at least one of these enhancers and promoters function together to direct V-to- segments, and capture of the other RSS to form productive (D)-J recombination, it is logical to assume that the enforce- synaptic cleavage complexes. Recent advances in techniques ment of allelic exclusion could involve mechanisms that mod- and reagents have enabled methods to investigate higher-order ulate the activities of these cis elements. Support for this http://www.jimmunol.org/ structural conformations of Ag receptor loci (29), RAG bind- notion is provided by the observation that IgH allelic exclu- ing to chromatin over genomic locations (30), RAG activity at sion depends upon the IgH intronic enhancer maintaining a particular gene segment (31), and RAG cleavage within high expression of V-D-J-CH genes during the pre-B to im- individual Ag receptor loci (32). Unfortunately, these assays mature B cell transition (36). have inherent limitations that temper conclusions and can measure only a single mechanistic step required for V-to- Initiation of allelic exclusion (D)-J rearrangement. Such difficulties are substantial obstacles The V-to-(D)-J recombination step of Ag receptor loci subject for elucidating the precise mechanisms that control V-to-(D)- to allelic exclusion is thought to occur on one allele at a time, J recombination within the contexts of feedback regulation with the assembly and expression of a functional TCR or Ig by guest on October 2, 2021 and allelic exclusion. gene from the first allele inhibiting V-to-(D)-J rearrangement Ag receptor locus transcriptional enhancers and promoters within the corresponding locus on the second allele (37). To are required for V-to-(D)-J recombination; however, the pre- enforce allelic exclusion by feedback inhibition, only one al- cise mechanisms by which these cis elements direct V rear- lele can initiate V-to-(D)-J recombination during the time rangements and their potential role in allelic exclusion remain window required for feedback signals to exert their cellular unknown. The IgH and Igk loci each contain two enhancers, effects. Monoallelic initiation of V rearrangement could occur whereas the TCRb locus contains only one known enhancer at any of the mechanistic steps required for V-to-(D)- (Fig. 1). Promoters are known to reside upstream of each V J recombination. segment within these loci, as well as upstream of the DQ52, The correlation between transcription and rearrangement Jk1, Db1, and Db2 segments (Fig. 1). The molecular mech- led to studies suggesting that the differential positioning of anisms by which enhancers and promoters direct V-to-(D)-J TCRb, IgH, and Igk loci at nuclear regions known to repress rearrangements are understood most for the TCRb locus. The transcription might affect monoallelic initiation of V-to-(D)-J TCRb enhancer (Eb) and the Db1 promoter (pDb1) form recombination (Fig. 2) (29). For example, TCRb loci associ- a holoenzyme complex that directs D-to-Jb1 and V-to-D-Jb1 ate with inner nuclear membrane lamina or pericentromeric 2 2 rearrangements, as well as germline transcription and chro- heterochromatin at a higher frequency in CD4 /CD8 matin accessibility of Db1andJb1 segments (33). Neither (double-negative [DN]) thymocytes than in embryonic stem Eb nor pDb1 controls Vb germline transcription or chroma- cells, B lineage cells, or CD4+/CD8+ (double-positive [DP]) tin accessibility (34), which appear regulated at least in part by thymocytes (38, 39). TCRb alleles with an ectopic enhancer Vb promoters (35). Collectively, these data suggest that Eb that promotes TCRb allelic inclusion are localized less fre- and pDb1 may direct Vb rearrangements only through pro- quently at nuclear membrane lamina and pericentromeric moting accessibility of D–Jb complexes so the RAG proteins heterochromatin (39), providing indirect evidence that asso- can bind 59Db RSSs and capture Vb RSSs. The IgH and Igk ciation of Ag receptor loci with these transcriptional repressive enhancers and promoters most likely direct V-to-(D)-J re- nuclear regions may suppress V-to-(D)-J rearrangement. De- combination through similar regulation of D–JH and Jk seg- spite frequent association of TCRb loci with nuclear mem- ments. Consistent with this notion, experiments using a brane lamina and pericentromeric heterochromatin, D-to-Jb cleavage-incompetent Rag1 protein have demonstrated that rearrangements, germline Vb transcription, and Vb RAG RAG binding is detectable over germline D and J, but not accessibility each occur on both TCRb alleles in developing V, segments within TCRb, IgH, and Igk loci (30). Yet, defin- thymocytes (31, 37). In addition, the association of IgH loci 3804 BRIEF REVIEWS: Ag RECEPTOR ALLELIC EXCLUSION Downloaded from

FIGURE 2. Multiple redundant and successive mechanisms most likely cooperate to control Ag receptor allelic exclusion. Monoallelic initiation of V-to-(D)-J http://www.jimmunol.org/ 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. with pericentromeric heterochromatin does not inhibit tran- primary V-to-(D)-J rearrangements within these loci. Germ- by guest on October 2, 2021 scription of germline or rearranged alleles (32, 40). Collec- line and/or D–J-rearranged TCRb, IgH, and Igk loci exhibit tively, these data suggest that the positioning of TCR and Ig monoallelic contraction by chromosome looping between V alleles at inner nuclear membrane lamina or pericentromeric and D/J segments at a higher frequency in lymphocytes of the heterochromatin may suppress V-to-(D)-J rearrangements by lineage and stage where these loci rearrange as compared with inhibiting the juxtaposition of V and D–J segments, rather in other cells (29, 38, 39). Data revealing that rearrangements than through suppressing transcription or RAG accessibility of Vb and VH segments inserted just upstream of Db or DH (Fig. 2). Consistent with this notion, germline and D–Jb- segments cause allelic inclusion and/or are not subject to rearranged loci positioned at pericentromeric heterochromatin normal feedback inhibition provide indirect evidence that do not exhibit contraction by looping between Vb and D/Jb locus contraction/decontraction may regulate the V-to-D-J segments as do unrearranged TCRb loci residing away from recombination step within TCRb and IgH loci (23, 44). these nuclear regions (38). Identification of the cis elements and These Ag receptor locus conformational changes may be reg- trans factors that control the association of TCR and Ig loci ulated by mechanisms that direct association of TCR and Ig with inner nuclear membrane lamina and pericentromeric het- loci with repressive nuclear regions and/or by distinct cis erochromatin is required to elucidate the potential function of elements and trans factors such as enhancer/promoter inter- nuclear positioning in regulating V-to-(D)-J rearrangements actions (45) and CCCTC-binding factor/cohesin proteins and allelic exclusion. Logical candidates have been provided (46). However, factors in addition to locus decontraction by the discoveries of cis elements between V and J segments must control the juxtaposition of accessible V and D–J seg- within the TCRb, IgH, and Igk loci (41–43), and the demon- ments within TCRb and Igk loci to regulate secondary V stration that this Igk Sis element binds the Ikaros transcrip- rearrangements across short distances and recombination of tional repressor, targets Igk transgenes to centromeric hetero- the Vb14 segment, which resides in close proximity to D–Jb chromatin, and inhibits V-to-Jk rearrangement (42). segments. Monoallelic initiation of V-to-(D)-J recombination also In addition to RAG accessibility and juxtaposition, intrinsic could be affected by developmentally regulated conformation properties of RSSs may contribute to affect allelic exclusion. changes of Ag receptor loci that control the juxtaposition of The inherent inefficiency of Vb and VH RSSs could restrain RAG-accessible V and D–J segments (Fig. 2). Because germ- the overall rate at which accessible and juxtaposed V and D–J line TCRb, IgH, and Igk loci span large chromosomal dis- segments bind RAG and/or form productive synaptic com- tances, the positioning of V and D–J segments in proximity plexes such that the frequency of synchronous V-to-D-J rear- by locus contraction most likely facilitates or is required for rangements between TCRb or IgH alleles is rare (47). Vbs The Journal of Immunology 3805

rearrange to D–Jb complexes with little or no Vb-to-Jb thymocytes or pro-B cells from G0/G1 and into S phase may joining due to beyond 12/23 compatibility RSS joining contribute to affect allelic exclusion by shortening the time restrictions (37). These RSS restrictions limit the number of window for additional V-to-D-J rearrangements (Fig. 2). In potential Vb rearrangements on each allele to 70 (35 Vbs 3 2 addition, considering that TCRb-driven cellular proliferation Dbs), as compared with the 840 (35 Vbs 3 2Dbs 3 12 Jbs) is required for progressive silencing of TCRg transcription potential Vb rearrangements allowed by the 12/23 rule. To- during the DN-to-DP transition (54), similar TCRb-and gether, joining restrictions and inefficiencies of TCRb RSSs IgH-dependent epigenetic mechanisms may silence germline may lower the probability that both alleles can initiate Vb V segment transcription to help suppress V-to-D-J rearrange- rearrangement in the time window required for feedback in- ments in DP thymocytes and pre-B cells. Finally, TCRb and hibition. Consistent with this notion, replacement of the IgH signals that inhibit RAG expression also could contribute Vb14 RSS with the 10-fold more efficient 39Db1 RSS, which to allelic exclusion by preventing DN or pro-B cells from rearranges to 59Db or Jb RSSs, short-circuits TCRb feedback continuing V-to-D-J recombination (49). signals, and increases the frequency of TCRb allelic inclusion (C.H. Bassing, unpublished observations). However, these data Maintenance of allelic exclusion also support the notion that TCRb feedback signals activate To maintain TCRb and IgH allelic exclusion, V-to-D-J rear- 59Db RSS-binding trans factors to prevent RAG accessibility rangements must remain suppressed on D–J-rearranged alleles and V-to-D–Jb rearrangements (48, 49), analogous to inhibi- following RAG re-expression in DP thymocytes and pre- tion of V-to-Db rearrangements by c-fos binding 39Db RSSs B cells. Evidence suggests that developmental stage-specific Downloaded from (37). Considering that TCRb and IgH, but not TCRd genes inhibition of RAG access to germline V segments and juxta- are assembled through D–J intermediates, such inhibition of position of V and D–J segments most likely cooperate to RAG access to 59D RSSs could account for why feedback maintain feedback inhibition of V-to-D-J recombination regulation inhibits the assembly of V-D-Jb and V-D-JH on D–J-rearranged alleles (Fig. 2) (37–39, 55, 56). Studies exons more stringently than V-D-Jd exons (Table I) (7, 18, investigating mechanisms that maintain TCRb feedback in- 20–22). hibition have been conducted using DP cells of RAG- http://www.jimmunol.org/ deficient mice either treated with anti-CD3 Abs or expressing Feedback inhibition aTCRb transgene to drive DN-to-DP differentiation. One The assembly and expression of a functional TCRb or IgH finding often ignored in these experiments is that germline gene in DN thymocytes or pro-B cells, respectively, activate Vb transcripts and Vb chromatin marks associated with ac- intracellular pathways that signal feedback inhibition, cessa- tive transcription are present at higher levels in DP cells of tion of RAG expression, cellular proliferation/expansion, and RAG-deficient mice treated with anti-CD3 Abs as compared differentiation into DP thymocytes or pre-B cells (Fig. 2). with DP cells from RAG-deficient mice expressing a TCRb Although the pathways and mechanisms through which transgene (41, 57). Germline Vb14 transcripts exhibit the TCRb and IgH chains signal these processes remain largely largest difference. These data indicate that anti-CD3 treat- by guest on October 2, 2021 unknown, experiments have revealed that DN thymocytes ment and TCRb transgenes do not equally activate pre- employ distinct pathway(s) to signal feedback inhibition TCR signaling pathways or thresholds required for Vb silenc- through mechanisms involving the E47 and Ets-1 transcrip- ing; determining which represents the more physiologic con- tion factors (49–51). TCRb-signaled downregulation of E47 dition would be important. Germline transcription of D–Jb appears to inhibit V-to-D-Jb rearrangements in DN cells segments in DP thymocytes has been interpreted that D–Jb through rendering Vb segment RAG inaccessible (50). complexes remain RAG accessible (41, 57); however, analysis Ets-1 binds to and represses Eb (52), which is not required of RAG binding to 59Db RSSs on alleles with preassembled for the expression of assembled V-D-J-Cb genes in DN cells D–Jb complexes is required for definitive conclusions. or TCRb-mediated DN-to-DP expansion and differentia- Maintenance of TCRb and IgH allelic exclusion in DP tion (53). Thus, TCRb feedback signals could modulate thymocytes and pro-B cells also must involve the suppression Ets-1 activity to inhibit Eb in DN cells and thereby down- of secondary V rearrangements on alleles that have assembled regulate RAG accessibility of 59Db RSSs on the D–Jb- out-of-frame V-D-J-C genes in DN or pro-B cells (Fig. 2). The rearranged allele while enabling continued TCRb expression 12/23 rule and beyond 12/23 restrictions inhibit, but do not from the V-D-Jb–rearranged allele. This hypothetical mech- block V-to-JH and V-to-Jb rearrangements in pro-B cells and anism also would explain how TCRb feedback signals sup- DN thymocytes (48, 58). Such RSS joining restrictions might press Vb14-to-D-Jb rearrangements without downregulating contribute to suppress V rearrangements over out-of-frame RAG accessibility of Vb14 segments (31). The use of alleles V-D-J-C genes in pre-B or DP cells (Fig. 2), particularly if with preassembled D–J complexes or V-D-J-C genes along with the downstream J segments remain RAG accessible. Because assays of RAG binding, juxtaposition, and synapsis should fa- Vb rearrangements to Db2–Jb2 complexes are not restricted cilitate elucidation of the mechanisms through which TCRb by RSS joining constraints, additional factors must suppress and IgH feedback signals inhibit V-to-D-J rearrangements in such recombination events on alleles that have assembled out- DN thymocytes and pro-B cells. of-frame V-D-J-Cb1 genes in DN thymocytes (Fig. 2). In addition to the activation of mechanisms that directly Germline Vb segments located immediately upstream of V- inhibit V-to-D-J rearrangements in DN or pro-B cells, TCRb D-J-Cb genes remain transcribed in DP thymocytes (37, 59), and IgH signals may indirectly promote feedback inhibition yet Vb rearrangements to Db2–Jb2 complexes are largely through cessation of RAG expression, cellular proliferation/ suppressed (37). This data indicate that V-to-D-Jb rearrange- expansion, and differentiation into DP thymocytes or pre- ments must be controlled by developmental stage-specific fac- B cells. For example, TCRb and IgH signals that drive DN tor(s) that controls RAG binding to RSSs, juxtaposition, and/ 3806 BRIEF REVIEWS: Ag RECEPTOR ALLELIC EXCLUSION or synapsis in either DN or DP thymocytes (37). One such could arise because the mechanisms that control monoallelic candidate is the E47 transcription factor because forced initiation and feedback inhibition of Vb rearrangements are expression of this factor promotes Vb rearrangements to Db2– only effective in 90–99% of DN thymocytes, as evidenced by Jb2 complexes in DP cells (50). Notably, V-to-Db2 recombi- TCRb allelic inclusion and biallelic in-frame V-D-J-Cb nation intermediates are detectable in DP thymocytes of wild- rearrangements in 1–10% of ab T ;cells (20). Failure to en- type mice, and intermediates involving Vb14, which resides force monoallelic initiation or feedback inhibition of V-to-D-J close to Db–Jb segments, are observed in DP cells of TCRb rearrangements at normal levels could result in the generation transgenic mice (55, 60). Although such V(D)J recombination of RAG DSBs, whereas TCRb or IgH signals are driving de- intermediates could arise from contaminating DN cells, these veloping lymphocytes from G0/G1 through G1 and into S observations suggest that Vb rearrangements to Db2–Jb2 phase. This would lead to increased elimination of DN or complexes on alleles with V-D-J-Cb1 genes are not com- pro-B cells that express functional TCRb or IgH chains and pletely blocked in DP thymocytes. Such secondary Vb rear- compromise host immunity by shrinking the repertoire of Ag rangements on alleles with out-of-frame V-D-J-Cb1 genes receptors expressed on mature lymphocytes. Because RAG could lead to TCRb allelic inclusion either before or after DSBs that persist into S phase can result in genomic instability, positive selection of ab TCRs containing TCRb-chains from deficiencies in these mechanisms controlling monoallelic initi- the other allele. In addition, secondary Vb rearrangements on ation and feedback inhibition of V-to-D-J rearrangements also alleles with in-frame V-D-J-Cb1 genes during positive selection might cause an increased predisposition to lymphomas driven could lead to the loss of an ab TCR or the replacement of by TCRb or IgH translocations. Accordingly, monoallelic ini- a selected ab TCR with an autoreactive receptor. The ability tiation and feedback inhibition of V-to-D-J rearrangement may Downloaded from of DNA sequences to form boundaries between active and have evolved through pressure to generate broad Ag receptor inactive Vb chromatin domains upstream of assembled V-D- repertoires and restrain oncogenic translocations by enforcing J-Cb1 genes may have evolved to suppress the frequency of such regulation upon a random process. In this context, the more deleterious Vb rearrangements in DP thymocytes (59). stringent allelic exclusion observed at the IgH locus as com- Interaction of self-Ags with autoreactive immature B cells or pared with the TCRb,TCRg, and TCRd loci (Table I) may naive ab T lymphocytes sustains or reinduces RAG expres- have evolved in response to the greater oncogenic potential of http://www.jimmunol.org/ sion, respectively, to promote V rearrangements that replace RAG DSBs introduced at the IgH locus than at these other loci in-frame V-Jk or V-D-Jb exons. To maintain allelic exclusion (63). In the B cell lineage, similar pressure combined with of TCRb,Igk, and possibly IgH loci during ab TCR revision greater cellular proliferation upon the rearrangement and ex- and BCR editing, V rearrangements need to be restricted on pression of IgH genes than IgL genes may have led to more alleles with in-frame V-(D)-J-C genes. Studies of mechanisms strict enforcement of allelic exclusion at the IgH locus as com- that potentially suppress V-to-(D)-J rearrangements on alleles pared with the Igk and Igl loci. lacking or containing out-of-frame V-(D)-J-C genes to maintain allelic exclusion during BCR editing and ab TCR revision Evidence for lateral inhibition of V(D)J recombination by guest on October 2, 2021 are lacking. Yet, one study indicates that V-to-Jk rearrange- Since the discovery that allelic exclusion is regulated by ments occur at equal frequency on both alleles during BCR feedback inhibition, many experiments have been conducted ∼ editing, leading to allelic inclusion in 10% of cells (5). Con- to evaluate whether the monoallelic initiation of V rear- sidering that the frequency of ab T cells exhibiting TCRb rangements occurs through deterministic versus stochastic allelic inclusion progressively increases as mice age (8), V-to- mechanisms. TCRb, IgH, and Igk loci each replicate asyn- D-Jb rearrangements also might occur with equal probability chronously in lymphocytes, with the order between alleles on both alleles during TCRb revision. Because BCR editing randomly determined and clonally maintained (64). The early and ab TCR revision will regenerate self-reactive receptors that could be edited/revised or restrained by peripheral tolerance checkpoints, the immunologic consequences of allelic inclusion during these processes may not exert physiologic pressure to restrict V rearrangements on alleles with in-frame V-(D)-J-C genes.

Potential physiologic consequences of defects in allelic exclusion Defects in mechanisms that control Ag receptor allelic exclusion could have deleterious consequences for host organisms in addition to causing autoimmunity. TCRb and IgH gene rearrangements proceed through the programmed induction of RAG DNA double-strand breaks (DSBs) in G1 phase DN thymocytes or pro-B cells with expression from in-frame V-D-J-C genes driving cells into S phase and through multiple FIGURE 3. Potential lateral inhibition of V(D)J recombination may cell cycles. The ataxia telangiectasia mutated (ATM) and p53 contribute to enforce allelic exclusion. RAG cleavage of Ag receptor loci in tumor suppressor proteins inhibit the persistence of RAG DSBs developing lymphocytes activates ATM-dependent signals that may transiently inhibit additional V(D)J recombination events. This regulation may enable throughout the cell cycle (61); however, some fraction of RAG time for DNA repair, transcription, translation, surface expression, and signal- DSBs generated during V-to-D-Jb rearrangements normally ing required for feedback inhibition. These putative ATM signals could di- evades the G1/S checkpoint and induces apoptosis of DN rectly prevent V-to-(D)-J rearrangements or indirectly, such as through thymocytes at the G2/M checkpoint (62). These RAG DSBs downregulation of RAG activity. The Journal of Immunology 3807 replicating Igk allele is preferentially demethylated, rendered will need to consider potential regulation at all mechanistic RAG accessible, and selected for V-to-Jk rearrangement in steps, not just the one being assayed. In the long-term, experi- pre-B cells (64), yet no connection has been reported between ments that manipulate all of the redundant and successive asynchronous TCRb or IgH locus replication and determina- mechanisms that regulate Ag receptor allelic exclusion will be tion of monoallele V-to-D-J rearrangement. In contrast, avail- required to reveal the biological relevance of this phenomenon able data suggest that stochastic association of TCRb alleles that has mystified immunologists for half a century. with inner nuclear membrane lamina and pericentromeric heterochromatin reduces the probability that biallelic V-to- Disclosures D-Jb rearrangements occur before feedback inhibition (37). The authors have no financial conflicts of interest. These Igk deterministic and TCRb stochastic models each assume that a static time window exists between monoallelic initiation and feedback inhibition of the V-to-(D)-J recom- References 1. Pernis, B. G., G. Chiappino, A. S. Kelus, and P. G. H. Gell. 1965. Cellular lo- bination step. calization of immunoglobulins with different allotypic specificities in rabbit lym- One neglected proposal of the original feedback inhibition phoid tissues. J. Exp. Med. 122: 853–876. 2. Mostoslavsky, R., F. W. Alt, and K. Rajewsky. 2004. The lingering enigma of the model is that V(D)J recombination events on one allele could allelic exclusion mechanism. Cell 118: 539–544. activate signals that transiently inhibit rearrangements on the 3. Zakharova, I. S., A. I. Shevchenko, and S. M. Zakian. 2009. Monoallelic gene expression in mammals. Chromosoma 118: 279–290. second allele (65). In response to RAG DSBs, the ATM kinase 4. Barreto, V., and A. Cumano. 2000. Frequency and characterization of phenotypic phosphorylates numerous proteins and activates a genetic Ig heavy chain allelically included IgM-expressing B cells in mice. J. Immunol. 164:

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