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Central B-Cell Tolerance: Where Selection Begins

Roberta Pelanda and Raul M. Torres

Integrated Department of , National Jewish Health and University of Colorado Denver School of Medicine, Denver, Colorado 80206 Correspondence: [email protected]

The development of an adaptive based on the random generation of receptors requires a stringent selection process that sifts through receptor specificities to remove those reacting with self-. In the B-cell lineage, this selection process is first applied to IgMþ immature B cells. By using increasingly sophisticated mouse models, investigators have identified the mechanisms that negatively select auto- reactive immature B cells and prevent inclusion of their antigen receptors into the peripheral B-cell pool. Additional studies have uncovered mechanisms that promote the differentiation of nonautoreactive immature B cells and their positive selection into the peripheral B-cell population. These mechanisms of central selection are fundamental to the generation of a naı¨ve B-cell repertoire that is largely devoid of self-reactivity while capable of reacting with any foreign insult.

-cell generation in the of adult stage that the BCR is tested for the first time Bmammals occurs through a tightly con- for reactivity against autoantigens. This test de- trolled developmental process (Fig. 1). Produc- termines whether the immature and the tive rearrangement of immunoglobulin heavy it expresses on the surface will be se- (IgH) and light (IgL) chain gene segments in B lected into the peripheral B-cell repertoire. Cen- precursor cells, in addition to the tral B-cell tolerance, in fact, refers to the process expression of Ig-a (CD79a) and Ig-b (CD79b), that negativelyselects newlygenerated immature result in the generation and expression on the B cells that react with a self-antigen in the bone cell surface of a mature B-cell antigen receptor marrowenvironment.Thisisconsideredthe first (BCR). Whereas the combination of Ig H and checkpoint of B-cell tolerance, and the results of L chains determines the antigenic specificity of this checkpoint are fundamental to the genera- the newly formed BCR, their association with tion of a naı¨ve repertoire that contains foreign Ig-a and Ig-b allows transduction of a signal in- reactive and is largely devoid of self- side the cell that directs cell fate. Developing B reactive specificities. cellsfirstexpressamatureBCRonthe cellsurface On passing this central checkpoint, imma- in the form of IgM and as such are classified as ture B cells continue to differentiate into transi- immature B cells (Fig. 1) (Hardy et al. 1991; Pe- tional and mature B cells before and after they landa et al. 1996). It is at the immature B-cell travel to the spleen (Loder et al. 1999; Allman

Editors: Diane Mathis and Alexander Y. Rudensky Additional Perspectives on available at www.cshperspectives.org Copyright # 2012 Cold Spring Harbor Laboratory Press; all rights reserved; doi: 10.1101/cshperspect.a007146 Cite this article as Cold Spring Harb Perspect Biol 2012;4:a007146

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R. Pelanda and R.M. Torres

pro-B pre-B pre-B Immature B Transitional B (large) (small) IgM IgD

lgM lgM

+ + + + DH->JH VDJ VDJ VDJ VDJ + + VH->DJH VL->J L VJ VJ

VDJC lgh

lgκ lgλ

Figure 1. Schematic representation of B-cell development and Ig loci in mice. Large pro-B cells initiate Ig gene rearrangement at the IgH locus. Expression of a H chain following a productive VHDHJH recombination event promotes the differentiation of large pre-B cells in which the expression of pre-BCR (H chain pairing with surrogate light chains) results in the clonal expansion of H chain-positive pre-B cells and the development of small pre-B cells. Expression of conventional L chains following productive rearrangements at the IgL chain loci in small pre-B cells promotes the development of a diverse population of IgMþ immature B cells, which then differentiate into IgMþIgDþ transitional B cells. The scheme of mouse Ig H, k, and l loci (not to scale) indicate the presence of V (white rectangles), D (black vertical lines), J (brown vertical lines; a dashed line in- dicates a nonfunctional element), and C (black rectangles; a gray rectangle indicates a nonfunctional element) gene segments. The scheme does not represent the number of VH, DH, and Vk gene segments in the actual Ig loci.

et al. 2001; Su and Rawlings 2002; Tarlinton et al. defect in central (and/or peripheral) B-cell se- 2003). Analysis of the bone marrow early imma- lection. Thus, it seems important that the devel- ture B-cell repertoire indicates that a staggering opment of autoreactive immature B cells be con- 50%–75% of these cells express BCRs that are strained to prevent the potential occurrence of specific for self-antigens, both in mice and hu- . However, there are also reasons mans (Grandien et al. 1994; Wardemann et al. to believe that the high frequency of autoreactive 2003). Similar studies performed on cell popula- specificities generated during primary Ig gene re- tions at the other end of this central checkpoint, arrangements may be necessary for the genera- namely, transitional and naı¨ve mature B cells in tion of the peripheral B-cell repertoire (Pelanda spleen and blood, show a much lower frequency et al. 1997; Kohler et al. 2008). Indeed, a fraction (20%–40%) of cells expressing autoreactive anti- of autoreactive immature B cells, those manifest- bodies (Grandien et al. 1994; Wardemann et al. ing a low level of self-reactivity,do bypassthe cen- 2003), demonstrating the stringency and limita- tral checkpoint of tolerance and differentiate into tion of this initial selection step. Moreover, indi- mature B cells (Hayakawa et al. 2003; Warde- viduals affected by autoimmune disease such as mann et al. 2003; Wenet al. 2005). The inclusion lupus erythematosus or rheumatoid arthritis of these weakly self-reactive B cells in the periph- bear many more autoreactive cells in their new eral B-cell repertoire may allow recognition of a emigrant and naı¨ve B-cell populations (Samuels broader spectrum of foreign molecules, poten- et al. 2005; Yurasov et al. 2005), indicating a tially decreasing the negative impact of infec-

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Central B-Cell Selection

tions, especially at early stages (Mouquet et al. brane-bound protein, it arrests in differentiation 2010). and undergoes within 2–3 d (Nema- What are the rules that govern the selection zee and Burki 1989; Hartley et al. 1991, 1993). In of immature B cells? Most studies of central tol- contrast, low avidity interactions with self-an- erance have been conducted by following the tigens permit further differentiation, but also selection of B cells expressing BCRs display- cause a shortened lifespan resulting in cell dele- ing well-defined reactivity for natural or syn- tion in the periphery (Goodnow et al. 1988). thetic self-antigens. This has been accomplished From these early in vivo studies with anti-Ig through the use of Ig transgenic mice in which treatment and conventional Ig transgenic mice, developing B cells have been altered to carry it was concluded that clonal deletionwas a major prerearranged Ig H and L chain genes encoding mechanism mediating central tolerance of de- antibodies of defined antigen specificity and veloping autoreactive B cells, guaranteeing the reactivity. Here we review some of these studies, elimination of autoreactive clones, and prevent- what we have learned from them, and open ing immune responses against self. However, questions that still await answers. later studies showed that is not the primary mode of autoreactive immature B- cell selection, but rather a default mechanism CLONAL DELETION IN CENTRAL B-CELL that operates when another system, namely, re- TOLERANCE: A DEFAULT MECHANISM ceptor editing, fails. The theory proposed by Burnet at the end of the 1950s suggested that clones RECEPTOR EDITING AS A MECHANISM of reactive with self-antigens are OF CENTRAL B-CELL TOLERANCE eliminated to prevent immune responses against self (Burnet 1959). A variety of experiments In 1993, three papers were published in The were conducted thereafter to test this theory. Journal of Experimental Medicine by the groups Throughout the 1970s and 1980s, studies were of David Nemazee (then at the National Jewish performed injecting antibodies reactive toward Center for Immunology and Respiratory Medi- Ig chains into newborn mice and rabbits with cine in Denver) and Martin Weigert (then at the the purpose of testing whether newly generated Fox Chase Cancer Center in Philadelphia) pro- B cells reacting with a “self-antigen,” here mim- posing what was then considered a radical mode icked byan anti-Ig antibody, would be deleted or of action by which the immune system could modulated. Indeed, injection of anti-IgM, anti- dispose of antibodies reacting with high avid- IgD, and even anti-idiotypic antibodies in new- ity self-antigens while preserving the cells that born animals resulted in the apparent elimina- originally produce these specificities (Gay et al. tion of IgM, IgD, and -expressing B 1993; Radic et al. 1993; Tiegs et al. 1993). In cells, respectively (e.g., Lawton et al. 1972; Man- their Ig transgenic mouse models in which the ning and Jutila 1972; Gordon et al. 1975; Toku- self-antigens were either MHC class I Kb or hisa et al. 1981; Finkelman et al. 1983; Takemori DNA, autoreactive immature B cells were shown and Rajewsky 1984; Cerny et al. 1986; Gause to “edit” their antigen receptors away from au- et al. 1987). In the 1980stransgenic mice became toreactivity. Specifically, autoreactive immature an experimental option and some of the first Ig B cells were shown to reactivate their Ig gene re- transgenic mice, anti-hen egg lysozyme (HEL), arrangement program at the Ig L chain loci re- and anti-H-2Kk,b, were developed with the sulting in the expression of a new L chain that purpose of testing whether newly generated au- paired with the existing H chain to form a non- toreactive B cells were either eliminated or sup- autoreactive BCR, an event that promoted the pressed on recognition of their specific self- selection of these edited B cells into the periph- antigen. These studies indicated that when an ery (Fig. 2A). The concept of “receptor editing” immature B cell reacts with a self-antigen with was then born. However, like many other new high avidity, such as a highly expressed mem- ideas, it took some years before receptor editing

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R. Pelanda and R.M. Torres

A Bone marrow Spleen 30–40% Anergy/ignorance

30–35%

Receptor editing ? lgκ Clonal deletion lgh Peripheral deletion

B CB17(IgMb) 3–83lgi (lgMa)

Nonreactive Autoreactive H-2d H-2b Mix bone marrow Clonal deletion

Receptor

(3–83lgi) editing a d b

H-2 H-2 IgM nonautoreactive autoreactive Both

Measure frequency of b IgM (wt) lgMa and lgMb B cells

Figure 2. Receptor editing in central B-cell selection. (A) Schematic representation of central B-cell tolerance. Immature B cells reacting with low to high avidity self-antigens undergo receptor editing, here represented by a secondary rearrangement at the Igk allele. Immature B cells reacting with low avidity self-antigens can alter- natively further differentiate and migrate into the spleen as anergic or ignorant B cells. Clonal deletion that oc- curs at a frequency that is presently unknown, but that is likely very low, is represented as a by-product of cells undergoing failed receptor editing. B cells encountering self-antigen in the periphery are represented under- going peripheral deletion. (B) Experimental setup that tested the relative contribution of receptor editing and clonal deletion to central tolerance of developing 3-83Igþ B cells (Halverson et al. 2004). Bone marrow cells from wild-type IgMb congenic and 3-83Igi IgMa congenic mice were mixed at equal proportion and injected into lethally irradiated recipient mice of H-2d and H-2b genetic backgrounds. The frequency of IgMa and IgMb B cells in the total B-cell population was measured in mixed bone marrow chimeras of the two experimental groups. The scheme on the right represents the expected outcomes of this analysis if all anti-Kb 3-83Igþ B cells had undergone clonal deletion (top panel), receptor editing (middle panel), or a combination of either tolerance mechanism (bottom panel) in mice expressing the self-antigen (H-2b), and relative to nonautoreactive mice (H-2d). The blue rectangle indicates the experimental findings.

would be commonly accepted as a main mech- physiological loci (at sites where naturally rear- anism of central B-cell tolerance rather than a ranged V[D]J sequences are found), unequivo- transgenic artifact. cally showed that receptor editing is a promi- The generation of Ig knockin mice, which nent mechanism of central tolerance (Fig. 2A), bear the rearranged V(D)J sequences at their occurring in all developing high avidity auto-

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Central B-Cell Selection

reactive immature B cells and capable of gener- generation of the variable region of an Ig H chain ating a nonautoreactive B-cell population of requires the rearrangement of a D element to a J normal size (Pelanda et al. 1997; Hippen et al. element followed by that of a V to arearranged DJ 2005; Huang et al. 2006). We now appreciate gene segment, and this is based on the 12/23 rule that depending on the avidity of the BCR for of the V(D)J recombination process (Sakano its own specific self-antigen, editing B cells can et al. 1981). Because of this configuration, the re- completely down-modulate surface expression arrangement of a VDJgene segment results in the of IgM, thus resembling pre-B cells (Pelanda deletion of all remaining D elements, thus pre- et al. 1997; Hippen et al. 2005), or express low venting a secondary canonical rearrangement to relatively normal levels of sIgM (Gay et al. event on the same allele. Although the second al- 1993; Hippen et al. 2005; Huang et al. 2006). lele can be rearranged instead (Liu et al. 2008), In addition to secondary rearrangements at this could cause the generation of B cells coex- the L chain loci, receptor editing can also take pressing two H chains, a rare event in wild-type place at the Ig H chain alleles, in the form of mice (Barreto and Cumano 2000). In fact, it H chain replacement (Chen et al. 1995a). How- hasbeen shownthat editingat theH chainoccurs ever, the Igk locus of mice is particularly suited via replacement of the VH element with a new for this process because of the following rea- VH gene segment by recombination of an up- sons: (1) the variable regions of L chains do stream VH gene either into a RS-like sequence not include D elements as found in the H chain that is located in the 30 region of most VH genes (Fig. 1) and thus permit a secondary recom- (Fanning et al. 1998; Zhang et al. 2003) or di- bination event between an upstream Vk and a rectly into a JH element (Koralov et al. 2006) in downstream Jk element; (2) there are 95 Vk violation of the 12/23 rule. VH replacement, and four Jk functional gene elements (Zachau however, has a low probability of generating a 2000; Martinez-Jean et al. 2001; Brekke and Gar- productive and functional H chain (Koralov rard 2004) allowing multiple secondary recom- et al. 2006) and would not be favored because bination events on the same allele; (3) most the IgH locus has already acquired a low accessi- primary Vk-Jk rearrangement events occur on ble state by the time an immature B-cell tests its Jk1 and Jk2 (Yamagami et al. 1999), leaving Jk4 BCR (Inlayet al. 2006; Hewitt et al. 2008). Recep- and Jk5 downstream, and Vk gene segments up- tor editing at the Igl locus is also not favored in stream of a Vk-Jk rearrangement (in addition to mice, because the locus has only three Vl gene the second Igk allele) as substrates of secondary segments that are located in two independent re- recombination events; (4) recombination of the arrangement clusters with theirown constant re- Igl locus follows that of Igk (Persiani et al. 1987; gions (Fig. 1) (Carson and Wu 1989). Thus, once Muller and Reth 1988; Arakawa et al. 1996; van an immature B cell expresses an autoreactive l der Burg et al. 2001) and can provide addition- chain, alternative modes of tolerance must be al L chains in cases of persistent autoreactivity used to deal with this persistent self-reactivity, with k chains; and (5) recombining sequence and these include the rearrangement of an addi- (RS) recombination elements located 30 of the tional l chain that may dilute the expression of Igk enhancers can recombine with upstream re- autoreactive BCRs (Doyle et al. 2006). combination signals to inactivate the Igk allele by deletion or inversion of the Ck and Igk intron RELEVANCE OF RECEPTOR EDITING enhancer intervening sequences (Moore et al. IN CENTRAL B-CELL TOLERANCE 1985; Shimizu et al. 1991) removing expression of an autoreactive k chain in l-expressing cells After receptor editing was found to occur fre- (Pelanda et al. 1997). quently in autoreactive Ig knockin mice, the In contrast to the relative ease by which Igk relevance of this mechanism was not initially alleles undergo secondary recombination events, clear relative to clonal deletion and within a poly- the IgH locus possesses several features that limit clonal B-cell population. In particular, it was its use as a substrate for editing. Specifically, the difficult to envision why a mechanism would

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R. Pelanda and R.M. Torres

exist to salvage an autoreactive B cell when the Additionalstudies have investigated whether immune system had already provided many ex- receptor editing operates in polyclonal B-cell amples of waste (i.e., cell death) in developing a populations. The analysis of receptor editing in vast repertoire. To address this issue, bone mar- 3H9 and derivative IgH chain transgenic mice row chimeras were generated that harbored a bearing a polyclonal Ig L chain repertoire indi- mix of autoreactive knockin B cells (3-83Igi in cated that receptor editing is operative whenever this system) and congenic wild-type B cells, a developing B cell rearranges an endogenous and the frequency of peripheral B cells derived L chain that pairs with the H chain to form an from the autoreactive B-cell precursors was anti-DNA specificity (Chen et al. 1995b). An- then contrasted to that derived from the wild- other study investigating the frequency of recep- type reference population (Fig. 2B) (Halverson tor editing in developing B cells bearing a pre- et al. 2004). Results were then compared to sim- rearranged knockin Ig L chain (three different ilar mixed bone marrow chimeras in which the Igk chains were tested) with the endogenous Ig Ig knockin B cells developed in the absence of H chain repertoire indicated that 25% of im- self-antigen (Fig. 2B) (Halverson et al. 2004). mature B cells had sufficient self-reactivity to in- These studies showed that the frequency of duce receptor editing (Casellas et al. 2001). This knockin-derived B cells was the same in the pre- frequency may be very close to the actual occur- sence or absence of self-antigen and indicated rence of receptor editing at the Ig L chain loci that all developing autoreactive B cells undergo in a wild-type B-cell population. Because VH receptor editing without cell loss and even replacements have been estimated to occur in when comprising only 10% of the developing 5%–10% of developing B cells (Zhang et al. B-cell population. Thus, it was concluded that 2003; Koralov et al. 2006), this brings the overall receptor editing is a very efficient process that estimated frequencyof receptorediting in a poly- can, theoretically, provide a new nonautoreac- clonal wild-type B-cell population to 30%– tive Ig L chain to any autoreactive immature B 35%. Finally, an elegant study by Nemazee and cell. These studies resulted in three additional colleagues showed that wild-type immature B important conclusions: (1) the Igk locus is es- cells are capable of undergoing receptor editing sential for establishing a successful and efficient on engagement of a self-antigen. In this study, receptor editing process, as shown by the fact a synthetic membrane antigen (k-macroself-Ag) that removal of downstream Jk substrates halves was engineered to react with all kþ B cells with the capacityof receptorediting to procure a non- high avidity, demonstrating the efficient induc- autoreactive specificity (Halverson et al. 2004); tion of receptor editing in wild-type kþ imma- (2) nonproductive receptor editing defaults to ture B cells with minimal or undetectable levels clonal deletion as shown by the fact that partial of clonal deletion (Ait-Azzouzene et al. 2005). or complete inhibition of receptor editing re- Overall, the findings of these studies suggest sults in reduced or absent B-cell output (Halver- that 30%–35% of newly generated B cells in son et al. 2004), respectively, consistent with wild-type mice interact with self-antigens that previous observations in conventional Ig trans- deliver a signal sufficient for the induction of re- genic mice (Spanopoulou et al. 1994; Chen ceptor editing. Furthermore, receptor editing is et al. 1995b; Xu et al. 1998); and 3) autoreactive extremely efficient at providing an autoreactive immature B cells from conventional Ig trans- B cell with a new Ig L chain (or less frequently genic mice most often default to clonal deletion a H chain) encoding a nonautoreactive specific- because Ig gene recombination events at their ity, and clonal deletion is a default mechanism endogenous Ig loci do not lead to inactivation that ensures the elimination of autoreactive of the Ig transgenes bearing the autoreactive immature B cells that fail receptor editing (Fig. specificity.Thus,thefrequencyofclonaldeletion 2A). Clonal deletion may occur when the H is artificiallyamplified (experimentally and con- chain contributes considerably to self-reactivity, ceptually)in the developing B-cell population of when the B cell has exhausted rearrangement conventional Ig transgenic mice. options at the k loci, or when the B cell remains

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Central B-Cell Selection

autoreactive after a productive Ig l chain rear- these findings suggest that some of the editing rangement. The actual extent of clonal deletion B cells in the wild-type repertoire may be low in developing B cells is presently unclear and avidity autoreactive immature B cells (Fig. 2A). may be insignificant based on the following rea- The reasons for why some low avidity auto- soning. Repertoire studies have indicated that reactive immature B cells undergo receptor edit- 50%–70% of immature B cells in mice react ing whereas others develop anergy are still un- with self-antigens (Grandien et al. 1994), which resolved, but may relate to cell competition for is compatible with estimates in humans (War- self-Ag in selective marrow microenvironments. demann et al. 2003). Given that 30%–35% of the immature B cells undergo receptor edit- POTENTIAL HAZARDS OF ing, the remaining autoreactive immature B cells RECEPTOR EDITING (15%–35%) must undergo a different selection process, which is clonal deletion, anergy, or ig- The initial proposal of receptor editing as a norance. Because 30%–40% of transitional B mechanism of tolerance was controversial also cells remain autoreactive after selection in the because it implied that autoreactive B cells bone marrow (Grandien et al. 1994; Wardemann would survive while editing occurred, with the et al. 2003), this suggests that those autoreac- subsequent risk that these cells may be selected tiveimmatureBcellsthat donotundergo editing into the peripheral B-cell compartment. In fact, are selected via ignorance or anergy to self- further studies in mouse models in which au- antigen, and very few undergo clonal deletion toreactive B cells undergo extensive receptor ed- (Fig. 2A). iting indicates that this process can generate in- termediary cell types that coexpress autoreactive and nonautoreactive BCRs (Fig. 3). Under cir- CENTRAL B-CELL TOLERANCE FOR LOW cumstances that are yet to be established, some AVIDITY INTERACTIONS of these haplotype-included B cells are able to Early studies based on conventional transgenic proceed in differentiation and are selected into mice also indicated that low avidity interactions the peripheral and mature B-cell populations between immature B cells and self-antigens re- (Li et al. 2002; Liu et al. 2005; Huang et al. sult in the development of anergic peripheral 2006). The frequency of peripheral haplotype- B cells (Goodnow et al. 1988; Benschop et al. included autoreactive B cells in these mouse 2001). Given that the extent of clonal deletion models varies from 15% to 30% of the peri- was overrepresented by the use of conventional pheral B-cell pool. Assuming that this frequency Ig transgenic mouse models, it was important reflects the probability of generating haplotype- to revisit central B-cell tolerance toward low included B cells from receptor editing, and that avidity self-antigens in a more physiological 30%–35% of wild-type B cells are generated context. This question was addressed by study- via receptor editing (Fig. 2A), we would expect ing the fate of H chain transgenic þL chain 4%–10% of wild-type peripheral B cells to be knockin anti-HEL B cells developing in the pres- haplotype-included and potentially still auto- ence of soluble HEL (Hippen et al. 2005). In this reactive. In fact, 3%–10% of B cells in wild- model, only half of the low avidity autoreac- type mice have been shown to coexpress two tive B cells developed anergy, whereas the other Ig L (or more rarely two H) chains (Giachino half underwent receptor editing (Hippen et al. et al. 1995; Barreto and Cumano 2000; Rezanka 2005), in contrast to conventional Ig H and L et al. 2005; Casellas et al. 2007; Velezet al. 2007), chain transgenic mice in which most B cells and data exist that suggest that these cells more were anergic (Goodnow et al. 1988). A similar frequently react with self-antigens relative to dichotomy in tolerance induction has been ob- single antibody-expressing B cells (Casellas et al. served in wild-type B cells that develop in the 2007). Precisely how many of these haplotype- presence of low concentrations of a k-macro- included B cells are self-reactive, whether they self-Ag (Ait-Azzouzene et al. 2006). Together are potential mediators of autoimmunity, and

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κ κ κ κ κ κ ′ V V -J 2 E iEC 3

Vκ Jκ EκiECκ κ3′

Generation of haplotype- Receptor included B cells editing (estimated at 3–10%)

Vκ Vκ-Jκ2 EκiECκ κ3′

Vκ Vκ-Jκ4EκiECκ κ3′

Figure 3. Receptor editing generates a small population of haplotype-included B cells. During receptor editing, a potential rearrangement at the second Igk allele (intact arrow), or at the IgH or Igl alleles, results in the gen- eration of cells coexpressing two or more types of H and L chains. Some of these haplotype-included B cells are selected into the peripheral B-cell population expressing both autoreactive and nonautoreactive antibodies. Note that if receptor editing occurs on the original rearranged Igk allele (dotted arrow), the previously used V-J sequence would be deleted.

what are the self-antigens they recognize, are 1990; Spanopoulou et al. 1994; Young et al. questions still awaiting clear answers. 1994). Moreover, targeting the Ig-a/Ig-b hetero- Finally,the evolutionary purpose of receptor dimer to the cell membrane promotes B-cell editing remains obscure, especially given its as- development in the absence of Ig H and L chains sociated risk of promoting the development of (Bannish et al. 2001), suggesting that cell surface autoreactive B cells. However, it can be argued assembled BCRs transduce signals that promote that receptor editing may be important for res- differentiation of immature B cells in the ab- cuing those pre-B cells that have undergone a sence of antigen binding. This antigen- productive IgH chain rearrangement event and, independent BCR signal is referred to as a tonic thus, to promote repertoire diversification (Pe- or basal signaling. landa et al. 1997; Kohler et al. 2008). Further studies investigating the nature and function of tonic BCR signaling in immature B cells have found that the ongoing differentiation POSITIVE SELECTION OF IMMATURE B of nonautoreactive immature B cells is depen- CELLS: AN ACTIVE PROCESS dent on a certain threshold of tonic BCR signal- Immature B cells that display nonautoreactive ing (Heltemes and Manser 2002; Wang et al. BCRs continue to differentiate and progres- 2004; Rowland et al. 2010a). Specifically, expres- sively acquire expression of surface markers typ- sion of BCRs on the cell surface of immature B ical of more mature B cells, such as IgD, CD21, cells must reach a certain level such that their and CD23, before and after they travel to the concerted tonic signal achieves the threshold spleen (Loder et al. 1999; Allman et al. 2001; required for initiating cell differentiation into Su and Rawlings 2002; Tarlinton et al. 2003). the transitional stage (Fig. 4) (Rowland et al. The surface expression of a mature BCR is an 2010a). Even a 30% reduction of wild-type BCR absolute requirement for this differentiation level has a negative impact on the differentiation event, because genetically altered pre-B cells un- of immature into transitional and mature B cells, able to express mature BCRs do not differentiate and this impact is more severe with still lower or leave the bone marrow (Reichman-Fried et al. BCR expression (Rowland et al. 2010a). We have

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Central B-Cell Selection

Bone marrow Spleen

Immature B cells Transitional/mature B cells

Positive selection

lgD, CD21, CD23

Tonic BCR signal (pErk) BAFFR Ras

Mek

Erk

Figure 4. Positive selection of nonautoreactive immature B cells into the peripheral B-cell compartment requires a threshold level of tonic BCR signaling. Differentiation of nonautoreactive immature B cells into transitional and mature B cells and entry into the peripheral B-cell population depends on a certain threshold of BCR ex- pression and tonic BCR signaling, which is translated by the Ras-Mek-Erk signaling pathway. BAFFR expression correlates with BCR surface expression and tonic signaling, and BAFFR signaling contributes to the differentia- tion of immature into transitional B cells. The scheme is based on data from Rowland et al. 2010a,b.

found that tonic BCR signaling that promotes et al. 2005; Schilling et al. 2009), suggesting that cell differentiation requires phosphorylation of this molecular pathway is generally used for the the Erk MAP kinase (Fig. 4), and that activation generation of mature hematopoietic cells from of the Ras-Mek-Erk signaling pathway in im- their lineage precursors. mature B cells mimics tonic BCR signaling pro- Interruption of tonic BCR signaling in im- moting their differentiation into transitional B mature B cells causes a developmental regres- cells (Rowland et al. 2010a). Moreover, enforc- sion characterized by the reexpression of genes ing the survival of immature B cells (e.g., with associated with pro-B and pre-B-cell stages of a Bcl-2 transgene) is not sufficient to promote development and the reactivation of V(D)J re- their differentiation (Rowland et al. 2010a), in- combination (Tze et al. 2005; Verkoczy et al. dicating that basal BCR signals are translated 2007; Schram et al. 2008). Thus, tonic BCR sig- onto a cell differentiation program that is inde- naling is important for the cessation of both pendent of cell survival. These findings support Rag expression and V(D)J recombination and, those from other studies in which reduced Erk consequently, promotes allelic exclusion and phosphorylation correlated with arrested devel- ensures that each naı¨ve B cell is monospecific. opment of, and sustained Ig gene rearrangement The signaling cascade downstream from tonic in, CD19-deficient immature B cells (Diamant BCR signaling appears to be qualitatively differ- et al. 2005). Thus, this active signaling pathway ent, at least in part, from that induced by self- promotes positive selection of nonautoreactive antigen. Support for this is evidenced by the immature B cells into the naı¨ve B-cell reper- finding that some immature B cells expressing toire. In an analogous fashion positive selection both an autoreactive and nonautoreactive BCR of and the differentiation of eryth- do not undergo tolerance but instead are al- rocytes are also dependent on the activation of lowed to differentiate into mature B cells (Li the Ras-Erk pathway (Alberola-Ila et al. 1996; et al. 2002; Liu et al. 2005; Huang et al. 2006). Werlen et al. 2000; Fischer et al. 2005; McNeil This indicates that tonic BCR signaling, under

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R. Pelanda and R.M. Torres

certain circumstances, can override self-antigen- mature B cells, synergizing with antigen-medi- mediated BCR signaling. Anergic B-cells may ated and tonic BCR signaling and directing fall into this category, which is cells that experi- developing B cells toward the proper selection ence levels of tonic BCR signals that are suffi- pathway. cient for inhibiting further Ig gene recombi- BAFF, known to promote peripheral B-cell nation and promote additional differentiation survival (Mackay et al. 2003), may be an exam- into the transitional B-cell stage despite some ple of such a that contributes to imma- level of self-antigen-mediated BCR signaling. ture B-cell selection. Work from our laboratory Immature B cells that react with self-antigen has recently shown that nonautoreactive im- internalize their engaged BCRs (Pelanda et al. mature B cells express the receptor for BAFF, 1997). Thus, these cells experience both anti- BAFFR, at levels that correlate with BCR expres- gen-mediated BCR signaling and a lack of tonic sion and tonic BCR signaling (Fig. 4) (Rowland BCR signaling. Given that the absence of tonic et al. 2010b). Further, treatment of nonauto- BCR signaling is sufficient to cause Ig gene re- reactive immature B cells with BAFF promotes combination (Tze et al. 2005; Verkoczy et al. their differentiation into transitional B cells 2007; Schram et al. 2008), it has been speculated (Fig. 5) (Rowland et al. 2010b). In contrast, that receptor editing is activated not by antigen- autoreactive immature B cells express minimal mediated BCR signaling but rather by the ab- levels of BAFFR (correlating with an absence sence oftonic BCR signaling (Schram etal. 2008). or minimal tonic BCR signaling) and are unable Based on these findings, we propose that to respond to BAFF (Rowland et al. 2010b). newly generated immature B cells in a wild-type On expression of an autoreactive antigen repertoire undergo a dynamic process of selec- receptor immature B cells have been shown to tion during which each cell experiences some survive for at least 2–3 d, during which time tol- level of antigen-mediated and tonic BCR sig- erance is implemented (Hartley et al. 1993; Mel- nals. The balance between these signals leads amed and Nemazee 1997; Casellas et al. 2001; to cell differentiation or retention and toler- Hippen et al. 2005). This period would ensure ance. Factors that influence these signals may that autoreactive immature B cells have suffi- include avidity for self-antigen, level of surface, cient time to make several attempts at receptor and unengaged BCR expression, and possibly editing. However, it remains to be established certain bone marrow microenvironments. which signals sustain the survival of autoreactive immature B cells during receptor editing. Given that IL-7 is a prosurvival cytokine important AND THE CENTRAL for earlier B-cell developmental stages, we asked SELECTION NICHE whether this cytokine may also contribute to Whether defined niches exist in the bone mar- autoreactive immature B-cell survival. Findings row in which newly generated immature B cells from these preliminaryexperiments suggest that test their BCR and undergo B-cell negative and IL-7 acts on autoreactive, but not nonautore- positive selection similar to developing T cells, active, immature B cells to extend cell survival is not clear. It has only recently been shown in vitro (K. Tuttle,R. Torres, and R. Pelanda, un- that bone marrow stromal cells exist that publ.). Thus, this cytokine mayserve to promote uniquely secrete CXCL12, IL-7, or galectin-1, receptorediting overclonal deletion during cen- and that developing early B lineage cells interact tral B-cell tolerance (Fig. 5). serially with each of these cell types (Tokoyoda Recent investigation of the bone marrow et al. 2004; Mourcin et al. 2011). Thus, it is rea- environment has determined that immature B sonable to consider the existence of additional cells partition between parenchyma and sinus- stromal cells specialized at presenting self-anti- oids in response to lipids recognized by the gens to immature B cells. These stromal cells cannabinoid receptor 2 (Pereira et al. 2009) may also provide specific cytokines and chemo- and sphingosine 1-phosphate receptor 3 (Do- attractants that signal through receptors on im- novan et al. 2010). In recent studies we found

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Central B-Cell Selection

Cytokine receptor

Ag-mediated Tonic BCR BCR signal + signal + BAFF cytokine (IL-7?) receptor signal receptor signal

Receptor editing Differentiation and with limited cell selection into survival periphery

Figure 5. Autoreactive and nonautoreactive immature B cells undergo different fates during bone marrow selec- tion. An autoreactive immature B-cell (on the left) experiences antigen-mediated BCR signaling in addition to the absence of tonic BCR signaling, and these events promote ongoing Ig gene rearrangements (receptor edit- ing). Cytokines, such as IL-7, may be able to sustain limited cell survival during the editing process. A nonau- toreactive immature B cell (on the right) experiences tonic BCR signaling in addition to BAFFR signaling, and these events promote further cell differentiation and selection into the peripheral B-cell compartment.

that only nonautoreactive immature B cells, in- toire. Future challenges will be to extend our cluding those generated after receptor editing, current knowledge to the human immune sys- localize in the sinusoids (Donovan et al. 2010). tem to determine whether human autoreactive In contrast, autoreactive immature B cells are re- immature B cells are also capable of undergoing stricted to the parenchyma of the bone marrow receptor editing, and whether clonal deletion is (Donovan et al. 2010), suggesting that sinusoid a default mechanism or rather a primary toler- localization before exiting the bone marrow ance process in human central B-cell tolerance. may be important for proper B-cell differentia- Another challenge will be to increase our knowl- tion. Whether BAFF and/or other promatura- edge of the bone marrow microenvironment to tion cytokines are differentially expressed in establish whether a specific niche exists for self- the sinusoids of the bone marrow tissue is not to newly generated B cells, known, but we predict that more detailed anal- and for the processes of negative and positive se- ysis of this particular environment will reveal lection. In particular, we will need to define the defined niches in which developing B lympho- cytokines and chemoattractants that influence cytes compartmentalize for proper differentia- immature B-cell selection and their distribu- tion and selection. tion in the marrow environment. Another ma- jor challenge will be to determine what factors regulate tonic BCR signaling in immature B CONCLUDING REMARKS cells, and particularly what defines the signaling The studies of central B-cell tolerance per- threshold that separates negative and positive formed in the last 30 years in mice have given selection. Finally, these findings will have to be us an understanding of what are the general applied to our understanding of diseases, to de- forces that shape the murine naı¨ve B-cell reper- termine whether individuals prone to B-cell-

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R. Pelanda and R.M. Torres

mediated autoimmunity harbor immature au- Carson S, Wu GE. 1989. A linkage map of the mouse im- toreactive B cells that have abnormal BCR tun- munoglobulin l light chain locus. Immunogenetics 29: 173–179. / ing and or aberrant bone marrow microenvi- Casellas R, Shih TA, Kleinewietfeld M, Rakonjac J, Nemazee ronments. D, Rajewsky K, Nussenzweig MC. 2001. Contribution of receptor editing to the antibody repertoire. Science 291: 1541–1544. ACKNOWLEDGMENTS Casellas R, Zhang Q, Zheng NY, Mathias MD, Smith K, Wilson PC. 2007. Igk allelic inclusion is a consequence Wethank present and past members of the Torres of receptor editing. J Exp Med 204: 153–160. and Pelanda laboratories who have contributed Cerny A, Heusser C, Sutter S, Huegin AW,Bazin H, Hengart- ner H, Zinkernagel RM. 1986. 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Central B-Cell Tolerance: Where Selection Begins

Roberta Pelanda and Raul M. Torres

Cold Spring Harb Perspect Biol 2012; doi: 10.1101/cshperspect.a007146 originally published online February 28, 2012

Subject Collection Immune Tolerance

Regulatory T Cells and Immune Tolerance in the Regulatory T Cells and Immune Tolerance in the Intestine Intestine Oliver J. Harrison and Fiona M. Powrie Oliver J. Harrison and Fiona M. Powrie Dendritic Cells: Arbiters of Immunity and Microbiota and Autoimmunity Immunological Tolerance Alexander V. Chervonsky Kanako L. Lewis and Boris Reizis Current and Future Immunomodulation Strategies Treg Cells, Life History, and Diversity to Restore Tolerance in Autoimmune Diseases Christophe Benoist and Diane Mathis Jeffrey A. Bluestone and Hélène Bour-Jordan T-Cell Tolerance: Central and Peripheral Infectious (Non)tolerance−−Frustrated Yan Xing and Kristin A. Hogquist Commensalism Gone Awry? Jesse C. Nussbaum and Richard M. Locksley Central B-Cell Tolerance: Where Selection Begins Historical Overview of Immunological Tolerance Roberta Pelanda and Raul M. Torres Ronald H. Schwartz The Immunogenetic Architecture of Autoimmune Tolerance: Control by Self or Disease Self-Control? An Goris and Adrian Liston Baptiste N. Jaeger and Eric Vivier

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