Pre-B and pre-t-cell receptors: conservation of strategies in regulating early lymphocyte development

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1 Stefan A. Muljo Mark S. Schlissel Pre-B and pre-t-cell receptors: conservation of strategies in regulating early lymphocyte development Authors addresses Stefan A. Muljo 1, Mark S. Schlissel 2, 1Graduate Program in Immunology, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA. 2Department of Molecular and Cell Biology, The University of California, Berkeley, California, USA. Correspondence to: Mark S. Schlissel Department of Molecular and Cell Biology The University of California 439 Life Sciences Addition Berkeley CA USA Fax: mss@uclink4.berkeley.edu Immunological Reviews 2000 Vol. 175: Printed in Denmark. All rights reserved Copyright Munksgaard 2000 Immunological Reviews ISSN Summary: Early lymphocyte development is characterized by the regulated activity of the V(D)J recombinase and the positive and negative selection of cells based on the structure of their assembled antigen receptor genes. Developing B and T cells use remarkably similar signaling complexes, the pre-b-cell receptor (pre-bcr) and the pre-t-cell receptor (pre-tcr) respectively, to monitor the progress of antigen receptor gene assembly. This review will compare and contrast the regulation and activities of the pre-bcr and pre-tcr signaling complexes. In addition, we will consider a number of critical but as yet unanswered questions prompted by such an analysis. Introduction The goal of lymphocyte development is to generate a large repertoire of cells expressing diverse receptors that enable them to recognize and react to a broad array of foreign antigens. The genes encoding these antigen receptors are unusual in that they are comprised of a series of V (variable), D (diversity), and J (joining) gene segments which require assembly by a site-specific DNA recombination reaction known as V(D)J recombination (1). As postulated by Alt and colleagues, gene rearrangements occur in an ordered sequence as development proceeds (2). In developing B cells, assembly of the immunoglobulin heavy-chain (IgH) gene occurs before that of the immunoglobulin (Ig) light-chain genes. Within the IgH locus, rearrangement begins on both alleles with joining of a D H to a J H gene segment (Fig. 1). These pro-b cells then go on to recombine a V H segment with one of the partially assembled DJ H alleles. The recombinase is ignorant of translational reading frame, however, so only one out of three joints is productive (i.e. can encode a complete functional heavy-chain protein). Following productive VDJ H rearrangement, the IgH allele is transcribed and its message translated resulting in the production of the transmembrane form of heavy-chain protein (Igµ). This protein is assembled into a signaling complex known as the pre-b-cell antigen receptor (pre-bcr). In addition to a homodimer of Igµ, the pre-bcr contains the products of the 80

2 Fig. 1. A diagrammatic comparison of early B and T-cell development. Top: ordered assembly of Ig genes during B-cell development. D-to-J H rearrangement on both heavy-chain alleles precedes V-to-DJ H rearrangement in pro-b cells. Cells with an in-frame heavy-chain gene rearrangement (pre-b cells) assemble the pre-bcr, containing Igµ protein, surrogate light chains (SLC) and Igα/Igβ. Pre-BCR expression is associated with the inactivation of allelic heavy-chain gene rearrangement and the activation of Igκ light-chain gene rearrangement (for simplicity, only Igκ is depicted here and Igλ is not considered). Successful light-chain rearrangement allows the immature B cell to assemble its BCR. Bottom: ordered assembly of TCR genes during T-cell development. In a fashion highly analogous to B-cell development, D-to-J β rearrangement in CD4/CD8 double-negative (DN) pro-t cells precedes V-to-DJ β rearrangement. Productive V-to-DJ β rearrangement results in the assembly of the pre-tcr, consisting of the β chain, pre-tα and the CD3 chains. These pre-t cells become CD4/CD8 double positive (DP). The pre-tcr mediates TCRβ locus allelic exclusion and activates V-to-J α rearrangement. Expression of a mature αβ TCR (and appropriate positive and negative selection) results in progression to the CD4 or CD8 single-positive (SP) stage of development. In both diagrams, genes required for normal development are indicated above the stage at which null mutations affect development (see text). surrogate light-chain (SLC) genes λ5 and VpreB, and a heterodimeric signaling module comprised of the transmembrane proteins Igα and Igβ (CD79a and CD79b). The SLC complex is required for the proper folding and membrane transport of Igµ in the absence of conventional light chains (3). In the absence of SLCs, Igµ is trapped in the endoplasmic reticulum (ER) bound to the chaperone protein BiP (4, 5). Examples exist of in-frame VDJ H rearrangements that encode an Igµ that cannot associate with the SLCs (6, 7). These heavy chains are not transported to the cell surface and are incapable of supporting further B-cell development. The Igα/β heterodimer associates with Igµ in the plasma membrane and is required for signaling by the pre-bcr (8 10). Igα and Igβ contain cytoplasmic tails that associate with signaling molecules such as the Src-family tyrosine kinases (11). In addition, their cytoplasmic tails contain immunoreceptor tyrosine-based activation motifs (ITAMs), which, when phosphorylated by Srcfamily kinases, facilitate docking of Syk, another tyrosine kinase. Six Src-family protein tyrosine kinases are expressed in the B lineage: Blk, Fyn, Lyn, Lck, Fgr and Hck (12). By extrapolating from what is known about signaling by the mature B- cell antigen receptor (BCR), signal transduction by the pre- BCR would presumably lead to activation of the Src-family kinases, which then phosphorylate key tyrosine residues within the kinases themselves and within the ITAMs of Igα and Igβ. This leads to the recruitment of Syk, which also gets phosphorylated and activated by Src kinases, and it in turn activates Bruton s tyrosine kinase (Btk), phospholipase-c gamma (PLCγ) and phosphoinositide 3-kinase (PI3K) (13). In addition, the Ras/Raf/mitogen-activated protein kinase (MAPK) pathway is also activated (13). The roles of some of the signaling molecules in the highly analogous pre-bcr and pre-t-cell antigen receptor (pre-tcr) signaling pathways will be discussed further below. Immunological Reviews 175/

3 Assembly of the pre-bcr by a pro-b cell leads to a series of effects on the developing cell collectively referred to as the pre- B-cell transition. These effects include the promotion of cell survival, proliferation, differentiation, and a change in the targeting of the V(D)J recombinase (14, 15). Pro-B cells have a limited time span in which to generate a productive heavychain gene rearrangement. When heavy-chain gene assembly fails due to non-productive VDJ H rearrangements on both alleles, pro-b cells undergo programed cell death (16). A developing B cell that produces an Igµ protein that gets incorporated into a pre-bcr is rescued from apoptosis and enters the pre-bcell compartment. Thus, the pre-bcr acts as a biological sensor that informs pro-b cells that they have succeeded in making a functional Ig heavy chain. In addition to promoting cell survival, pre-bcr-expressing cells undergo three to five rounds of cell division, generating clones of cells expressing a common heavy chain but with the potential to express a diverse array of light chains. During this period of proliferation, expression of RAG1 and RAG2, the lymphoid specific components of the V(D)J recombinase, is greatly diminished (17), and a variety of other genes are activated or repressed (18 23). When the nascent pre-b cells stop proliferating and increase expression of the recombinase, their pattern of recombinase activity has changed dramatically heavy-chain V-to-DJ H rearrangement has ceased and light-chain gene rearrangement has increased significantly (24, 25). This retargeting of the recombinase assures that an individual B cell will express only one functional Ig heavy chain (a phenomenon known as allelic exclusion). Although changes in chromatin structure have been implicated in this pre-bcr-mediated retargeting of the recombinase, the precise mechanism remains obscure (26, 27). Productive light-chain gene rearrangement allows the developing B cell to assemble a complete IgM molecule which is transported to the immature B-cell surface in association with Igα and Igβ, forming the BCR. B cells which express a self-tolerant BCR inactivate the recombinase and exit the bone marrow to join the peripheral B-cell population (14, 15, 28). These later stages of B-cell development have been reviewed elsewhere and are beyond the scope of this discussion (14). T-cell development follows a course remarkably similar to that of B cells (Fig. 1). In progenitor CD4 CD8 (double-negative (DN)) thymocytes, D β -to-j β rearrangement precedes V β -to- DJ β rearrangement. Productive T-cell receptor β (TCRβ) gene rearrangement results in the formation of the pre-tcr signaling complex comprised of a TCRβ chain, a pre-tα surrogate α chain, and the multisubunit CD3 signaling complex consisting of CD3ε/γ and CD3ε/δ heterodimers and a homodimer of CD3ζ (29). The structure and function of the pre-tcr has uncanny similarities to that of the pre-bcr. Assembly of the pre-tcr on the cell surface of DN thymocytes promotes their survival, signals them to progress to the CD4 + CD8 + (double-positive (DP)) stage of development and causes them to undergo significant clonal expansion (30). The pre-tcr is also responsible for enforcing allelic exclusion of TCRβ gene rearrangement by redirecting recombinase activity away from the TCRβ locus and towards the TCRα locus (31). This limits the probability that any given T cell will simultaneously express two functional TCRβ chains. Thus the pro-t (DN) to pre-t (DP)-cell transition appears to be highly analogous to the pro-b to pre-b-cell developmental transition and leads to the supposition that the two may use similar molecular strategies. Although considerable progress has been made in understanding the pre-b and pre-t-cell transition at the molecular level, some basic questions merit further investigation. How is the signal initiated? What intracellular molecular events must occur to transduce the pre-bcr or pre-tcr signal? In particular, what mechanisms are responsible for mediating survival, proliferation, allelic exclusion, and progression to the pre-b or pre- T-cell stage of development. Finally, how do the pre-bcr and pre-tcr signals alter target gene expression? Ligand requirement? Targeted deletion of the transmembrane exon of IgH prevents membrane assembly of the pre-bcr and is associated with a block in B-cell development at the pro-b to pre-b transition (32). Likewise, mutation of the λ5 gene imposes a severe block on B-cell development at that same transition, proving that membrane assembly of the pre-bcr is essential for developmental progression (33). Due perhaps to its low level of expression, the pre-bcr has proven difficult to detect by flow cytometry on the surface of developing murine bone marrow B cells (34). The question of whether the pre-bcr requires interaction with an extracellular ligand in order to transmit its developmental signal remains unanswered. No group has reported the existence of an extracellular ligand, which is required for pre-bcr activation. This failure has led to the hypothesis that the pre-bcr (and by analogy the pre-tcr) may not require ligand engagement. Several recent studies have lent support to this idea. One such study utilized an N-terminal truncated heavy-chain transgene product (missing its variable domain as well as C H 1 and part of C H 2) that was capable of surface expression in the absence of light chain. This truncated µ protein was sufficient to deliver the pre-bcr signal during bone marrow B-cell development even in the absence of λ5 (35). A second group reported a similar result using a human 82 Immunological Reviews 175/2000

4 heavy-chain disease mutant transgene (36). One may have predicted that if a ligand existed for the pre-bcr, it would bind to an invariant portion of the receptor, such as the SLC. These data suggest that this is not the case. However, it remains possible that the putative ligand recognizes other invariant portions of the pre-bcr, such as the C H 3 and/or C H 4 domains of Igµ. One shortcoming of both of these studies is the possibility that the mutant heavy chains were abnormally structured, leading them to aggregate on the cell surface. This hypothetical aggregation might relieve the requirement for ligand-induced cross-linking of the pre-bcr. In a similar fashion to λ5 deficiency in B cells, targeted mutation of the pretα gene results in a developmental block at the pro-to-pre-t-cell transition (37). Most notably, thymuses from pretα / mice are markedly hypocellular. The question of whether the pre-tcr signal requires an extracellular ligand has been addressed by two recent sets of experiments. The most compelling experiment utilized mutant TCRβ and pre-tα transgenes lacking all of their extracellular Ig-like domains (38). This truncated pre-tcr was still capable of signaling the DNto-DP transition as well as enforcing TCRβ chain allelic exclusion. An analogous experiment with the pre-bcr is probably not possible since complete truncation of the extracellular domains of Igµ heavy chain would remove its potential to dimerize through disulfide bond formation, thereby preventing the assembly of the pre-bcr. In the case of the pre-tcr, it is fortuitous that the disulfide bond between the TCRβ chain and the pre-tα chain lies just above the plasma membrane; thus, it was possible to remove all of the Ig-like ectodomains of the pre-tcr heterodimer without interfering with the interchain disulfide linkage. Although this result strongly suggests that ligand binding by the ectodomain of the pre-tcr is not required for delivery of the pre-tcr signal in vivo, it remains possible that this is an artifact due to the abnormal structure or assembly of the mutant signaling complex (as in the case of the truncated µ transgene described above). For example, a TCRβ transgene can partially rescue T-cell development and enforce β chain allelic exclusion in pretα-null mouse (39, 40). Since pre- Tα is a vital component of the pre-tcr (see below), one might assume that the pre-tcr is not formed in such a situation and this may be a non-physiological capability of TCRβ manifested only when expressed as a transgene (41). Remarkably, a TCRβ transgene is still capable of rescuing thymocyte development in pretα / RAG / mice. Thus, precocious TCRα chain expression is not entirely responsible for the activity exhibited by the TCRβ transgene in pretα / mice. In contrast, no significant rescue of B-cell development occurs when an Igµ transgene is expressed in λ5 / B cells (35). The second set of experiments involved a mutant TCRβ transgene that expressed an ER retention signal at its extreme C- terminus (42). This protein failed to be transported to the cell surface and failed to signal the pro-to-pre-t-cell transition. These workers concluded that the pre-tcr had to exit the ER in order to transmit its developmental signal. While this result is consistent with the requirement for an extracellular ligand, it does not prove that one is necessary. To explain how the pre-bcr and pre-tcr might be able to signal independently of ligand, we offer the following hypothesis (Fig. 2). Antigen receptors on mature lymphocytes become activated when protein tyrosine kinases associated with the cytoplasmic domains of their accessory chains cross-phosphorylate one another. This cross-phosphorylation is increased by ligand engagement. The phosphorylation of ITAMs leads to the recruitment of additional tyrosine kinases whose activation by phosphorylation triggers multiple signaling pathways. Balancing the activity of kinases are phosphatases that remove the phosphate groups introduced by the kinases. The balance of kinase and phosphatase activities establishes the threshold for receptor signaling. We propose that in pre-b and pre-t cells, there is a relatively low abundance of tyrosine phosphatase activity. The assembly and surface transport of the pre-bcr or pre-tcr signaling complex may be sufficient to initiate the cascade of phosphorylation events required to propagate the signal. Later in development, the activity of certain phosphatases may be higher, resulting in a much higher threshold requiring ligand engagement for receptor signaling. In vitro, phosphatases can be inhibited by treating cells with pervanadate. Using a myeloid cell line treated with pervanadate, one group demonstrated with an inducible system that upon BCR expression on the cell surface, SLP-65 and Syk become phosphorylated without ligand engagement (43). Such a result is consistent with our proposed model for ligand-independent pre-bcr signaling. Additional data consistent with this hypothesis exist for developing B cells. CD22 is a B-cell specific transmembrane glycoprotein whose cytoplasmic domain contains several immunoreceptor tyrosine-based inhibitory motifs (ITIMs). CD22 associates with the BCR on activated mature B cells and is a negative-feedback regulator of BCR signaling (44). Upon recruitment to the BCR complex, CD22 s ITIMs get phosphorylated by the protein tyrosine kinase Lyn (45). This results in the recruitment of the cytoplasmic tyrosine phosphatase PTP1C/SHP-1 onto CD22 s ITIMs near the inner membrane surface and dephosphorylation of nearby phosphotyrosines on Syk and the ITAMs of Igα/β, resulting in feedback inhibition of BCR signaling. Both CD22-null and PTP1C-mutant mice show increased Immunological Reviews 175/

5 Fig. 2. The ratio of kinase to phosphatase activity may be critical for the initiation of the pre-bcr signal. As described in the text, we propose a speculative model for activation of the pre-bcr based on the ratio between cellular kinases and phosphatases. In pro- Bcells, we assume that there are very low levels of membrane-proximal protein tyrosine phosphatase (PTP) and kinase (PTK) activities. However, there is presumably some basal level of signaling from the Igα/Igβ/calnexin complex on the surface of pro- B cells as evidenced by the pro-b-cell arrest observed in Igβ-deficient mice. The level of membrane-proximal PTK activity may be much greater in association with the pre-bcr in pre-b cells, and this activity is largely unopposed by PTP activity in the cytoplasm. In mature Bcells, CD22 associates with the BCR during signaling. The cytoplasmic domain of CD22 recruits PTP to the vicinity of the BCR, leading to a situation where physiologically relevant engagement of the BCR by ligand (antigen) is required to overcome this higher threshold established by CD22/PTP. B-cell signaling and alterations in cellular selection (46). Interestingly, CD22 is not expressed on the surface of pro-b cells, but is first detected in pre-b cells only after the pre-bcr has signaled (35). Therefore, unlike the BCR, the pre-bcr is not under the negative regulation of CD22 and PTP1C. This low threshold hypothesis could be tested by artificially raising the pre-bcr signaling threshold. For instance, one could overexpress CD22 or a constitutively active mutant of PTP1C in the plasma membrane of pro- and pre-b cells by transgenesis and determine whether developing cells arrest at the early pre-bcell stage. No obvious candidate for the CD22 analog in the T lineage is apparent. However, there is a compelling demonstration that the signaling threshold can be artificially lowered to result in precocious T-cell development independent of TCR gene rearrangements. Carboxy-terminal Src kinase (Csk) is a kinase that negatively regulates the Src-family kinases (47, 48). In its absence, thymocytes harbor increased Lck and Lyn kinase activity. In TCRβ-deficient mice, αβ T-cell development is arrested at the DN stage, but when those TCRβ-deficient thymocytes are also engineered to be Csk-null, they not only progress to the DP stage (mimicking the pre-tcr signal), but they also develop into CD4 + single-positive (SP) cells (mimicking perhaps positive selection) (49). The analogous experiment has not been performed in the B lineage to confirm that the requirement for pre-bcr signaling can be relieved by simply lowering the cellular signaling threshold. An interesting alternative experiment would be to raise the signaling threshold by overexpressing Csk and determine if the resultant altered signaling threshold can antagonize pre-bcr or pre-tcr signaling or lymphocyte development. The role of surrogate chains Single-cell PCR analysis of thymocytes sorted from pretα-null and control mice showed that in the mutant, a higher percentage of thymocytes contained two productively rearranged TCRβ alleles (41). This suggests that pre-tα is important for TCRβ locus allelic exclusion. A similar analysis was performed on λ5- null mice leading to the conclusion that λ5 is likewise involved in IgH locus allelic exclusion by the pre-bcr (50). The block in B-cell development in λ5 knockout mice is severe but not absolute. Precocious rearrangement and expression of an Ig lightchain gene is thought to mediate rescue of some B-cell development in these mutants. Interestingly, despite evidence of allelic inclusion at the genetic level, dual antigen receptorexpressing T or B cells cannot be detected in either the pretα / or the λ5 / mice (7, 40, 50). Can allelic exclusion in the mutants be enforced by a mechanism which takes effect after productive gene rearrangement? Indeed, productive biallelic V- to-dj H rearrangements in λ5 mutant mice can only be detected through single-cell PCR in the pro-b-cell population and not in more mature B-cell populations. Presumably, these cells never assemble a pre-bcr, never undergo the pre-b-cell transition, undergo programed cell death and are lost from the B-lymphocyte pool. The few cells that do undergo the pre-b-cell transi- 84 Immunological Reviews 175/2000

6 tion in mutant mice are those that are rescued by a precociously rearranged Ig light-chain gene (51). Light chain, in this case, might replace the need for SLC and allow assembly of a functional pre-bcr. This precocious light-chain gene rearrangement presumably precedes any V-to-DJ H rearrangement, since these rescued cells only have one productive V-to-DJ H rearrangement (52). One additional interesting feature of the V-to-DJ H rearrangements observed in the λ5 / mice is that a higher proportion utilize D H reading frame 2 (RF2) (50). These are rarely found in wild-type mice because cells that make a D-to-J H rearrangement in RF2 express the Dµ protein which leads to their counterselection before they ever complete their attempt at V- to-dj H rearrangement (53). This observation suggests that Dµ protein must associate with λ5 in order to affect B-cell development. Since Dµ-expressing cells are not negatively selected in λ5 / mice, their DJ H allele can be used for joining to a V H segment despite being in RF2. The other component of the SLC, VpreB, is encoded by either of two closely linked genes, VpreB1 and VpreB2 (54). Inactivation of the VpreB1 gene alone results only in a mild block in pre-b-cell development (55). Presumably, both VpreB genes would have to be disrupted in order to abrogate pre-bcr formation. This should be feasible but may require a special strategy, since the two genes are adjacent to each other in the genome. In the T lineage, it has been postulated that a VpreT gene exists by analogy to VpreB. Like the composite nature of the SLC in the pre-bcr, the surrogate α chain in the pre-tcr most likely requires another gene product to pair with the variable Ig domain of TCRβ. Since, normally, the variable Ig domain of TCRβ is paired with the variable Ig domain of TCRα, it may not fold properly and be unstable by itself. A VpreT gene has yet to be identified. The role of ITAM-containing proteins The Igβ (B29) / mice display a striking arrest of B-cell development at the pro-b-cell stage (56). Perhaps surprisingly, analysis of the rearrangement status of the IgH locus in this arrested population of mutant B cells revealed normal levels of D-to-J H rearrangements but the near absence of V-to-DJ H rearrangements. This observation suggests that Igβ may have a function in development prior to any role in transducing the pre-bcr signal. Igβ-deficient pro-b cells express normal levels of RAG mrnas and germline heavy-chain gene transcripts (which usually correlate with active heavy-chain gene rearrangement (57, 58)). The presence of the latter marker transcripts suggests that the IgH locus should be accessible to the V(D)J recombination machinery. An alternative interpretation of this data is that Igβ-deficient pro-b cells do generate productive V(D)J H rearrangements, but since these cells cannot assemble a functional pre-bcr they undergo rapid programed cell death. This interpretation is not likely to be correct because, in mice that harbor a deletion of the transmembrane domain of Igµ (µmt / mice), V(D)J H rearrangements can be detected easily, although developing B cells do not progress beyond the pro-b-cell stage (32, 59). In addition, forced expression of an anti-apoptotic bcl-2 transgene in Igβ / pro-b cells does not increase the frequency of detectable V-to-DJ H rearrangements (60). It is interesting to note that D-to-J H but not V-to-DJ H rearrangement, in addition to being a hallmark of Igβ-deficient B-cell development, is also observed during normal T-cell development and during B-cell development in Ig heavy-chain transgenic mice that allelically exclude endogenous IgH rearrangement (24, 35, 61, 62). These observations lead us to suggest that the V-to-DJ H step in B-cell development is stringently regulated by a mechanism which requires an Igβ-dependent activity. The requirement for Igα in pre-bcr signaling has been partially resolved. Through gene targeting, the cytoplasmic tail of Igα has been deleted and the resulting mb-1 c/ c mice contain 3- to 6-fold less pre-b cells in their bone marrow (63). Presumably, the tailless Igα can still heterodimerize with Igβ and support pre-bcr assembly, although it cannot itself contribute to signaling. Additionally, Igβ may be able to form homodimers and support pre-bcr assembly independent of Igα. It appears that the ITAM of Igα is redundant and dispensable, and the ITAM of Igβ is sufficient for pre-bcr signaling. The determination of Igβ s sufficiency in pre-bcr signaling awaits the generation and analysis of Igα nullizygous mice. It would also be interesting to determine the developmental phenotype of B cells expressing a mutant Igβ which is missing only its cytoplasmic tail. Targeted gene disruption has been used to test the roles of the various CD3 components in pre-tcr signaling. CD3δ is dispensable for pre-tcr assembly and signaling (64). CD3ζ / mice display a partial defect in the pre-t-cell transition which indicates that it too may not be absolutely required for pre-tcr signaling (65). However, in CD3ζ / thymocytes, allelic exclusion of endogenous TCRβ by a TCRβ transgene does not occur and TCRα rearrangement is limited (65, 66). T-cell development in CD3γ / mice is inefficient and characterized by extremely low thymocyte numbers and very few DP and SP cells (67). Thus, CD3γ probably plays an important function in the pre-tcr. However, CD3ε is absolutely required for pre-tcr signaling (68). CD3ε is capable of heterodimerizing with either CD3δ or CD3γ, although the above observations suggest that the Immunological Reviews 175/

7 CD3ε/γ heterodimer plays a dominant function in the pre-tcr. Notably, unlike the Igβ knockout mice, there is no observed V- to-dj β gene rearrangement defect in CD3-deficient mice (66). However, by analogy with B-cell development, we predict the existence of a regulatory mechanism which licenses pro-t cells to perform V-to-DJ β rearrangement. IL-7 signaling during the prelymphocyte transition Thymocytes are rendered more susceptible to apoptosis by inactivation of the common cytokine receptor γ chain (γ c ) gene (69). This γ c chain is required for signaling by the interleukin- 7 receptor (IL-7R). Interruption of IL-7 signaling in γ c / mice does not diminish the frequency of V-to-DJ β rearrangement (70). Similarly, as noted above, levels of V-to-DJ β rearrangements are normal in the pretα / mice (70). However, when the two mutations are combined, a curious defect in V-to-DJ β rearrangements manifests along with a thymocyte developmental arrest at the DN stage (70). Interestingly the γ c / pretα / DN thymocytes arrest at the earlier CD44 + CD25 + stage instead of the CD44 CD25 + stage as commonly observed, for example, in Rag-deficient mice. In the TCRβ enhancer-deleted mouse, thymocytes also fail to acquire V-to-DJ β rearrangements, although it is peculiar that double-stranded recombination signal sequence-dependent breaks are generated yet fail to undergo joining (71). It would be interesting to determine if the same is true in γ c / pretα / thymocytes. V-to-DJ H rearrangements seem to occur less efficiently in pro-b cells from IL-7Rα-deficient mice (58). The molecular basis of this defect is not known. It would be of interest to determine the phenotype of a λ5 / IL-7Rα / double mutant mouse which might manifest a more complete loss of V-to-DJ H rearrangement events and a more complete B-cell developmental arrest. Signal coupling from the plasma membrane to the cytoplasm Interestingly, prior to expression of the pre-bcr, the Igα/β heterodimer is already expressed on the surface of pro-b cells in complex with the chaperone protein calnexin (72). Similarly, in pro-t cells, the CD3 complex is expressed on the cell surface in association with this same chaperone protein (73). Crosslinking of the CD3 complex using anti-cd3ε antibodies is a well exploited experimental technique used to study early thymocyte differentiation (74 77). However, it was only recently that an analogous technique was developed to manipulate early B-cell differentiation. Nagata et al. took advantage of a monoclonal antibody directed to the ectodomain of Igβ by using it to cross-link Igα/β heterodimers on the surface of primary Igµnegative pro-b cells (72). Such a manipulation, ex vivo or in vivo, induced those pro-b cells to differentiate into pre-b cells as if they had received the pre-bcr signal. As expected, cross-linking of Igβ resulted in the phosphorylation of a number of proteins, such as Igα and Syk. Curiously, it was also shown that Src homology 2 (SH2) domain-containing leukocyte protein (SLP)-76 is one of the proteins that become phosphorylated (although it was previously reported to be absent in B cells). Other downstream effectors that were phosphorylated included PI3K (the p110 catalytic subunit), Vav and extracellular signalregulated kinase (ERK)1. Indeed among the MAPKs, kinase activity of ERK1 was shown to be induced, whereas kinase activity of c-jun N-terminal kinase (JNK)1 or p38 was not activated. The Src-family kinases The role of Src-family kinases in pre-tcr signaling and thymopoiesis has been firmly established. Loss- and gain-of-function genetic experiments reveal that the Src kinase-family member Lck plays a dominant role in these processes. Lck-null mutant thymuses contain reduced cell numbers and do not support development past the DP stage. The fact that pre- Tcells were still generated suggests that Lck is not absolutely required for pre-tcr signaling, and another Src kinase may provide a redundant function. Targeted gene disruption of the Src kinase fyn does not affect T-cell development. When Fyn deficiency is combined with Lck-deficiency, however, thymocyte differentiation is completely arrested at the DN stage, just like other mutant mice deficient in pre-tcr assembly (RAG /, TCR β / or CD3ε / mice) (78, 79). This strongly suggests that Lck and Fyn play some redundant roles in pre-tcr signaling. Expression of a constitutively active lck Y505F transgene at levels similar to endogenous lck expression promotes development of near wild-type numbers of DP thymocytes in RAG1 / mice (80) and mediates precocious allelic exclusion in wild-type mice (as indicated by suppression of V-to-DJ β rearrangements and premature recombination of the TCRα locus) (81, 82). In contrast, expression of a constitutively active fyn Y528F transgene only partially rescues DP thymocyte development in RAG1 / mice (78). The transgene could, however, completely restore T-cell development in lck / mice. This is consistent with the finding that a dominant-negative lck transgene arrests thymocyte development at the DN stage (83), whereas a dominant-negative fyn transgene has no effect on the pre-t-cell transition (84). Thus, the pre-tcr demonstrates a greater reliance on Lck in comparison to Fyn, which can subserve similar functions if Lck is limiting. 86 Immunological Reviews 175/2000

8 Presumably, the Src-family kinases have similarly important roles in pre-bcr signaling and B-cell development. However, experimental data in support of this notion are lacking. Mice deficient in individual members of the Src kinase family have been reported not to display obvious problems in early B-cell development. B lymphopoiesis is severely impaired only in the blk, fyn and lyn triple mutant (A. Tarakhovsky, personal communications). Surprisingly, some residual signaling capacity remains in these triple mutant pro-b cells that can be revealed upon cross-linking with anti-igβ antibodies ex vivo (A. Tarakhovsky, personal communications). Unfortunately, dominantnegative transgenes of blk, fyn, lyn or lck expressed in the B lineage have not been reported. A constitutively active blk Y495F transgene has been generated and shown to cause malignant transformation of early lymphoid progenitors of both the B and T lineages (85). The preferential expression of Blk in the B lineage predicted a critical function for this kinase during B-cell development similar to what has been observed for Lck in T-cell development (21, 86, 87). However, the blk Y495F transgene is not sufficient to mediate the pro-to-pre-b-cell transition in Rag-deficient mice (S. Desiderio, personal communication). It is noteworthy that the Epstein Barr virus multispanning membrane protein low molecular mass polypeptide (LMP) 2A is capable of mimicking the pre-bcr signal (88). LMP2A has an ITAM motif in its cytoplasmic tail and it aggregates spontaneously in the plasma membrane, resulting in activation of Lyn and Syk. Given the lack of clarity of these experiments, it may be informative to generate constitutively active fyn, lyn and/or lck transgenes that are expressed in early developing B lymphocytes. Such a gain-of-function candidate gene approach may allow the identification of the Src-family kinase that is chiefly responsible for pre-bcr signaling. Two or more gain-of-function mutants may need to be combined in order to bypass the need for the pre-bcr. The Syk/ZAP-70 kinases Another tyrosine kinase implicated in pre-bcr signaling is Syk. Syk-deficient mice exhibit perinatal lethality (89, 90). Therefore, B-cell development in this mutant was analyzed by in vitro culture of fetal liver cells on stromal cells or by using radiation chimeras generated by adoptive transfer of fetal liver cells. These approaches both showed marked decreases in the pro-topre-b transition in Syk-deficient B-cell progenitors. Using a PCR-based assay, these workers were able to detect V-to-DJ H and even V-to-J κ rearrangements, although these were not carefully quantified (89). B220 + CD25 + CD43 pre-b cells and IgM + immature B cells were not generated in these cultures, however (89). Furthermore, similar to what was observed in λ5 / and Igβ / mice discussed previously, D-to-J H rearrangements in RF2 that result in production of the Dµ protein are not selected against in syk / mice (90). It is interesting that both Igβ and Syk are required for counterselection of Dµ-expressing cells, yet Igβ but not Syk seems to be required to license V-to-DJ H rearrangement. Evaluation of Ig heavy-chain allelic exclusion in syk / mice has not been reported to date. It would also be feasible to analyze adult B-cell development in the context of Syk deficiency by deriving and utilizing syk / embryonic stem cells in the more elegant Rag-deficient blastocyst complementation assay (91) or conditionally inactivating syk only in the B lineage using cre-lox gene targeting technology (92). ZAP-70, a relative of Syk, is also expressed along with Syk during thymocyte development. As would be predicted, ZAP- 70 and Syk can carry out similar functions and both are implicated in pre-tcr signaling. Flow cytometric analyses of thymocytes from newborn mice do not indicate any defects in T- cell development in either syk / or zap-70 / mice (93). However, when the two mutations are combined, a severe reduction in thymocyte numbers and a developmental arrest at the DN stage results (93). V-to-DJ β rearrangements are easily detected in syk / zap-70 / mice, but it is not known whether TCRβ locus allelic exclusion occurs in the double mutant. The role of adapter proteins The SH2 domain-containing leukocyte protein SLP-76 is vital for coupling the pre-tcr to downstream effector pathways (94). It fits in the category of adapter proteins, because it possesses no intrinsic enzymatic activity, but instead harbors several modules that mediate interaction with various other proteins and thus functions like a scaffold. SLP-76 constitutively interacts with Grb2, another adapter protein, which couples to the Ras pathway via Sos. SLP-76 becomes phosphorylated rapidly upon TCR activation. This induces Vav to dock via its SH2 domain onto SLP- 76. SLP-76 has its own SH2 domain, which mediates inducible association with SLP-76-associated phosphoprotein of 130 kda (SLAP-130). In Slp-76 / mice, thymocyte development is arrested at the CD44 CD25 + DN stage, which is indicative of a defect in pre-tcr signaling (95, 96). The pre-tcr is fully assembled and transported to the cell surface in Slp-76 / pro-thymocytes, but it is uncoupled from its downstream signaling pathways. As expected, allelic exclusion of TCRβ locus rearrangement does not occur in Slp-76 / thymocytes (97). Thus, although it is not known whether Syk/ZAP-70 is required for enforcing allelic exclusion, a factor downstream of Syk/ZAP-70 has already been shown to be critical. The challenge now is to Immunological Reviews 175/

9 identify a SLP-76-dependent pathway which prevents V(D)J recombinase activity in the TCRβ locus but is distinct from the MAPK pathway, which is sufficient to activate recombinase activity in the TCRα locus (see below). SLP-76 plays a unique role in channeling the signal initiated by Src-family kinases to the various downstream pathyways which emanate from the pre-tcr. Linker of activated T cells (LAT) is a palmitoylated integral membrane protein expressed in T cells but not in B cells (98). Upon TCR activation, LAT is phosphorylated on multiple tyrosine residues by ZAP-70 or Syk (99). Two tyrosines have been identified as being critical for LAT function, perhaps by mediating its interactions directly or indirectly with SLP-76, Grb2, PLCγ1, Cbl, Vav or PI3K, each of which is induced upon receptor activation. In LAT / mice, thymocyte development is arrested at the CD44 CD25 + DN stage just like Slp-76 / thymocytes (100). TCRβ rearrangements occur in LAT / thymocytes, but assembly and surface expression of the pre-tcr has not been directly demonstrated and allelic exclusion has not yet been evaluated. The in vivo role of adapter proteins in pre-bcr signaling was recently determined. An SLP-76 B-cell homolog, SLP-65/B-cell linker, has been cloned (101, 102). Two groups showed that mice deficient in SLP-65 display a partial but significant block in development at the pre-b-cell stage (103, 104). Allelic exclusion has yet to be assessed in these mice. It has been reported that SLP-76 is not expressed in the B lineage (105), and indeed Slp-76 / mice do not display any defects in B-cell development or function (95, 96). However, a second study showed that it is present and phosphorylated in pro-b cells following Igβ cross-linking (72). If so, SLP-76 may perform a redundant function in Slp-65 / precursor B cells. The Slp-65 / mice will now need to be crossed to the Slp-76 / mice. As yet, no B-cell-specific LAT homolog has been cloned. LAT is reportedly not expressed in B cells, and LAT deficiency has no effect on B-cell development or function (98, 100). Interestingly, B and T lymphocytes have evolved to use different adapter molecules. The Src-family kinases, ZAP-70/Syk and the Ras/Raf/MAPK pathway are clearly coupled to both pre-bcr and pre-tcr signaling, but perhaps different adapters are required to activate lineage specific functions downstream of receptor activation in each lineage. The Ras/Raf/MAPK pathway The Ras/Raf/MAPK pathway is presumed to be an important effector pathway downstream of pre-tcr activation. However, a dominant-negative transgene of either Ha-ras S17N (106), c-raf1 (107) or the MAPK kinase gene MEK1 K97A (108) affected positive selection of thymocytes but did not have an effect on the earlier pro-to-pre-t-cell transition. In contrast, in studies using fetal thymic organ cultures, overexpression of a dominant-negative MEK1 S217A transgene completely blocked the development of DP cells (109). A constitutively active Ha-ras V12 transgene, expressed in Rag-deficient thymocytes, was sufficient to promote the differentiation of DN pro-t cells into DP pre-t cells, restore thymic cellularity and activate germline transcription of the TCRα locus in a fashion similar to a TCRβ transgene expressed in Rag-deficient thymocytes (110, 111). It would be worthwhile to confirm the implications of this result by breeding the Ha-ras V12 transgene onto the TCRβ / background to confirm that rearrangement of TCRα is also activated. A caveat in the interpretation of this result is that Ha-ras is not expressed in hematopoietic cells, which instead co-express N- and K-ras. Unlike the lck Y505F or a TCRβ transgene, the Ha-ras V12 transgene is not capable of mediating TCRβ locus allelic exclusion (111). Similar results were obtained using constitutively active c-raf1 or MEK1 LN3,S118,122E transgenes (112). Almost certainly, Lck is the kinase responsible for mediating the allelic exclusion component of pre-tcr signaling. Ras lies downstream of Lck in the pathway and is capable of mediating the pre-t-cell transition without enforcing allelic exclusion. Therefore, the pre- TCR, via Lck, must activate a Ras-independent pathway responsible for allelic exclusion. Again, the roles of Syk/Zap-70 and LAT in TCRβ allelic exclusion has not been determined, but SLP- 76 is involved. Downstream of MAPK/ERK kinase (MEK) in the MAPK pathway are the MAPKs themselves, ERK1 and ERK2. Their role in pre-tcr signaling has not been directly demonstrated as yet. Upon phosphorylation by MEK, these MAPKs have the ability to enter the nucleus and phosphorylate nuclear substrates, such as the transcription factors that may mediate changes in gene expression that follow pre-tcr signaling. For example, the Ets family of transcription factors is believed to be one class of factors involved in rapidly inducing expression of immediate early genes without de novo protein synthesis following MAPK stimulation (113). One immediate-early gene that is upregulated downstream of antigen receptor and MAPK stimulation encodes for the zinc finger transcription factor Egr-1 (114, 115). Indeed, an Ets factor is capable of regulating the egr-1 promoter in vitro (116). The observation that egr-1 gene expression is low in pro-t cells and is induced in pre-t cells is consistent with the notion that it is a nuclear target downstream of pre-tcr signaling (117). Remarkably, overexpression of egr-1 in thymocytes by transgenesis can bypass the block at the CD44 CD25 + DN stage of thymocyte development in Rag-deficient mice (117). This is not the final key to the puzzle, since Egr-1 is not sufficient to 88 Immunological Reviews 175/2000

10 mediate rescue all the way to the DP pre-t-cell stage, but only to the preceding CD8 + immature single positive stage. Thus, Egr-1 is capable of mediating some of the cell surface marker changes downstream of pre-tcr signaling, but it is still not clear what other aspects of the signal it is responsible for. The Ras/Raf/MAPK signaling pathway also seems to lie downstream of the Src-family kinases in pre-bcr signaling. The Ha-ras V12 transgene, when expressed in either Ig heavy-chaindeficient J H / or RAG1 / pro-b-cells, promoted certain aspects of the pre-b transition independent of pre-bcr expression (118, 119). In J H / mice, B-cell development is arrested at the pro-b stage and, accordingly, Ig light-chain gene rearrangements are found only rarely (120). Upon expression of the Haras V12 transgene, however, both Igκ and Igλ light-chain gene rearrangements become abundant (119). Furthermore, a constitutively active c-raf1 transgene can also partially rescue B-cell development in RAG2 / mice (121). The rescue, however, was not as complete as that observed with the Ha-ras V12 transgene. Amongst other differences, the Ha-ras V12 transgene induced expression of cell surface markers expressed by mature B cells, such as CD23 and CD21/CD35, while the c-raf1 transgene did not. Technical limitations prevented an analysis of the influence of the Ha-ras V12 transgene on allelic exclusion of heavy-chain gene rearrangement in these experiments. The c-raf1 transgene did not mediate allelic exclusion. The in vivo roles of the MEKs and the MAPKs ERK1 and ERK2 in pre-bcr signaling need to be evaluated. Termination of the signal Transcription of λ5 and VpreB decreases abruptly following assembly of the pre-bcr (18, 20), and as the levels of SLCs decrease, pre-bcr signaling is extinguished. The mechanism of this feedback autoregulation of pre-bcr expression is not likely to rely on the MAPK pathway, since, in the Rag-deficient B cells rescued by the Ha-ras V12 transgene, λ5 transcripts are still abundant, despite other markers that indicate that the cells are at a more mature stage (118). Pre-BCR-expressing cells undergo a brief period of clonal expansion but then exit the cell division cycle. It is presumed that the pre-bcr is responsible for the initiation of this clonal expansion but it is not known whether termination of clonal expansion is due to cessation of the pre-bcr signal. A recent study involving the conditional deletion of an expressed Ig heavy-chain gene showed that mature B cells depend upon the continuous expression of the BCR for their survival (122). It is possible that pre-b cells share this requirement. As the pre-bcr is disappearing from the cell surface, pre- B cells undergo active V(D)J recombination in their light-chain loci. If light-chain gene rearrangement is successful, the cell can express a complete IgM, the critical component of its BCR, leading to its survival. We think it is likely that the time-dependent loss of the pre-bcr signal limits the length of time a pre- B cell has to make a complete BCR. This would provide a way for the lymphopoietic system to eliminate cells with non-productive rearrangements at each of its light-chain alleles. In the absence of such a mechanism, cells with non-productive lightchain gene rearrangements would accumulate in the marrow and possibly serve as targets for leukemic transformation. Such a mechanism may be less important for pre-t cells since the TCRα locus contains approximately 50 J α gene segments, making it very unlikely that a cell would be unable to produce a functional αβ TCR. Events in the nucleus Changes in expression of several critical target genes coincide with expression of the pre-bcr. For instance, transcription of both RAG1 and RAG2 genes is transiently diminished (17). Transcription of terminal deoxynucleotidyl transferase (TDT), λ5 and VpreB also cease (18), but germline transcription of the Igκ locus is activated (18 20, 24). Little is known about the nuclear factors (NF) that mediate such changes in gene expression and how these factors are regulated. Expression of germline transcripts from the Igκ locus has been studied as a model of gene regulation by the pre-bcr. Previous work had shown that the κ locus is associated with two transcriptional regulatory elements termed the intronic and 3' enhancers based on their locations within the locus. Using an in vivo footprinting assay on primary pro-b and pre-b cells, one group reported that the NF-κB binding site in the intronic enhancer was similarly occupied in both pro- and pre-b cells (123). The pattern of binding site occupancy in the 3' enhancer did undergo a striking change during that transition, however (123). In pro-b cells, a factor was bound to a DNA element previously shown to bind B-cell-specific activator protein (BSAP)/Pax-5 (124), but this factor appeared to be displaced following the pre-b transition. BSAP has been reported to possess both transcriptional activating and repressing capabilities (125, 126). If the bound factor is indeed BSAP, then it might act as a repressor when bound to the 3' κ enhancer in pro-b cells. In pre-b cells, the BSAP site is vacant, but an adjacent PU.1 site is occupied. The obvious challenge now is to determine how these changes in enhancer binding are brought about by signaling molecules downstream of the pre-bcr and how these changes activate germline transcription and V(D)J recombination of the Immunological Reviews 175/

11 Igκ locus. A similar study of the TCRα locus in pro-t and pre- T cells has been reported (127). Another approach to understanding the mechanisms of transcriptional regulation by the pre-bcr and pre-tcr has involved the introduction of transgenes encoding factors which may lie downstream of the pre-bcr and pre-tcr into the RAG / background. The mutant transgenic mice are then analyzed for rescue of pre-bcr or pre-tcr signaling phenotypes. As discussed above, an egr-1 transgene expressed in Rag-deficient thymocytes was capable of promoting T-cell differentiation past the DN stage thereby bypassing the pre-tcr (117). When the same approach was tested with B cells, Egr-1 also seemed to bypass the pro-b-cell arrest in Rag-deficient mice, although further characterization will be required since the degree of rescue was only modest (128). Since Egr-1 is a transcription factor, it will be important to identify its targets and to determine what roles those gene products serve during the transition. A similar approach has implicated Ikaros, a zinc finger transcription factor, as a negative regulator downstream of the pre-tcr (129). Thymocytes from doubly mutant RAG / Ikaros / mice were capable of differentiating to the DP stage even without expression of a pre-tcr, and some cells even progressed to become CD4 + SP T cells. This result is reminiscent of the developmental phenotype of TCRβ / csk / thymocytes, which was attributed to a lowering of the membrane-proximal signaling threshold (49). In the case of Ikaros, these workers propose the novel notion that Ikaros, a nuclear protein, sets a membrane-distal signaling threshold for the pre-tcr. Again, not all aspects of the pre-t transition were recapitulated. For example, thymocyte numbers remained low. It remains to be determined whether the pre-bcr can be bypassed in a similar fashion. The roles of Aiolos and Helios, other members of the Ikaros family, will also need to be assessed. Concluding remarks The pre-bcr and pre-tcr mediate remarkably similar changes in developing B and T lymphocytes. These changes involve alterations in transcriptional regulation, proliferation, cell survival and V(D)J recombination. The challenge for the future will be to understand how activation of the membrane-proximal components of various signaling pathways eventually result in these alterations. Ultimately, the developmental program controlling this differentiation step is determined by the activation of certain genes and the repression of others. Which genes are key in this process? Differentially expressed genes need to be identified systematically (perhaps by the application of rapidly improving gene array technologies) and their functional role(s) determined (by transgenesis and targeted disruption). The transcription factors downstream of the pre-bcr and pre- TCR signaling pathways that are responsible for the regulated expression of these key genes must be identified. Finally, we must understand how these critical transcription factors are regulated by pre-bcr and pre-tcr signaling. References 1. Schatz DG, Oettinger MA, Schlissel MS. V(D)J recombination: molecular biology and regulation. Annu Rev Immunol 1992;10: Alt FW, et al. Ordered rearrangement of immunoglobulin heavy chain variable region segments. EMBO J 1984;3: Melchers F. Fit for life in the immune system? Surrogate L chain tests H chains that test L chains. Proc Natl Acad Sci USA 1999;96: Lassoued K, Illges H, Benlagha K, Cooper MD. Fate of surrogate light chains in B lineage cells. J Exp Med 1996;183: Lee YK, Brewer JW, Hellman R, Hendershot LM. BiP and immunoglobulin light chain cooperate to control the folding of heavy chain and ensure the fidelity of immunoglobulin assembly. Mol Biol Cell 1999;10: Keyna U, Beck-Engeser GB, Jongstra J, Applequist SE, Jack HM. Surrogate light chain-dependent selection of Ig heavy chain V regions. J Immunol 1995;155: Ten Boekel E, Melchers F, Rolink AG. Precursor B cells showing H chain allelic inclusion display allelic exclusion at the level of pre-b cell receptor surface expression. Immunity 1998;8: Clark MR, Friedrich RJ, Campbell KS, Cambier JC. Human pre-b and B cell membrane µ-chains are noncovalently associated with a disulfide-linked complex containing a product of the B29 gene. J Immunol 1992;149: Papavasiliou F, Misulovin Z, Suh H, Nussenzweig MC. The role of Ig β in precursor B cell transition and allelic exclusion. Science 1995;268: Cronin FE, Jiang M, Abbas AK, Grupp SA. Role of µ heavy chain in B cell development. I. Blocked B cell maturation but complete allelic exclusion in the absence of Ig α/β. J Immunol 1998;161: Clark MR, et al. The B cell antigen receptor complex: association of Ig-α and Ig-β with distinct cytoplasmic effectors. Science 1992;258: Satterthwaite A, Witte O. Genetic analysis of tyrosine kinase function in B cell development. Annu Rev Immunol 1996;14: Kurosaki T. Genetic analysis of B cell antigen receptor signaling. Annu Rev Immunol 1999;17: Rajewsky K. Clonal selection and learning in the antibody system. Nature 1996;381: Melchers F, et al. Positive and negative selection events during B lymphopoiesis. Curr Opin Immunol 1995;7: Immunological Reviews 175/2000