Cell, Vol. 66, 1081-1094,

September20, 1991, Copyright 0 1991 by Cell Press

Molecular and Cellular Origins of B Lymphocyte Diver& Antonius Rolink and Fritz Melchers Base1 Institute for Immunology Easel Switzerland

The diverse repertoire of antibodies or immunoglobulins (Igs) in the immune system is created by B lymphocytes. The specificity of an lg for an antigen is made up by three complementarity-determining regions (CD%) in the variable (V) regions of the heavy(H) and light (L) chains. Each B lymphocyte displays a single combination of H and L chains with a unique set of CDRs, out of millions of possible combinations in the total repertoire of lg molecules. At the molecular level, the diversity of antigen-binding V regions of lg molecules is generated by successive rearrangements and potentially by replacement events at both alleles of the H and L loci, as well as by N and P regions in H but not L chains, which are introduced in the joining reaction. At every step in this generation of diversity, it appears that cells of the B lymphocyte lineage check whether they have rearranged nonproductively or productively by depositing the product of the productively rearranged gene in the surface membrane. The importance of this process for pre-B cells is indicated by the fact that products of certain genes expressed specifically in these cells mediate the deposition. At the cellular level, proliferation and differentiation of progenitor B cells occur in close contact with a microenvironment provided by the primary lymphoid organ in which this diversity is generated. Elements of this microenvironment, known as the stroma, include adventitial cells, reticular cells, endothelial cells, fibroblasts, macrophages, adipocytes, and an extracellular matrix. The influence of stromal cellson B progenitors is mandatory for B cell development (reviewed by Kincade et al., 1989) and is exerted through ceil-cell contact and through cytokines secreted by stromal cells upon contact with lymphocytes. It remains unclear whether lg and Ig-like molecules on the surfaces of B lineage cells participate in contacts with the environment. That is, does the environment also check the productively rearranged lg deposited on the surface of B lineage cells? Differentiation Pathway of B Lymphocytes B lymphocytes are derived from pluripotent stem cells that also give rise to the erythroid and myeloid lineages of blood cells and to T lymphocytes (Smith et al., 1991). T and B lymphocytes are postulated to have a common lymphoidcommitted stem cell (S-lymphoid) (Keller et al., 1985; Fulop and Phillips, 1989; Phillips, 1989) that differentiates from the pluripotent stem cell in unknown waysat unknown sites. Commitment to the T or B lineage of lymphocytes occurs before or as progenitors enter the primary organs of lymphocyte differentiation. The first descendant of the pluripotent stem cell that is committed to the B lineage is termed a pro-B cell (see Figure 1). This pro-B cell does not

Review

express B lineage-specific markers and has not yet begun to rearrange the lg genes (Davidson et al., 1984, 1988; Muller-Sieburg et al., 1986). During embryogenesis, B lymphocyte development occurs in waves: first in placenta and embryonic blood, then in fetal liver, and finally in spleen and bone marrow (Melchers, 1979). B lymphocytes are continuously generated throughout life. Every day, an adult mouse is estimated to generate 5 x 1 O7cells from B lineage progenitors in bone marrow. Of these, 2-3 x lo6 enter the peripheral pooi of 5 x 1 O8mature, antigen-sensitive B cells in the circulation and secondary organs, such as spleen and lymph nodes (Osmond, 1990a, 1990b, and references therein). Therefore, stem cells with the capacity for self-renewal must exist; the question is whether these stem cells are pluripotent, restricted tothe lymphoid lineage, or further restricted to the B lymphocyte lineage. After colonizing the primary lymphoid organ, progenitors committed to the B lineage probably first expand there. In bone marrow they fill the extravascular spaces between large sinusoids in the shaft of long bones. Proliferation of the most immature B lineage progenitors is highest near the inner surface of the bone in the subendosteal reticulum of bone marrow (Hermans et al., 1989; Jacobsen and Osmond, 1990; Jacobsen et al., 1990). As B lineage progenitors become more differentiated precursors, they move toward the center of the marrow either to die in situ or to exit into the peripheral circulation via the sinuses and the central venous sinus. Molecular Markers for Different Stages of B Cell Development A series of molecular markers and cellular properties define different stages in B cell development (Figure 1; see also Figures 3 and 4). For example, pluripotent stem cells not only are characterized by certain molecular markers, but the pattern of expression of these markers (e.g., low levels of Thy-l, expression of Sea-1 , and the absence of lineage-specific surface antigens such as 8220 or BP-l) is used as a means of enriching populations for these cells (Spangrude et al., 1988). However, not every cell in such enriched populations can give rise to all the lineages of the blood system. Thus, these populations may also contain cells committed to a particular subset of blood cell lineages, such as the lymphoid stem cell. The existence of a lymphoid-committed stem cell has been difficult to prove, because it is not yet distinguishable by a specific set of surface markers. Nevertheless, a single cell, marked genetically either by a specific chromosomal translocation or by a retroviral insertion (Keller et al., 1985), can reconstitute T and B lymphoid functions but not myeloid functions in irradiated hosts (Fulop and Phillips, 1989). These cells can be maintained in long-term Whitlock-Witte cultures (Denis and Witte, 1989). The B lineage has been characterized by the expression of lineage-related markers, notably B220, BP-l, PB-76, terminal deoxynucleotidyl transferase (TdT), and Thy-l.

Cell 1082

New markers and functions have been added to this analysis: RAG-7 and RAG-P, genes encoding proteins that activate recombination of the variable (V), diversity (D), and joining (J) gene segments (Oettinger et al., 1990); and oncogenes such asabl, N-myc, c-myb, and bcl-2(reviewed by Potter, 1990). Subtractive hybridization of cDNA libraries constructed from 6 lineage cells to sequences also expressed in T lineage cells has led to the identification of several other 6 lineage-related genes, some of which are discussed in the following section. The earlier, morphologically oriented definition of cellular stages of B cell development can now be refined with the help of these new morphological, biochemical, and functional markers (Figure 1). B Lineage-Related Markers Function in the Surface Deposition of lg in Pre-B and B Cells Although the binding of antigen to surface lg molecules activates B cells and stimulates them to proliferate and mature, for a long time no association could be found between surface lg and signal-transducing molecules. This is in contrast to T cell antigen receptors, which are associated with the CD3 complex of membrane proteins. Such a complex may now have been found in mature B cells. IgM molecules become surface bound only when associated with the product of the mb-7 gene (Sakaguchi et al., 1988; Hombach et al., 1988; Kashiwamura et al., 1990), which is linked by disulfide bonds to the product of the 829 gene (Hombach et al., 1990a, 1990b). Both MB-1 and 829 proteins (also called IgM-a and lg-8) are transmembraneglycoproteins with extracellular lg-like domains and cytoplasmic tails with overall structural similarities to CD3 components of the T cell receptor complex. Their transmembrane regions contain an amino acid that might be in contact with an amino acid of opposite charge in the transmembrane portion of the IgM heavy(uH) chain. Deletion of the third and fourth constant (C) region domains of the uH chain abolishes both its association with MB-l and 829 and its surface deposition (Hombach et al., 1990b). A similar protein complex is found in human B cells (van Noesel et al., 1990). By analogy to the T cell receptor complex, the MB-lB29-uH complex may be associated with other proteins, such as r; chain-like molecules and tyrosine kinases b/k and /yn (Dymecki et al., 1990; Yamanishi et al., 1991). However, the complex has not been demonstrated to function as a signal transducer in mature B cells. The H chain of IgD is also associated with adisulfide-linked heterodimeric complex. It is not yet clear whether the slightly different size of the IgD-a chain relative to the IgM-a chain is due to a difference in glycosylation or protein structure (Parkhouse, 1990; Campbell and Cambier, 1990; Chen et al., 1990; Wienands et al., 1990). A Surrogate L Chain Selectively Expressed in Pre-B Not B Cells B lymphocyte development has traditionally been divided into two phases (Figure 1). The first phase is thought to be independent of foreign antigen and terminates when lg is expressed from rearranged H and L chain genes on the

surface of immature 6 cells. At that point B cells with lg specific for self antigens are either deleted (Nemazee and Biirki, 1989) or anergized (Goodnow et al., 1989). In the second, antigen-dependent phase, the remaining surface lg+ cells can be positively selected in the peripheral circulation and colonize the secondary organs. Depending upon the nature of the antigen, these cells can be stimulated either independently of T cells or with the cooperation of helper T cells (reviewed by Melchers, 1989). This view of B cell development is based on the assumption that V regions of lg loci are not expressed on the surface of pre-B cells (Siden et al., 1981). The lack of expression was inferred from the observations that the gene segments encoding L chains are not yet rearranged in pre-B cells (Maki et al., 1980; Alt et al., 1981; Perry et al., 1981) and that L chains are required for H chains to be transported to the surface (Thorens et al., 1985). It was found that uH chains are expressed in the cytoplasm in pre-B cells (Figure 1; Maki et al., 1980; Levitt and Cooper, 1980). However, their expression on the surface (Melchers, 1977; Rosenberg and Parish, 1977) remained controversial. The expression of rearranged H or L chain transgenes in pre-B cells inhibits the rearrangement of the corresponding endogenous loci (reviewed by Storb et al., 1989). The inhibition is dependent on the expression of membranebound chains and does not occur with secreted chains. Expression of rearranged H and L chains inhibits the rearrangement of both the endogenous H and L loci. While it is possible that the rearranged H or L chain signals the rearranging enzyme directly to turn off its activity at the appropriate locus, the necessity of surface expression of the transgenic lg chain might indicate that it is the membrane-bound H chain that signals the cell to stop rearrangements. A signaling role for the H chain from the surface of pre-B cells, however, appeared implausible, since the PH chain was thought not to be expressed on the surface. This view changed with the discovery of genes expressed selectively in pre-B cells that encode proteins with the potential to associate noncovalently to form a surrogate L chain (Sakaguchi and Melchers, 1986; Kudo and Melchers, 1987). Subsequently, the products of these genes, known as h5 and Vprbe,were found to be associated with j.rH chains on the surface of pre-B cells (Pillai and Baltimore, 1987, 1988). Both Vpre.eand 15 genes are expressed from the earliest stages of lg gene rearrangements, i.e., in DHJH-rearranged pre-B-l cells (Sakaguchi and Melchers, 1986; Rolink et al., 1991) to the transition from a pre-B cell to a surface lg+ B cell, when lg can occur on a cell together with uH chains with surrogate L chains (Cherayil and Pillai, 1991). The products of the h5 and uH genes form a disulfide-linked complex to which Vpre-8 is noncovalently attached (Karasuyama et al., 1990; Tsubata and Reth, 1990). Proteins encoded by Vpre.8 and h5 can also form a noncovalently bound L chain-like structure without PH chains. The sequences of the Vpre.8genes are highly conserved among mammals (Bauer et al., 1988). In mice and hu-

y;$.v:

Figure 1.

Origins of B Lymphocyte

Markers, Functions,

Diversity

Pool Sizes, and Turnover Rates of B Lineage Compartments

The nomenclature of Osmond is given (Osmond, IQQOa, lQQOb, and references therein). slgkl, conventional surface lg molecules with H and L chains expressed from rearranged loci (not @like molecules made of H chains with surrogate V,,,,,- b5 L chains). Transformation by Abelson virus is shown according to Phillips (1989). Pristane injection and antigenic stimulation increase the pool sizes and turnover rates of early and intermediate pro-B cells; pristane alone decreases those of late pro-B and large pre-Ei cells (Osmond et al., 1990a. 19QCIb). For the determination of the blocks of differentiation in normal and scid mice with and without rearranged H and/or L transgenes, see Fulop et al. (1988) Ritchie et al. (1984) Manz et al. (1988) Storb et al. (1989) Osmond et al., (lQQOa), and Reichmann-Fried et al. (1990). Pro-B to pre-El-l is defined by the rearrangement of the H chain loci from germline to DHJH. Pre B-I to pre-B-II is defined by the turnoff of TdT activity.

two VPre.8genes and one h5 gene are located on the that carries the hL chain loci. In the 9-12 million years since the various Mus genera diverged, the h5 and Vpre.Bgenes have changed little. Restriction fragment length polymorphism analysis reveals that a Vpre-~ gene has been maintained within IO kb of the h5 gene in the Mus genera (D’Hoostelaere et al., 1989). Also highly conserved is the genomic organization of the mouse h5 gene (Kudo et al., 1987a) and its human homolog, gene 14.1 (Evans and Hollis, 1991; Mattei et al., 1991). Both genes, as well as another gene in the human hL chain locus, lg*,, have three exons, which can be expressed without rearrangement by splicing of the primary transcript (Evans and Hollis, 1991). In mature 6 cells, however, lgl, has the potential to be rearranged to a canonical VJC hL chain gene.

mans,

chromosome

A Surrogate H Chain Expressed in Pre-B Cells There are genes in the Vn locus with a structure very similar to Vpre.~.Germline VH transcripts are selectively expressed in pre-B cells (Yancopoulos and Alt, 1985; Reth and Alt, 1984). An intriguing possibility is that the products of these genes encode proteins that are not only structurally analogous to VW but also functionally analogous. In such a model, these proteins might associate with the DHJHCp protein expressed on the surface of pre-B-I cells (Gu et al.,

1991). The C&J&,, protein cannot be expressed together with V p,,e-h5 surrogate L chain on the pre-B cell surface unless a VH region is added noncovalently to the complex (Tsubata et al., 1991); it is attractive to speculate that the products of the germline VH transcripts provide the necessary VH region. It is likely that DHJHClrchains and, later, VHDHJHCllchains are anchored in the surface membrane of pre-B cells by the MB-l-B29 protein complex, as 829 is expressed throughout the B lineage (Hermanson et al., 1988), while mb-7 is expressed in immature and mature B cells but not in Ig-secreting plasma cells (Sakaguchi et al., 1988). The MB-l-B29 complex appears to function in signaling, since antibodies against MB-1 stimulate calcium fluxes in pre-B lymphoma cells (Nomura et al., 1991). The Process of Rearrangement of lg Gene Segments During B cell differentiation, the genes encoding H and L chains are assembled by sequential rearrangements (Tonegawa, 1983) (see Figure 2). First, DH segments are joined to JH segments, followed by VH to DHJ,.,,then V, to J,, and finally VI to JI segments. If the rearrangement preserves the reading frame, it is said to be productive; out-of-frame rearrangments result in nonproductive H or L chain genes. Since cells without surface lg but with mark-

Cell 1084

G,A --DJ G H -DJ

RA RA

VH-~&,JHRA

V~I~MID”JH

RP

S-J,

RA

DHJH

TdT-

‘.~ 1 1 1 $fs diverse

Figure 2. Cascade of Successive RA = rearrangement;

lg Gene Segment

Rearrangements

TdT-

+

WJE,

and Replacements

RP = replacement.

ers characteristic of mature B cells are absent from the peripheral pool of mature B cells in secondary organs, it appears that surface lg- B cells generated from nonproductively rearranged lg H and/or L chain genes die in situ within the primary organ. The site-specific recombination enzyme complex involved in these DNA rearrangements has not yet been isolated; however, its mechanism has been studied by the analysis of alterations of transfected recombination substrates and the recombination defect in the mouse mutant known as scid. The recombinase is apparently targeted by conserved recombination signal sequences flanking the 3’ ends of V segments, the 5’ ends of J segments, and the 5’ and 3’ ends of D segments (Rathbun et al., 1989). Endonucleolytic scissions are first observed between coding and recombination signal sequences, followed by exonucleolytic degradation of the coding but not the signal sequences to various lengths. Both DNA strands may then be resynthesized by a template-independent activity, TdT, which creates N region sequences at the joints between D,+ and JH or between VH and DHJH. Finally, the two coding strands (of Dn and JH1and of Vn and DHJ~) are ligated, as well as the two recombination signal sequences. If the coding sequences of the two segments to be joined have the same polarity on the chromosome, the intervening sequences are joined in a circle and deleted. If they have opposite polarity, the corresponding part of the locus is inverted. Joining of VL to JL is imprecise but does not generate N region diversity (Tonegawa, 1983). Some of the proteins responsible for these activities may be present in many cell types, but others are specific for lg and Tcell receptor gene recombination. The RAG-7 and RAG-2 genes are expressed in T and B lineage cells in

the primary lymphoid organs precisely when these cells rearrange lg and T cell receptor gene segments (Oettinger et al., 1990). It is not yet known whether the products of these two genes indirectly activate rearrangement activities or whether they directly rearrange VDJ recombination substrates. The two genes are adjacent but are not on mouse chromosome 16, which carries the scid mutation (Bosma et al., 1988) the XL chain loci, and the pre-B cellspecific genes Vpre.8and h5 (Kudo et al., 1987b). This is yet another indication that rearrangements of the lg and T cell receptor loci require more than the expression of RAG-1 and RAG-2. Mice homozygous for the scid mutation are impaired in VDJ recombination (Bosma et al., 1989, and references therein). The scid recombinase can make appropriate scissions between coding and recombination signal sequences but aberrantly rearranges coding segments to generate nonfunctional genes. Joinings of recombination signal sequences are relatively unaffected (Blackwell et al., 1989). However, it should be emphasized that the defect in scid mice is probably in a general recombination or repair activity that is not specific to V region recombination, because repair of UV-induced dimers is also defective in scid mice (Fulop and Phillips, 1990; Biedermann et al., 1991; Hendrickson et al., 1991). N Region Diversity during Ontogeny Although it is common in adult lymphocyte repertoires, N region diversity at the junctions of D and J and the junctions of V and DJ is rare if not totally absent in prenatal and neonatal repertoires of B cells (Holmberg et al., 1989; Gu et al., 1990; Meek, 1990; Feeney, 1990) and y6 T cells (Elliott et al., 1988; Lafaille et al., 1989; Asarnow et al.,

v;evw

Origins of 6 Lymphocyte

Diversity

1989). N regions are thought to be added by TdT; the lack of N region diversity correlates with low levels of this enzyme in fetal liver (Gregoire et al., 1979). Absence of N region diversity limits the heterogeneity of V regions of the antigen-binding receptors; some waves of y8 Tcell development consist of a single V,V, combination, resulting in virtually monoclonal binding specificity. The appearance of these germline-encoded specificities early in ontogeny leads to speculation that they recognize self antigens in the environment in which these lymphocytes function. The self antigens recognized by 6 cells could be the idiotypes of germline-encoded lg molecules that set up a network of idiotypic-anti-idiotypic interactions (Vakil and Kearney, 1986; Vakil et al., 1986; Coutinho, 1989; Holmberg et al., 1989). With their very limited spectrum of variability, pre-B cells with N- uH chains and surrogate L chains might already participate in such a network and in the generation of the B cell repertoire. In adult B cells, N region diversity is generally greater at Vn to DH joins than at O H to JH joins; this correlates with N region diversity appearing first at VH to DH joins during ontogeny (Gu et al., 1990). This could be accounted for by differential activity of TdT at the two types of joins. Alternatively, pre-B cells that have already rearranged Dn to JH when there is little or no N region diversity may rearrange VH to D JH when TdT activity is increased to generate N sequences. Rearrangements of DH to JH Segments There are more than ten D segments, each encoding most of CDRB of the VH domain. Flanked by recombination signal sequences, D segments in the mouse are located within 80 kb of four JH segments (Ichihara et al., 1989, and references therein). It is not completely clear at which stage progenitor cells of the B cell lineage begin DHJH rearrangements. Abelson virus-transformed pro-B cell lines still have both H chain genes in the germline configuration (Scherle et al., 1990). However, earlypre-B cell lines growing on stromal cells in the presence of recombinant interleukin 7 (IL-7) have both H chain genes in DHJHconfiguration (Rolink et al., 1991). In the case of T cells, DHJH rearrangements might be initiated in lymphoid stem cells, since 100/o-20% of T cells carry DHJH-rearranged chromosomes (Gu et al., 1991). Primary DHJH rearrangements use preferentially the 5’-most and 3’-most D elements (Tsukada et al., 1990) and the ?-most JH segment (Gu et al., 1990). However, a single H chain locus may rearrange O H to JH more than once; such secondary rearrangements are seen in IO%-20% of the subclones of Abelson virus-transformed pre-B cell lines analyzed (Reth et al., 1986a, 1986b, and references therein). This might indicate that such successive DHJH rearrangements end in the use of either the Y-most D or the 3’-most J element. Normal pre-B cells in the DHJH-rearranged state that still exhibit long-term proliferative capacity “in vitro” are prime candidates for performing secondary DJH rearrangements (Rolink et al., 1991). Secondary DHJ,, rearrangements are detectable within a 2 week culture period, using an Abelson virus-transformed pre-B cell line with one H chain locus nonproduc-

tively rearranged and one in the germline configuration, which underwent continuous DJH rearrangement. In another Abelson virus-transformed pre-B cell line with a previously productively rearranged H chain locus, when the ability to produce uH chain was lost, the other allele, which was previously DHJH rearranged, restarted VH to DHJH rearrangement (Maeda et al., 1989). Thus, successive DHJH rearrangement may not be subject to allelic exclusion, i.e., it can occur on both chromosomes in the progeny of a single cell. These data also suggest that VH to DHJH rearrangement is suppressed in uH chain-producing cells to maintain allelic exclusion, while VH to VHDHJHreplacement is not suppressed (Kleinfeld et al., 1986; Reth et al., 1986a). It should be noted, however, that these results apply to transformed cell lines; their validity needs to be reevaluated with normal cells when such experiments become feasible.

DJ-C, Protein and Its Potential Function The 5’ regions of most D elements contain promoters, a transcription initiation site, and an AUG start codon (see Gu et al., 1991, and references therein) that might function in the expression of the DHJH-rearranged locus in pre-B cells. A strong bias for reading frame 1 is seen for VHDHJH-rearranged H chain loci of conventional Lyl- B cells (Gu et al., 1990) whereas early in ontogeny, Lyl+ B cells use all three reading frames (Gu et al., 1990). The biased usage of DH elements is determined at the stage of DHJH joining and is therefore an antigen-independent process (Gu et al., 1990, 1991). Short stretches of homology present in DH and JH segments may mediate the alignment of the recombining DNA strands after they have been cut endonucleolytically at the base of the recombination signal sequences. This mechanism can select for reading frame 1 as long as no N region sequences are introduced, i.e., early in ontogeny. In addition, there is counterselection against reading frame 3: at least 70% of DH segments have stop codons that render rearrangements nonproductive. In reading frame 2, however, many D elements allow the expression of a DHJHC,,protein. As a possible explanation for the absence of reading frame 2 in lg molecules, one could speculate that expression of the product of reading frame 2 on the surface of pre-B cells (possibly with Vp,e.8.L-X5surrogate L chain, Vpre-B.H, and the MB-l-B29 anchoring and signaling complex of proteins) results in selection against further development of those cells to VHDHJH-rearranged pre-B and B cells. In such a negative-selection model, surface deposition of the DnJnC, protein appears mandatory-T cells, which are incapable of surface deposition because they lack anchoring molecules, have no bias in reading frame usage. Furthermore, there are mice incapable of producing membrane-bound DHJHCpprotein and full uH chains from a mutated H chain allele (which has recombined with a uH chain gene lacking the transmembrane portion) that produce surface Ig+ B cells from their normal alleles. In such mice, if the mutant allele is at the DHJH stage, it is rearranged at equal frequencies in all three reading

Cell 1066

frames. Again, this indicates that selection against reading frame 2 depends on membrane deposition of the DHJ& protein.

VH to DnJH: Rearrangements and Replacements In mice and humans, between 100 and 1000 VH segments are organized in families of related sequences (Rathbun et al., 1989). Most VH family members are grouped, although some are interspersed in other families. Some are pseudogenes. As determined by deletion mapping, the orientation of all known VH segments on the chromosome is such that rearrangement leads to deletion of the intervening sequences. Early in ontogeny, the 3’-most VH segments (in particular, the VH81X of the VH7183 family) are preferentially rearranged. Randomization is achieved later in life, so that the frequencies of representation of VH families in adult repertoires correspond roughly to those in the germline. This need not be true for every member of a family, so that a given VH gene may dominate part of the adult repertoire. A productively rearranged VHDHJHallele gives rise to PH chains that can be deposited in the surface membrane together with the surrogate L chain Vpre.BL-h5 (Pillai and Baltimore, 1987,1988; Kerret al., 1989; Kudoet al., 1989). Early in ontogeny, this lg-like molecule might have very limited diversity, since it preferentially uses VH81X and has very little, if any, N region diversity. Surface expression, probably in association with MB-l and 829, might signal the cell to stop additional VH to DHJH rearrangements. In pre-B cells expressing a transfected VHDHJH-rearranged ~IH chain (with other VH segments and N region diversity), endogenous VH rearrangements are turned off (reviewed in Storb et al., 1989). If signaling is achieved by insertion into the surface membrane, it may be the MB-l-B29 complex that senses occupancy by the uH chain. If signaling involves interactions with stromal cells, the “constant” binding sites of the surrogate Vprg.8.L-h5L chain are likely candidates for mediating this interaction. Rearrangements useVH pseudogene segments and can occur out of frame; thus, many alleles will be nonproductively rearranged. Such pre-B cells may be capable of secondary rearrangements on the other allele (or, in cases of inversions, on the same allele). Even when both alleles have been rearranged nonproductively, a productive uH chain can be generated by VH gene replacement (Kleinfeld et al., 1988; Reth et al., 1988a); the same mechanism can achieve the opposite effect, rendering apreviouslyproductive allele nonproductive (Maeda et al., 1989). Vn replacement is mediated by heptamer joining sequences 7 nucleotides upstream of the 3’end of the coding regions of most VI+segments. The replacement occurs by alignment of the heptamer in the VHDHJn-rearranged site with the heptamer in the nonrearranged Vn. Since productively rearranged alleles can be replaced to become nonproductive, the replacement activity is not controlled by surface membrane-bound lglike PH-V,,~.~.~-W molecules (which are present earlier in ontogeny).

Differential Control of D-J and V to DJ Rearrangements In transgenic mice carrying Vr,, Dg, and JB segments of the T cell antigen receptor 8 locus in germline configuration, Dr,J, rearrangement occurs in both T and B cells (Ferrier et al., 1990). However, VB to DBJB rearrangement is restricted toT cells. The DBJBrearrangements are dependent on the presence of the enhancer of the IgH locus (Ew). Therefore, two separate controls operate in D-J and V to DJ rearrangements of the T cell receptor 8 locus and probably also in the lg H locus. First, a dominant cis-acting element within the Eucontaining DNA segment initiates D-J rearrangements in lymphoid cells. The second element acts specifically in either T or B cells to control V to DJ rearrangements. It correlates in activity with the sterile expression of the unrearranged V segments. NFuNR, a trans-acting factor that binds to suppressor sites flanking the Ep region, could play a role in regulating V to DJ rearrangement. The factor is present in nuclear extracts of non-B lineage cells and of DHJH-rearranged pre-B cell lines; but, notably, it is not detectable in nuclear extracts of VHDHJH-rearranged pre-B cell lines, mature B cell lines, and plasmacytomas (Scheuermann and Chen, 1989). Two of the four NFtrNR-binding sites overlap with consensus sequences for matrix attachment sites. In view of this, it will be interesting to determine whether attachment of the H chain gene to the nuclear matrix via the enhancer is necessary to stimulate transcription and rearrangement and whether NFuNR inhibits this attachment to the nuclear matrix. VL to JL Rearrangements In most pre-B cells, VL segments are only rearranged to JL segments once VH segments have been rearranged to DHJH segments. A uH chain expressed on the surface of a pre-B cell might not only turn off additional VH to DHJH rearrangements but also induce VLJL rearrangements. V,J, rearrangements most often precede V,J1 rearrangements (Selsing et al., 1989, and references therein). Between 100 and 300 V, segments organized in at least 18 families rearrange to 5 J, segments in mice and humans. V, gene segments occur in both orientations relative to J, and contain many pseudogenes (Zachau, 1989). Primary rearrangements during ontogeny use the 5’-most J, most often and the 3’-most J, least often (Harada and Yamagishi, 1991, and references therein). V,4 segments are preferentially rearranged in mouse pre-B cell lines transformed by Abelson virus (Kalled and Brodeur, 1990). In contrast, in primary mature B cell repertoires, V,4 is used much less frequently and probably as often as it is represented in the germline V, segment repertoire (Lawler et al., 1989; Kalled and Brodeur, 1990). Although the organization of the V, segments on the chromosome is not yet fully understood, V,4 is apparently not the 3’-most gene family, unless the family is so dispersed that some members are at the 3’end of the V, region. V,4 might be more accessible to primary recombination, because it contains highly conserved noncoding sequences that are strongly recombinogenic (Tutter and Riblet, 1989). This

Review: Origins of B Lymphocyte 1087

Diversity

might generate an initial inversion, leading to secondary rearrangements (Shapiro and Weigert, 1987). No N region diversity is introduced at the VL-JL junctions, because TdT activity is turned off at this stage of B cell development. A productively rearranged KL chain gene leads to expression of KL on the surface of a cell that has a productively rearranged H chain allele. The surface deposition of an assembled lg molecule is believed to signal the cell to stop additional KL chain gene rearrangements. Multiple V,J, rearrangements have been observed in cell lines (for example, Feddersen and van Ness, 1990, and references therein). These secondary rearrangements delete both productively and nonproductively rearranged KL chain genes (Haradaand Yamagishi, 1991). This result suggests that feedback inhibition by an expressed KL chain of additional KL chain gene rearrangements cannot function at all stages of B cell development. It remains to be elucidated when secondary VJJ, rearrangements can occur during B cell development. It is unresolved whether KL and hL chain loci are activated for rearrangement concurrently or sequentially (i.e., A after K) (Selsing et al., 1989; Cohn and Langman, 1990). hL chain-producing B cells often have both KL chain loci rearranged to a recombination signal sequence that is 1 O20 kb downstream of the C, exon and have thereby deleted C,, while many kL-producing cells have the other allele and the hL chain loci in germline configuration. Therefore, sequential rearrangement of first KL and then hL chain loci appears to be frequent. Cell Types of the Molecular Steps of 6 Cell Differentiation Cell types in which the cascade of lg gene rearrangements and replacements is enacted have been defined either in transformed cell lines obtained by virus infection (e.g., human Epstein-Barr, mouse Abelson) or in tumors of the early stages of the B lineage (B-ALL, B-CLL in humans; virally and chemically transformed B lymphomas in mice) (reviewed by Potter, 1990; Sawyers et al., 1991; Figures 1 and 2). They have also been identified by surface and cytoplasmic markers and have been localized in the bone marrow by histocytological methods; the pool sizes and rates of turnover of these cell types have been measured. Although some transformed cell lines continue to rearrange lg loci, the rates at which they do so are not those of normal precursor B cells. Furthermore, viral and malignant transformation not only arrests theirstateof differentiation, as judged by their pattern of marker expression and lg synthesis and secretion, but also deregulates their control of proliferation. It is hoped that studies with normal progenitors in tissue culture can offer a way to measure the proliferative and differentiative potential of B lineage cells at different stages of their development. Normal Progenitors and Precursors in Long-Term Culture Long-term lymphoid cultures from bone marrow and fetal liver (Denis and Witte, 1989, and references therein) have allowed the study of Tand B lymphopoiesis (Phillips, 1989)

in culture and in repopulation experiments in which cultured cells are transferred into suitable hosts. These longterm culture systems allow interactions between stromal cells of the primary lymphoid organs and lymphoid progenitor cells. The cell contacts established between these cells result in the production of stimulator-y cytokines (Kincade et al., 1989; Dorshkind, 1990). Monocytes and macrophages in the primary organs also play a cooperating role in B lymphopoiesis (Gisler et al., 1987; King et al., 1988). Progenitors and stromal cells are likely to make multiple molecular contacts, analogous to the adhesive contacts between lymphocytes and their environment (Springer, 1990). Several molecules(Sanderson et al., 1989; Thomas et al., 1988), including fibronectin (Bernardi et al., 1988), VLA4 (Miyake et al., 1991) Pgp-l/CD44 (Miyake et al., 199Oa), and hyaluronate (Miyake et al., 1990b), mediate these contacts and are required for B lymphopoiesis. Some of the cellular and molecular elements of the original Whitlock-Witte long-term bone marrow cultures have now been identified, particularly for murine B cell development. Progress has been made particularly in the cloning and expression of recombinant cytokines, the establishment of clones and linesof stromal cells active in B lymphopoiesis (reviewed in Kincade et al., 1989; Dorshkind, 1990), and the identification of progenitors of the B lineage with long-term proliferative capacity. IL-CDependent and IL-!&Dependent Progenitors From a Whitlock-Witte culture of B220- bone marrow cells, cell lines can be established under the stimulatory influence of IL-4. They can be transformed with Abelson virus, express the B lineage-specific BP-l marker, and are DHJHrearranged but not yet VHDHJHrearranged (Peschel et al., 1989). Differentiation to surface lg’ cells is not observed. It is not clear from these experiments whether IL-4 acts directly on the progenitors or indirectly via stromal cells. Other experiments note both stimulatory (King et al., 1988) and inhibitory (Billips et al., 1990) effects on development of IL-4 (and IL-l) on similar pre-B cell cultures. These effects have been studied with clones of stromal cells but not yet with clones of progenitors. Two IL-5dependent Lyl’ precursor B cell lines were established from long-term cultures of young bone marrow on a line of stromal cells (ST2; developed by Ogawa et al., 1988) in the presence of recombinant IL-5 One of the two lines also responded to IL-3. The expression by these lines of high and low avidity receptors for IL-5 indicates a means by which recombinant IL-5 can act directly on the progenitors. The H chain loci are DHJ,, rearranged, while L chain loci are in germline configuration. The pre-B cell-specific gene 15 is expressed in all ILd-responsive cell lines. In several other ILd-responsive cell lines, H and L chain gene loci are in germline configuration but continue to rearrange DH to Jn (and VH to DHJH) upon prolonged culture in the presenceof ST2stromalcellsand recombinant IL-S(Katoh et al., 1990). After treatment with 5-azacytidine, these clones differentiate to Lyl’ cells expressing either surface lg (indicat-

Cell 1088

ing that they have become B cells) or Mac-l (indicating that they have become macrophages). Prolonged culture on ST2 cells and granulocyte-macrophage colonystimulating factor (GM-C%) results in the development of Lyl’ Mac-l+ macrophages. The results of these experiments emphasize the close relationship between the myeloid and pre-B lymphoid Lyl+ pathways of differentiation (Davidson et al., 1988, and references therein).

IL-7-Dependent Progenitors There are B cell progenitors from fetal liver, bone marrow, and neonatal spleen that proliferate in Whitlock-Witte cultures and that are capable of repopulating the B cell compartment of scidmice(Phillips, 1989). These cells continue to proliferate under the stimulatory influence of IL-7 (Namen et al., 1988) and in contact with cloned stromal cell lines (Collins and Dorshkind, 1987; Whitlock et al., 1987; Nishikawa et al., 1988). Cell cloning experiments have distinguished three stages of B cell development. The earliest precursor proliferates without added IL-7 on stromal cells that do not produce IL-7. The intermediate precursor proliferates either on stromal cells producing IL-7 or with exogenously added recombinant IL-7; the late precursor proliferates without stromal cells in the presence of recombinant IL-7 (Hayashi et al., 1990). The number of late precursors is lowered in the bone marrow of KL chain transgenic mice, while the numbers of late and intermeditate precursors are lowered in bone marrow of PH chain transgenic mice (Era et al., 1991) indicating that primary VH to DHJH rearrangement might be performed in the intermediate precursor and primary L chain gene rearrangement in the late precursors. In an extension of these experiments, lines and clones of B lineage precursors in serum-substituted cultures on different stromal cell lines in the presence of recombinant IL-7 can be recloned with high plating efficiencies and can be maintained for nearly 1 year. No other interleukins, including IL-3, IL-4, and IL-5 can replace IL-7. These clones show variable expression of 8220 and BP-l; all express the pre-B cell-specific genes Vp,e.~.Land h5. The proliferating cells are DHJHrearranged on both alleles, with an occasional nonproductive VHDHJHallele but no productive VHDnJH H chain loci; thus, none produce PH chains. Compared with the precursors defined by Hayashi et al. (1990) most of these appear to be intermediate precursors (Rolink et al., 1991). Within 2 to 3 days after removal of recombinant IL-7 or stromal cells, the capacity to proliferate on stromal cells and IL-7 is lost. This leads to the induction of VH to DnJn rearrangements, L chain locus rearrangements, the appearance of surface lg+ cells, and the death of many cells by apoptosis. Loss of cells at the stage of L chain gene rearrangements can also be seen in situ (Deenen et al., 1990). Many of the surface lg+ cells become reactive to mitogens and can thus differentiate into lg-secreting plasma cells in culture. Therefore, under these conditions the late precursor defined by Hayashi et al. (1990) is not observed. The differentiation to surface lg+ B cells can also occur

after transfer of these precursors into scid mice (Rolink et al., 1991). The population in the spleen and the gutassociated lymphoid tissues is stable for months, and the B cells generated in vivo can be stimulated by polyclonal activators to proliferate and secrete lg. No T cells or macrophages of donor phenotype can be detected after differentiation to surface lg+ B cells. Therefore, these DHJH-rearranged precursor B cells are prime candidates for B lineage-committed stem cells. IL-7 receptor expression on precursor B cells appears to be mandatory for the development of at least Lyl- B cells. The receptor is involved in the up-regulation of the B lineage-specific antigen BP-118C3 on precursor B cells (Welch et al., 1990; Sherwood and Weissman, 1990). Interestingly, the promoter of the murine IL-7 receptor gene contains a potential binding site for interferon regulatory factor 1 (IRF-1). If IRF-1 induces the expression of the IL-7 receptor gene as it does the interferon a and 8 genes (Harada et al., 1990), constitutive overexpression of IRF-1 might deregulate the expression of the IL-7 receptor and its response to IL-7. Such overexpression would be expected when IRF-1 is under the control of the H chain enhancer Ep and might explain why Eu-IRF-1 mice are specifically depleted of B cells (Yamada et al., 1991). Lyl’ B Cells The Lyl’ B cell lineage (reviewed by Hardy, 1990) is characterized by its distinctive surface phenotype, its bias toward B cells involved in autoimmune responses, its minor contribution to spleen and lymph node B cell compartments and major contribution to peritoneal compartments, and its presence early in life and absence later. Lyl+ B cells have increased levels of hL chain expression, can be reactive to IL-5 and remain suppressed in anti-lg-treated animals for life, while Lyl- B cells repopulate the B cell compartments. The two compartments appear to influence each other on the progenitor-precursor level. It appears likely that Lyl+ B cells are a separate lineage that derives from early progenitors that have both H and Lchain genes in germline configuration (Katoh et al., 1990, and references therein); they are reactive to IL-3 and/or IL-5 in contact with specific stromal cells, such as ST2 (Figure 3). Progenitors of the Lyl’ lineage differentiate to either pre-B or myeloid cells, depending on their environment of stromal cells and the cytokines acting on themIL-5 induces pre-B cells, while GM-CSF induces myeloid cells (Katoh et al., 1990). In contrast to the Lyl- B cell lineage, D&C, protein expression does not inhibit further development of Lyl+ B cell precursors, since all reading frames are used with nearly equal frequencies in the repertoire generated early in ontogeny (Gu et al., 1990,199l). Usage of VH segments in the Lyl+ B cell repertoire is skewed toward VH1 1 (Carmack et al., 1990) indicating antigenic selection of unknown, possibly autoreactive specificities. Notably, Lyl+ B cells with V,.,l 1 use mainly JHI and some JH2segments, in contrast to peripheral Lyl- B cells, which use the 5’-most JH segments. These Lyl’ 6 cells might therefore derive from progenitors and precursors that have not undergone secondary Dn to Jn rearrangements.

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Figure 3. Model of the Cellular Stages of Development of Lyl’ and Lyl Ei Cells

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Several IL-d-dependent progenitor lines and clones have been reported to give rise to slg’ B cells that are both Lyl and Lyl +, as well as GM1 2’ Mac-i + myeloid cells when cultured on heterogeneous stromal cells of either fetal liver or bone marrow (Kinashi et al., 1988, and references therein). The development of f3 cells from IL-3-reactive progenitors is still debated, and controversial results may be due to a low frequency of such cells in normal fetal liver or bone marrow.

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Are Late Pro-B-Large Pre-B Cells Precursors That Repopulate the Adult I3 Cell Compartments and Generate Diversity by VH Replacements and Primary and Secondary L Chain Gene Rearrangements? The pool sizes and rates of turnover of the late pro-B-large pre-B cells are approximately 10 times those of the early and intermediate pro-B cells (Figure l), so they could well generate 5 x 10’ surface lg’ and surface lg- immature B cells per day. Their expression of Vpre.&Land h5 is undetectable by in situ hybridization and Northern blot analyses. This contrasts with the observation that Abelsontransformed cell lines and human and mouse pre-B lymphomas express similar levels of Vpre.~.~ and h5 at the pre-B-l and pre-B-II cell stages (Bauer et al., 1991, and references therein). Furthermore, late pro-B-large pre-B cells are TdT- (Osmond, 1990; Figure l), indicating that they do not rearrange DH to J,., or VH to DnJH. As precursor B cells, they should therefore be involved in VH replacements and in primary and secondary L chain gene rearrangements, none of which involve TdT. If this is the case, it follows that adult bone marrow generates over 90% of all surface lg+ and surface lg- immature B cells from VHDHJH-rearranged precursors whose Lchain loci are either in germline configuration or have undergone primary V, to J, rearrangement. The pre-B lymphoma 7OZ/3 (Paige et al., 1978) may be the transformed counterpart of such a precursor cell. Terminal rearrangements of V, to recombination signal sequences that induce hL chain gene rearrangement or productive KL chain rearrangements might terminate this state. In culture, the late precursor (Hayashi et al., 1990) is so far the only candidate that could have the expected proliferative capacity. Macrophages regulate B cell generation in the bone marrow, possibly through contacts with precursors and by producing both stimulatory and inhibitory cytokines (Billips et al., 1990, and references therein). They might well influence the continuous generation of B cells in adult bone marrow at that stage.

The Simplified Picture of Cellular Development In Vivo The picture of B cell development that emerges from these investigations is still complex (Figures 3 and 4), with many uncertainties to be resolved and many alternatives that precursors may use to generate the B cell repertoire. In a simple view of B cell generation in the bone marrow, all available sites of the stroma that are capable of cell-cell contacts with progenitors and inducible to cytokine production by these contacts are occupied by nonrearranged pro-B cells and DHJH-rearranged pre-B-l cells. Further proliferation of these cells pushes the precursors away from stromal cell contacts and appropriate cytokine supply and thereby releases these cells into V,, to DHJH and VJL rearrangements. A wave of B cell development in fetal liver may be generated by colonization of the organ by progenitors between days 12 and 13 of gestation, followed by an expansion of D&-rearranged progenitors completed around day 14. When the sites are filled, the cells generated by division are pushed off the stromal cells to differentiate to surface lg+ B cells (Strasser et al., 1989). The development of B cells in these organs can be inhibited by mechanisms that may involve the action of inhibitory cytokines such as IL-l, IL-4, and TNF-8 (Billips et al., 1990, and references therein). From Abelson virus-transformed cell lines, an intermediate stage of differentiation is predicted: VHDHJH-rearranged, L chain genes in germline configuration. While this might be contained in Osmond’s late pro-B-large pre-B compartments, it has not yet been identified as a normal precursor cell with proliferative capacity. Productive rearrangements lead to surface lg+ B cells, nonproductive rearrangements to surface lg- B cells; the latter appear to die by apoptosis in situ. The immature surface lg+ cells may then be subjected to selection by self antigens. The best-fitting ones are eliminated (Nemazee and Biirki, 1989) or anergized (Goodnow et al., 1989) whereas those that fit less well may even be

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Figure 4. Stages of B Cell Development From a progenitor presumed to express membrane-bound protein X in association with a surrogate Vp,,s- 15 L chain, pre-B-l cells differentiate by DnJH rearrangement in these cells, expressing a DnJ&, protein, possibly in association with V p,bs.nand with the surrogate L chain, anchored by MB-l and 829 proteins. Pre-B-l cells using reading frames 1 and 3 proliferate, because they still express X-surrogate L chain receptors. Upon VH to DHJH rearrangement, pre-B-II cells are generated. Cells in reading frame 3 die because they cannot express PH chains, while cells in reading frame 1 continue VH replacements and VJL rearrangements. At this intermediate stage, cells coexpress surface lg and PH chains with surrogate L chains. Cells that have nonproductively rearranged loci remain surface lg- and die by apoptosis. Surface lg’ immature cells are negatively and positively selected and enter the pool of mature, antigen-sensitive B cells. These mature B cells can respond to T-independent antigens by proliferation and maturation to IgM-secreting cells without much lg class switching or hypermutation of the lg V gene regions. Alternatively, they can respond to T-dependent antigens, again by proliferation and maturation, first to IgM secretion, then to surface deposition, and later to secretion of other classes of lg. T cell-dependent responses induce not only class switching but also hypermutation in the V region segments of lg H and L chain genes. This reaction occurs in secondary lymphoid organs in so-called germinal centers. Centroblasts at the stage of hypermutation and class switching will die by apoptosis if the result of the mutation is an lg- cell (MacLennan and Gray, 1966). Centrocytes will fall back into a resting cell from which a memory B cell response can be recalled at a second encounter with the same antigen.

positively selected for export into the peripheral mature B cell pool. Positive selection is supported by the finding that most B cells in germ-free and neonatal mice produce so-called multireactive natural autoantibodies (Dighiero et al., 1985; Prabhakar et al., 1984). Later in life, foreign antigens, with the help of T cells, will select and expand their best-fitting cells and thereby dilute the original multireactive B cells. The extensive clonal proliferation of B precursors capable of colonization and differentiation (Rolink et al., 1991) opens many possibilities to investigate the molecular requirements and the cellular dynamics of B lineage differentiation from a committed stem cell to an Ig-secreting cell. The functional roles of genes in B cell development can now be tested by transfection and by assaying the recombinants for their capacity to proliferate and differentiate and for their life span. Genes of interest include those that encode rearranged lg H and L chains, cytokine receptors, cytokines, signal-transferring molecules, and oncogenes active in the B lineage. The isolation of precursor B cells from different strains of mice with defects leading to autoimmune disease, immunodeficiencies, or malignancies will be useful in the identification of the cellular stage(s) and the molecular bases for these defects. Once comparable normal cell

lines and clones can be established from human bone marrow or fetal liver, a wide variety of human regulatory disorders the 6 cell compartment will become amenable to the same analysis. Although this review has concentrated mainly on B cell development in the mouse, the molecular events in human lg gene rearrangements and some of the associated cellular stages appear to be comparable. B cell development in amphibians might follow similar rules, but fish and birds have their lg gene loci organized very differently. The differentiation of 6 cells in these organisms is known to be comparable in part, but it is expected that other molecular mechanisms and other cellular stages will also be used. Acknowledgments We thank Drs. Steven Bauer, Antonio Lanzavecchia, Roland Gisler, Klaus Karjalainen, and Jean-Claude Weill for discussions and critical reading of the manuscript. We thank Hans-Peter Stahlberger for illustrations and Nicole Schoepflin and Janette Millar for preparing the manuscript. The Base1 Institute for Immunology was founded and is supported by F. Hoffmann-La Roche, Basel, Switzerland. References Alt, F., Rosenberg, N., Lewis, S., Thomas, E., and Baltimore, D. (1961). Organization and reorganization of immunoglobulin genes in A-MuLV-

Review: Origins of B Lymphocyte 1091

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Bauer, S. R., Kudo, A., and Melchers, F. (1988). Structure and pre B lymphocyte-restricted expression of the V,,es gene in humans and conservation of its structure in other mammalian species. EMBO J. 7, 111-116.

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