B-cell lymphopoiesis in mouse and man

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B-cell lymphopoiesis in mouse and man Tucker W LeBien Functional immunoglobulin gene rearrangement is a sine qua non for successful B cell development in mammalian

bone marrow, but other factors are also important. Studies reported during the past year have contributed new insight into the surface receptor complexes and signaling outcomes that influence the fate of B cell precursors. Identification and characterization of secreted and membrane-associated stromal cell products, and their actions on B-cell precursors, was a parallel area of ongoing investigation.

Addresses Department of Laboratory Medicine/Pathology, University of Minnesota Cancer Center, Center for Immunology, University of Minnesota Medical School, Box 806 Mayo, 420 Delaware St. SE, Minneapolis, MN 55455, USA; e-mail: lebieO01 @tc.urnn.edu Current Opinion in Immunology 1998, 10:188-195 http:/Ibiornednet.cornlelecref1095 2791501000188 © Current Biology Ltd ISSN 0952-7915 Abbreviations BM bone marrow IL-7R interleukin-7receptor RAG recornbination-activatinggene RT-PCR reverse transcriptase polyrnerase chain reaction SClD severecombined imrnunodeficiency SLC surrogateimrnunoglobulin light chain SDF-1 strornalcell-derived factor-1

Introduction

T h e development of B cell precursors (i.e. B-lineage cells that lack expression of conventional surface immunoglobulin) is governed by endogenous patterns of gene expression and cell--cell, cell-matrix, or cytokine cues from the fetal liver and bone marrow (BM) microenvironments. Endogenous patterns of gene expression are tightly governed by unique transcription factors, and by the increasingly complex recombinase machinery (see contributions in this issue by Reya and Grosschedl [pp 158-165] and by Gawunder, West and Lieber [pp 172-180]). T h e objective of this review is to summarize progress in characterizing the developmental biology of B-lineage cells, with a particular emphasis on the role of cell surface receptors and regulation by the BM microenvironment. Highlighted studies were selected from published work that appeared between December, 1996 and November, 1997, the annual period of review. The pre-BCR:

the plot thickens?

Two major B cell developmental checkpoints are phenotypically defined: firstly, expression of the pre-BCR, consisting of immunoglobulin it HC, surrogate immunoglobulin light chain (SLC) and the Ig-(MIg-13 signaling heterodimer on pro/pre-B cells; and secondly expression of the BCR consisting of Ix HC, conventional LC and

Ig-ct/Ig-13 on immature and mature B cells [1]. T h e importance of a successfully expressed pre-BCR for B cell development to proceed normally is well documented. Mice with a targeted disruption of the immunoglobulin light chain-related k5 gene encoding a component of the SLC exhibit a disruption of B cell development at birth, that lessens in severity as the mice age [2]. Mice harboring mutations in the membrane exon of it HC [3] or Ig-~ [41 exhibit a more severe disruption in B cell development. A major unresolved issue is whether the pre-BCR functions as a receptor that binds a specific ligand, or as a molecular initiating point for signal transduction that functions in the absence of ligand binding. T h e function of the BCR beyond classical antigen recognition was elucidated by the recent report of Lain and colleagues [5°°]. The powerful Cre recombinase methodology was employed to ablate the expression of a transgenic BCR specific for a defined hapten, and mice with the deleted BCR exhibited a rapid death of mature B cells [5°°]. In an accompanying editorial, Neuberger [6°°] suggested that continuous (relatively weak?) signaling through the BCR provides a 'persistence signal' that does not culminate in cell cycle entry, but does preserve survival of the mature B cell [6°°]. T h e nature and strength of the BCR cross-linking signal may distinguish a persistence signal (leading to survival) from an activation signal (leading to cell cycle entry). This new insight into BCR function is interesting. Does the paradigm apply to the pre-BCR as well? In other words, could the pre-BCR function by transducing persistence or activation signals that culminate in survival or proliferation of pre-B cells? In addition, what are the precise functions of the individual components of the pre-BCR (it HC, SLC, Ig-ct/Ig-13) relative to the overall function of this molecular complex? T h e hypothesis that the pre-BCR recognizes a ligand in the fetal liver or BM microenvironment has been a frequently proposed but unproven function ever since ~.5 was discovered over 10 years ago [7]. There is currently no direct evidence that a ligand derived from stromal ceils binds to the pre-BCR and transduces a signal to pro-B or pre-B cells. Quite the contrary, it HC transgene products can transmit signals that lead to developmentally important changes in gene expression in B cell precursors (e.g. downregulation of expression of terminal deoxynucleotidyl transferase, a marker of immature lymphocytes), in the absence of a BM microenvironment [8]. Development of the V H repertoire can be potentially influenced by the physical disposition of V, D and J) genes, and by preferential pairing of Ix HC with SLC. Data generated by single cell analysis of the V H repertoire in murine B cell precursors was used to conclude that the pre-BCR itself can mediate changes in the VH repertoire [9"]. Assuming that the capacity of the pre-BCR to mediate a V H repertoire shift relies on stable

B-cell lymphopoiesis in mouse and man LeBien

expression of pre-BCR on the cell surface, then soluble pre-BCR could conceivably alter the shift by competing with membrane anchored pre-BCR for ligand binding [10]. Somewhat in contrast, a separate study concluded that although the nature of the expressed V H gene imparted selective survival to individual B cell precursors, some of the V H genes selected against (i.e. V H gene usage reduced in frequency or not detected in B cells present in secondary lymphoid tissue) were capable of associating with SLC and being expressed on the cell surface [11]. In an effort to elucidate the function of IX HC in the absence of SLC, Shaffer and Schlissel [12"'] developed transgenic mice expressing a truncated IX HC that could not associate with SLC, but was expressed on the cell surface. Surprisingly, B-lineage cells expressing this truncated IX HC exhibited developmental changes indistinguishable from mice harboring intact IX HC that associated with SLC. T h e authors proposed, reasonably, that SLC might function as a chaperone facilitating assembly of IX HC into the pre-BCR, rather than playing a role in ligand binding as a component of the pre-BCR. An additional level of complexity in the function of pre-BCR components was revealed by the detection of Ig-o~/Ig-13 heterodimers complexed with calnexin on the surface of pro-B cells from recombination-activating gene (RAG)-2-deficient mice [13"']. Cross-linking Ig-13 in vivo in these RAG-2-deficient mice induced signals for differentiation which were comparable to those induced by conventional pre-BCR, and similar results were observed using ~.5-deficient mice [13"']. From these results and prior work, there may be at least four distinct cell surface complexes containing pre-BCR components on pro-B and pre-B cells (Figure 1): firstly, conventional pre-BCR (IX HC, SLC, Ig-(~Ig-13); secondly, the newly discovered complex of Ig-(~/Ig-13--calnexin (with no SLC); thirdly, SLC associated with the so-called 'surrogate heavy chain' [14]; and fourthly, the truncated heavy chain termed DIX, associated with Ig-~/Ig-13 and SLC [15]. Given the fact that Ig-13-deficient mice exhibit a disruption at the stage of V H to DJ rearrangement [4], it seems plausible (as proposed by Nagata et al. [13"] that Ig-13 could function in a signaling pathway prior to expression of conventional pre-BCR. C o n t r o l o f B-cell survival

T h e past year has witnessed additional progress in elucidating the signaling pathways that regulate the apoptotic fates of B-lineage cells. Studies in many laboratories have detailed the essential nonredundant role of IL-7 in the development and expansion of murine B cell precursors [16], but we still have an incomplete understanding of how signaling through the IL-7 receptor (IL-7R) is eventually integrated into proliferation, differentiation, or survival (anti-apoptotic) pathways that regulate the development of B cell precursors. New insight into survival effects was gleaned from analysis of the Bcl-2 pathway in mice deficient in the expression of IL-7R complex genes. Enforced expression of Bcl-2 in mice deficient in the

189

common cytokine y-chain [17"] or in the IL-7R ct-chain [18] failed to reconstitute B cell development, but did rescue T cell development. These results suggest that IL-7 signaling transduces a survival signal, mediated at least in part by Bcl-2, to developing T cells in the thymus. In contrast, IL-7/IL-7R interaction does not result in a Bcl-2-mediated, anti-apoptotic signal being delivered to B cell precursors. One explanation for this difference may be that IL-7 signaling is coupled to distinct anti-apoptotic pathways in B-cell and T-cell progenitor populations. An intriguing candidate for transduction of survival signals in B cell precursors would be BcI-X L. Mice expressing a BcI-X L transgene exhibit a dramatic expansion (or rescue) of pro-B ceils, and V(D)J rearrangements in these pro-B cells are almost exclusively nonproductive [19]. It will be most interesting to determine if enforced expression of BcI-X L in y-chain-deficient or IL-7R(~-deficient mice restores B-cell lymphopoiesis. T h e Bcl-2-transgenic mice continue to provide insight into signaling pathways that facilitate proliferation or differentiation of B cell precursors. RAG-2-deficient mice expressing a transgenic bt HC undergo pro-B to pre-B differentiation, and the extent of this differentiation is enhanced by the co-expression of a Bcl-2 transgene [20"]. Similarly, transgenic expression of Bcl-2 increased the number of early B-lineage cells in mice with severe combined immunodeficiency (SCID), which were also RAG-l-deficient, but continued (albeit limited) differentiation occurred only in mice with SCID and a Bcl-2 transgene [21]. Furthermore, overexpression of Bcl-2 can inhibit the transition of B cell precursors between states of quiescence and activation, independent of their differentiation stage [22]. Effects o f t h e b o n e m a r r o w m i c r o e n v i r o n m e n t

The collective effects of BM microenvironmental signals on B cell development reflect the sum of diverse stimuli that may promote survival or death. T h e role of contact with stromal cells and soluble mediators in regulating B cell development was a theme of continuing investigation in 1997. T h e essential, nonredundant requirement of IL-7 for normal murine B cell development is indisputable [23]. Mice deficient in both IL-7Ro~ and the common y chain exhibit a more severe disruption in B cell development than mice deficient in IL-7 [16]. One explanation for this difference suggests that a distinct cytokine that also binds to IL-7Rc~ could be involved in early B-cell precursor signaling, but it lacks formal proof [16]. Several reports provide additional insight into stromal cell production and B cell precursor responsiveness to IL-7. T h e gradual reduction in B-cell lymphopoiesis observed in aging mice may be attributable to impaired release of IL-7 from BM stromal cells [24], or to changes in IL-7R-mediated signaling pathways [25]. Enhanced production of murine stromal cell IL-7 following direct contact with lymphoid cells was reported several

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Lymphocyte development

Figure 1

D~t Pre-BCR

Calnexin Pre-BCR

Conventional Pre-BCR

Surrogate HC Pre-BCR

Ca++mobilization Protein tyrosine kinase activation AIlelic exclusion Light chain rearrangement Multiple changes in gene expression Current Opinion in Immunology

"'c°ex

'rD urr°e Lc

0CanexnI'rr°ae"c

Receptor complexes on B ceil precursors that contain different components of the pre-BCR. Conventional pre-BCR contains Ix HC, SLC, and Ig-cdlg-13. The Dla pre-BCR [15] contains a truncated ~t HC. The newly described cainexin pre-BCR contains calnexin associated with Ig-cdlg-13 [13"']. The surrogate HC pre-BCR contains (at least) a poorly characterized glycoprotein of -130 kDa [14] that is apparently not associated with Ig-cdlg-13.The presence of Ig-a/Ig-13 in any complex implies a signaling capability. Evidence from many laboratories has indicated that calcium flux, protein tyrosine kinase activation, allelic exclusion, onset of light chain rearrangement, and other changes in gene expression are outcomes of signals transduced by the pre-BCR. Less certain is a signaling function for the surrogate HC pre-BCR, given the apparent absence of Ig-odlg-I~ from this receptor complex. It is important to emphasize that a complete characterization of the expression and function of these four complexes on normal murine and human B cell precursors has not been accomplished.

years ago [26]. Although conceptually attractive, the notion that B cell precursors can influence their fate by enhancing the production of stromal cell IL-7 has not received confirmation and extension in the literature. Indeed, our own studies indicate that lymphoid cell/stromal cell contact does not enhance IL-7 production by human BM stromal cells [27]; however, transforming growth factor-13 can suppress IL-7 mRNA expression and IL-7 protein secretion in at least some human BM stromal cells [28]. Although IL-7 is not essential for human B cell development [29], IL-7 can downregulate RAG-I/RAG-2 mRNA expression in human fetal BM pro-B cells [30]. Murine IL-7 inhibits immunoglobulin gene rearrangement and B-cell precursor maturation [31], and may also influence V(D)J recombination by post-transcriptional regulation of RAG-I/RAG-2 [32]. With respect to the reduced importance (compared to the mouse) of IL-7 for expansion of human pro-B cells and normal human B cell development, the question remains as to the identity of the molecule(s) that promote the growth of human

B cell precursors. The combination of Fh3 ligand (that binds to the Flt3 tyrosine kinase receptor and has a broad range of effects on hematopoietic stem cells) and IL-7 provides a greater proliferative stimulus to human pro-B cells than does IL-7 alone [33]. Flt3 ligand does not, however, deliver a growth stimulus to pro-B cells [33], so this molecule by itself cannot substitute for IL-7 in humans with the mutations in the common ~t chain that results in X-linked SCID. Other molecules must therefore be involved. Identification of cytokines that stimulate Polycomb gene products [34°] might provide fresh leads. Efforts continue to characterize other soluble molecules or molecules associated with stromal cells that regulate B cell development. SC1/ECM, a calcium-dependent matrix glycoprotein, co-operates with IL-7 to facilitate the clonogenicity of fresh pre-B cells and pre-B cell lines [35°]. The chemokine stromal-cell-derived factor-1 (SDF-1), originally defined as pre-B cell growth-stimulating factor [36], has now been shown to exert chemoattractant effects

B-cell lymphopoiesis in mouse and man LeBien

on pro-B and pre-B cells [37"]. This exciting observation provides experimental support for a long-sought mechanism to explain how developing B cells 'migrate' within or between BM stromal cell niches. Furthermore, as proposed by the authors [37"], SDF-1 may act to retain B cell precursors in the BM microenvironment to ensure adequate transduction of growth and differentiation signals. T h e complex relationship between extramedullary endocrine/neuropeptide function and B-cell lymphopoiesis has been further explored. B cell development was shown to depend upon an intact pituitary/thyroid axis [38], and vasoactive intestinal peptide was reported to suppress the B-cell precursor response to IL-7 by stimulation of interferonqx production [39]. A surprisingly complex potential relationship between B-cell lymphopoiesis and bone loss was revealed by the observation that female mice treated with IL-7 exhibit a marked reduction in bone mass, which resembles the bone loss that follows ovariectomy [40"]. Lymphoid cell/stromal cell communication may reflect activation of signal transduction pathways in both directions (i.e., lymphoid cell ~ stromal cell or stromal cell lymphoid cell). Studies in two laboratories [41",42 °] demonstrated that stromal cells can suppress apoptosis of freshly isolated murine B220 ÷ surface bt- B cell precursors, consistent with a prior report [43] indicating that stromal cells suppress apoptosis of normal and leukemic human B cell precursors. Furthermore, stromal cells may exhibit some heterogeneity in their capacity to suppress apoptosis [42"]. Studies in two laboratories reported that stromal cell contact and complex cytokine mixtures can induce the expression of RAG-1 or RAG-2 mRNA (based on RT-PCR) in freshly isolated human BM mononuclear cells or primitive stem cells [44,45]. Lymphoid cells can also activate signaling pathways in stromal cells, resulting in tyrosine phosphorylation of substrates in the cytoskeleton and the Ras/ERK2 pathway of the stromal cells [46"]. At least one protein tyrosine phosphatase in BM stromal cells also appears to be important for normal B lymphopoiesis [47].

Gene expression during B-cell lymphopoiesis Analysis of both immunoglobulin and non-immunoglobulin gene expression in murine and human B cell development has continued to yield new insight into the developmental biology of B lymphocytes. T h e widely used scheme developed by Hardy et al. to study B cell development has been refined [48°]. Multiparameter flow cytometry was used to purify CD19-- (fraction A) cells into three additional fractions designated A0, A! and A2. Fraction A0 cells do not express genes associated with B-lineage commitment, and may represent a common lymphoid progenitor. Fractions A 1 and A2 express high levels of germline g transcripts, and variable levels of RAG-I/RAG-2 and Ig-l~ transcripts [48"]. Fractions A0, A 1 and A2 represent a probable hierarchy of developmental commitment to the B cell lineage, and collectively share

191

some developmental commonality with the CD10 + CD19-common lymphoid progenitor in humans [49]. It is possible that the capacity of IL-7, IL-10, IL-11, and Flt3 ligand to induce B cell differentiation from immature lymphohematopoietic progenitor fractions [50-52] may at least partially reflect direct stimulation of fraction A0 cells. Although expression of cell surface CD19 has been the primary phenotypic marker of human B-lineage cells for many years, the precise function of CD19 on human and murine 13 cell precursors has remained elusive. CD19 is known to regulate signaling thresholds through the BCR on mature B cells [53], and CD19-deficient mice are hyporesponsive to a variety of external signals [54,55]. New data indicate that expression of a human CD19 transgene in CD19-deficient mice restores B cell function, and 2-3-fold differences in cell-surface CD19 expression dramatically affect B cell development and B cell responses [56]. In addition, the cytoplasmic tail of CD19 is essential for CD19 function in vivo [57]. An external ligand for CD19 has not been found and may not exist. Mounting evidence for its co-operative role in transducing signals through the BCR [58] and other molecules such as CD38 [59] suggests a critical function that does not involve ligand binding or signal transduction at all stages of 13 cell development. My colleagues and I [60] have shown that signaling initiated by cross-linking of CD19 leads to preferential activation of DNA-binding elements NF-~B or AP-1 in pre-B and immature human B cell lines, respectively. Several reports have provided new insight into the timing and nature of immunoglobulin gene rearrangement/expression in early human ]3 cell development. CD34+ CD19-- progenitors express germline g transcripts, DJ rearrangements, Ig-13, and RAG-1 [61"-63"]. These collective results provide strong evidence that commitment to the B lineage precedes expression of cell-surface CD19 in humans, as also occurs in the mouse. RT-PCR analysis of single CD19 ÷ human BM B-lineage cells demonstrated a profile of RAG, SLC, and immunoglobulin expression similar to that previously characterized in the mouse [64"]. Reports using murine [65] and human [62"] BM stromal cells demonstrated that signals derived from stromal cells are crucial for differentiation and expansion of hematopoietic stem cells or primitive lymphoid progenitors into the B lineage, consistent with earlier reports [29,66]. These models may be useful in evaluating mechanisms that regulate V gene usage during human B cell development in vivo [67], thereby complementing the in vitro model for evaluating V H repertoire development in the mouse [68].

Conclusions T h e importance of individual recombinase, immunoglobulin and immunoglobulin-associated gene products in normal 13-cell lymphopoiesis has been convincingly demonstrated through the analysis of gene-deleted mice. Forced expression of genes that inhibit or promote apoptosis

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in a background of cytokine-signaling deficiency will continue to be a fruitful line of investigation. Identification and characterization of the collective stromal-cell gene products that regulate the proliferation, differentiation and apoptosis of B cell precursors is still at a relatively early stage of delineation and will constitute an area of future emphasis.

Acknowledgements E-mail discussions with Ted Bertrand and Mark Schlissel are deeply appreciated. I thank Adelia Falk and Julie Pribyl for assistance with the manuscript. Work in my laboratory is supported by National Institutes of Health grants R01-CA31685 and R01-CA76055.

References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: • ••

of special interest of outstanding interest

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2.

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5. =•

Lam KP, K~ihn R, Rajewsky K: In vivo ablation of surface immunoglobulin on mature B cells by inducible gene targeting results in rapid cell death. Cell 1997, 90:1073-1083. This study was a technical and conceptual tour de force. Mice harboring a Cre recombinase transgene under the control of a type I interferon-inducible promoter were crossed with mice expressing a BCR transgens specific for the hapten 4-hydroxy-3-nitrophenyl acetyl (NP). Treatment of these mice with recombinant od~ interferon ablated expression of the transgenic BCR through activation of Cre recombinase. The so-called 'receptorless' B cells underwent apoptosis in vivo, and cell death could be delayed, but not prevented, by constitutive expression of Bcl-2. The study provides powerful support for the argument that signaling through the BCR is essential for survival, although the biophysical nature of the cross-linking event or the biochemical nature of the signaling event in this model is unknown. It is unlikely that a naturally occurring in vivo 'NP homologue' was responsible for BCR cross-linking. 6. Neuberger MS: Antigen receptor signaling gives lymphocytes a *• long life. Cell 1997, 90:971-973. This minireview provides insightful comment on the study by Lam and colleagues [5"'], and discusses differing states of BCR signaling, culminating in preservation of B cell survival or antigen-driven B cell activation. 7.

Sakaguchi N, Melchers F: X5, a new light-chain-related locus selectively expressed in pre-B lymphocytes. Nature 1986, 324:579-582.

8.

WassermanR, Li Y-S, Hardy RR: Down-regulation of terminal deoxynucleotidyl transferase by Ig heavy chain in B lineage cells. J Immunol 1997, 158:1133-1138.

9. •

Ten Boekel E, Melchers F, Rolink AG: Changes in the VH gene repertoire of developing precursor B lymphecytes in mouse bone marrow mediated by the pre-B cell receptor. Immunity 1997, 7:357-368. The technology of single-cell PCR was employed to analyze immunoglobulin VH gene usage in B-lineage cells from normal and X5-deficient mice. PreB cells from X5-deficient mice and a rare c-kit+ cytoplasmic I~+ population of pre-B-I cells from normal mice preferentially used D-gene proximal VH genes in V(D)J gene rearrangements. Approximately 50o/0 of the I.¢ heavy chains encoded by these V(D)J rearrangements could physically associate with surrogate light chains and form a pre-BCR. In contrast, D-proximal VH usage was suppressed in productive rearrangements in c-kit- large pre-B-II cells. The authors concluded that the pre-BCR mediates this repertoire shift, although it is unclear whether this shift occurs by a signaling mechanism transduced through the pre-BCR.

10.

BornemannKD, Brewer JW, Perez E, Doerre S, Sita R, Corley RB: Secretion of soluble pre-B cell receptors by pre-B cells. J/mmuno/1997, 158:2551-2557.

11.

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Shaffer AL, Schlissel MS: A truncated heavy chain protein relieves the requirement for surrogate light chains in early B cell development. J Immuno11997, 159:1265-1275. This study approached the problem of the role of the surrogate light chain (SLC) in pre-BCR function by generating two different strains of transgenic mice: one with a truncated rnurine p. transgene missing the heavy chain (HC) domains VH and CHt, as well as half of the CH2 domain; the other with a full-length human IXtransgene. Both strains were bred onto a recombinationactivating gene (RAG)-1-deficient background. The full-length human p. HC protein could not be detected on the surface of RAG-l-deficienUl~-transgenic B-lineage cells, whereas the truncated murine I~ HC protein was readily detected. It was assumed that the full-length human p. HC associated with routine SLC, although direct evidence was not presented. The truncated routine p. HC protein was expressed on the surface without SLC. Remarkably, B-lineage cells expressing truncated routine p. HC underwent developmental changes (i.e. changes in surface markers, transcriptional regulation, and recombinase retargeting) indistinguishable from those occurring in mice expressing full-length human p. HC. A thoughtful discussion considered the ramifications of results obtained in these genetically altered mice, and argued for a chaperone function as the primary role for SLC in the function of the pre-BCR. 12. •,

13. •o

Nagata K, Nakamura T, Kitamura F, Kuramochi S, Taki S, Campbell KS, Karasuyama H: The Ig(~/Igl~ heterodimer on p.-negative proB cells is competent for transducing signals to induce early B cell differentiation. Immunity 1997, 7:559-570. The authors used pro-B cell lines and early B-lineage cells from recombination-activating gene-2-deficient mice to identify stable cell-surface complexes consisting of the Ig-(:dlg-~ signaling heterodimer and the molecular chaperone calnexin. At least some of the Ig-(x/Ig-~ complexes did not contain p. heavy chain (HC) or surrogate light chain (SLC). An in-depth series of experiments confirmed that cross-linking Ig-~ on these cells induced the tyrosine phosphorylation of several intracellular signaling proteins (e.g. Syk, PI-3 kinase, ERK) in vitro and induced pro-B to pre-B cell differentiation in vivo. It remains to be determined, however, whether Ig-cu'lg-~ complexes devoid of p. HC and SLC exist on the surface of pro-B cells in normal mice. 14.

KarasuyarnaH, Rolink A, Melchers F: A complex of glycoprotelns is associated with VpreB/X5 surrogate light chain on the surface of p_ heavy chain-negative early precursor B cell lines. J Exp Med 1993, 178:469-478.

15.

Home MC, Roth PE, DeFranco AL: Assembly of the truncated immunoglobulin heavy chain Dp. into antigen receptor-like complexes in pre-B cells but not in B cells. Immunity 1996, 4:145-158.

16.

Candelas S, Muegge SK, Durum S: IL-7 receptor and VDJ recombination: trophic versus mechanistic actions. Immunity 1997, 6:501-508.

17. •

Kondo M, Akashi K, Domen J, Sugamura K, Weissman IL: Bcl-2 rescues T lymphopoiesis, but not B or NK cell development, in common ¥ chain-deficient mice. Immunity 1997, 7:155-162. This study crossed mice deficient in the common 7 chain subunit of the IL2/-4/-7/-9/-15 receptors with mice expressing a Bcl-2 transgene under the control of the Ep. or H2K promoters. The forced expression of Bcl-2 led to a significant, albeit incomplete, restoration of thymocyte expansion and thymic positive selection, but had no detectable effect on B-cell or natural killer-cell development. These data argue that signaling through, for example, the IL-7 receptor (x chain/¥ chain complex may lead to different survival outcomes in individual lymphoid lineages. 18.

AkashiK, Kondo M, von Freeden-Jeffry U, Murray R, Weissman IL: Bcl-2 rescues T lymphpoiesis in interleukin-7 receptordeficient mice. Cell 1997, 89:1033-1041.

19.

FangW, Mueller DL, Pennell CA, Rivard JJ, Li Y-S, Hardy RR, Schlissel MS, Behrens TW: Frequent aberrant immunoglobulin gene rearrangements in pro-B cells revealed by a bcl-x L transgene. Immunity 1996, 4:291-299.

20. •

YoungF, Mizoguchi E, Bhan AK, AIt FW: Constitutive bcl-2 expression during immunoglobulin heavy chain-promoted B cell differentiation expands novel precursor B cells. Immuni~" 1997, 6:23-33. The potential cooperativity of Bcl-2 and p. heavy chain (HC) in mediating survival, proliferation or differentiation of B cell precursors was examined. The Bcl-2 and p. HC transgenes were introduced individually, or together, into recombination-activating gene-2-deficient mice. Bcl-2 alone promoted expansion, but not differentiation, of pro-B cells (that, somewhat surprisingly, was more marked in spleen than bone marrow). On the other hand, the p.HC

B-cell lymphopoiesis in mouse and man LeBien

transgene promoted pro-B to pre-B differentiation as assessed by acquisition of CD22 and low-level surface p.. This effect was enhanced by co-expression of the Bcl-2 transgene. The pre-B cells in the Bcl-2/l~ HC double transgenics exhibited somewhat of a precocious immature B-cell phenotype, as assessed by unexpectedly high cell-surface levels of CD21, CD22, CD23 and CD40. This interesting Bcl-2/l~ HC double transgenic may have revealed a rare population of transient cells at the pre-B to immature B cell interface. 21.

Tadinton DM, Corcoran LM, Strasser A: Continued differentiation during B lymphopoiesis requires signals in addition to cell survival. Int Immunol 1997, 9:1481-1494.

22.

O'Reilly LA, Harris AW, Tarlinton DM, Corcoran LM, Strasser A: Expression of a bcl-2 transgene reduces proliferation and slows turnover of developing B lymphocytes in vivo. J Immuno/ 1997, 159:2301-2311.

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Von Freeden-Jeffry U, Vieira P, Lucian LA, McNeil T, Burdach SE, Murray R: Lymphopenia in interleukin (IL)-7 gene-deleted mice identifies IL-7 as a nonredundant cytokine. J Exp Med 1995, 181:1519-1526.

24.

Stephan RP, ReUly CR, Witte PL: Impaired ability of bone marrow stromal cells to support B-lymphopoiesis with age. Blood 1998, 91:75-88.

25.

Stephan RP, LilI-Elghanian DA, Witte PL: Development of B cells in aged mice: decline in the ability of pro-B cells to respond to IL-7 but not to other growth factors. J Immunol 1997, 158:15981609. Sudo T, Ito M, Oyawa Y, lizuka M, Kodama H, Kunisoda T, Hayaski SI, Ogawa M, Sakal K, Nishikawa S, Nishikawa Sl: Interleukin 7 production and function in stromal cell-dependent B cell development. J Exp Med 1989, 170:333-338.

26.

27

Jarvis II, LeBien TW: Stimulation of human bone marrow stromal cell tyrosine kinases and IL-6 production by contact with B lymphocytes. J Immuno11995, 155:2359-2368.

28.

Tang J, Nuccie BL, Ritterman I, Liesveld JL, Abboud CN, Ryan DH: TGF-~ down-regulates stromal IL-7 secretion and inhibits proliferation of human B cell precursors. J/mmuno11997,

29.

Pribyl JA, LeBien "I'W: Interleukin 7 independent development of human B cells. Proc Nat/Acad Sci USA 1996, 93:10348-10353.

30.

Billips LG, Nunez CA, Bertrand FE Ill, Stankovic AK, Gartland GL, Burrows PD, Cooper MD: Immunoglobulin recombinase gene activity is modulated reciprocally by interleukin 7 and CD19 in B cell progenitors. J Exp Med 1995, 182:973-982.

31.

Malamed D, Kench JA, Grabstein K, Rolink A, Nemazee D: A functional B cell receptor transgene allows efficient IL-7independent maturation of B cell precursors. J/mmunol 199?, 159:1233-1239.

32.

Dobbeling U: The influence of IL-7 V(D)J recombination. Immunology 1996, 89:569-572.

33.

Namikawa R, Muench MO, De Vries JE, Roncarolo M-G: The Rk2/FIt3 ligand synergizes with interleukin-7 in promoting stromal-cell-independent expansion and differentiation of human fetal pro-B cells in vitro. Blood 1996, 87:1881-1890.

159:117-125.

34. •

Akusaka T, Tsuji K-I, Kawakira H, Kanno M, Harigaya K-I, Hu L, Ebihara Y, Nakahata T, Tetsu O, Taniguchi M, Koseki H: The role of reel-18, a mammalian polycomb group gene, during IL-7dependent proliferation of lymphocyte precursors. Immunity 1997, 7:135-146. This intriguing paper introduces Polycomb group (Pc-G) gene function into the 11.-7proliferation equation. Pc-G genes in Drosophila melanogaster are transcription factors that regulate expression of HOM-C genes, which in turn are induced by segmentation gane products such as Kruppel and hunchback. The outcome of these complex interactions includes the determination of anterior-posterior segment identity in the fly. Vertebrate homologs of Pc-G have been identified, and include reel-18 and bmi-l. Evidence from several laboratories suggested a possible role for bmi-1 in hematopoietic cell proliferation. The current study examined lymphoid growth in mel-18-deficient mice. In addition to skeletal and smooth muscle abnormalities, mel18-deficient mice exhibited defects in B-cell and T-cell lymphopoiasis that resembled the defects which occur in IL-7-deficient mice. IL-7-dependent proliferation of thymocytes and pre-B-cell colony formation were reduced in mel-18-deficient mice; however, analysis of the expression and function of IL-7 signaling pathway molecules (¥ chain, Jak3, STATS) revealed no detectable deficiencies in reel-18-deficient mice. Thus, mel-18 is important in IL-7 dependent proliferation, but acts potentially downstream of, or parallel to, Jak/STAT function. 35. •

Oritani K, Kanakura Y, Aoyama K, Yokota 1, Copeland NG, Gilbert D J, Jenkins NA, Tomiyama Y, Matsuzawa Y, Kincade PW: Matrix

193

glycoprotein SC1/ECM2 augments B lymphopoiesis. Blood 1997, 90:3404-3413. Oritani and Kincade have previously developed a novel cloning strategy that selects for stromal cell molecules that bind to the surface of pre-B cells. This study confirms the utility of that cloning strategy, by demonstrating that secreted and membrane forms of a matrix glycoprotein designated SCI/ECM2 can potentiate the clonogenicity of IL-7-dependent pre-B cells. 36. Nagasawa T, Kikutani H, Kishimoto T: Molecular cloning end structure of a pre-B-cell growth-stimulating factor. Proc Nat/ Acad Sci USA 1994, 91:2305-2309. 37. D'Apuzzo M, Rolink A, Loetscher M, Hoxie JA, Clark-Lewis I, • Melchers F, Baggiolini M, Moser B: The chemokine SDF-1, stromal cell-derived factor 1, attracts early stage B cell precursors via the chemokine receptor CXCR4. Eur J Immuno/ 1997, 27:1788-1793. Freshly isolated B220 + murine bone marrow cells, along with murine and human B cell lines at varying developmental stages, were examined for their capacity to undergo directed migration in response to SDF-f. Within the B220 + population, CD19- (i.e., Hardy fraction A) cells exhibited the strongest chemotactic response to SDF-I. Analysis of human pro-B leukemic, pre-B leukemic and Burkitt lymphoma cell lines revealed both a chemotactic response and a transient rise in intracallular calcium response to SDF-1 in the pro-B and pre-B cells. Importantly, an antibody to the CXCR4 chemokine receptor for SDF-1 blocked migration. This report provides the first evidence that B cell precursors exhibit chemotactic responses to a specific chemokine. 38. Montecino-Rodriguez E, Clark RG, PowelI-Braxton L, Dorshkind K: Primary B cell development is impaired in mice with defects of the pituitary/thyroid axis. J Immunol 1997, 159:2712-2719. 39.

Shimozato T, Kincade PW: Indirect suppression of IL-7responsive B cell precursors by vasoactive intestinal peptide. J /mmuno/1997, 158:5178-5184.

40. •

Miyaura C, Onoe Y, Inada M, Maki K, Ikuta K, Ito M, Suda T: Increased B-lymphopoiesis by interleukin 7 induces bone loss in mice with intact ovarian function: similarity to estrogen deficiency. Proc Nat/Acad Sci USA 1997, 94:9360-9365. Working models of B-cell lymphopoiesis are generally based on a consideration of fetal liver or bone marrow (BM) microenvironment factors alone. However, long range effects mediated by extramedullary endocrine function is also important. Previous studies had demonstrated that estrogen deficiency stimulates B lymphopoiesis, whereas pregnancy (with elevated estrogen in plasma) suppresses B lymphopoiesis. In the current study, the authors found that ovariectomy and accompanying estrogen deficiency led to pronounced bone loss (due to osteoclast-mediated bone resorption) and increased number of B220 + B-cells in BM. Furthermore, IL-7 administration enhanced B-cell lymphopoiesis and stimulated bone loss in female mice. Mice deficient in IL-7-rsceptor exhibited increased trabecular bone volume compared with wild-type littermates. These results identify a previously unrecognized relationship between bone metabolism and lymphopoiesis. B cell precursors or IL-7 could directly or indirectly promote osteoclast activation and bone resorption. It is noteworthy that lymphoid cells can stimulate BM stromal cells to produce IL-6 [25], a potential mediator of bone resorption. 41.

Lu L, Osmond DG: Apoptosis during B lymphopoiesis in mouse bone marrow. J Immuno/1997, 158:5136-5145. rashly isolated murine adult bone marrow B-lineage cells were evaluated for apoptotic characteristics before and after short-term culture. Large B220+/surface IgM- cells that encompassed a transition between nonexpression and expression of cytoplasmic p. exhibited the highest incidence of apoptosis. The population exhibiting the second-highest incidence of apoptosis was immature surface IgM+ IgD- cells. These data are consistent with loss of B-lineage cells via activation of apoptotic pathways in cells that have made nonfunctional H chain rearrangements, and immature B cells undergoing negative selection. A provisional model was presented that included estimates of dally B-lineage cell production and loss (through apoptosis) in vivo, as a function of the stage of B cell development. 42. •

Borghesi LA, Smithson G, Kincade PW: Stromal cell modulation of negative regulatory signals that influence apoptosis and proliferation of B lineage lymphecytes. J Immunol 1997, 159:4171-4179. The objective of this study was to analyze murine stromal cell lines established from various hematopoietic tissues for their capacity to inhibit apoptosis of B cell precursors. Most stromal cells inhibited apoptosis through a mechanism dependent on contact, but not on vascular cell adhesion molecule-I, although one bone marrow (BM) stromal cell line appeared to enhance apoptosis. Lymphoid cell apoptosis induced by glucocorticoids and cytokines (e.g. IL-lo¢) was reduced by stromal cell contact. These results support the hypothesis that interactions between multiple lymphoid cells and stromal cell receptors and ligands probably mediate apoptosis (through activation or inhibition) in the BM microenvironment. 43. Manabe A, Murti GK, Coustan-Smith E, Kumagal M-A, Behm FG, Raimondi SC, Campana D: Adhesion-dependent survival of

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Lymphocyte development

normal and leukemic human B lymphoblasts on bone marrow stromal cells. Blood 1994, 83:758-766. 44.

Tagoh H, Kishi H, Okumura A, Kitagawa T, Nagata T, Mori K, Muraguchi A: Induction of recombination activating gene expression in a human lymphoid progenitor cell line: requirement of two separate signals from stromal cells and cytokines. Blood 1996, 88:4463-4473.

45.

Gaffney PM, Lund J, Miller JS: FLT-3 ligand and marrow stroma derived factors promotes CD3¥, CD3~, CD3~, and RAG-2 gene expression in primary human CD34+ Lin-DR- marrow progenitors. Blood 1998, 91:1662-1670.

JarvisLJ, Maguire JE, LeBian TVV: Contact between human bone marrow stromal cells and B lymphocytes enhances very late antigen-4/vascular cell adhesion molecule-l-independent phosphorylation of focal adhesion kinase, paxillin, and ERK2 in stromal cells. Blood 199'7, 90:1628-1635, This study is an extension of a prior report [25] demonstrating that human VLA-4-deficient lymphoid cells can induce tyrosine kinase activation and IL-6 production by human bone marrow (BM) stromal cells. An experimental strategy was developed that facilitates the detection of specific tyrosine-phosphorylated substrates in stromal cells 'stimulated' by lymphoid cell contact. Lymphoid cell contact induced tyrosine phosphorylation of focal adhesion kinase and paxillin in normal BM stromal cells within five minutes, a time period less than that usually required to achieve a steady-state adhesive interaction between these two cell types. Lymphoid cell contact also induced the tyrosine phosphorylation of ERK2 and the ERK2 substrate Elk1 (a transcription factor). This report demonstrates the feasibility of studying the activation of signaling pathways dependent on tyrosine kinases in stromal cells, and should facilitate a more detailed exploration of the biochemical changes that occur in the nonhematopoietic component of the BM.

54.

Rickert RC, Rajewsky K, Roes J: Impairment of T-cell dependent B-cell responses and B-1 cell development in CD19-deficient mice. Nature 1995, 376:352-355.

55.

Engel P, Zhou L-J, Ord DC, Sato S, Koller B, Tedder TF: Abnormal B cell development, activation, and differentiation in mice that lack or overexpress the CDt9 signal transduction molecule. Immunity 1995, 3:39-50.

56.

Sato S, Steeber DA, Jansen PJ Tedder TF: CD19 expression levels regulate B lymphocyte development: human C D 1 9 restores normal function in mice lacking endogenous CD19. J/mmuno/1997, 158:4662-4669.

57.

Sato S, Miller AS, Howard MC, Tedder TF: Regulation of B lymphocyte development and activation by the CD19/CD21/CD81/Leu 13 complex requires the cytoplasmic domain of CD19. J Immunol 1997, 159:3278-3287.

58.

Li X, Sandoval D, Freeberg L, Carter RH: Role of CD19 tyrosine 391 in synergistic activation of B lymphocytes by coligation of CD19 and membrane Ig. J Immuno/1997, 158:5649-5657.

59.

Kitanaka A, Ito C, Coustan-Smith E, Campana D: CD38 ligation in human B cell progenitors triggers tyrosine phosphorylation of CD19 and association of CD19 with lyn and phosphatidylinositol 3-kinase. ) Immuno/1997, 159:184-192.

60.

Wang W-K, Shah N, O'Brien D, Van Ness B, LeBien TW: Differential induction of DNA-binding activities following C D 1 9 cross-linking in human B-lineage cells. J Immuno11997, 159:5509-5508.

46. •

47.

You-TenKE, Muise ES, Iti~ A, Michaliszyn E, Wagner J, Jothy S, Lapp W S, Tremblay M: Impaired bone marrow microenvironment and immune function in T cell protein tyrosine phosphatsse-deficient mice. J Exp Med 1997, 186:683693.

48. •

Li YS, Wasserman R, Hayakawa K, Hardy RR: Identification of the earliest B lineage stage in mouse bone marrow. Immunity 1996, 5:527-535. Hardy's laboratory used their well-known expertise in multiparameter flow cytometry to provide new insight into the earliest stages of B cell development in the mouse. Antibodies recognizing B220, the stem cell antigen AA4.1, heat stable antigen (HSA or CD24), and CD4 were used to subdivide fraction A cells into three fractions (Ao, A t and A2). These fractions were then sorted and analyzed for expression of genes specific for, or associated with, the B- cell lineage by reverse transcriptase polymerase chain reaction. Fraction Ao cells (CD41owAA4.1+ B220-) expressed low or undetectable levels of germline i~, recombination-activating gene (RAG)-1 , RAG-2, Ig(x or Ig~ gens transcripts. These cells probably represent a heterogeneous population of lymphoid progenitors. Fraction A t (CD4 I°w AA4.1 + B220 +) cells expressed germline ~t and low levels of RAG-l, RAG-2 and Igl~. Fraction A 2 cells (CD4-AA4.1 + B220 +) expressed germline p., RAG-l, RAG-2, Ig(x, Ig~ and X5 gene transcripts. This refinement of the earliest stages of B cell development provides potential opportunities for investigating whether cytokines or stromal cell membrane-associated molecules can activate transcription of germline ~t, RAG-l, RAG-2 and Ig-~ genes. 49.

Galy A, Travis M, Cen Z, Chen B: Human T, B, natural killer and dendritic cells arise from a common bone marrow progenitor cell subset. Immunity 1995, 3:459-473.

50.

Ray RJ, Palge C J, Furlonger C, Lyman SD, Rottapel R: Fit3 ligand supports the differentiation of early B cell progenitors in the presence of interleukin-11 and interleukin-Z Eur J Immuno/ 1996, 26:1504-1510.

51.

Veiby OP, Lyman, SD, Jacobsen SEW: Combined signaling through interleukin-7 receptors and fit3 but not c-kit potently and selectively promotes B-cell commitment and differentiantion from uncommitted murine bone marrow progenitor cells. Blood 1996, 88:1256-1265.

52.

Veiby OP, Borge O J, M.~rtensson A, Beck EX, Schade AE, Grzegorzewski K, Lyman S, M~rtensson IL, Jacobsen SEW: Bidirectional effect of IL-10 on early murine B cell development: Stimulation of flt3-1igand plus IL-7-dependent generation of CD19" proB cells from uncommitted bone marrow progenitor cells, and growth inhibition of CD19 + proB cells. Blood 1997, 90:4321-4331.

53.

Carter RH, Fearon DT: CD19: lowering the threshold for antigen receptor stimulation of B lymphocytes. Science 1992, 256:105-107.

61. •

Bertrand FE, Billips LG, Burrows PD, Gartland GL, Kubagawa H, Schroeder HW: Ig DH gene segment transcription and rearrangement before surface expression of the pan-B-cell marker CD19 in normal human bone marrow. Blood 1997, 90:?36-?44. The authors examined the onset of immunoglobulin gene transcription and rearrangement in fluorescence-activated cell sorter-purified CD34 + CD19and CD34 + CD19 + human fetal and adult bone marrow cells. Germline ~. gene and germline DHQ52 transcripts were detected in CD34+CD19- cells isolated from fetal and adult bone marrow. DJ gene rearrangements involving DHQ52 were found in CD34 + CD19- fetal cells only. V(D)J rearrangements between VH3 or VH6 families were present only in CD34 +CD19 + pro-B cells. These results convincingly show that CD19 surface expression occurs subsequent to the initial stages of immunoglobulin gane rearrangement/expression. Hence, CD19 expression is probably an indication or manifestation of B-lineage commitment, rather than a mediator of commitment. 62.

RyanDH, Nuccie BL, Ritterman I, Liesveld JL, Abboud CN, Insel RA: Expression of interleukin-7 receptor by lineage-negative human bone marrow progenitors with enhanced lymphoid proliferative potential and B-Uneage differentiation capacity. Blood 1997, 89:929-940. A population of CD34 + CD19- cells expressing the IL-7 receptor (IL-7R)-c¢ chain were purified by fluorescence-activated cell sorting, and examined for expression of genes involved in early lymphoid cell development and clonogenic capacity in short-term culture. The CD34 + CD19-IL-?Rc¢+ cells were uniformly terminal deoxynucleotidyl transferase+, expressed the Ig-~ signaling molecule, recombination-activating gene-1 products and PAX-5 (a B-cell specific transcription factor critical to early B cell development) mRNAs, and differentiated into pro-B cells, in contrast, CD34 + CD19-IL-?Rct- cells had greatly reduced clonogenic capacity and failed to differentiate into pro-B cells. These results suggest a potential role for signaling through the IL-TR in promoting the entry of lymphoid progenitors into the B-cell lineage. These CD34 + CD19- IL-?R(~+ cells formed colonies expressing CD19, indicating a precursor-progeny relationship between CD19- and CD19 + cells in this culture system. •

63.

Davi F, Faili A, Gritti C, Blanc C, Laurent C, Sutton L, Schmitt C, Merle-Beral H: Early onset of immunoglobulin heavy chain gene rearrangements in normal human bone marrow CD34 + cells. Blood 1997, 90:4014-4021. Similar to the data reported in [61 "], this group detected DJ gene rearrangements in CD34+CD19 - human bone marrow cells and V(D)J gene rearrrangements in CD34 + CD19 + pro-B cells. One difference is that the current study reported DJ rearrangements in adult bone marrow CD34 + CD19cells, whereas the authors of [61"] did not. This might be explained by the fact that further sorting in the current study revealed that DJ rearrangements were detected in CD34 + CD10 + CD19- cells, but not CD34 + CD10" CD19cells. 64. Ghia P, ten Boekel E, Sanz E, de la Hera A, Rolink A, Melchers • F: Ordering of human bone marrow B lymphocyte precursors by single-cell polymerese chain reaction analyses of the rearrangement status of the immunoglobulin H and L chain gene loci. J Exp Med 1997, 184:2217-2229. A comprehensive analysis of gene expression in human CD19 + bone marrow B-lineage cells was accomplished using a combination of multiparameter •

B-cell lymphopoiesis in mouse and man LeBien

analysis/cell sorting and single cell reverse transcriptase PCR. Monoclonal antibodies made against human Vpre-B were crucial reagents in this study. These antibodies detected Vpre-B epitopes (Vpre-B and ~.5 make up the surrogate light chain of the pre-BCR that is essential for B cell development) on CD34 + and CD34- B-lineage cells. DJ gene rearrangements were first detected in CD34+CD19 + pro-B cells-V(D)J rearrangements were not analyzed- and VLJL gene rearrangements were first detected in large CD34-VpreB- cells. Ghia and colleagues quite convincingly demonstrated the existence of three subpopulations of cytoplasmic p_+ pre-B cells, based on staining by anti-Vpre-B, cell cycle analysis, and VLJL gene rearrangements. The authors ordered these three populations into Vpre-B+/large pre-B II, Vpre-B-/large pre-B II and Vpre-B-/small pre-B I1. Definitive placement of these three cytoplasmic i~+ populations into a developmental hierarchy will necessitate purification of cells by fluorescence-activated cell sorters and in vitro assays that support differentiation of the three pre-B populations. 65.

Berardi AC, Meffre E, Pflumio F, Katz A, Vainchenker W, Schiff C, Coulombel L: Individual CD34+CD381owcD19-CD10 progenitor

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cells from human cord blood generate B lymphocytes and granulocytes. B/ood 1997, 90:3554-3564. 66.

Rawlings DJ, Quan SG, Kato RM, Witte ON: Long-term culture system for selective growth of human B-cell progenitors. Proc Nat/Acad Sci USA 1995, 92:1570-1574.

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Kraj P, Rao SP, Glas AM, Hardy RR, Milner ECB, Silberstein LE: The human heavy chain Ig V region gene repertoire is biased at all stages of B cell ontogeny, including early pre-B cells. J Immunol 1997, 158:5824-5832,

68.

Marshall AJ, Paige C J, Wu GE: V(H) repertoire maturation during B cell development in vitro: differential selection of Ig heavy chains by fetal and adult B cell progenitors. J Immuno/ 1997, 158:4282-4291.