An antibody VH gene that promotes marginal zone B cell development and heavy chain allelic inclusion
Lynn Heltemes-Harris1,*,
Xiaohe Liu* and
Tim Manser
Department of Microbiology and Immunology and Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA, USA
1 Present address: Laboratory of Immunology, National Institute on Aging, National Institutes of Health, Baltimore, MD, USA
Correspondence to: T. Manser; E-mail: manser{at}mail.jci.tju.edu
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Abstract
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The Ig heavy (H) chain plays a pivotal role in the regulation of primary B cell development through its association with a variety of other proteins including Ig
and Igß, the surrogate light chain components and bona fide L chains, to form transmembrane signaling complexes. Little is known about how alterations in the structure of the H chain variable region influence association with these proteins, or the signaling capacity of the complexes that form. Here we describe a line of VH knockin mice in which the transgene-encoded VH region differs by eight amino acid residues from the VH region in a VH knockin line we previously constructed and characterized. The transgenic H chain locus in the line of mice we characterized earlier efficiently promotes H chain allelic exclusion and all phases of primary B cell development, resulting in the generation of mature B1, marginal zone (MZ) and follicular (FO) B cell compartments. In contrast, the transgenic H chain locus in the new line fails to enforce allelic exclusion, as evidenced by the majority of peripheral B cells expressing two H chains on their surfaces. Moreover, this locus inefficiently drives bone marrow B lymphopoiesis and FO B cell development. However, this H chain locus does promote MZ B cell development, from precursors that appear to be generated during fetal and neonatal life. We discuss these data in the context of previous findings on the influence of Ig H chain structure on primary B cell development.
Keywords: antibody heavy chain, B cell, development, transgenic mice
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Introduction
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Mouse B lymphopoiesis takes place throughout life. Fetal and neonatal development occurs in the omentum, liver and then spleen. After birth, the major site of B lymphopoiesis shifts to the bone marrow (BM) and continuous production of B cells from this primary organ continues during adult life (14). The peripheral lymphoid organs and sites are seeded by several distinct types of mature B cells including B1, marginal zone (MZ) and follicular (FO) B cells. If adult B lymphopoiesis is blocked by inactivation of the Rag-1 gene at birth, or due to an IL-7 deficiency, B1 and MZ, but not FO, B cells stably populate the periphery (5, 6). These results are in accord with numerous studies indicating that much of the murine FO B cell compartment is derived from adult lymphopoiesis in the BM, while a major phase of B1 B cell development takes place in the fetus (15). By comparison, additional data regarding the developmental origin of MZ B cells are limited and often contradictory (7, 8).
Ig heavy (H) chains play critical roles in controlling multiple stages of B cell development. Mice deficient in the membrane form of the µ H chain lack mature B cells due to a developmental block at the pre-B cell stage (9). Association of the H chain with the Ig
and Igß signaling co-receptors results in formation of a complex whose function is absolutely required for normal developmental progression (10, 11). Moreover, the VH region of this complex also can associate with the surrogate light chain (SLC) components
5 and V-pre-B, creating the pre-BCR (12). One function of the pre-BCR appears to be to promote the proliferative stage of adult pre-B cell development (13).
During the pre-B cell phase of development, most B cells become committed to the expression of only one of the two available antibody H chain loci, a phenomenon termed allelic exclusion (14). While the mechanism responsible for H chain allelic exclusion remains incompletely understood, and has been debated for years (1517), it seems clear that several factors contribute to this end result. These include the low probability of productive rearrangement of the two H chain loci in a single cell (15), and the action of feedback and feed forward developmental mechanisms that regulate the recombination and transcriptional activity of the Igh loci and are triggered by the expression of H chain protein (17). The discovery of the pre-BCR prompted speculation that it was this form of the µ H chain that mediates H chain allelic exclusion (18). However, recent data obtained from mice deficient in all conventional components of the SLC complex suggest that expression of a functional pre-BCR is not required for this process (19). While it has been shown that surface expression of two transgenic H chains is compatible with the development of mature B cells (20), the frequency of B cells in normal mice that express both Igh alleles in the form of BCRs is extremely low (21), despite the fact that pre-B cells containing two productively rearranged Igh loci are readily detected in normal mice (22).
The structural and functional components of the Ig H chain necessary for promoting primary B cell development have been extensively investigated. Recent studies in which chimeric forms of the Ig
and Igß co-receptors (23), or surrogate receptors that signal in a similar fashion (24), have been shown to promote many phases of this development have indicated that the most critical role of the H chain is to allow expression of these co-receptors in the form of an integral membrane-signaling complex. Given these findings, previous studies where expression of altered or unnatural forms of the H chain was found to fail to recapitulate the role of the µ H chain in primary development (reviewed in 25) can be explained by the inability of these H chains to nucleate the assembly of functional pre-BCR- or BCR-signaling complexes.
In this regard, studies on the expression of the VH81X gene segment during B cell development first suggested that not all VH structures are compatible with efficient adult B lymphopoiesis. This variable (V) gene is frequently expressed among fetal B cells and adult pre-B cells but is grossly under-represented in the adult peripheral B cell pool (26, 27). Subsequent biochemical studies demonstrated that several VH81X H chains associate poorly, if at all with the SLC components (28). More recent studies have shown that VH81X is not idiosyncratic in this regard. H chains with VH regions derived from members of the small VH11 and VH12 families also promote fetal, but not adult, B lymphopoiesis and do not, in general, appear to associate well with the SLC components (29, 30). These results suggest that the regulatory influences of the pre-BCR differ during adult and fetal/neonatal B lymphopoiesis (29).
We have described a line of VH knockin mice, termed HKI65, that express a transgene-encoded VH region derived from a B cell clonotype that dominates the immune response of A/J mice to the hapten arsonate (Ars). This VH gene includes a J558 family VH segment that lacks somatic mutations, and is thus representative of the form of this V gene expressed by primary B cells in normal mice. The HKI65 knockin locus promotes efficient development of B1, MZ and FO subsets and H chain allelic exclusion (31). Here, were report the construction and analysis of a new line of VH knockin mice, termed HKI71, containing a VH gene that differs from the HKI65 VH gene at only eight amino acid positions. Six of these amino acid substitutions are the result of somatic hypermutation during an immune response to Ars and two appear to be due to junctional diversity. Strikingly, the majority of adult splenic B cells in mice hemizygous for the HKI71 locus expresses surface IgM (sIgM) encoded by both HKI71 and the endogenous Igh loci, that is these B cells are H chain allelically included. Moreover, mice capable of expressing only the HKI71 locus contain predominantly MZ B cells and many of these MZ cells appear to be derived from precursors generated during fetal and neonatal life. These data have important implications for our understanding of the structure of VH domains compatible with allelic exclusion and fetal and neonatal versus adult B lymphopoiesis, as well as the developmental origin of MZ B cells.
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Methods
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Mice
Construction of the HKI65 line of mice has been described previously (31, 32). The HKI71 line was generated in an identical fashion (see Fig. 1) and was progressively bred from a mixed 129/C57BL/6 background to a C57BL/6 background. These mice were crossed to JHD (C.B17 background), µMT (C57BL/6 background) or V
1060 (C57BL/6 background) transgenic mice, and the H chain and L chain genotypes of offspring evaluated by PCR of DNA obtained from tail clips. The V
1060 line was constructed as described (33) and contains a conventional transgenic array encoding a V
10A-J
1 kappa light (L) chain. Mice were housed in a barrier rodent facility in micro-isolator cages and were given autoclaved food and water. In most experiments, adult mice of 812 weeks of age and of both sexes were used. For the analysis of neonatal mice, timed matings were set up and time of conception was assayed by vaginal plugs. Seven days after birth, mice were sacrificed and livers and spleens were removed for analysis. Tail clips were also taken for retrospective genotyping by PCR. All experiments using mice were approved by the Institutional Animal Care and Use Committee.

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Fig. 1. The HKI71 knockin locus. Panel A: the structure of the HKI71 VH knockin targeting vector and the recombination pathways leading to alteration of the endogenous Igh locus and subsequent deletion of the neomycin resistance gene are illustrated. The transcriptional orientation of the neomycin resistance (NeoR) gene is indicated by an arrow; IE = intronic enhancer, DT = diphtheria toxin gene. Panel B: primary structures of the HKI65- and HKI71 VH-coding regions. The location and type of amino acid changes that distinguish the two VH regions are shown using the one-letter code (HKI65 amino acids indicated first). Amino acid numbering is from the mature amino terminus of the H chain.
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Flow cytometry
Single-cell suspensions were prepared from lymphoid organs and peritoneal lavages and immunostained following protocols described before (31, 32). Mild acid stripping of splenocytes was performed according to Jimenez et al. (34). Combinations of the following antibodies (FITC, PE or biotin conjugates unless otherwise noted) were used: anti-IgM (Jackson ImmunoResearch Laboratories, West Grove, PA, USA), anti-IgD (1126) (Southern Biotechnology Associates, Birmingham, AL, USA), AA4.1-PE (eBioscience, San Diego, CA, USA), anti-IgMa (DS-1), anti-IgMb (AF6-78), anti-B220 (6B2), anti-CD1d (1B1), anti-CD5 (53-7.3), anti-CD19 (1D3), anti-CD21/35 (7G6), anti-CD23 (B3B4), anti-CD24 (HSA, M1/69, 30-F1), anti-CD43 (S7), anti-CD45R (RA3-6B2; eBioscience) or anti-idiotypic mAb E4-biotin (prepared in-house). All antibodies were obtained from BD PharMingen (San Diego, CA, USA) unless otherwise indicated. SACyChrome or SAPerCP Cy5.5 (BD PharMingen) was used to detect biotinylated antibodies. Cells were assayed on an EPICS Elite (Coulter, Hialeah, FL, USA) or a FACSCalibur (BD Immunocytometry Systems, San Jose, CA, USA), and data were analyzed using FlowJo software (Treestar, San Carlos, CA, USA).
Immunohistochemistry and immunofluorescence
Spleens from 8- to 12-week old naive mice were frozen, and cryosections were prepared and processed as previously described (35). Sections were stained with combinations of various mAbs (described above), as well as anti-MOMA-1FITC (SeroTech, Raleigh, NC, USA), analyzed on a fluorescence microscope and digital images were captured. For confocal microscopy, dissociated splenocytes were adjusted to 5 x 105 cells ml1 and placed on glass slides by cytospin centrifugation at 80 x g min. After fixation with 4% PFA in PBS, the slides were washed with PBS and blocked with Tris-buffered saline/BSA/Tween 20, rewashed and stained with IgMaPE and IgMbFITC in a moist chamber for 1 h at room temperature. After extensive washing, slides were imaged with a confocal laser-scanning microscope (Zeiss 510 Meta).
Autoreconstitution
Adult mice were exposed to a sub-lethal dose of whole-body gamma irradiation (500 or 550 rad) as described before (36) and were allowed to rest for 3 or more weeks. Cells were then obtained from lymphoid organs, labeled and analyzed by flow cytometry as described above.
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Results
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The methods used to generate the VH knockin locus in the HKI71 mouse line were identical to those used to generate the HKI65 knockin locus (31, 32) and are illustrated in Fig. 1(A). As a result, the HKI71 line differs from the HKI65 line only in the structure of its knocked in VH gene. The location and type of amino acid differences that distinguish these two VH genes are illustrated in Fig. 1(B).
Transgenic H chain expression and allelic exclusion in the knockin mice
BM and spleen cells from HKI65 and HKI71 adult (812 week old) transgene hemizygous mice, as well as non-transgenic littermates were stained with anti-B220 in combination with allotype-specific anti-IgM reagents to evaluate transgene expression and allelic exclusion. Figure 2(A) shows that, as previously reported (31), whereas the majority of HKI65 BM B cells expresses the knockin H chain allotype (IgMa), approximately half of the splenic B cells in these mice express an endogenous (IgMb) H chain. This phenomenon has been observed in several other VH knockin mice (31, 37, 38) and appears to result predominantly from the inactivation of the knockin locus by Rag-mediated recombination events, allowing subsequent rearrangement of the endogenous locus (37). Despite this, H chain allelic exclusion is maintained among HKI65 B cells, as individual BM or splenic B cells expressing both sIgMa and sIgMb are not apparent.

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Fig. 2. sIgM expression and allelic exclusion and inclusion in HKI71 and HKI65 hemizygous mice. Panel A: BM and spleen cells were isolated from the indicated mice (pooled from two animals of each genotype, littermates were B6 x Balb/c F1s), stained with anti-B220 and allotype-specific anti-IgM mAbs and cells analyzed by flow cytometry. Levels of expression of IgMa and IgMb in the B220+ lymphocyte gate are shown. The HKI knockin loci encode IgMa antibodies. Gates were drawn to include the major sub-populations of sIgMa-, sIgMb- and sIgMa/sIgMb-expressing lymphocytes, and the values next to each gate indicate the percentage of cells in that gate relative to the total number of B220+ cells. Panel B: spleen cells (pooled from two mice of each genotype) from HKI65 (upper plot) and HKI71 mice (lower plot) hemizygous for HKI knockin loci from which the neomycin resistance gene had been removed were analyzed as in Panel A, but anti-CD19 was used to elaborated B cells. Panel C: spleen cells from HKI71 and HKI65 hemizygous mice lacking the neomycin resistance gene were stained with the indicated anti-IgM allotype reagents and analyzed by confocal microscopy. In the left panels, white arrows indicate cells that were stained with both reagents. All data are representative of multiple experiments.
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Analysis of the BM of HKI71 hemizygous (HKI71/+) mice showed that the B cell compartment was heterogeneous, consisting of a minor sub-population expressing sIgMa, and major sub-populations expressing both sIgMa and sIgMb, or sIgMb only (Fig. 2A). In spleen, three major sub-populations of B cells were observed, one that expressed moderate levels of sIgMb and high levels of sIgMa, one that expressed roughly equal levels of these two alleles and one that expressed sIgMb only. These data suggest that as in HKI65 mice, inactivation of the HKI71 knockin locus allows a sub-population of sIgMb-expressing B cells to develop but, in contrast to the situation in HKI65 mice, many peripheral B cells display surface expression of both the transgenic-encoded H chain and a H chain encoded by the endogenous Igh locus. Similar results were obtained after excluding cellular aggregates from the analysis using forward-scatter area versus pulse width parameter gating. Moreover, mild acid treatment of spleen cells to remove cytophilic antibody before staining and flow cytometric analysis did not alter the results obtained (data not shown).
As the neomycin resistance (Neo) gene had not been removed from the HKI65 and HKI71 Igh loci, we considered that this gene was affecting allelic exclusion by these loci. To test this idea, we deleted this gene by crossing to a Cre-expressing transgenic line (see Fig. 1), and repeated the above analyses. Figure 2(B) shows that this resulted in a reduction in the frequency of sIgMb-expressing B cells in both adult HKI65 and HKI71 spleens. Thus, the Neo gene promotes the development of B cells expressing the endogenous H chain allele. This could result from an inhibitory effect of expression of this gene on transcription levels of the adjacent H chain gene due to promoter competition (39, 40). Also, transcription of the Neo gene might enhance the frequency of Rag-mediated events that inactivate the knockin locus by keeping the upstream D region in an open chromatin conformation (41, 42). Nonetheless, the majority of splenic B cells in HKI71Neo hemizygous mice expresses both sIgMa and sIgMb. To corroborate this finding, spleen cryosections and cell suspensions from HKI71 and HKI65 mice were stained with anti-IgMa and anti-IgMb reagents and analyzed by fluorescence microscopy or confocal microscopy, respectively. These analyses revealed a high frequency of cells with fluorescence emissions consistent with staining by both anti-IgM reagents in HKI71, but not HKI65 mice (results from cell suspension staining shown in Fig. 2C). HKI71Neo mice were also found to be mildly B lymphopenic, displaying
40% and 3-fold reductions in B cell numbers in spleen and LN, respectively (data not shown).
Further analysis of the subset distribution of the peripheral B cells in HKI71 mice lacking the Neo gene showed that splenic sIgMa-high/sIgMb-low cells predominantly resided in an MZ phenotypic compartment by virtue of the fact that they were sIgMhigh, sIgDlow, CD21high, CD23low, CD24int and CD1d+, while the splenic FO compartment (sIgDhigh, CD21low, CD23high, CD24low, CD1d) was enriched in cells expressing roughly equal amounts of sIgMa and sIgMb. In contrast, sIgMa- and sIgMb-expressing B cells were similarly represented in the MZ and FO compartments of HKI65 mice (Fig. 3A and data not shown). In the peritoneal cavity, CD5+ B1a cells expressed either both sIgMa and sIgMb or sIgMb only in HKI71 mice, and either sIgMa or sIgMb in HKI65 mice (Fig. 3B).

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Fig. 3. Mature B cell subset representation in H chain allelically excluded and included B cell sub-populations in HKI71Neo and HKI65Neo hemizygous mice. Panel A: spleen cells (pooled from two mice of each genotype) were stained with anti-IgM allotype as well as anti-CD21/35 and CD23 mAbs and analyzed by four-color flow cytometry. Primary gates were set around the major sIgM allotype-expressing sub-populations (left plots), and levels of CD21 and CD23 were evaluated on the cells in each gate (indicated with dashed arrows). Panel B: peritoneal lavage cells from the indicated mice were stained with anti-CD5 and anti-IgM allotype mAbs and analyzed by three-color flow cytometry. Percentages of CD5+ cells in the sIgM+ quadrants are indicated. Results are representative of at least two independent experiments.
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The splenic B cell compartment of HKI71 mice lacking a functional endogenous H chain locus is composed predominantly of MZ B cells
The above data suggested that the HKI71 H chain preferentially promotes development to MZ phenotype and, thus, generation of FO B cells in HKI71 mice requires the co-expression or exclusive expression of BCRs encoded by the endogenous Igmb locus. To examine this possibility, HKI71 mice were crossed to mice with targeted alterations of the H chain locus (µMT or JHD) (9, 43) that preclude the expression of endogenous µ H chains, and splenic and LN B cell compartments in the resulting mice were analyzed by flow cytometry. Figure 4(A) shows that the majority of splenic B cells in HKI71/JHD mice has an MZ cell-surface phenotype. Analogous results were obtained from HKI71/µMT mice (data not shown). In addition, B cell numbers in these mice were moderately (
2-fold) and dramatically (
10-fold) reduced in the spleen and LNs, respectively. As previously reported (31), near-normal numbers of both MZ and FO B cells were found to be present in the spleens, and FO B cells in the LNs of HKI65/JHD(µMT) mice (data not shown).

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Fig. 4. Splenic B cells in HKI71/JHD mice are predominantly of MZ phenotype and locale. Panel A: splenic B cells from the indicated mice (pooled from two mice of each genotype) were stained with anti-B220 and combinations of the two other indicated reagents and analyzed by three-color flow cytometry. The numbers in various quadrants or next to gates indicate the percentage of the B220+ cells in that quadrant or gate. Panel B: splenic cryosections from the indicated mice were stained with mAbs specific for the indicated markers and analyzed by fluorescence microscopy. Magnification of original images was x25. The data are representative of multiple experiments.
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To corroborate these flow cytometric studies, immunohistology was performed on the spleens of adult HKI65/JHD and HKI71/JHD mice. Figure 4(B) shows that HKI65/JHD spleens exhibited an expected white pulp substructure with two major B cell areas: an MZ area outside of the ring of MOMA-1+ metallophiles and an FO area, inside (relative to the position of T cell areas and central arterioles) this ring (data not shown). Staining with anti-IgM, anti-IgD and anti-CD1d indicated that B cells in the MZ areas were sIgMhigh, sIgDlow and CD1d+ whereas B cells in the FO areas were sIgMlow, sIgDhigh, CD1d. In marked contrast, whereas large B cell regions were found in HKI71/JHD spleens at locations expected of follicles, the majority of B cells in these areas was located outside of the MOMA-1+ ring, indicating a substantial expansion of the MZ. As in HKI65/JHD mice, B cells in these MZ areas were sIgMhigh, sIgDlow, CD1d+. Unlike in HKI65/JHD mice, however, the B cells in the follicle-like areas of HKI71/JHD mice were CD1d+.
The HKI71 H chain locus inefficiently promotes adult BM B lymphopoiesis
As previous studies have shown that MZ B cells can develop and be maintained in the periphery under conditions in which adult BM B cell development is blocked (3, 4), we next addressed whether the HKI71 H chain locus could promote adult BM B cell development. Total B cell numbers in the BM of HKI71/JHD, but not HKI65/JHD, mice were severely reduced relative to littermate mice (>5-fold, data not shown). In addition, whereas relative numbers of the B220low, CD43+ (pro-B/pre-BI) sub-population were fairly normal in HKI71/JHD mice, there was a 2-fold reduction in the B220low, CD43 (pre-BII/immature B) sub-population, and an even more severe reduction in the B220high, CD43 (mature, recirculating) sub-population (Fig. 5). More strikingly, the frequency of sIgM+ B cells in the B220low/CD43 (immature B cell) fraction of HKI71/JHD BM was substantially reduced. We previously found that in HKI65/JHD mice, all the various stages of BM B cell development are well represented (31). Less detailed analysis of the splenic transitional B cell compartment using the mAb AA4.1 revealed a >10-fold reduction of AA4.1+ splenic B cells in the HKI71/JHD mice as compared with HKI65/JHD and non-transgenic mice as well (data not shown).

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Fig. 5. Inefficient late adult BM B cell development in HKI71/JHD mice. BM cells were isolated from the indicated mice, stained with anti-B220, anti-CD43 (S7) and anti-IgM and analyzed by three-color flow cytometry. Gates were set around the major B cell developmental sub-populations (left plots) according to Hardy and colleagues (2); pro- and pre-B fractions, AC, right gates; pre-B and immature B fractions, D and E, lower left gates and mature B cell fractions F, upper left gates, and sIgM levels in the more mature (CD43) sub-populations evaluated (right plots). In the right plots, the percentages of cells in the CD43 gates illustrated in the left plots that have phenotypes characteristic of late pre-B (B220low, CD43, sIgM), mature (B220high, CD43, sIgM+) and immature (B220low, CD43, sIgM+) B cells are indicated. The results shown are from individual mice but are representative of three independent experiments.
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B cell development in neonatal mice
Taken together, the data above suggested that the MZ B cell population that predominates in the spleens of HKI71/JHD(µMT) mice might be derived from fetal B lymphopoiesis. To test this idea, we analyzed the B cell compartment in day 7 neonatal liver and spleen from HKI71/JHD mice by flow cytometry. HKI71/JHD neonatal liver contains large percentages of B220+ and sIgM+ lymphocytes, and sIgM+ lymphocytes are only
2-fold reduced in number as compared with controls (Fig. 6). Interestingly, however, these cells generally express lower levels of B220 and higher levels of CD5 as compared with control neonatal B cells. In the neonatal spleen, the number of B220+ cells is reduced in HKI71/JHD mice, but as in liver substantial numbers of sIgM+ cells are present that also express low levels of CD5. In both neonatal liver and spleen, B cells expressing high levels of CD21 and low levels of CD23 or high levels of CD1d (data not shown) could not be detected, indicating that mature MZ B cells had not yet developed. This is, perhaps, not surprising, as the MZ of the spleen does not physically arise until more than 2 weeks after birth (7, 8).

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Fig. 6. B lymphopoiesis in neonatal HKI71/JHD mice. Liver and spleen cells were isolated from the indicated mice 7 days after birth, stained with anti-B220 and mAbs specific for the other indicated markers and analyzed by three-color flow cytometry. The left histograms show levels of B220 expression on cells in the lymphocyte gate. The right plots show the levels of staining for the indicated markers on B220+ lymphocytes. Data are from individual mice but are representative of those obtained from four mice of each genotype.
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B lymphopoiesis in adult autoreconstituting mice
To further test the possibility that the MZ B cells that populate the spleen of HKI71/JHD(µMT) mice might be derived from fetal B lymphopoiesis, an autoreconstitution approach was taken. In adult mice, given a sub-lethal dose of whole-body irradiation, mature B cells originating from both fetal and adult lymphopoiesis are ablated, and the B cell compartment is reconstituted over a period of weeks by radiation-resistant hematopoietic stem cells in the BM (36).
In one experiment, HKI71/JHD and non-transgenic littermate mice were given 500 rad whole-body irradiation, and allowed to autoreconstitute for 3 weeks. Splenic B cell populations were then analyzed by flow cytometry. Figure 7 (upper panels) shows that in control mice total splenic B cell numbers and subset distributions had returned to normal. In contrast, HKI71/JHD mice displayed severe splenic mature B lymphopenia. The majority of the B cells in the spleen at this time was B220low, HSAhigh, sIgM, presumably pre-B cells from the BM. More strikingly, of the few mature B cells that were present in the spleens of these mice at this time, most were of the FO phenotype.

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Fig. 7. Poor autoreconstitution of the mature and MZ B cell compartments of HKI71/JHD and µMT mice. Mice of the indicated genotypes were given the indicated doses of whole-body irradiation, allowed to autoreconstitute for the indicated times and then spleen cells were isolated, stained with mAbs specific for the indicated markers and analyzed by flow cytometry. The level of mature (B220high) B cell lymphopenia in the autoreconstituting mice are illustrated in the left histograms. The upper and lower groups of panels present data from two different experiments (see text). The data are from individual mice but are representative of those obtained from two mice of each genotype.
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In a second experiment, HKI71/µMT mice were given 550 rad and allowed to reconstitute for 7 weeks to determine if slow reconstitution due to uncharacterized transgene effects might be influencing the results. At this time after irradiation, HKI71/µMT mice were still mature B lymphopenic, but less so than had been observed in the previous experiment. Nonetheless, a predominant MZ compartment was not reconstituted in these mice. Collectively, these data show that when only adult B lymphopoiesis is operative, the large MZ B cell compartment characteristic of HKI71/JHD(µMT) mice does not develop.
The HKI71 locus can promote adult BM B lymphopoiesis when co-expressing with a transgenic kappa L chain
We next evaluated whether the inability of the HKI71 locus to promote H chain allelic exclusion and adult B cell development was due to inappropriate levels of H chain expression or generic toxic effects of the HKI71 H chain. HKI71 mice were crossed to a line of conventional transgenic mice expressing a V
10A-J
1 L chain, known to pair with the 36-71 H chain and promote efficient antibody expression in hybridoma cell lines (44). The BM and splenic B cells of the resulting double-transgenic mice were then analyzed for H chain allelic exclusion and development of various B cell stages and peripheral subsets, as described above.
Figure 8(A) illustrates that in contrast to HKI71 mice, the HKI71/V
10A double-transgenic mice (both loci hemizygous) contain splenic B cell compartments in which the majority of B cells expresses only the transgenic H chain locus. This compartment is comprised of two sub-populations, expressing high and low levels of sIgM. Analysis of the sIgMlow sub-population showed it to be sIgDhigh, CD21low, CD23high and CD1d, a phenotype consistent with FO B cells (data not shown). Use of the anti-clonotypic mAb E4 that detects V regions composed of the HKI71VH-V
10A-J
1 combination (45) revealed that essentially all E4+ B cells expressed only the transgenic H chain and were of the FO phenotype. Further flow cytometric analysis of the sIgMhigh sub-population of B220+ cells showed that it contains cells of the MZ phenotype (sIgDlow, CD21high, CD23low, CD24int and CD1d+, data not shown). Therefore, in these mice B cells expressing both the transgenic H and L chains predominantly reside in the FO compartment, whereas B cells expressing the transgenic H chain and, presumably, an endogenous L chain, reside in the MZ compartment.
Analysis of B cell compartments in the BM of HKI71/V
10A double-transgenic mice, as described above, revealed all expected precursor sub-populations, with a 2-fold increase in the size of the pro-B/pre-BI sub-population (Fig. 8B). Similar results were obtained from spleen and BM of HKI71/µMT/Vk10A mice (data not shown), supporting the idea that L chain editing allows the MZ compartment to form, and this may explain the expansion of the pro-B/pre-BI compartment in HKI71/V
10A BM. These data demonstrate that the HKI71 knockin locus can drive relatively efficient BM B cell development when co-expressed with the V
10A transgene.
To evaluate the developmental origins of the two major splenic sub-populations of B cells in HKI71/V
10A(µMT) mice, the autoreconstitution protocol described above was employed. Figure 9 shows that 7 weeks after receiving 550 rad whole-body irradiation, the mature splenic B cell compartment was well reconstituted; however, reduction in the MZ compartment was evident. Analyses using the E4 mAb indicated that most, if not all, E4+ B cells were resident in the FO compartment (data not shown). This indicates that, as in un-irradiated HKI71/V
10A(µMT) mice, B cells expressing endogenous L chain genes had given rise to the MZ compartment.

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Fig. 9. Efficient autoreconstitution of adult B lymphopoiesis in HKI71/V 10A double-transgenic mice. Mice of the indicated genotypes were given the indicated doses of radiation, rested and spleen cells were analyzed by flow cytometry as described in Fig. 7 (cells pooled from two mice of each genotype). The upper plots illustrate the lack of mature B lymphopenia in the autoreconstituting mice. The data are representative of two experiments.
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Discussion
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Expression of a functional Ig H chain is absolutely required for most stages of B cell development, as well as for the maintenance of immature and mature B cell viability. How structural differences in the VH region may influence the diverse assortment of activities required of the H chain protein in this capacity are just beginning to be appreciated, however. Two such functions appear particularly important at present: the ability to pair with SLC components, resulting in the formation of a signaling-sufficient pre-BCR (12, 13, 30), and influence on BCR specificity for autoantigens that may mediate positive selection of immature B cells into the long-lived peripheral pool (30, 4650). The other major, and better-understood effect of the H chain on B cell development, peripheral stability and activity takes place via the operation of tolerance pathways. The mechanistic interrelationship of negative selection and positive selection of B cells by autoantigens is a current subject of debate (51, 52). Nonetheless, it is generally believed that the nature of antigen receptorautoantigen interactions that mediate these two processes must be quantitatively and perhaps qualitatively distinct, and may also differ during fetal and adult B lymphopoiesis (4653).
Superimposed on this complexity is the influence of homeostatic pathways that function to maintain a relative constant number of each class of lymphocyte in the periphery under steady-state conditions (54). The most important such pathway for MZ and FO B cells seems to be mediated by the tumor necrosis factor family cytokine BAFF (55). Moreover, it has been reported that autoreactive, anergic B cells are more reliant on BAFF for peripheral survival than non-autoreactive B cells (56, 57), indicating that BCR- and BAFF-R-signaling pathways overlap in a manner that collectively determines MZ and FO B cells functional status and peripheral life span (49).
While all the above factors must be considered as potential explanations for the characteristics of the B cell compartment that develops in HKI71 mice, some seem more probable than others. The perturbation of primary adult B lymphopoiesis in HKI71/JHD(µMT) mice at the immature B cell stage could be explained by a tolerance checkpoint that acts via clonal deletion, or induction of BAFF dependence at this juncture. However, this idea is inconsistent with the finding that fetal B cell development is less severely affected and MZ B cells do develop in these mice, and does not account for why most B cells in HKI71/+ mice are H chain allelically included. Moreover, to explain the results of our autoreconstitution experiments on HKI71/JHD and µMT mice, it would have to be assumed that nearly all HKI71:L chain combinations are strongly autoreactive, and the B cells expressing these combinations subjected to clonal deletion or dramatically reduced peripheral life span. This seems unreasonable and indeed in HKI71/V
10A double-transgenic mice B cell development appears fairly normal.
A checkpoint that functions via receptor editing is more compatible with the data. However, while editing mechanisms that operate via L chain and H chain replacements, and L chain allelic inclusion have been reported by many groups (5862), there have been only a handful of reports suggesting a role for H chain allelic inclusion in the regulation of autoreactive B cell development (63, 64). This is not to say that receptor editing does not take place in HKI71 mice in a sub-population of B cells that initially express H:L combinations that are autoreactive or incompatible with positive selection into various peripheral subsets. Indeed, L chain editing may be required to allow the efficient formation of the HKI71 MZ compartment, as indicated by the fact that the BCRs expressed by most MZ B cells in HKI71/V
10A double-transgenic mice are not recognized by the anti-clonotypic E4 mAb.
A more likely, but not mutually exclusive, possibility is that structural differences in the HKI71 as compared with the HKI65 VH region preclude efficient formation or signaling function of the pre-BCR or other complexes that include the µ H chain and that regulate H chain allelic exclusion. The existence of the latter complexes has been supported by data showing that B cells in mice deficient in all SLC components still display H chain allelic exclusion (19). This could explain why essentially all B cells in HKI71/+ mice express either sIgMa and sIgMb, or sIgMb alone. Interestingly, this notion implies that these differences in µ H chain function have a less severe impact on fetal, as compared with adult B lymphopoiesis, as previously proposed by Hardy and colleagues (29). In addition, while the action of tolerance pathways seem an incomplete explanation for the influence of the HKI71 H chain on B cell development and subset distribution, a strong influence on the specificity of the BCRs partially encoded by this H chain and their level of surface expression is likely. This was made apparent by the fact that most MZ B cells in HKI71/+ mice are sIgMa-high, sIgMb-low, while the FO B cells in these mice express similar levels of the knockin and endogenous locus-encoded µ H chains or only the µb chain on their surfaces. Moreover, whether or not the periphery of mice in which HKI71 B cells are developing is B lymphopenic appears to influence the B cell subset distribution that results.
Given all these considerations, we propose a model to account for the data we have presented in this study (Fig. 10). This model is based on three as yet untested assumptions: (i) the HKI71 µ H chain inefficiently drives the early stages of B cell development and allelic exclusion due to failure to physically or functionally associate with invariant proteins that form signaling regulatory complexes; (ii) the HKI71 VH region preferentially pairs with L chains that create BCRs with specificities compatible with development of the MZ, but not the FO and B1, B cell compartments and (iii) this MZ development is enhanced during fetal and neonatal periods in the spleen and, perhaps, other lymphoid sites.

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Fig. 10. Model to explain the effects of HKI71 H chain locus expression on mouse B lymphopoiesis. The size and mature subset composition of the B cell compartment during the fetal/neonatal and adult periods of development of the three types of mice indicated are illustrated schematically. In the case of HKI71 hemizygous mice, the development of MZ and FO B cells in mice with the form of the HKI71 knockin locus either containing or lacking the neomycin resistance gene (Neo) are illustrated using solid and stippled filled objects, as indicated. The predominant BCR structures expressed by each sub-population are indicated within the object illustrating the development of that sub-population (Dual = BCRs encoded by both the HKI71 and endogenous H chain loci, Endo Ls = L chains encoded by the endogenous L chain loci).
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In HKI71 hemizygous mice (left side of Fig. 10), fetal B lymphopoiesis first seeds the neonatal spleen with precursors that ultimately expand and develop into MZ B cells. Whether these precursors are the B220low, CD5+, sIgM+ cells observed in our experiments (Fig. 6) remains to be formally tested. This pathway is favored in early development due to the pre-functional status of the HKI71 knockin locus, positive selection by self-antigens and perhaps the lymphopenic nature of the neonatal spleen. These MZ cells persist through adult life due to their capacity to self-renew (7, 8). Concurrently, the inability of the HKI71 µ chain to enforce H chain allelic exclusion allows B cells that are H chain allelically included to develop, and cells that express sufficient levels of endogenously encoded BCRs with specificities capable of promoting FO B cell development gradually accumulate. In the context of the previous signal strength models of Pillai and colleagues (50), one could speculate that the level of signaling via HKI71 locus-encoded BCRs is insufficient to drive FO B cell development, necessitating high-level surface expression of BCRs encoded by the endogenous allele to allow this pathway to operate efficiently.
When the expression of the endogenous Igh locus is blocked in HKI71 mice by the µMT or JHD mutations, only the fetal/MZ pathway operates productively, and the developmental lymphopenia in these mice combined with the inability of the HKI71 H chain to promote adult B lymphopoiesis ultimately results in an adult splenic B cell compartment of reduced size composed nearly entirely of MZ B cells and their putative precursors (Fig. 10, middle). If these cells are eliminated by sub-lethal irradiation, the inefficient promotion of adult B cell development by the HKI71 H chain results in a persistent B lymphopenic state. Interestingly, despite the fact that these autoreconstituting adult mice are severely B lymphopenic, the MZ compartment is not reconstituted. This reinforces the idea that many of the precursors to the MZ compartment in HKI71 mice are generated during fetal/neonatal life.
The introduction of the V
10A transgene rescues both fetal and adult B lymphopoiesis in HKI71 mice by enforcing expression of a functional BCR-signaling complex on the cell surface. This renders the regulatory pathways normally sub-served by the µ chain in association with other proteins such as the SLC components less important (6567). In addition, this particular H:L combination either encodes a BCR specificity that is incompatible with efficient MZ B cell development or its expression results in a developmental intermediate necessary for MZ development being bypassed. As such, during both fetal and adult B lymphopoiesis, B cell production is robust, and peripheral lymphopenia never develops. Consequently, a relatively normal number of MZ B cells accumulate in these mice, but this requires expression of an L chain other than that encoded by the V
10A transgene, presumably via L chain editing processes (Fig. 10, right). When these double-transgenic mice are sub-lethally irradiated, the peripheral B cell compartment is rapidly reconstituted by cells largely destined to become FO B cells. This results in a reduction in the MZ compartment, perhaps due to inefficient editing of the V
10A transgene, a fetal origin of most of the MZ precursors or both.
While this model is hypothetical, and future studies will clearly be required to test its major tenets, it emphasizes the cumulative nature of the effects of alterations in H chain function by the V region at different stages of B cell and organismal development, and the influence of homeostatic forces on the penetrance of such effects. In addition, it supports previous conclusions that MZ B cells can be derived from fetal/neonatal hematopoiesis (3, 4), and that the regulatory requirements of the H chain differ at this stage of B cell development as compared with during adult B lymphopoiesis (29). Perhaps most importantly, it reinforces the notion that the mature B cell subset composition and BCR repertoire of the adult B cell compartment are determined by an extremely dynamic process in which Ig V regions play a pivotal regulatory role (4149).
It should be emphasized that this model does not adequately account for the regulation of development of the B1 B cell population in HKI71 mice. A large body of data suggests that the generation and maintenance of the B1 B cell sub-population depend on pathways distinct from those that regulate MZ and FO B cell development and activity (3, 4, 30, 6870). In HKI71/+ mice, peritoneal B1 cells do not express the HKI71 (IgMa) locus alone, and the majority of these cells expresses sIgMb only. This indicates that the HKI71 µ chain does not support B1 development and is compatible with the idea that only a limited number of H chain V region structures can efficiently drive this process (29, 30). However, in apparent contradiction of this idea, in HKI71/µMT (JHD) mice, levels of CD5+ and other B1 type B cells are not reduced in the peritoneal cavity (data not shown). Characterization of the developmental origin and BCR repertoires of the B1 compartments that arise under these two conditions may provide insight into this issue.
It is surprising that the transgenic expression of two VH genes that differ by only eight amino acids can have such dramatically distinct influences on multiple stages of primary B cell development. Nonetheless, the previous studies of Storb and colleagues have shown that even a single amino acid difference in the V domain of the BCR can dramatically alter primary B cell development (71). In this regard, we should reiterate that at least six of the eight amino acid differences between the HKI65 and HKI71 VH regions resulted from somatic hypermutations during an antigen-driven immune response; that is at a time when the only presumed functional requirements of the VH region were to pair with an L chain to form a BCR that lacked autoreactivity and had affinity for the driving antigen. Future studies of the amino acid differences responsible for the various phenotypic characteristics displayed by HKI65 and HKI71 B cell compartments are likely to provide new insight into the VH region structural requirements for regulation of several key steps in primary B cell development.
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Acknowledgements
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We thank Randy Hardy for the JHD mice and for conducting some pilot flow cytometry studies, Larry Wysocki and Katja Aviszus for the V
1060 mice and Kerstin Kiefer for a review of the manuscript. Technical support was provided by the Flow Cytometry and Transgenic/Knockout Facilities of the Kimmel Cancer Center. We also thank Scot Fenn for technical help and all members of the Manser laboratory for their indirect contributions to this work. This studies was funded by research grants from the National Institutes of Health (NIH; AI23739 and AI38965) to T.M., and an NIH training grant (CA72318) supported L.H.
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Abbreviations
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Ars | arsonate |
BM | bone marrow |
FO | follicular |
H | heavy |
L | light |
MZ | marginal zone |
NIH | National Institutes of Health |
sIgM | surface IgM |
SLC | surrogate light chain |
V | variable |
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Notes
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* These authors contributed equally to this work. 
Transmitting editor: J. Ravetch
Received 26 July 2005,
accepted 22 August 2005.
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