Selection of stereotyped VH81X-µH chains via pre-B cell receptor early in ontogeny and their conservation in adults by marginal zone B cells
Yohei Kawano,
Soichiro Yoshikawa,
Yoshiyuki Minegishi and
Hajime Karasuyama
Department of Immune Regulation, Tokyo Medical and Dental University Graduate School, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519, Japan
Correspondence to: H. Karasuyama; E-mail: karasuyama.mbch{at}tmd.ac.jp
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Abstract
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The pre-B cell receptor (preBCR) plays critical roles in early B cell differentiation. It has been shown that not all µH chains are capable of pairing with surrogate light (SL) chains to form preBCR. Here, we established a novel system to differentially identify two types of early pre-B cell populations in bone marrow and fetal liver of mice, one producing SL-pairing µH chains and the other producing SL-non-pairing µH chains. The former population accounted for 80% of all the early pre-B cells in adult bone marrow, while it accounted for only 20% of those in fetal liver. Comparison of the two types of pre-B cell populations in fetal liver revealed the structural difference between SL-pairing and -non-pairing µH chains encoded by the VH81X segment that was most frequently utilized in fetal liver pre-B cells but rarely expressed by B cells generated in adults. PreBCR played an important role in the positive selection of VH81X-µH chains carrying the characteristic sequences of the complementarity-determining region 3 with little or no nibbling or N nucleotide addition, leading to their predominance in neonatal splenic B cells. These fetal-type VH81X-µH chains were also detected in adult spleen, but almost exclusively in marginal zone (MZ) B cells in contrast to the adult-type VH81X-µH chains. This strongly suggests that neonatally generated and selected B cells expressing the stereotyped VH81X-µH chains are maintained in the adult MZ and could function as innate-like lymphocytes.
Keywords: B cell development, B cell repertoire, B cell subsets, immunoglobulin, fetal liver
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Introduction
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B cell development occurs in fetal liver during embryonic development and is subsequently maintained in the bone marrow of adult animals. During B cell development, V(D)J recombinations take place at IgH and IgL chain loci in an ordered fashion to create a diverse repertoire of B cell receptors (BCRs) (1). The pre-B cell receptor (preBCR), composed of µH chains, invariant VpreB/
5 surrogate light (SL) chains and signal transducing module Ig
/Igß heterodimers, plays critical roles during the early stage of B cell development (2, 3). Deficiency of preBCR formation or its signaling results in severe impairment of B cell differentiation at the transition from pro-B to pre-B cell stages in both humans and mice (4).
The combination of VH, DH and JH gene segments with nucleotide addition or deletion at the junctions creates enormous variation at the complementarity-determining region (CDR) 3 of the H chain to increase the diversity of antigen specificity (1). On the other hand, the recombination often results in non-productive rearrangements that cannot encode H chain protein. This drawback can be overcome by preBCR-mediated proliferation of cells (5, 6). Even though the frequency of productive rearrangement is rather low, the product of successful rearrangement induces clonal expansion through formation of preBCR to ensure sufficient numbers of pre-B cells that subsequently undergo rearrangements at the L chain locus.
It has been shown that not all of the µH chains produced through productive VDJ rearrangements are able to pair with SL chains to form preBCR (7, 8). Such µH chains incapable of pairing with SL chains were shown to be deficient in pairing with conventional L chains (9). Therefore, the invariant SL chain of the preBCR functions as a prototype of various types of L chains to assess the ability of µH chains to pair with future partners in advance. Pre-B cells that produce SL-non-pairing µH chains are not favored during early B cell development since they do not receive preBCR signals for their survival and proliferation (6). Thus, the quality of µH chains is assessed at the preBCR stage of B cell development.
It remains largely unknown as to what structural aspect of µH chains determines their ability to pair with SL chains. In this sense, µH chains using the VH81X gene, a member of the VH7183 gene family, are particularly interesting (10). VH81X is frequently incorporated into VDJ rearrangements during B cell development (10, 11) but is rarely expressed in mature peripheral B cells in adult mice (1217). It has been clearly demonstrated that VDJ rearrangements using particular VH segments such as VH81X and VHQ52 are extremely prone to produce µH chains incapable of forming preBCR (7, 8). Interestingly, however, such disfavoring of VH81X in adults has not been observed in neonatal spleen (14, 1820). An intriguing model has been proposed in that pre-B cells expressing SL-non-pairing µH chains have the proliferative advantage over those expressing SL-pairing µH chains in fetal liver, opposed to what happens in the bone marrow (21). Some studies indicated the importance of the particular CDR3 sequences in the selection of VH81X-µH chains (20, 22), whereas another study could not define any sequence constraint in functional VH81X-µH chains (23). Thus, the molecular mechanism underlying the selection of VH81X-µH chains during B cell development remains to be clarified.
In this study we have established a novel system to directly analyze individual early pre-B cells in bone marrow and fetal liver in terms of whether their µH chains were paired with SL chains to form preBCR in vivo. This approach allows us to identify two types of early pre-B cell populations, one positively selected and the other negatively selected at the preBCR checkpoint, and further investigate their kinetics during B cell development early in ontogeny. Structural differences of the CDR3 in SL-pairing and -non-pairing µH chains utilizing an identical VH81X segment became evident. µH chains with stereotyped VH81X sequences were found to be positively selected through preBCR formation early in ontogeny and incorporated in neonatal splenic B cells. We further addressed the question of whether neonatally generated and selected B cells with such stereotyped VH81X-µH chains were maintained in adults. We found that such B cells did indeed exist in adult spleen but were restricted to the marginal zone (MZ), suggesting the fetal origin of some MZ B cells.
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Methods
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Animals
Adult (812 weeks old) and pregnant C57BL/6 mice were purchased from CLEA Japan Ltd (Tokyo, Japan). Rag-2/ (24) and Rag-2/ with a rearranged VHDHJH (B1.8) knock-in gene (25) of the C57BL/6 strain were bred and maintained under specific pathogen-free conditions in our animal facility. Heterozygous JHT (
) mice (26) of the C57BL/6 strain were kindly provided by Ohmori (Okayama University, Okayama, Japan). All the experiments in this study were performed according to the Guidelines for Animal Use and Experimentation as set out by our University.
Antibodies
Alexa488 (Molecular Probes, Eugene, OR, USA)-conjugated anti-preBCR mAb (SL156) (27) and PE (PROzyme, San Leandro, CA, USA)-conjugated anti-
5 mAb (LM34) (28) were prepared according to the manufacturer's instructions. FITCanti-CD21/35, PEanti-CD23 and allophycocyanin (APC)anti-µH chain (II/41) were purchased from BD PharMingen (San Diego, CA, USA).
Flow cytometry and cell sorting
sIg+ cells were depleted with sheep anti-mouse IgG (H + L)-conjugated beads (Dynal, Skoyen, Norway). B220-positive cells were enriched by using anti-B220-conjugated beads (Miltenyi Biotech, Bergisch Gladbach, Germany). Cell purity was >95%. Isolated cells were first stained for cell-surface markers for 30 min and subsequently fixed and permeabilized with Cytofix/Cytoperm (BD PharMingen) and were then stained intracellularly for a further 30 min. Stained cells were analyzed with FACSCalibur (BD Bioscience, San Jose, CA, USA) or were sorted with FACS Vantage (BD Bioscience).
Genomic DNA isolation, amplification and sequencing
Cells were treated with PCR lysis buffer [10 mM Tris (pH 8.3), 50 mM KCl, 1.8 mM MgCl2, 0.5% Tween-20] containing 100 µg ml1 proteinase K (Roche, Basel, Switzerland) at 56°C for 1 h, and then heated to 95°C for 10 min. The lysates were used directly for PCR. DNA amplification was carried out in two rounds of PCR. The following primers were used for primary PCR: VH universal sense 5'-GGACTAGT(C/G)A(A/G)GT(A/G/C/T)(A/C)AGCTG(C/G)AGTC-3' and JH4 3'-intron antisense 5'-AGGCTCTGAGATCCCTAGACAG-3'. PCR was performed at 95°C for 1 min, followed by 30 cycles of 95°C for 45 s, 55°C for 60 s and 72°C for 120 s. In the second PCR, 1 µl of the first PCR product was re-amplified with VH7183 family-specific sense primer carrying a PstI restriction site (5'-AAAACTGCAGGAGTCTGGGGGAGGCTTAG-3') and each JH-specific antisense primers carrying HindIII restriction sites as follows: JH1, 5'-TTTGGGAAGCTTTGACTCTCTGAGGAAACGGTGACCGTGG-3'; JH2, 5'-TTTGGGAAGCTTTGACTCTCTGAGGAGACTGTGAGAGTGG-3'; JH3, 5'-TTTGGGAAGCTTTGACTCTCAGCAGAGACAGTGACCAGAG-3'; JH4, 5'-TTTGGGAAGCTTTGACTCTCTGAGGAGACGGTGACTGAGG-3'.
PCR was performed at 95°C for 1 min, followed by 30 cycles of 95°C for 20 s, 55°C for 30 s and 72°C for 60 s. The products of the second-round PCR were digested with PstI and HindIII, cloned into the PstI/HindIII site of the pBS-µH vector as described below and sequenced. In the experiments shown in Fig. 6, 5'-primers specific to VH81X, 5'-GGAGGCTTAGTGCAGCCTGGAGAG-3' and 5'-TCCCTGAAACTCTCCTGTGAATCC-3', were utilized for primary and secondary PCR, respectively. PCR products were then digested with EcoRI/HindIII and inserted into the corresponding restriction sites of pBluescript II (Stratagene, La Jolla, CA, USA) for sequence analysis. The site mutation was generated by PCR techniques to replace an amino acid at position 1 of the CDR3 of some µH clones with histidine (His).

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Fig. 6. The role of His at position 1 of the CDR3 in the SL-pairing ability of VH81X-µH chains. (A) Two VH81XDHJH1 clones shown in Fig. 5, one carrying the CDR3 sequence of HYYGSSYWYFDV isolated from the SL156+ early pre-B cell population (upper panels) and the other carrying the CDR3 sequence of YYYGSSYWYFDV isolated from the SL156 early pre-B cell population (lower panels), were analyzed for their capacity to associate with SL chains to form preBCR as shown in Fig. 2B. Staining profiles of infected GFP+ cells (thick histogram) and uninfected GFP cells (thin histogram) were overlaid. (B) A representative VH81XDHJH4 clone carrying the CDR3 sequence of RWFYYAMDY isolated from the SL156 early pre-B cell population (Fig. 5) and its mutant clone in that Arg at position 1 of the CDR3 was replaced with His by site-directed mutagenesis were analyzed in the same way.
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Construction of pBS-µH
cDNA was synthesized from total RNA isolated from C57BL/6 splenocytes by reverse transcription using ReverTra Ace (Toyobo, Osaka, Japan). The full length of the membrane form of the µH chain containing the VH leader sequence was amplified by PCR using the following primers: 5'-GGAATTCCACCATGGGATGGAGCTGTATC-3' (sense) and 5'-CCGCTCGAGCGGTCAATAGCAGGTGCCGCC-3' (antisense). The amplified products were digested with EcoRI and XhoI, and ligated into the corresponding restriction sites of pBluescript II. A HindIII site was created in the third and fourth codons of Cµ, and a PstI site within the Cµ gene was deleted by silent mutation to obtain pBS-µH such that any of the VH7183DHJH-rearranged fragments obtained from PCR could be inserted into pBS-µH between the PstI site in the FR1 of VH region and the artificial HindIII site.
Retroviral expression vector and infection to pro-B cell line 38B9
pBS-µH was digested with BamHI/XhoI, and the purified fragment coding for the full-length clone of the membrane form of the µH chain was inserted into the corresponding restriction sites of the retroviral vector pMX-IRES-GFP (29) which allowed bicistronic expression of green fluorescent protein (GFP) and a gene of interest. The ecotropic packaging cell line Plat-E (29) (2 x 105 cells) was cultured in 1 ml of Plat-E medium in 24-well plates for 24 h, and then transfected with 1 µg of retroviral plasmid DNA using Effectene (QIAGEN, Hilden, Germany). Forty-eight hours after transfection, 0.5 ml of recovered viral supernatant was used to infect 1 x 106 cells of pro-B cell line 38B9 by using 10 µg ml1 polybrene (SigmaAldrich, St Louis, MO, USA). Two to five days after infection, cells were harvested and analyzed for µH chain expression by flow cytometry.
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Results
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Differential identification of primary pre-B cells expressing µH chains capable versus incapable of forming preBCR
A combination of three mAbs was employed for flow cytometric analysis to examine whether µH chains of individual early pre-B cells were paired with SL chains to form preBCR in vivo: LM34 specific for the
5 SL chain (28), II/41 specific for the µH chain and SL156 which recognized the conformational epitope of the µH chain/SL chain complex (preBCR) but not each component (27). Since levels of surface expression of preBCR on normal pre-B cells in vivo were extremely low and the vast majority of preBCR was detected in their cytoplasm (2, 5), we stained cells intracellularly with the antibodies to obtain fluorescence intensity sufficient for analysis. In this system,
5 SL chain-expressing cells (pro-B cells and early pre-B cells) were gated and analyzed for the expression of µH chains and preBCR (SL156 epitope) to identify preBCR+ and preBCR early pre-B cells that express µH chains capable and incapable of pairing with SL chains, respectively (Fig. 1A and B). As a model to assess this detection system, we first analyzed B220+ bone marrow cells from RAG-2/ mice (24) and those with a productively rearranged IgH knock-in gene (25) (Fig. 1C). In RAG-2/ mice, as expected, virtually no µH+ cells and hence no SL156+ cells were detected among
5+ cells (Fig. 1C, top panels). In IgH knock-in RAG-2/ mice, most of the
5+ cells were found to express µH chains, demonstrating the efficient transition from pro-B to pre-B cells (Fig. 1C, middle panels). In this case, the vast majority of these
5+µH+ cells were positive for SL156, indicating that the transgenic µH chain had the ability to pair with SL chains to form preBCR.
Analysis of B220+sIg bone marrow cells from normal mice revealed that
20% of the
5+µH+ cells remained unstained with SL156 even though they produced both components (SL chains and µH chains) of preBCR (Fig. 1C, bottom panels). The specificity of SL156 predicted that these SL156 cells produced µH chains incapable of pairing with SL chains to form preBCR. This was validated by the in vitro association assay in that µH chain clones isolated from the SL156+ or SL156 fraction of
5+µH+ early pre-B cells were transfected to SL+µH pro-B cells to assess the assembly and surface expression of preBCR. µH chains from the SL156+ fraction were expressed on the cell surface together with SL chains, whereas µH chains from the SL156 fraction were retained in the cytoplasm (some examples are shown in Fig. 2). Thus, flow cytometric analysis with the combination of the three mAbs allowed us to identify and distinguish two types of early pre-B cells ex vivo, one expressing SL-pairing µH chains and the other expressing SL-non-pairing µH chains.
Kinetics of preBCR+ and preBCR early pre-B cell populations during B cell development in fetal and neonatal liver
We next applied this detection system to pre-B cells that are produced during ontogeny (Fig. 3). In fetal liver of Day 14 gestation when VDJ rearrangements had just begun, only 21% of
5+µH+ early pre-B cells was positive for SL156 (Fig. 3, top panels). The proportion of the SL156+ fraction increased up to
50% by Day 16 of gestation, and had reached 84% by 2 days after birth, a level comparable to that observed in adult bone marrow (Fig. 3, middle and bottom panels). Though the cell size of the preBCR+ and preBCR cells was comparable in Day 14 fetal liver, the former became larger than the latter in Day 16 fetal liver and even more in Day 2 neonatal liver as judged by their profile of forward scatter (Fig. 3, right panels). We found in bone marrow that the preBCR+ cells were larger in size and more actively cycling than the preBCR cells as determined by 5-bromo-2-deoxyuridine uptake (data not shown). These results suggest that preBCR-driven proliferation lead to the expansion of the preBCR+ population during B cell development in fetal/neonatal liver.

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Fig. 3. Kinetics of preBCR+ and preBCR early pre-B cell populations during B cell development in fetal liver. B220+sIg cells in fetal and neonatal liver at the indicated developmental time points were stained and analyzed as described in Fig. 1(C). Profiles of forward scatter of cells and their mean values in the SL156+ and SL156 populations are shown in the right panels. Representative data from four independent experiments are shown.
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Selection of stereotyped VH81X-encoded µH chains via preBCR during B cell development in fetal liver
The VH repertoire of fetal B lineage cells is largely restricted to the VH7183 family (11, 30). To identify possible structural differences between SL-pairing and SL-non-pairing µH chains, SL156+ and SL156 early pre-B cells were sorted separately from Day 16 fetal liver, and productively rearranged VH7183 genes were cloned from each cell population. In the SL156 population, 39 out of 40 independent clones examined utilized VH81X (VH7183.1b) (Fig. 4A). This is consistent with previous observations that most of the VH81X-bearing µH chains expressed in adult bone marrow and spleen were SL-non-pairing (7, 8). Interestingly, however, as many as 41% (23/56) of clones in the SL156+ population also utilized VH81X. This indicated that VH81X-µH chains produced at this developmental stage were not necessarily SL non-pairing unlike in adult bone marrow and spleen.

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Fig. 4. Characterization of VH segments and CDR3 utilized in SL156+ and SL156 early pre-B cells in Day 16 fetal liver. (A) The SL156+ and SL156 fractions in Day 16 fetal liver as shown in Fig. 3 were sorted separately, and productively VDJ-rearranged fragments encoding the VH7183 family were cloned from each fraction and sequenced. Isolated clones were categorized according to which member of the VH7183 family was utilized. (B) Clones using the VH81X segment isolated from SL156+ and SL156 fractions were further categorized according to their usage of the JH segment. Filled bars indicate clones carrying His at position 1 of the CDR3 while open bars indicate those carrying other amino acids at that position. The same analysis was applied to the previously reported CDR3 sequences of VH81XDHJH clones isolated from sIgM+ splenic B cells of neonatal mice (13, 23).
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Comparison of the CDR3 region of the VH81XDHJH clones revealed a clear contrast between the SL156+ and SL156 populations (Fig. 5, summarized in Fig. 4B). First of all, the usage of JH segments was different. In the SL156+ population, JH4 was most frequently used (51%) while JH2 was rarely used (2%) among 41 clones examined. In contrast, in the SL156 population, JH2 was most frequently utilized (43%) while JH3 was rarely utilized (2%) among 46 clones examined. Secondly, the first position of the CDR3 (position 95 in VH) was extremely biased to His regardless of JH usage in the SL156+ population. Eighty-five percent of the clones (35/41) utilized His in the SL156+ population compared with only 28% (13/46) in the SL156 population. There was no significant difference between the two populations with regard to DH usage in CDR3 (Fig. 5).

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Fig. 5. Analysis of the CDR3 of VH81XDHJH clones isolated from the SL156+ and SL156 early pre-B cells in Day 16 fetal liver. Amino acid sequences and length of the CDR3, DH usage and its reading frame (RF), as well as JH usage of each clone are shown. Some of the µH chain clones isolated from each cell population were assayed for SL-pairing capacity in vitro, and the results are shown as + (SL pairing) or (SL non-pairing) in the column on the right-hand side of each panel.
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The in vitro SL-pairing assay confirmed that the SL-pairing capacity of each VH81X-µH chain isolated from the early pre-B cell populations correlated well with the SL156-staining pattern of the populations (Figs 5 and 6A). All the 18 productively rearranged VH81X-µH clones randomly selected from the SL156+ population showed the capacity to form preBCR (Fig. 5). This suggested that the frequency of cells harboring two productively rearranged H chain genes, one producing SL-non-pairing µH chains and the other producing SL-pairing µH chains, could be <6%, in accord with the previous report (31). Thus, the presence of such double producers in the SL156+ early pre-B cell population at the low frequency, if any, did not seem to disturb the comparison between the SL156+ and SL156 populations significantly.
Possessing His at position 1 of the CDR3 appeared critical for VH81X-µH chains to acquire the SL-pairing capacity typically when JH1 or JH4 was utilized (Fig. 4B). VH81X-µH chains carrying the CDR3 sequence of HYYGSSYWYFDV in conjunction with JH1 isolated from the SL156+ population (Fig. 5) formed preBCR on the cell surface when introduced into SL+µH pro-B cells (Fig. 6A, upper panels). In contrast, VH81X-µH chains carrying the CDR3 sequence of YYYGSSYWYFDV in conjunction with JH1 isolated from the SL156 population (Fig. 5) could not form preBCR (Fig. 6A, lower panels). Furthermore, the mutational replacement of the first position of the CDR3 (RWFYYAMDY) from Arg to His in a representative VH81X-µH chain clone carrying JH4 isolated from the SL156 population (Fig. 5) converted it from SL non-pairing to SL pairing (Fig. 6B). Thus, His at position 1 of the CDR3 conferred the SL-pairing capacity on VH81X-µH chains. Interestingly, however, this did not appear to be the case when VH81X-µH chains utilized JH2. In the SL156 population, the clones carrying the His-bearing CDR3 almost exclusively utilized JH2, which was seldom utilized in the SL156+ population.
N nucleotides were rarely found in the VD junctions of the VH81XDHJH clones in either population (Table 1), in accord with the fact that there was little or no terminal deoxynucleotidyl transferase (TdT) activity in fetal liver (32). Addition of P nucleotides at the 3'-end of the VH gene segment was much more frequently found in the SL156+ population (61.0%) than in the SL156 population (13.0%). The 3'-end of the VH81X segment possesses the nucleotides CA in its germ line configuration. Therefore, P nucleotide addition to this end creates CAT that encodes His at position 1 of the CDR3. Overlapping of more than two nucleotides between VH and DH gene segments, including P nucleotides, was found more often in the SL156+ population (51.2%) than in the SL156 population (10.9%). In contrast, nibbling (loss of nucleotides) at the 3'-end of VH was much less frequently found in the former (14.6%) than in the latter (65.2%). Consistent with this, the average length of the CDR3, using JH4 for example, was 9.0 amino acids in the former and 7.4 in the latter. These results indicated that VDJ rearrangements using germ line-encoded sequences including P nucleotides with little or no deletion at the junction were enriched in the SL156+ population.
Importantly, Marshall et al. have reported that all the 17 productive VH81XDHJH clones isolated from neonatal spleen possessed His at position 1 of the CDR3 (20). Since this analysis focused only on clones utilizing JH4, we compiled and analyzed the previously reported CDR3 sequences of VH81XDHJH clones isolated from neonatal splenic B cells (13, 23). As summarized in the right panel of Fig. 4(B), 41 out of 46 (93%) clones carry His at position 1. JH4 was most frequently utilized (48%) while JH2 was not utilized at all. The average length of the CDR3 using JH4 was 9.3 amino acids. These patterns were very similar to those of the SL156+ population in Day 16 fetal liver pre-B cells but were distinct from those of the SL156 population. This strongly suggested that SL156+, but not SL156, early pre-B cells were positively selected to differentiate into mature B cells, most likely through preBCR formation.
Expression of stereotyped VH81X-µH chains with a fetal phenotype in adult spleen but restricted to MZ B cells
It has been reported that B cells in the transgenic mice expressing a fetal-type VH81X-µH chain are favored to become IgMhighCD21high and enriched in the MZ of the spleen (33, 34). The question whether such MZ B cells are generated during ontogeny or in adult life remains to be answered. Therefore, we next tried to examine the possibility that the stereotyped VH81X-µH chains found in fetal/neonatal B lineage cells might be expressed in adult spleen, particularly MZ B cells, in normal mice. However, this type of experiment was hampered by the fact that the majority of rearrangements utilizing VH81X isolated from adult splenic B cells were non-productive (1216). To overcome this difficulty,
mice (26) were employed, in that one of the IgH chain alleles is destroyed and therefore all VDJ rearrangements isolated from sIg+ B cells must be productive.
On the basis of CD21 and CD23 expression, three B cell subsets were separately sorted from sIgM+ splenic B cells: MZ B cells, follicular (FO) B cells and newly formed (NF) B cells as shown in Fig. 7(A). VH81XDHJH4-rearranged fragments were cloned from each subset. The frequency of clones carrying the His-bearing CDR3 was not drastically different in the three B cell subsets, 62% (13/21) in MZ B cells, 62% (13/21) in FO B cells and 48% (10/21) in NF B cells (Fig. 7B). Moreover, no apparent bias in the amino acid sequences of their CDR3 was recognized among the three subsets. Importantly, however, a marked contrast was observed in terms of N nucleotide addition at the VDJ junctions. In FO and NF B cells, almost all the VH81XDHJH4 clones examined showed abundant N nucleotides at the VD and/or DJ junctions, 100% of the clones (21/21) from the former and 95% of the clones (20/21) from the latter (summarized in Fig. 7B, see the details in Supplementary Fig. 1, available at International Immunology Online). This was consistent with the previous report by Hayden et al. in that all of the 30 VH81XDHJH4 clones isolated from bone marrow immature B cells and all of the 13 clones from IgMlowIgDhigh splenic B cells exhibited N nucleotides at the VDJ junctions (23). In contrast, as many as 52% of the clones (11/21) isolated from MZ B cells in the present study possessed no N nucleotides at the VDJ junctions. The frequency of clones lacking N nucleotides in MZ B cells increased to 63% (17/27) when one took account of the fact that some of these clones were repetitively identified in separate experiments (Fig. 7B), perhaps as a consequence of clonal expansion of B cells bearing these particular sequences. Moreover, all of the clones lacking N nucleotides carried the His-bearing CDR3. The average length of the CDR3 in these clones was 8.9 amino acids. These characteristics of the CDR3 were the same as those observed in preBCR+ early pre-B cells in fetal liver and neonatal splenic B cells, suggesting that a substantial fraction of MZ B cells expressing VH81X-µH chains were generated early in ontogeny rather than differentiating from NF B cells in adults.
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Discussion
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The proportion of the preBCR population among early pre-B cells was much higher than what we had expected, particularly in fetal liver. As many as 80% of early pre-B cells in Day 14 fetal liver were found to produce SL-non-pairing µH chains. This indicates that an unexpectedly large proportion of productive VDJ rearrangements made at this developmental stage are non-functional. It has been shown that approximately half of the µH chains expressed in a particular bone marrow pre-B cell population at the stage before the preBCR checkpoint were SL non-pairing (8). The much higher proportion of preBCR population in Day 14 fetal liver could be attributed to the predominant usage of the D-proximal VH gene segments, particularly VH81X, that are often incompetent for forming preBCR, as compared with the more randomized VH usage in adult bone marrow. The proportion of the preBCR fraction in fetal liver continuously decreased along the ontogeny down to 16% by 2 days after birth, a level comparable to that observed in adult bone marrow. This shift appears to be at least in part due to the preferential expansion of the preBCR+ population over the preBCR population during B cell development in fetal liver that occurs in a single synchronous wave (35).
The vast majority of preBCR early pre-B cells in Day 16 fetal liver was found to utilize VH81X among the VH7183 family. More importantly,
40% of preBCR+ cells in Day 16 fetal liver utilized VH81X as well. Since the proportion of preBCR+ and preBCR populations was almost equal, it was calculated that as many as 30% of the VH81X-µH chains were SL pairing at this developmental stage, in sharp contrast to the observations in adult bone marrow and spleen (7, 8). The structural difference in the CDR3 between SL-pairing and -non-pairing VH81X-µH chains became evident in the present study. There was no obvious similarity in the CDR3 sequence among SL-non-pairing VH81X-µH chains, whereas those with His at position 1 of the CDR3 utilizing JH1, JH3 or JH4, but not JH2, were almost exclusively enriched in the preBCR+ population. Furthermore, the vast majority of VH81X-µH chains expressed by sIgM+ B cells in neonatal spleen were found to possess these characteristic CDR3 sequences. These results clearly indicate that the stereotyped VH81X-µH chains are positively selected via preBCR formation during fetal B cell development, in contrast to the model proposed for the selection of VH11-µH chains (21).
Previous work by others drew a similar conclusion from the comparison of VH81XDHJH4 clones isolated from neonatal spleen of normal mice versus Day 16 fetal liver of preBCR-deficient µmT mice (20). All of the 17 productive sequences from the former encoded the His-bearing CDR3, whereas only 66.7% (6/9) of those from the latter did so. However, we found that this comparison did not necessarily warrant the conclusion. VH81X-µH chains in Day 16 fetal liver undergo preBCR-mediated selection as shown in the present study, whereas those in neonatal spleen have already passed through preBCR- and BCR-mediated selection. From our results, we calculated the percentage of His-bearing clones among VH81XDHJH4 µH chains in Day 16 fetal liver of normal mice to be 45.5%. Thus, the lower frequency of His-bearing clones in µmT fetal liver compared with those in normal neonatal spleen cannot be attributed simply to the absence of preBCR-mediated selection.
Intriguingly, when JH2 is utilized, VH81X-µH chains are almost exclusively SL non-pairing, even if position 1 of the CDR3 is His. Two different VH81X-µH chain transgenic mice have been established, one showed intact B cell development (36) while the other showed complete blockage of B cell development at the pro-B cell stage (9). Although both transgenic µH chains possessed the His-bearing CDR3, the former utilized JH1 whereas the latter utilized JH2. This follows the principle of SL pairing that we have found in fetal liver. It remains to be determined what structural aspect of VH81X-µH chains using JH2 restricts their pairing with SL chains.
Marshall et al. have reported the reduction of His-bearing VH81X-µH clones in the neonatal spleen of TdT-transgenic mice, indicating that the N nucleotide addition diminished the chance to create the stereotyped VH81X-µH chains as observed in B cell development in adults (20). The present study revealed that even under conditions of N nucleotide paucity, the stereotyped chains were only 30% of all the VH81X-µH chains expressed in Day 16 fetal liver, suggesting that their predominance in neonatal spleen was not simply a consequence of the homology-directed recombination (18, 20, 37). Such stereotyped chains were positively selected via preBCR. The frequent appearance of the His-bearing CDR3 among SL-pairing VH81X clones in Day 16 fetal liver can be attributed mainly to the addition of P nucleotides to the 3'-end of the VH81X gene segment that creates CAT coding for His. Nibbling of nucleotides at the 3'-end of the VH81X gene segment was frequently observed in SL-non-pairing clones, resulting in shorter length of the CDR3 and a much reduced chance to create the His-bearing CDR3. Thus, SL-pairing VH81X-µH chains appear to be preferentially created from VDJ rearrangements using limited germ line-encoded sequences including P nucleotides with little or no deletion or addition of nucleotides at the junctions.
We next addressed the question of whether neonatally generated and selected B cells expressing VH81X-µH chains were maintained in adults, as in the case of B1 cells. The fetal-type VH81X-µH chains carrying the stereotyped CDR3 without N nucleotides were indeed detected in adult spleen, but almost exclusively in MZ B cells. MZ B cells are a crucial component of the early immune response to blood-borne pathogens, even though they represent only
5% of splenic B cells (38, 39). It is generally believed that immature B cells generated in adult bone marrow migrate into the spleen where they differentiate to either FO or MZ B cells (39). In accord with this, VH81X-µH chains with abundant N nucleotides were detected in all the three B cell subsets examined. On the other hand, the stereotyped VH81X-µH chains without N nucleotides were expressed almost exclusively in the MZ B cell subset. This strongly suggests that MZ B cells are generated at least in two ways: one originates during fetal life whereas the other differentiates from NF B cells in adult life. In this sense, it appears to be necessary to carefully interpret the previous reports that the VH81X-µH chains with the His-bearing CDR3 of 9 amino acids in length were enriched in adult splenic B cells as a consequence of their positive selection in bone marrow via preBCR (20, 22).
There are a couple of observations favorable to the idea that some MZ B cells are generated during the fetal/neonatal period. When the RAG-2 gene was conditionally deleted at birth, namely in the absence of B cell influx from bone marrow after birth, a small number of B cells with the MZ phenotype were maintained in adult spleen (40). This was also the case in adult spleen of IL-7/ or IL-7R
/ mice in that B cell production takes place exclusively during fetal/perinatal life (41, 42). However, it remains to be determined whether this also occurs in normal mice in that NF B cells are continuously supplied from bone marrow to spleen. We cannot formally exclude the possibility that the MZ B cells expressing fetal-type VH81X-µH chains have originated from the fetal-type VDJ rearrangements that might occur in adult bone marrow, albeit at low frequency. Such rare B cells might have expanded only in MZ in response to certain blood-borne pathogens or self-antigens. However, this scenario seems unlikely. The frequency of the His-bearing clones among the VH81X-µH chains was almost comparable in all the three B cell subsets, and no apparent bias in the amino acid sequences of the CDR3 was observed between them regardless of the N nucleotide addition. It is difficult to imagine how bone marrow-derived B cells without N nucleotide addition, but not those with N nucleotides, have selectively expanded in MZ unless the BCRantigen interaction discriminates the DNA sequence rather than the amino acid sequence of BCR.
The developmentally programmed, restricted V gene usage and limited junctional diversity appear to be common features of innate-like lymphocytes including MZ B cells, B1 cells, natural killer T (NKT) cells and skin 
T cells, and perhaps endow these cells with natural memory to respond quickly to a limited number of conserved antigens (43, 44). Indeed, N nucleotide addition in fetal B cells has been shown to result in a loss of protection against pneumococcal infection in adult mice due to the lack of a T15 anti-phosphorylcholine antibody that is encoded by the canonical rearrangement of the VHS107 gene and secreted by B1 cells (45). Thus, N nucleotide addition in adult B cell development provides the vast adult-type repertoire at the expense of the production of these canonical antibodies by newly generated B cells. Instead, neonatally generated innate B cells appear to be maintained in specialized sites in adults such as the MZ and the peritoneum to preserve the early neonatal repertoire.
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Supplementary data
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Supplementary data are available at International Immunology Online
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Acknowledgements
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We thank F.W. Alt, S. Taki and H. Ohmori for providing Rag-2/, Rag-2/ with an IgH gene and
mice, respectively, and T. Kitamura for providing pMX-IRES-GFP and Plat-E. We are thankful to H. Nishikawa and H. Hayashi for their expert assistance with cell sorting, Y. Hayashi for preparing the manuscript and F. Melchers for critical reading of the manuscript. This work was supported by Grants-in-Aid 12051243, 14370110 and 16043218 from the Japanese Ministry of Education, Culture, Sports, Science and Technology.
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Abbreviations
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APC | allophycocyanin |
BCR | B cell receptor |
CDR | complementarity-determining region |
FO | follicular |
GFP | green fluorescent protein |
MZ | marginal zone |
NF | newly formed |
NKT | natural killer T |
preBCR | pre-B cell receptor |
SL | surrogate light |
TdT | terminal deoxynucleotidyl transferase |
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Notes
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Transmitting editor: M. Miyasaka
Received 17 February 2005,
accepted 11 April 2005.
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