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A V_H12 Transgenic Mouse Exhibits Defects in Pre-B Cell Development and Is Unable to Make IgM⁺ B Cells¹

Department of Microbiology and Immunology, University of North Carolina, Chapel Hill, NC 27599

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
V_H12 B cells undergo stringent selection at multiple checkpoints to favor development of B-1 cells that bind phosphatidylcholine. Selection begins with the V_H third complementarity-determining region (CDR3) at the pre-B cell stage, in which most V_H12 pre-B cells are selectively eliminated, enriching for those with V_HCDR3s of 10 aa and a fourth position Gly (designated 10/G4). To understand this selection, we compared B cell differentiation in mice of two V_H12 transgenic lines, one with the favored 10/G4 V_HCDR3 and one with a non-10/G4 V_HCDR3 of 8 aa and no Gly (8/G0). Both H chains drive B cell differentiation to the small pre-BII cell stage, and induce allelic exclusion and L chain gene rearrangement. However, unlike 10/G4 pre-B cells, 8/G0 pre-B cells are deficient in cell division and unable to differentiate to B cells. We suggest that this is due to poor 8/G0 pre-B cell receptor expression and to an inability to form an 8/G0 B cell receptor. Our findings also suggest that V_H12 H chains have evolved such that association with surrogate and conventional L chains is most efficient with a 10/G4 CDR3. Thus, selection for phosphatidylcholine-binding B-1 cells is most likely the underlying evolutionary basis for the loss of non-10/G4 pre-B cells.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
The first Ig receptor expressed by B-lineage cells is the pre-B cell Ag receptor (pre-BCR).⁵ It is composed of at least three parts: the Ig µH-chain, the surrogate L chain components 5 and V_preB (1, 2, 3, 4, 5), and the transmembrane signal transduction molecules Ig and Ig (6, 7). Not all pre-B cells express a pre-BCR complex upon productive rearrangement of the H chain V_H, D, and J_H gene segments. The inability of a pre-B cell to display a pre-BCR results in cell death, be it due to the inability to make an H chain that can pair with surrogate L chain, the inability to make the surrogate L chain, or the inability to signal through the pre-BCR (8, 9, 10, 11, 12, 13). The pre-BCR is also essential for mediating allelic exclusion (11, 12, 14), and for initiating changes associated with differentiation to a pre-BII cell, including L chain gene rearrangement (15, 16, 17). Whether all of these events occur as a result of just one signal by the pre-BCR or multiple signals is unknown.

B cell development follows a set pathway involving changes in expression of a variety of cell surface and cytoplasmic proteins, and rearrangement of Ig H and L chain genes (14, 18, 19, 20). Pre-BI cells are the first B-lineage cells to have undergone an Ig gene rearrangement. These cells have a D to J_H rearrangement on one or both H chain alleles, but lack V_H and V_L gene rearrangements (18, 21). They undergo V_H to DJ_H rearrangement, and those that acquire a productive (in-frame) rearrangement express cytoplasmic µ (22, 23). Not all H chains are able to associate with surrogate L chain (11, 24), but those cells that have a H chain that can associate with surrogate L chain express pre-BCRs on their surface. These cells are pre-BII cells and they are the most abundant pre-B cell type in the mouse bone marrow (20). Cells that enter this compartment are initially pre-BCR⁺, large, and cycling, but transition into smaller noncycling pre-BCR^- cells as they mature (18, 25). Small pre-BII cells undergo L chain gene rearrangement (18, 25), and those that express an L chain that can pair with the H chain express surface IgM and are defined as immature B cells. These cells exit the bone marrow and migrate to the spleen, where they differentiate to mature recirculating B cells.

We have followed the differentiation of B cells expressing a single V_H gene segment, V_H12, because V_H12 B cells provide an unusual window on B cell development. Most V_H12 B cells in adult mice bind the common phospholipid phosphatidylcholine (PtC) and are B-1 (26, 27). All V_H12 H chains from these cells have a 10/G4 third complementarity-determining region (CDR3) and pair with V4/5H L chains (26, 27, 28). There is a strong bias for the differentiation of V_H12 B cells with the ability to bind PtC. First, the majority (95%) of V_H12-expressing cells are selectively lost during the transition from pre-BI to pre-BII (29). V_H12 pre-B cell survival appears to be dependent on the structure of the H chain CDR3; those with a 10/G4 CDR3 are favored for survival, while those of other CDR3 sequences (designated non-10/G4) are generally disfavored. Non-10/G4 V_H12 H chains can associate with surrogate L chain and be expressed on the cell surface as a pre-BCR in cells of a pre-B cell line (29), indicating that the inability to support pre-B cell differentiation is not necessarily due to an inability to form a pre-BCR. Second, 10/G4 V_H12 H chains are unable to associate with most L chains (30), and V4/5H is one of the few L chains with which it will pair. This bias in association in part creates a high frequency of B cells that can bind PtC. These cells are then selected into the B-1 subset (26, 31, 32). The combination of selection for V_HCDR3 and V4/5H strongly implies that PtC-specific B-1 cells have important survival value. Indeed, anti-PtC Abs have been demonstrated to provide protection against certain bacterial infections (33).

To understand the events that affect the loss of non-10/G4 pre-BII cells, we compared B cell differentiation between 10/G4 and non-10/G4 V_H12 transgenic (Tg) mice. The 10/G4 V_H12 Tg mice (6-1 mice) were previously generated and described (26), and produce B cells of both the conventional and B-1 subsets (26). We report in this work that non-10/G4 V_H12 Tg mice carrying an 8/G0 V_H12 rearrangement are similar to 10/G4 V_H12 Tg mice in that both drive differentiation to the pre-BII cell stage, exclude endogenous gene rearrangement, and initiate L chain gene rearrangement. However, unlike 10/G4 pre-B cells, 8/G0 pre-B cells are deficient in cell division at the large pre-BII cell stage and are unable to generate Tg-expressing B cells. Thus, 8/G0 B cell differentiation is deficient at both the pre-B and B cell stages.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mice

V_H12 (10/G4 (6-1)) (26) and 2-12H (34) Tg mice have been previously described. V1 H chain Tg mice were kindly provided by J. Kenny (National Cancer Institute, Frederick, MD), and recombination-activating gene (RAG)-1^-/- mice were purchased from The Jackson Laboratory (Bar Harbor, ME). The 8/G0 Tg mice were generated using a non-10/G4 V_H12-D-J_H1 construct designated 8/G0. The construct used is identical with that used to make 10/G4 (6-1) Tg mice, except that the CDR3 is 8 aa in length and lacks a Gly (29). The 8/G0 Tg mice were produced by the University of North Carolina Transgenic Mouse Facility by microinjection of the construct into fertilized eggs of (C57BL/6 x SJL)F₂ mice. Mouse lines carrying the 8/G0-Cµ were identified by PCR analysis of tail DNA using an oligonucleotide complementary to a sequence of the V_H12 (5'-CTTCCTTACCTGCTCTATTACTGGTTTCC-3') and an oligonucleotide complementary to a sequence 3' of J_H1 exon (5'-TGAGGAGACGGTGACCGTGGTC-3'). DNA was prepared by incubating tail snips in 50 mM Tris-HCl (pH 8), 100 mM EDTA, 100 mM NaCl, and 1% SDS with 1 µg/µl proteinase K at 55°C overnight. PCR was performed as described (34). The 10/G4 (6-1) and 8/G0 Tg mice have been maintained by backcrossing male Tg⁺ mice with female C.B17 mice. Mice were bred and maintained in our own pathogen-free mouse colony at University of North Carolina.

Antibodies

mAbs against B220 (RA3-6B2), IgM^a (DS-1), IgM^b (AF7-78), CD43 (S7), CD25 (7D4), CD2 (RM2-5), and c-kit (2B8) were obtained from BD PharMingen (San Diego, CA), and were either directly conjugated to FITC, R-PE, or Cy-Chrome, or were biotinylated. Unlabeled and FITC-conjugated goat anti-mouse µ- or -chain Abs were purchased from Southern Biotechnology Associates (Birmingham, AL).

Flow cytometry

To detect membrane molecules, single cell suspensions were prepared in HBSS (without Ca²⁺, Mg²⁺, and phenol red) containing 3% FCS and 0.1% sodium azide (CHBSS). FcR were blocked by incubation with mAb 2.4G2 (purified from 2.4G2 hybridoma culture supernatant). Cells were then stained with appropriate concentrations of the above Abs in a volume of 50 µl and incubated at 4°C in the dark for 20 min. Biotinylated mAbs were revealed with streptavidin-conjugated Cy-Chrome (BD PharMingen). For the detection of intracellular µH- or L-chains, bone marrow cells were first stained with B cell phenotype-specific Abs, followed by fixation with 1% paraformaldehyde. Cells were then permeabilized with 0.04% saponin (Sigma, St. Louis, MO) in 0.5% BSA/PBS buffer and stained with FITC-conjugated goat anti-mouse µ- or -chain Abs for 30 min at 4°C. After washing twice with saponin buffer and once with CHBSS, cells were analyzed using a FACScan (BD Biosciences, Mountain View, CA) with acquisition computer and software from Cytomation (Fort Collins, CO). All data represent cells falling within the lymphocyte gate determined by forward and 90°C light scatter. All contour plots are 5% probability.

For cell-sorting experiments, 5–10 x 10⁷ adult (8–20 wk) bone marrow cells were stained with Abs recognizing B220, IgM^a/b, and CD43. B220⁺ IgM^a/b- CD43^- fraction D cells were sorted on a MoFlo high speed sorter (Cytomation). Sorted populations were always >95% pure. The cells were then fixed with 70% ethanol and stained with a buffer containing 100 µg/ml propidium iodide and 250 µg/ml RNase A (Boehringer Mannheim, Indianapolis, IN) overnight at 4°C. The DNA content was analyzed by FACScan, as described.

Analysis of transcripts

Bone marrow pre-B cells were purified in a two-step process using magnetic beads. Bone marrow cells were stained with biotin anti-IgM and incubated with streptavidin-coated Dynabeads M-280 magnetic beads (Dynal, Lake Success, NY). The IgM⁺ cells were removed by magnet. Bone marrow cells depleted of IgM⁺ cells contained <1% IgM⁺ cells. These cells were incubated with anti-B220-coated beads (Miltenyi Biotec, Auburn, CA) and B220⁺IgM^- cells separated by magnet. These cells were >95% pure based on flow cytometry analysis. Total RNA was extracted from purified cells, and RT-PCR of Cµ transcripts was done by 5'-RACE (Life Technologies, Gaithersburg, MD), according to the manufacturer’s instructions. cDNA was prepared using an oligonucleotide complementary to the first exon of Cµ (5'-ATCCTTGAAGGTTCAG-3'). The PCR was performed using a poly(G) oligonucleotide supplied by the manufacturer and a Cµ oligonucleotide (5'-TTCACCTGGAACTACCAGAAC-3'), which is internal to the Cµ oligonucleotide used to generate the cDNA. The RT-PCR products were cloned into the pAMP vector (Life Technologies) and subject to DNA sequencing, as described previously (29).

Cell transfection

To assess pre-BCR formation, a µ-chain-deficient pre-B cell line Bine 4.8, kindly provided by H.-M. Jack (Loyola University of Chicago, Maywood, IL), was transfected with H chain constructs, as described previously (29, 30). Briefly, cells were washed with PBS and resuspended in a Gene Pulser cuvette (0.4-cm electrode) in 0.45 ml PBS with 10 µg DNA linearized with SfiI (Life Technologies). Electroporation was done using a Bio-Rad Gene Pulser apparatus (Hercules, CA). Cells were cultured in RPMI 1640 medium supplemented with 10% FCS, 2 mM glutamine, 100 U/ml penicillin, and streptomycin for 24 h and then plated in 24-well plates in the presence of 0.6 mg/ml G418 (Life Technologies). After 7–9 days, cells from individual wells were used for analysis of H chain expression and pre-BCR formation.

To assess the ability of V_H12 H chains to associate with conventional L chains, L chain-only hybridoma cells were transfected with the 8/G0 construct, as described (30). In the case of V4/5H and V21C, the 8/G0 construct was cotransfected with L chain constructs into P3-X63-Ag8.653, as described (30). The bulk cultures of G418-selected cells were used for testing secretion of IgM molecules.

ELISA

To quantify the expression levels of cytoplasmic µH-chains in transfected pre-B cell lines, a cell lysate ELISA was used, as described previously (29). Briefly, 96-well microtiter plates coated with polyclonal goat anti-mouse µH-chain (Southern Biotechnology Associates) were incubated with 5000 cells in lysis buffer containing 1% Nonidet P-40, 10 mM Tris, pH 7.4, 10 mM NaCl, 0.3 mM MgCl₂, 200 µg/ml PMSF, and 2 µg/ml aprotinin. After extensive washes, the plates were incubated with alkaline phosphatase-labeled polyclonal goat anti-mouse µH-chain Ab (Southern Biotechnology Associates), followed by p-nitrophenyl phosphate (Sigma) to develop the reaction. OD readings were determined by an automated plate reader (Molecular Devices, Sunnyvale, CA).

To test whether a complete Ig molecule was formed by L chain-only cell lines, supernatant was subjected to ELISA using microplates coated with polyclonal goat anti-mouse µH-chain (Southern Biotechnology Associates) and alkaline phosphatase-labeled polyclonal goat anti-mouse L-chain (Southern Biotechnology Associates) to develop the reaction (30). In those cases in which Ig secretion was not detected, the production of H and L chains was confirmed by ELISA using cell lysates and the polyclonal goat anti-mouse µH- or L-coated plates, as above. The former were developed with phosphatase-labeled polyclonal goat anti-mouse µH-chain to detect H chain, and the latter were developed with phosphatase-labeled polyclonal goat anti-mouse L-chain to detect L chain (30). OD readings were determined with an automated plate reader (Molecular Devices).

5-bromo-2'-deoxyuridine (BrdU) labeling

Adult mice were BrdU labeled in vivo using the method of Allman et al. (35). Briefly, BrdU (Sigma) was administered in drinking water at 0.5 mg/ml with 1 mg/ml dextrose continuously for 2–3 days, or injected i.p. at 0.6 mg per mouse every 12 h for 24 h. At each time point, mice were sacrificed and bone marrow cells were isolated for staining with anti-IgM PE and anti-B220 CyChrome, as described above. Subsequent permeabilization followed by treatment with DNase (Sigma) and staining with anti-BrdU-FITC (BD Biosciences) allowed use of FACS analysis to assess the fraction of BrdU-labeled B cells.

Statistical analysis

The paired Student’s t test and the independent Student’s t test were used to assess the significance of the observed differences in the number of B cells in each subpopulation and in pre-BCR expression levels. p < 0.05 was considered significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
To understand the loss of non-10/G4 V_H12 pre-B cells, we have generated Tg mice using an 8/G0 V_H12-D-J_H1 construct. The 8/G0 H chains can associate with both the 5 and V_preB components of the surrogate L chain, and are expressed on the cell surface of a pre-B cell line (29). Comparison of B cell development between these mice and our previously generated 10/G4 (6-1) Tg mice permits the identification of developmental differences responsible for the loss of most non-10/G4 V_H12 pre-B cells. Two 8/G0 founder mice were generated and backcrossed to C.B17 (IgH^b) mice. The characteristics of one are described in detail in this work.

The 8/G0 H chains are unable to support B cell development

Adult peripheral B cells in 8/G0 Tg mice were examined for expression of endogenous (IgH^b allotype) and transgene (IgH^a allotype) H chains. There are no detectable IgH^a B cells in the bone marrow of these mice (Fig. 1A), nor are there B cells expressing either allotype in the neonatal spleen (Fig. 2). To exclude the possibility that the absence of B cell development is due to a lack of 8/G0 H chain expression, cells were stained for cytoplasmic µH-chain. As shown in Fig. 3, nearly all small 8/G0 pre-B cells stain brightly for cytoplasmic µH-chain. This level is not different from that in small pre-BII cells of non-Tg littermates. Anti-allotypic reagents do not recognize cytoplasmic H chains of pre-B cells (36), and therefore cannot be used to confirm that these H chains are of transgene origin. However, using RT-PCR on purified B220⁺ IgM^- bone marrow cells, we could only detect 8/G0 transcripts (Table I), suggesting that the cytoplasmic µH-chain in these mice is of 8/G0, not endogenous, origin. That 8/G0 H chains are produced was definitively demonstrated using 8/G0/RAG-1^-/- mice. As these mice cannot undergo VDJ rearrangement, any µ protein present must be transgene encoded. As shown in Fig. 3, 8/G0/RAG-1^-/- small pre-B cells have the same cytoplasmic µH-chain level as non-Tg littermate mice. Therefore, we conclude that the 8/G0 transgene encodes the majority, if not all, cytoplasmic µH-chains in 8/G0 pre-B cells.

View larger version (30K): [in this window] [in a new window]   FIGURE 1. The 8/G0 pre-B cells differentiate to the pre-BII cell stage. Bone marrow cells from 8/G0 Tg, 10/G4 (6-1) Tg, and non-Tg littermate mice were stained with FITC-conjugated anti-B220, PE-conjugated anti-IgMa or IgMb, and biotinylated anti-c-kit, CD2, CD25, or CD43. Biotinylated reagents were visualized with CyChrome streptavidin. A, Cells stained with B220 and IgMa or IgMb (top row) were gated on lymphocytes according to forward and side light scatter. All other histograms are also gated on IgM- cells. Gates R1 through R6 indicate the criteria used to measure cell numbers in each subset presented in Table II. The percentage of cells in gates R2 and R3 of total lymphocytes is given. B, Size of cells in the pre-BII compartment. B220+, IgMa/b-, and CD25+ cells were gated (R4 in A). The horizontal line is in the same position in all three histograms. C, CD43 expression level by pre-BII cells. The pre-BII cells were gated on B220+, IgMa/b- cells (R1 in A).

View larger version (38K): [in this window] [in a new window]   FIGURE 2. The 8/G0 splenic B cells do not express transgene H chains. Spleen cells from newborn and adult mice were stained with CyChrome-conjugated anti-B220, PE-conjugated anti-IgMa, and FITC-conjugated anti-IgMb. Contour plots are gated on the lymphocyte gate (adult) or B220+ cells in the lymphocyte gate (neonatal). Twenty thousand cells were analyzed from each mouse. The percentage of gated cells in each box is given for adult mice, and the percentage of cells in each of the indicated quadrants is provided for neonatal mice.

View larger version (15K): [in this window] [in a new window]   FIGURE 3. The 8/G0 pre-B cells express cytoplasmic transgene H chains. Bone marrow cells from 8/G0 Tg, 8/G0/RAG-1-/- Tg, and non-Tg littermate mice were stained with CyChrome-conjugated anti-B220 and PE-conjugated anti-IgMa and IgMb, followed by permeabilization and staining with FITC-conjugated anti-µH-chain Abs. The histograms are gated on small B220+, IgMa/b- cells, with the exception of those from RAG-1-/- mice, which are gated on both large and small B220+, IgMa/b- cells. The percentage of cytoplasmic µH-chain-positive cells (indicated by the horizontal bar) is given.

View larger version (24K): [in this window] [in a new window]   FIGURE 4. The V1 transgene rescues B cell development in 8/G0 Tg mice. Spleen cells from V1-only Tg, 8/G0 Tg, and V1/8/G0 Tg mice were stained and analyzed, as described in Fig. 2. The percentage of IgMa, B220+ cells is provided for each histogram.

Although 8/G0 H chains do not support B cell development, they appear to mediate allelic exclusion. The 8/G0 Tg mice have only small numbers of IgH^b-expressing B cells in adult bone marrow and neonatal spleen relative to non-Tg littermates (Figs. 1A and 2). The small size of these populations, coupled with the fact that these cells do not coexpress the IgH^a allotype, suggests that the 8/G0 H chain is an excellent excluder of endogenous H chain gene rearrangement. We cannot exclude the possibility that IgH^b rearrangements occur and that cells coexpressing both H chains are eliminated, but we think this is unlikely because we find no evidence of IgH^b transcripts in 8/G0 pre-B cells (Table I). Thus, the most likely basis for the absence of IgH^b-expressing B cells in 8/G0 Tg mice is allelic exclusion induced by 8/G0 H chains. The number of IgH^b B cells in adult spleen (Fig. 2) increases with age (data not shown), suggesting that a few IgH^b B cells are generated by the bone marrow and that they accumulate in the spleen over time.

The 8/G0 pre-B cells differentiate to small pre-BII cells, but undergo limited cell division at the large pre-BII cell stage

To examine 8/G0 pre-B cell development, comparison was made between B-lineage cells of 8/G0, 10/G4 (6-1), and non-Tg mouse bone marrow. The cell surface markers used to distinguish pro/pre-BI and pre-BII cells include CD43, CD25, CD2, and c-kit. Pro- and pre-BI cells are CD43^high, CD25^-, CD2^-, and c-kit⁺, while pre-BII cells are CD43^low/-, CD25⁺, CD2⁺, and c-kit^- (14, 18, 25).

Like non-Tg mice, 8/G0 and 10/G4 (6-1) Tg mice have B220⁺, CD43^high pro/pre-BI bone marrow cells (Fig. 1A). The remaining pre-B cells in mice of both 8/G0 and 10/G4 (6-1) exhibit a pre-BII phenotype. They are CD2⁺, CD25⁺, and c-kit^- (Fig. 1A). Although the significance of this is not yet understood, the levels of CD43 expression by 8/G0 and 10/G4 pre-B cells are intermediate (CD43^int) to pre-BI and pre-BII of wild-type mice (Fig. 1C). Also, the 8/G0 and 10/G4 pre-BII cells are equivalent in size to the pre-BII cells of non-Tg mice (Fig. 1B). Thus, both 8/G0 and 10/G4 pre-B cells differentiate to the small pre-BII cell stage.

The sizes of the pre-B cell populations are shown in Table II and, with one exception, are not significantly different. The one significant difference is that while control mice have equal numbers of pro/pre-BI cells and large pre-BII cells, 8/G0 Tg mice have 57% fewer large pre-BII cells than pro/pre-BI cells (Table II; p < 0.01). This indicates that the large pre-BII population of 8/G0 Tg mice is unusually small. This is also evident by comparison of the proportion of large and small pre-BII cells in these mice (Table III). The 8/G0 Tg mice have a smaller percentage of large pre-BII cells (p < 0.01) and a larger percentage of small pre-BII cells (p < 0.01) than either non-Tg or 10/G4 (6-1) Tg mice (Table III). A third H chain Tg mouse, 2-12H, that expresses a J558 H chain has a frequency of large and small pre-BII cells equivalent to those of non-Tg and 10/G4 (6-1) Tg mice. Because most large pre-BII cells from normal mice are in cell cycle (18, 25), the proportions of cycling pre-B cells in these mice were determined. As shown in Table III, 30% of pre-BII cells of non-Tg, 10/G4 (6-1), and 2-12 Tg mice are in the S/G₂M phase of the cell cycle, consistent with previous reports using non-Tg mice (18, 25). However, only 12.5% of 8/G0 pre-BII cells are in cycle, consistent with the reduced frequency and number of large pre-BII cells in these Tg mice (Tables II and III). This difference in distribution of pre-BII cells between large and small is also evident between 8/G0 Tg and 10/G4 (6-1) Tg mice that lack RAG-1 expression, indicating that this difference is due to the 8/G0 H chain and not to coexpression of an endogenous H chain. Together these data suggest that there is less clonal expansion at the large pre-BII cell stage in 8/G0 Tg mice than in 10/G4 (6-1) Tg mice.

View larger version (42K): [in this window] [in a new window]   FIGURE 5. The 8/G0 small pre-BII cells have a slower turnover rate than 10/G4 small pre-BII cells. BrdU incorporation into small B220+, IgM- cells of 8/G0 Tg, 10/G4 () Tg, and non-Tg littermate mice was measured at 1, 2, and 3 days following initiation of treatment, as described in Materials and Methods. Each time point represents three or more mice. The 8/G0 are significantly different from 10/G4 (6-1) and non-Tg pre-B cells at day 1 (*, p < 0.001) and at day 2 (#, p < 0.01). The 10/G4 (6-1) pre-B cells incorporate BrdU significantly faster than non-Tg B cells (**, p < 0.05).

H chain association with surrogate L chain is critical to pre-BII cell differentiation (11, 12, 13). We have previously shown that the 8/G0 H chain can associate with surrogate L chain in a pre-B cell line (29), and the fact that 8/G0 Tg mice have a pre-BII cell population indicates that it can associate with surrogate L chain in vivo. To more carefully assess the ability of the 8/G0 and 10/G4 H chains to form pre-BCRs, the 8/G0 and 10/G4 H chain constructs were transfected into the pre-B cell line Bine 4.8. As an H chain control, the J558 2-12H construct was also transfected. The Bine 4.8 pre-B cell line produces surrogate L chains, but lacks H chains, and therefore cannot produce a pre-BCR. Multiple cell lines from three independent transfections were compared for each H chain. As shown in Fig. 6 and Table IV, cells transfected with 10/G4 and 2-12H constructs have roughly twice as much cell surface pre-BCR as those transfected with the 8/G0 construct (p < 0.001). Comparison was made only between cell lines producing equal amounts of cytoplasmic H chain (Fig. 6 and Table IV). Thus, cell lines producing 8/G0 H chains are significantly less efficient at formation of the pre-BCR than those producing 10/G4 H chains.

View larger version (23K): [in this window] [in a new window]   FIGURE 6. The 8/G0 pre-BCR is expressed poorly by cells of a pre-B cell line. The µH-chain-loss pre-B cell line Bine 4.8 was transfected with 8/G0, 10/G4, or 2-12H constructs. The G418-resistant cells from separate wells were analyzed by flow cytometry. Staining with FITC-conjugated polyclonal goat anti-mouse IgM Abs revealed the pre-BCR expression levels on transfected cells. The intracellular Tg expression levels were measured by staining paraformaldehyde-fixed and membrane-permeabilized cells with the same Ab. The data shown are of representative lines. The light line is the unstained control, and the dark line is the staining by the anti-IgM Ab.

L chain rearrangement is initiated at the small pre-BII cell stage, and L-chains are present in the cytoplasm of a significant percentage of pre-BII cells of normal mice (38). To determine whether 8/G0 pre-BII cells initiate V gene rearrangement, 8/G0 pre-B cells were examined for the presence of cytoplasmic L-chains. Approximately 14% of IgM^- B220⁺ cells of non-Tg littermate mice are cytoplasmic L-chain positive (Fig. 7) in agreement with Pelanda et al. (39), and an equivalent number of 8/G0 pre-B cells are cytoplasmic L-chain positive (Fig. 7). J rearrangement was verified by ligation-mediated PCR (data not shown). Thus, the pre-BII cells of 8/G0 Tg mice have initiated L chain gene rearrangement, despite the fact that essentially none reach the immature B cell stage. We rule out the possibility that the L-chain gene rearrangement in 8/G0 pre-BII cells is driven by expression of an endogenous µH-chain, because no endogenous µH-chain transcripts are detected among 8/G0 pre-B cells (Table I), and all 8/G0 small pre-BII cells express the transgene H chain (Fig. 3).

View larger version (21K): [in this window] [in a new window]   FIGURE 7. Some 8/G0 pre-BII cells express cytoplasmic L-chains. Bone marrow cells from 8/G0 Tg, 8/G0/RAG-1-/- Tg, and non-Tg littermate mice were stained for B220IgMaIgMb, and cytoplasmic L-chains. Histograms are gated on B220+ IgMa- IgMb- cells. The percentage of cells inside the indicated gates representing the cells that express L-chains is given.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The low percentage of large cycling pre-BII cells in 8/G0 Tg mice relative to 10/G4 Tg mice is not due to an inability to express an 8/G0 pre-BCR in vivo. First, 8/G0 pre-BCRs can be expressed by cells of a pre-B cell line, albeit at lower than normal levels (Table IV). Second, 8/G0 pre-B cell differentiation advances beyond the pre-BI cell stage, and 8/G0 H chains are excellent excluders of endogenous H chain gene rearrangement. Neither can occur in the absence of a functional pre-BCR (9, 10, 42). Third, 8/G0 Tg, RAG-1^-/- mice have the same pre-BII-like population (Table III and data not shown), formally excluding the possibility that an endogenous H chain is responsible for pre-BII cell development. Nor is there a signaling pathway defect downstream of the pre-BCR that blocks all B cell development regardless of the H chain, because like V1-only Tg mice, 8/G0-V1 double Tg mice produce large numbers of B cells. Thus, we conclude that an 8/G0 pre-BCR is formed and expressed in vivo, but that it is deficient in some functions.

Our data suggest that the deficiency in 8/G0 pre-BCR function is due to a low expression level. The 8/G0 pre-BCRs are expressed at only half the level of 10/G4 pre-BCRs in a pre-B cell line (Table IV and Fig. 6). We presume that this is due to a poor ability of the 8/G0 H chain to associate with the surrogate L chain, and therefore that V_HCDR3 structure determines the ability of V_H12 H chains to form a pre-BCR. The most significant defect in 8/G0 pre-B cells is the reduced percentage of large pre-BII cells in cycle. The pre-BCR is required for large pre-B cells to undergo clonal expansion (9, 43, 44, 45), but this is the first demonstration of suboptimal clonal expansion in vivo. Assuming that the 8/G0 pre-BCR is expressed at subnormal levels in vivo, these data argue that the strength of the clonal growth signal is a function of the pre-BCR expression level. Less clonal growth of large pre-BII cells would explain why there are fewer large pre-BII cells than pro/pre-BI cells in 8/G0 Tg mice (Table II). The prolonged t_1/2 of small 8/G0 pre-BII cells is probably related to the reduced input of new cells from the large pre-BII compartment. It is suggested that small pre-BII cells do not differentiate to immature B cells until the small pre-BII compartment has been filled (19). Thus, a slower input of cells into this compartment would necessitate a longer t_1/2.

The pre-BCR is able to deliver signals through multiple pathways, providing a possible mechanism for partial pre-BCR function in vivo (46). Iritani et al. (47) have demonstrated that there is more than one signaling pathway leading from the pre-BCR. They find that constitutively activated Raf-1 drives B cell differentiation, but not allelic exclusion. The activation of these pathways could be dependent on the strength of the signals through the pre-BCR. Additional sites on a signaling molecule or additional components of a pathway may be phosphorylated with increasing strength of the pre-BCR signal, as seen in the -chain of the TCR complex and in an FcR (48, 49). We suggest that the separate pathways are differentially sensitive to receptor level, making it possible for the 8/G0 pre-BCR to signal allelic exclusion and differentiation to pre-BII, but not normal clonal growth. A greater sensitivity to induction of allelic exclusion than to cell division would ensure that only allelically excluded pre-B cells continue differentiation, thereby preventing the production of B cells with multiple H chains. Equal sensitivity to allelic exclusion and differentiation would likely result in an unacceptable frequency of pre-B cells in which complete differentiation is promoted in the absence of allelic exclusion, and resulting in the production of B cells that express two H chains.

The normal cycling observed for 10/G4 pre-BII cells, but not 8/G0 pre-BII cells, is consistent with the selective advantage of 10/G4 V_H12 pre-B cells over non-10/G4 V_H12 pre-B cells in non-Tg mice. However, the ability of 8/G0 H chains to drive differentiation to the pre-BII cell stage does not explain the tremendous loss of non-10/G4 V_H12 pre-B cells in non-Tg mice (29). One possible explanation is that other non-10/G4 V_H12 H chains are less efficient at driving large pre-BII proliferation than are 8/G0 H chains, or are unable to support differentiation beyond the pre-BI cell stage. This possibility is supported by the observation that Bine 4.8 pre-B cells transfected with two other non-10/G4 H chain expression constructs (14/G7 and 12/G3, 7) express less surface pre-BCR than cells transfected with the 8/G0 expression construct (29). These H chains may be poorer at association with surrogate L chain than 8/G0 H chains, and thus unable to support pre-BII cell differentiation, similar to H chains that cannot form a pre-BCR (11, 12, 13, 24). Thus, non-10/G4 H chains may exhibit a range of abilities to drive pre-BII cell differentiation based on their ability to associate with surrogate L chain, and thereby account for the absence of most non-10/G4 pre-BII cells from the repertoire (29).

The poor ability of 8/G0 H chains to associate with conventional L chains indicates that the V_HCDR3 limits the differentiation of V_H12 pre-BII cells to immature B cells. This is consistent with the function of the pre-BCR to perform a quality control function for L chain association (50). Thus, the absence of 8/G0 B cells in either the bone marrow or spleen may be due to the inability of 8/G0 H chains to associate with most conventional L chains. However, we cannot exclude other explanations for the inability of 8/G0 to drive differentiation to the B cell stage, because 8/G0 H chains can associate with at least one V10 L chain. Even a low frequency of B cell development, as in 5T mice (9), results in accumulation of significant numbers of splenic B cells. In addition, 10/G4 H chains are deficient in ability to associate with L chains, yet 10/G4 Tg mice (6-1) generate large numbers of B cells of both the conventional and B-1 subsets (26, 30). Thus, the absence of 8/G0 B cells could also be due to a defect at the pre-BII cell stage that blocks B cell differentiation despite an ability for 8/G0 H chains to associate with at least some L chains. For example, poor expression of the 8/G0 pre-BCR may be unable to mediate positive selection and turn off an ongoing programmed cell death pathway, as suggested previously (23). Such a possibility is supported by the observation that a 2b transgene shows a similar deficit in B cell production (40), despite a demonstrated ability to associate with conventional L chains (40, 41). Efforts are currently underway to resolve these two possibilities.

The V_HCDR3 selection at the pre-B cell stage favors the development of the V_H12 B-1 cell repertoire. Although 10/G4 pre-BII cell development appears to be normal, we have previously demonstrated a limitation at the pre-BII to immature B cell checkpoint for these cells (30). The 10/G4 V_H12 B cells that express a V4/5H L chain rearrangement are favored at this transition due to a limited repertoire of L chains with which 10/G4 H chains can associate (30), and to selective maturation to the B-2 cell stage of V4/5H-expressing 10/G4 B cells in the spleen (51). The inability of 8/G0 H chains to associate with V4/5H L chains (Table IV) indicates that the ability to associate with this L chain is determined by V_HCDR3. Thus, there is synergy between the selection for V_HCDR3 at the pre-BII cell stage and for the L chain at the immature B cell stage. This focuses the V_H12 B cell repertoire to a combination of H and L chain that can bind PtC. PtC-specific B cells are selected into the B-1 subset from B-2 cell precursors (26, 31, 32) and are responsible for a high level of anti-PtC Abs in circulation (21). This Ab, like other Abs produced by B-1 cells, probably provides an important early defense during bacterial infections, as anti-PtC Abs are protective against bacterial infections in acute peritonitis (33). We suggest that this extraordinary selection to produce anti-PtC V_H12 B cells with a 10/G4 V_HCDR3 and a V4/5H L chain is the underlying evolutionary basis for the selective loss of non-10/G4 V_H12 pre-B cells. Thus, V_H12 H chains have evolved such that association with surrogate L chain and conventional V4/5H L chains is most efficient with a 10/G4 CDR3.

	Acknowledgments

 
We gratefully acknowledge the Flow Cytometry Facility and the Transgenic Mouse Facility at the University of North Carolina for their assistance with this work. We are also indebted to Dr. James Kenny for his V1 Tg mice, and Anne Wolthusen for her assistance with mouse breeding and genetic analysis.

	Footnotes

 
¹ This work was supported by National Institutes of Health Grants AI29576 and AI43587 and a grant from the Arthritis Foundation to S.H.C.

² H.W. and J.Y. contributed equally to this work.

³ Current address: Curagen Corporation, 322 East Main Street, Branford, CT 06405.

⁴ Address correspondence and reprint requests to Dr. Stephen H. Clarke, Department of Microbiology and Immunology, CB#7290, 804 Mary Ellen Jones Building, University of North Carolina, Chapel Hill, NC 27599. E-mail address: shl{at}med.unc.edu

⁵ Abbreviations used in this paper: BCR, B cell Ag receptor; BrdU, 5-bromo-2'-deoxyuridine; CDR3, third complementarity-determining region; PtC, phosphatidylcholine; Tg, transgenic; RAG, recombination-activating gene.

Received for publication May 17, 2001. Accepted for publication May 23, 2001.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Tsubata, T., M. Reth. 1990. The products of pre-B cell-specific genes (5 and VpreB) and the immunoglobulin µ chain form a complex that is transported onto the cell surface. J. Exp. Med. 172:973.[Abstract/Free Full Text]
2. Nishimoto, N., H. Kubagawa, T. Ohno, G. L. Gartland, A. K. Stankovic, M. D. Cooper. 1991. Normal pre-B cells express a receptor complex of µ heavy chains and surrogate light-chain proteins. Proc. Natl. Acad. Sci. USA 88:6284.[Abstract/Free Full Text]
3. Kerr, W. G., M. D. Cooper, L. Feng, P. D. Burrows, L. M. Hendershot. 1989. µ heavy chains can associate with a pseudo-light chain complex (L) in human pre-B cell lines. Int. Immunol. 1:355.[Abstract/Free Full Text]
4. Karasuyama, H., A. Kudo, F. Melchers. 1990. The proteins encoded by the VpreB and 5 pre-B cell-specific genes can associate with each other and with µ heavy chain. J. Exp. Med. 172:969.[Abstract/Free Full Text]
5. Misener, V., G. P. Downey, J. Jongstra. 1991. The immunoglobulin light chain related protein 5 is expressed on the surface of mouse pre-B cell lines and can function as a signal transducing molecule. Int. Immunol. 3:1129.[Abstract/Free Full Text]
6. Nagata, K., T. Nakamura, F. Kitamura, S. Kuramochi, S. Taki, K. S. Campbell, H. Karasuyama. 1997. The Ig/Ig heterodimer on µ-negative proB cells is competent for transducing signals to induce early B cell differentiation. Immunity 7:559.[Medline]
7. Brouns, G. S., E. de Vries, C. J. van Noesel, D. Y. Mason, R. A. van Lier, J. Borst. 1993. The structure of the µ/pseudo light chain complex on human pre-B cells is consistent with a function in signal transduction. Eur. J. Immunol. 23:1088.[Medline]
8. Kitamura, D., J. Roes, R. Kuhn, K. Rajewsky. 1991. A B cell-deficient mouse by targeted disruption of the membrane exon of the immunoglobulin µ chain gene. Nature 350:423.[Medline]
9. Kitamura, D., A. Kudo, S. Schaal, W. Muller, F. Melchers, K. Rajewsky. 1992. A critical role of 5 protein in B cell development. Cell 69:823.[Medline]
10. Gong, S., M. C. Nussenzweig. 1996. Regulation of an early developmental checkpoint in the B cell pathway by Ig. Science 272:411.[Abstract]
11. ten Boekel, E., F. Melchers, A. G. Rolink. 1997. Changes in the V_H gene repertoire of developing precursor B lymphocytes in mouse bone marrow mediated by the pre-B cell receptor. Immunity 7:357.[Medline]
12. ten Boekel, E., F. Melchers, A. G. Rolink. 1998. Precursor B cells showing H chain allelic inclusion display allelic exclusion at the level of pre-B cell receptor surface expression. Immunity 8:199.[Medline]
13. Kline, G. H., L. Hartwell, G. B. Beck-Engeser, U. Keyna, S. Zaharevitz, N. R. Klinman, H. M. Jack. 1998. Pre-B cell receptor-mediated selection of pre-B cells synthesizing functional µ heavy chains. J. Immunol. 161:1608.[Abstract/Free Full Text]
14. Ehlich, A., V. Martin, W. Muller, K. Rajewsky. 1994. Analysis of the B-cell progenitor compartment at the level of single cells. Curr. Biol. 4:573.[Medline]
15. Tsubata, T., R. Tsubata, M. Reth. 1992. Crosslinking of the cell surface immunoglobulin (µ-surrogate light chains complex) on pre-B cells induces activation of V gene rearrangements at the immunoglobulin locus. Int. Immunol. 4:637.[Abstract/Free Full Text]
16. Spanopoulou, E., C. A. Roman, L. M. Corcoran, M. S. Schlissel, D. P. Silver, D. Nemazee, M. C. Nussenzweig, S. A. Shinton, R. R. Hardy, D. Baltimore. 1994. Functional immunoglobulin transgenes guide ordered B-cell differentiation in Rag-1-deficient mice. Genes Dev. 8:1030.[Abstract/Free Full Text]
17. Young, F., B. Ardman, Y. Shinkai, R. Lansford, T. K. Blackwell, M. Mendelsohn, A. Rolink, F. Melchers, F. W. Alt. 1994. Influence of immunoglobulin heavy- and light-chain expression on B-cell differentiation. Genes Dev. 8:1043.[Abstract/Free Full Text]
18. Hardy, R. R., C. E. Carmack, S. A. Shinton, J. D. Kemp, K. Hayakawa. 1991. Resolution and characterization of pro-B and pre-pro-B cell stages in normal mouse bone marrow. J. Exp. Med. 173:1213.[Abstract/Free Full Text]
19. Rolink, A., F. Melchers. 1993. Generation and regeneration of cells of the B-lymphocyte lineage. Curr. Opin. Immunol. 5:207.[Medline]
20. Osmond, D. G.. 1990. B cell development in the bone marrow. Semin. Immunol. 2:173.[Medline]
21. ten Boekel, E., F. Melchers, A. Rolink. 1995. The status of Ig loci rearrangements in single cells from different stages of B cell development. Int. Immunol. 7:1013.[Abstract/Free Full Text]
22. Karasuyama, H., A. Rolink, Y. Shinkai, F. Young, F. W. Alt, F. Melchers. 1994. The expression of Vpre-B/5 surrogate light chain in early bone marrow precursor B cells of normal and B cell-deficient mutant mice. Cell 77:133.[Medline]
23. Rolink, A., A. Kudo, H. Karasuyama, Y. Kikuchi, F. Melchers. 1991. Long-term proliferating early pre B cell lines and clones with the potential to develop to surface Ig-positive, mitogen reactive B cells in vitro and in vivo. EMBO J. 10:327.[Medline]
24. Keyna, U., S. E. Applequist, J. Jongstra, G. B. Beck-Engeser, H. M. Jack. 1995. Igµ heavy chains with V_H81X variable regions do not associate with 5. Ann. NY Acad. Sci. 764:39.[Medline]
25. Rolink, A., U. Grawunder, T. H. Winkler, H. Karasuyama, F. Melchers. 1994. IL-2 receptor chain (CD25, TAC) expression defines a crucial stage in pre-B cell development. Int. Immunol. 6:1257.[Abstract/Free Full Text]
26. Arnold, L. W., C. A. Pennell, S. K. McCray, S. H. Clarke. 1994. Development of B-1 cells: segregation of phosphatidylcholine-specific B cells to the B-1 population occurs after immunoglobulin gene expression. J. Exp. Med. 179:1585.[Abstract/Free Full Text]
27. Ye, J., S. K. McCray, S. H. Clarke. 1995. The majority of murine V_H12-expressing B cells are excluded from the peripheral repertoire in adults. Eur. J. Immunol. 25:2511.[Medline]
28. Pennell, C. A., K. M. Sheehan, P. H. Brodeur, S. H. Clarke. 1989. Organization and expression of V_H gene families preferentially expressed by Ly-1⁺ (CD5) B cells. Eur. J. Immunol. 19:2115.[Medline]
29. Ye, J., S. K. McCray, S. H. Clarke. 1996. The transition of pre-BI to pre-BII cells is dependent on the V_H structure of the µ/surrogate L chain receptor. EMBO J. 15:1524.[Medline]
30. Tatu, C., J. Ye, L. W. Arnold, S. H. Clarke. 1999. Selection at multiple checkpoints focuses V_H12 B cell differentiation toward a single B-1 cell specificity. J. Exp. Med. 190:903.[Abstract/Free Full Text]
31. Arnold, L. W., S. K. McCray, C. Tatu, S. H. Clarke. 2000. Identification of a precursor to phosphatidylcholine-specific B-1 cells suggesting that B-1 cells differentiate from splenic conventional B cells in vivo: cyclosporin A blocks differentiation to B-1. J. Immunol. 164:2924.[Abstract/Free Full Text]
32. Clarke, S. H., L. W. Arnold. 1998. B-1 cell development: evidence for an uncommitted immunoglobulin (Ig)M⁺ B cell precursor in B-1 cell differentiation. J. Exp. Med. 187:1325.[Abstract/Free Full Text]
33. Boes, M., A. P. Prodeus, T. Schmidt, M. C. Carroll, J. Chen. 1998. A critical role of natural immunoglobulin M in immediate defense against systemic bacterial infection. J. Exp. Med. 188:2381.[Abstract/Free Full Text]
34. Santulli-Marotto, S., M. W. Retter, R. Gee, M. J. Mamula, S. H. Clarke. 1998. Autoreactive B cell regulation: peripheral induction of developmental arrest by lupus-associated autoantigens. Immunity 8:209.[Medline]
35. Allman, D. M., S. E. Ferguson, V. M. Lentz, M. P. Cancro. 1993. Peripheral B cell maturation. II. Heat-stable antigen^hi splenic B cells are an immature developmental intermediate in the production of long-lived marrow-derived B cells. J. Immunol. 151:4431.[Abstract]
36. Stall, A. M. 1996. Mouse immunoglobulin allotypes. In Weir’s Handbook of Experimental Immunology, 5th Ed. L. A. Herzenberg, D. M. Weir, L. A. Herzenberg, and C. Blackwell, eds. Blackwell Science Publishers, Cambridge, p. 27.1.
37. Storb, U., C. Pinkert, B. Arp, P. Engler, K. Gollahon, J. Manz, W. Brady, R. L. Brinster. 1986. Transgenic mice with µ and genes encoding antiphosphorylcholine antibodies. J. Exp. Med. 164:627.[Abstract/Free Full Text]
38. Yamagami, T., E. ten Boekel, J. Andersson, A. Rolink, F. Melchers. 1999. Frequencies of multiple IgL chain gene rearrangements in single normal or L chain-deficient B lineage cells. Immunity 11:317.[Medline]
39. Pelanda, R., S. Schaal, R. M. Torres, K. Rajewsky. 1996. A prematurely expressed Ig transgene, but not VJ gene segment targeted into the Ig locus, can rescue B cell development in 5-deficient mice. Immunity 5:229.[Medline]
40. Kurtz, B. S., P. L. Witte, U. Storb. 1997. 2b provides only some of the signals normally given via µ in B cell development. Int. Immunol. 9:415.[Abstract/Free Full Text]
41. Roth, P. E., B. Kurtz, D. Lo, U. Storb. 1995. 5, but not µ, is required for B cell maturation in a unique 2b transgenic mouse line. J. Exp. Med. 181:1059.[Abstract/Free Full Text]
42. Loffert, D., A. Ehlich, W. Muller, K. Rajewsky. 1996. Surrogate light chain expression is required to establish immunoglobulin heavy chain allelic exclusion during early B cell development. Immunity 4:133.[Medline]
43. Ceredig, R., A. G. Rolink, F. Melchers, J. Andersson. 2000. The B cell receptor, but not the pre-B cell receptor, mediates arrest of B cell differentiation. Eur. J. Immunol. 30:759.[Medline]
44. Rolink, A. G., T. Winkler, F. Melchers, J. Andersson. 2000. Precursor B cell receptor-dependent B cell proliferation and differentiation does not require the bone marrow or fetal liver environment. J. Exp. Med. 191:23.[Abstract/Free Full Text]
45. Hess, J., A. Werner, T. Wirth, F. Melchers, H. M. Jack, T. H. Winkler. 2001. Induction of pre-B cell proliferation after de novo synthesis of the pre-B cell receptor. Proc. Natl. Acad. Sci. USA 98:1745.[Abstract/Free Full Text]
46. Guo, B., R. M. Kato, M. Garcia-Lloret, M. I. Wahl, D. J. Rawlings. 2000. Engagement of the human pre-B cell receptor generates a lipid raft-dependent calcium signaling complex. Immunity 13:243.[Medline]
47. Iritani, B. M., J. Alberola-Ila, K. A. Forbush, R. M. Perimutter. 1999. Distinct signals mediate maturation and allelic exclusion in lymphocyte progenitors. Immunity 10:713.[Medline]
48. Kersh, E. N., A. S. Shaw, P. M. Allen. 1998. Fidelity of T cell activation through multistep T cell receptor phosphorylation. Science 281:572.[Abstract/Free Full Text]
49. Torigoe, C., J. K. Inman, H. Metzger. 1998. An unusual mechanism for ligand antagonism. Science 281:568.[Abstract/Free Full Text]
50. Melchers, F.. 1999. Fit for life in the immune system? Surrogate light chain tests H chains that test L chains. Proc. Natl. Acad. Sci. USA 96:2571.[Free Full Text]
51. Tatu, C., S. H. Clarke. 2000. Selective maturation of V_H12 B cells in the spleen enriches for anti-phosphatidylcholine B cells: evidence for receptor editing. Curr. Top. Microbiol. Immunol. 252:77.[Medline]
52. Retter, M. W., R. A. Eisenberg, P. L. Cohen, S. H. Clarke. 1995. Sm and DNA binding by dual reactive B cells requires distinct V_H, V, and V_H CDR3 structures. J. Immunol. 155:2248.[Abstract]

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