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The Journal of Immunology, 2006, 177: 2412-2422.
Copyright © 2006 by The American Association of Immunologists

B Cells Expressing a Natural Polyreactive Autoantibody Have a Distinct Phenotype and Are Overrepresented in Immunoglobulin Heavy Chain Transgenic Mice1

Qi Tian2,*, Michael Beardall2,*, Yang Xu*, Ju Li*, David C. Parker{dagger}, Nina Casanova*, Anthony C. Bakke* and Ching Chen3,*,{dagger}

* Department of Pathology and {dagger} Department of Molecular Microbiology and Immunology, Oregon Health and Science University, Portland, OR 97239


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Despite stringent regulation of disease-associated autoantibodies, a substantial proportion of circulating Abs in sera of healthy individuals exhibit self-reactivity. These Abs are referred to as naturally occurring or natural autoantibodies (NAAs). To understand the origin and function of NAAs, we have generated a new site-directed transgenic mouse model in which a prerearranged VDJ gene coding for the H chain of a typical polyreactive NAA, ppc1-5, is inserted into the IgH locus. This H chain, when combined with its original L chain, the {lambda}1 L chain, yields a NAA that characteristically binds a variety of self and non-self Ags including ssDNA, actin, ubiquitin, and nitrophenyl phosphocholine. Despite their autoreactivity, B cells expressing ppc1-5H/{lambda}1 NAA are not negatively selected, but rather are overrepresented in the transgenic mice. The shift toward {lambda}1 expression mainly occurs during the transition of immature to mature B cells in the spleen, suggesting a BCR selection process. The ppc1-5H/{lambda}1 B cells exhibit a phenotype that is different from those of the known mature B cell populations, and they are located predominantly in the lymphoid follicles of the spleen and the lymph nodes. These B cells are functionally active, producing high levels of Abs in vivo and responding well to BCR stimulation in vitro. The findings indicate that the ppc1-5/{lambda}1 natural autoantibodies originate from a distinct B cell subset that may be positively selected by virtue of its poly/autoreactivity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Natural autoantibodies (NAAs)4 are present in healthy humans and rodents as well as in lower phylogenetic species (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13). These Abs appear very early in ontogeny (present in cord blood) and do not arise from actual Ag stimulation (present in germfree mice). Most of these Abs show a broad spectrum of specificities, capable of binding multiple structurally unrelated Ags such as proteins, nucleotides, polysaccharides, and lipids, many of which are self-constituents. Such Abs are thus also referred to as natural polyreactive autoantibodies. Although polyreactive, the binding is selective rather than nonspecific in that each NAA has its own binding profile and can distinguish between epitopes that are structurally similar (9). The majority of NAAs are IgM, but they may also be IgA or IgG.

The structure of NAA has been extensively studied (2, 10). Early studies indicate that the general biochemical and immunologic features of NAA are comparable to those of conventional, monospecific Abs. NAAs are usually encoded by germline Ig V region genes with little or no somatic mutation (2, 9, 14, 15), but they use the same spectrum of VH and VL genes as those used by conventional Abs (9, 16). Previously, we have shown that NAAs differ from Ag-induced Abs in the third hypervariable region of the H chain (9), a finding subsequently confirmed by others in human polyreactive Abs (17, 18, 19).

The exact cellular origin of NAA is unclear. Mature B cells can be divided into two major populations: B1 and B2 cells (20). B2 cells in the spleen are further divided into two subsets, follicular (FO) and marginal zone (MZ) B cells (21). B1 cells are of fetal origin, preferentially located in body cavities and are typically IgMhighIgDlowCD23lowCD5+; FO B cells constitute the majority of circulating B cells located in the follicles of spleen and lymph nodes and are IgMlowIgDhighCD23highCD5–; MZ B cells are confined to the splenic MZ and are IgMhighIgDlowCD23lowCD21highCD5–. B1 cell-derived Abs bear similarity to NAA in that they often recognize self-Ags such as phosphatidylcholine and carbohydrate epitopes on cell membrane glycoproteins, as well as common bacterial Ags such as phosphocholine. Thus, it is believed that B1 cells are a major source of natural autoantibodies (20, 22, 23). Recently, it has been shown that B cells with a low level of self-reactivity or with reactivity to bacterial wall components preferably reside in the MZ (24). It is thus possible that more than one B cell population contributes to the production of NAAs.

There is increasing evidence that B cells, like T cells, undergo both positive and negative selection during their maturation (25, 26, 27). Self-reactive B cells are negatively selected by receptor editing, clonal deletion, or anergy (28, 29, 30, 31). However, it is not well-understood how B cells are positively selected. Early evidence for positive selection of B cells came from observations of a significant difference in Ig gene usage between immature and mature B cells (32, 33, 34). BCR signaling appears to play a crucial role in B cell survival and positive selection; it was first demonstrated by the finding that peripheral ablation of BCR causes rapid B cell death (35). Subsequent examination of various mutants deficient in BCR signaling components has further confirmed this notion (24, 36, 37, 38). Studies on Ig transgenic (tg) mouse models have shown that BCR specificity directs B cell segregation into B1, B2, or MZ compartments (24, 39, 40, 41, 42, 43, 44, 45). In particular, using an anti-Thy-1 tg mouse model, Hayakawa et al. (46) have demonstrated that anti-Thy-1 Abs and CD5+ B cells develop only in the presence of Thy-1 Ag, providing direct evidence for self-Ag-mediated positive selection of B1 cells.

In this study, we report a new site-directed tg (sd-tg) mouse model in which a prototypic NAA H chain gene, VHppc1-5, is expressed. The ppc1-5 NAA is derived from the liver of a newborn BALB/c mouse, and has broad reactivity toward multiple self and foreign Ags including ssDNA, actin, ubiquitin, OVA, keyhole limpet hemocyanin (KLH), nitrophenyl phosphocholine, and trinitrophenol; yet, this binding is specific in that ppc1-5 NAA does not bind to free phosphocholine, DNP, mouse Ig, dextran or polysaccharides (9). The ppc1-5 NAA is encoded by the ppc1-5 VH (a member of the VH7183 family) together with a V{lambda}l gene, both unmutated and without N addition (9). In the H-only sd-tg mice, the {lambda}1 B cells represent NAA-producing B cells while the majority of {kappa}-expressing B cells are non-NAA and serve to maintain a relatively normal and diverse B cell repertoire. Using this model, we show that B cells producing ppc1-5H/{lambda}1 NAA are mainly located in the B cell follicles and have a distinct phenotype: IgMintIgDlowCD23brightCD21intCD5–CD43–, which is different from those of B1, MZ, or FO B cells. The number of ppc1-5H/{lambda}1 B cells increases with maturation, from <7% in immature B to nearly 30% in mature B compartment in the spleen. Thus, we propose that the ppc1-5H/{lambda}1 NAA B cells are positively selected during peripheral B cell maturation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Generation of sd-tg mice

The ppc1-5 VDJ sequence was isolated and cloned from a NAA-producing hybridoma, ppc1-5, as previously described (9). The 4.2-kb fragment of ppc1-5 VDJ was inserted into the IgH targeting vector we constructed previously (47) (see Fig. 1A). The targeting construct was then transfected into embryonic stem (ES) cells and the targeted insertion of ppc1-5 VDJ into the IgH locus was screened by PCR with specific primers and confirmed by Southern blot assay with a JH probe (see Fig. 1A). The targeted ES cells were then used for injection of blastocysts and production of sd-tg mice. Mice were maintained and bred at the Oregon Health and Science University. All animal care and procedures were conducted in accordance with the Animal Welfare Act.


Figure 1
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  		FIGURE 1. Generation of ppc1-5H sd-tg transgenic mouse. A, Structure of the ppc1-5 H chain targeting construct, germline IgH locus and the targeted IgH-ppc1-5 locus. The germline and the targeted IgH loci are expected to give rise to 6.4- and 7.2-kb EcoRI restriction fragments, respectively. B, PCR analysis of the targeted ES cell clone (ES-2E2) and the ES cell-derived mouse lines (ppc1-5A and B). The ppc1-5 PCR tests the presence of the ppc1-5 VH gene and the DH/neo PCR confirms the correct insertion of the targeting construct. C, Southern blot analysis of the targeted ES cell clone (ES-2E2) and the ES cell-derived ppc1-5A mouse line.

 
Flow cytometric analysis

Single-cell suspensions of spleen and bone marrow were obtained by pressing spleens between glass slides and flushing femoral bones with a syringe, respectively. Subsequently, cells were absolved of RBC by lysis with 0.14 M NH4Cl, followed by centrifugation over FBS and filtration through nylon mesh (74 µm; Small Parts). After washing with buffered medium (PBS with 0.2% sodium azide, 2% FBS), cells were harvested by centrifugation and resuspended to 5 x 107 cell/ml. Cells (106) were stained with combinations of the following anti-mouse Abs labeled with biotin, PE, FITC, or allophycocyanin: anti-B220 (RA3-6B2), anti-CD5 (53.7), anti-CD23 (B3B4), anti-CD21/CD35 (7G6), anti-CD43 (S7), anti-CD22.2 (Cy34.1), anti-CD69 (H1.2F3), AA4.1, anti-IA/IE (2G9), anti-{lambda}1 (R11-153), anti-CD24 (30-F1), anti-CD80 (16-10A1), anti-CD86 (GL1), anti-CD95 (Jo2), anti-CD62L (Mel-14), anti-IgMa (DS-1), anti-IgMb (AF6-78) (BD Pharmingen); and anti-{kappa} (187.1), anti-CD44 (KM201), anti-IgM (polyclonal), and anti-IgD (polyclonal) (Southern Biotechnology Associates). For secondary staining, biotin-labeled Abs were coupled to streptavidin-PerCP (BD Pharmingen). Stained cells were then analyzed for Ag expression using a FACSCalibur flow cytometry apparatus (BD Biosciences). Data were analyzed using CellQuest or FlowJo software.

Cell survival and proliferation

Splenic cell suspension was prepared as described above. CD19+ B cells were isolated using anti-CD19 magnetic beads (Miltenyi Biotec) following manufacturer’s instructions. Cell purity was over 95% as verified by flow cytometry. Cultures were started at ~5 x 105 cells/ml. Cells were harvested and counted using trypan blue exclusion at various time points. Cell survival is calculated as a percentage of their initial numbers. For proliferation studies, CD19+ cells were labeled with CFSE (Molecular Probes) as previously described (48). Briefly, CD19+ B cells were diluted to ~1 x 107 cells/ml and incubated with 0.1 µM CFSE at 37°C for 10 min. Labeled cells were washed twice with PBS and placed in culture for 72 h in the presence of anti-IgM F(ab')2 (20 µg/ml; Jackson ImmunoResearch Laboratories), anti-IgM F(ab')2 (10 µg/ml) plus anti-CD40 (5 µg/ml) (clone 1C10; Southern Biotechnology Associates), or LPS (2 µg/ml) (Escherichia coli 055:B5, Sigma-Aldrich). Cells were analyzed by flow cytometry and supernatants were collected for measurement of Ab production by ELISA.

ELISA

Ig isotype and concentration were measured by a solid-phase ELISA as described previously (9). Plates were coated with goat anti-mouse {kappa} and {lambda} (Southern Biotechnology Associates), incubated with sera or cell culture supernatants, and developed with alkaline phosphatase-labeled anti-IgM, -IgG1, -IgG2a, -IgG2b, -IgG3, -{kappa}, or -{lambda} (Southern Biotechnology Associates). Binding to various Ags was conducted by solid-phase ELISA as described previously (9). Histone is unfractionated calf thymus histone (Sigma-Aldrich), and, therefore, includes various subtypes of histone.

Immunohistochemistry

Mouse spleens were embedded in OCT and flash-frozen in liquid nitrogen. Five-micrometer sections of frozen tissue were fixed in 1% paraformaldehyde, quenched in 0.3% H2O2, blocked with 1% BSA and stained with anti-CD22-FITC and anti-{lambda}1-Biotin (Bachem Bioscience) for 45 min, followed by incubation with AP-labeled anti-FITC (Sigma-Aldrich), and avidin-biotin-complex (Vectastain Elite kit; Vector Laboratories) for 30 min. AP and HRP were developed with Fast Blue (Sigma-Aldrich) and diaminobenzidine (DakoCytomation), respectively.

BrdU-labeling assay

BrdU (Sigma-Aldrich) was administered to mice via a single initial i.p. injection (0.8 mg/mouse) followed by continuous feeding in drinking water (0.8 mg/ml). Mice were sacrificed at various time points and BrdU incorporation was measured using a BrdU kit (BD Pharmingen) following manufacturer’s instructions. Briefly, cells were stained with various surface markers as described above. After washing with PBS, cells were fixed and permeabilized with BD Cytofix/Cytoperm buffer, treated with DNase, and stained with FITC-labeled anti-BrdU Ab. The cells were then analyzed by flow cytometry.

RT-PCR and sequencing

RT-PCR and {kappa} L chain sequencing were conducted as described previously (49). cDNA was synthesized with a C{kappa} primer, and subsequently amplified with the C{kappa} and the degenerate V{kappa} primers. The PCR products were subjected to automated, fluorescent DNA sequencing (ABI 377; Applied Biosystems) using a nested C{kappa} primer.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Targeted insertion of ppc1-5 VH gene into the IgH locus

The ppc1-5 H chain gene (VHDHJH3) along with its regulatory sequences was cloned from the NAA-producing hybridoma ppc1-5 (9). The downstream JH4 gene segment was removed to reduce potential secondary VH gene rearrangement. The targeted insertion of ppc1-5 VH into the IgH locus in replacement of JH is shown in Fig. 1. One of the targeted ES cell clones (2E2) was used for the production of sd-tg mice, and the mice were crossed to C57BL/6 (B6) background for at least six generations before analysis.

Normal B cell development in ppc1-5H sd-tg mice

The ppc1-5 VH is joined to the Cµ region of the 129/Sv mouse strain and hence the transgene encoded BCR is of IgMa allotype while the endogenous B6 IgH is IgMb. Flow cytometric analysis using the allotype-specific reagents showed that over 90% of the splenic B cells in ppc1-5H/B6 mice expressed IgMa, ~4% expressed IgMb, and the remainder expressed both (Fig. 2A), indicating that the ppc1-5H sd-tg exerts good allelic exclusion. To determine whether the expression of ppc1-5H sd-tg would perturb normal B cell development, bone marrow B cell populations were examined. The percentages of pre-B (IgM–IgD–), immature B (IgM+IgD–), and mature/recirculating B cells (IgM+IgD+) were very similar between the ppc1-5H and the non-tg mice (Fig. 2B). The absolute B cell numbers in various developmental compartments both in the bone marrow and spleen were also comparable in both types of mice (Table I).


Figure 2
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  		FIGURE 2. B cell development in ppc1-5H sd-tg mice. A, H chain allelic exclusion in ppc-1-5H sd-tg mice. Splenic B cells were stained with allotype-specific reagents IgMa for ppc1-5 H chain and IgMb for the B6-derived H chain. The percentages were calculated based on total lymphocytes. B, Analysis of bone marrow B cells. The relative percentages of pre-B (IgM–IgD–), immature B (IgM+IgD–), and mature B (IgM+IgD+) were calculated based on B220+ B cells. The data are representative of five separate experiments and at least 10 mice in each group.

 

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  		Table I. Frequencies and absolute numbers of B cells in the bone marrow and spleen of ppc1-5H sd-tg mice

 
Analysis of Abs of ppc1-5H sd-tg mice

We have shown previously that ppc1-5 mAb binds multiple self and foreign Ags (9). To confirm the binding specificity and evaluate the relative binding avidity of ppc1-5 Ab, an ELISA was performed over a range of Ab concentrations. Fig. 3A shows that ppc1-5 mAb had high relative binding to actin, ssDNA, and KLH, moderate binding to ubiquitin, and little or no binding to unfractionated histone. In contrast, a prototypic disease-associated anti-DNA Ab, 3H9 (29), did not show reactivity to actin, KLH, or ubiquitin, but it bound ssDNA and histone. These results confirm the poly/autoreactivity of ppc1-5 Ab.


Figure 3
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  		FIGURE 3. Analyses of Abs from ppc1-5H sd-tg mice. A, Ag binding by ppc1-5 mAb. The figure shows the binding curves of ppc1-5 (solid line) and 3H9 (dotted line) mAbs at various concentrations. The Ags tested include actin, ssDNA, ubiquitin, KLH, and unfractionated histone. B, Serum titers of IgM, IgG1, IgG2a, IgG2b, IgG3, {kappa}, and {lambda} Abs of ppc1-5H sd-tg and non-tg B6 mice were determined by ELISA as described in Materials and Methods. Sera were titrated in a series of 1/3 dilutions starting at 1/1000 (=6.3 log3). Results are shown as the mean of five mice ± SD. C, Ag-binding specificity of serum {lambda} Abs. Ag binding was determined by ELISA. Sera were diluted at 1/40 and the ppc-1-5 hybridoma supernatant was diluted at 1/2. Sera from five mice in each group were pooled for the analysis. D, Ag binding by {kappa} and {lambda} hybridoma Abs derived from a ppc1-5H sd-tg mouse. Each symbol represents one hybridoma. Results are depicted as OD405 readings recorded 1 h after substrate development. Ubi, ubiquitin; ova, ovalbumin; his, histone; PC-his, phosphocholine-coupled histone.

 
Comparison of serum Ab levels of ppc1-5H sd-tg mice with those of non-tg B6 mice showed that both had similar amounts of IgM, IgG1, IgG2a, IgG2b, IgG3, {kappa}, and {lambda} Abs (Fig. 3B), indicating a relatively normal Ab repertoire in ppc1-5H sd-tg mice. The finding that the ppc1-5H mice have normal to slightly increased levels of {lambda} Abs is significant because these Abs represent NAA: binding studies showed that the serum {lambda} Abs from ppc1-5H mice but not non-tg mice had a poly/autoreactive binding pattern similar to that of the original ppc1-5 mAb (Fig. 3C). The level of serum {lambda} Abs in ppc1-5H mice may be an underestimate because these polyreactive Abs may be masked by self-Ag binding (50) and cleared rapidly (51). These results demonstrate that the ppc1-5H/{lambda}1 NAA is "naturally" produced in vivo.

It has been shown that H chain plays a central role in polyreactive Ab binding (17, 19). Therefore, it is possible that the ppc1-5H/{kappa} Abs might also be polyreactive. We examined several hybridoma mAbs derived from a ppc1-5H sd-tg mouse. To our surprise, only one of the seven {kappa} mAbs showed binding to three of the seven Ags tested while, as expected, all {lambda} mAbs exhibited polyreactivity (Fig. 3D). It appears that the {lambda} L chain is essential in polyreactive Ag binding of ppc1-5 NAA and that the majority of the ppc1-5H/{kappa} Abs are nonpolyreactive. Therefore, these {kappa} Abs provide a valuable internal control for {lambda}1 NAA in the subsequent analyses.

The number and location of ppc1-5H/{lambda}1 NAA B cells

In non-tg B6 mice, {lambda}1 B cells constituted <5% of the total B cells (Fig. 4A). Strikingly, in ppc1-5H/B6 sd-tg mice, {lambda}1 B cells comprised over 25% of splenic B cells, 39% of lymph node B cells, and a similar percentage of peripheral blood B cells (40%, data not shown). In the peritoneal cavity, {lambda}1 B cells were only slightly increased (11%). Immunohistochemical examination showed numerous {lambda}1 B cells in the spleen of ppc1-5H mice (Fig. 4B), and the majority of these B cells were located in the follicles with a few in the MZ and red pulp. Notably, the {lambda}1 B cells often formed small clusters (Fig. 4B, inset), suggesting that they might have undergone a number of divisions in this location.


Figure 4
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  		FIGURE 4. Number and distribution of ppc1-5H/{lambda}1 B cells. A, Increased {lambda}1+ B cells in ppc-1-5H sd-tg mice. Percentages of {lambda}1+ B cells were determined by flow cytometry using gated B220+ B cells. At least 10 mice from each group were tested. B, Immunostains of spleen sections of ppc1-5H sd-tg and non-tg B6 mice. CD22 stains all B cells and is visualized as blue, and {lambda}1+ B cells are shown as brown. Inset, A cluster of {lambda}1+ B cells. These are representative sections from five mice of each group. Original magnification, x200.

 
To determine the developmental stage at which {lambda}1 B cells become overrepresented, splenic B cells were divided into three compartments based on CD21 and CD23 expression (52, 53): newly emigrant immature B (or transitional B), mature FO B, and MZ B cells (Fig. 5A). In ppc1-5H sd-tg mice, <7% (6.3 ± 1.8%) of the immature B cells in the spleen were {lambda}1 whereas nearly 30% (27.1 ± 2.6%) of the mature FO B cells were {lambda}1 (Fig. 5A). The MZ compartment had ~10% (10.3 ± 2.5%) {lambda}1 B cells. In contrast, non-tg mice had low numbers of {lambda}1 B cells (3–5%) in all three compartments. Analysis of bone marrow B cells revealed similar results: in the AA4+ immature B stage the percentage of {lambda}1 B cells was slightly higher in ppc1-5H mice (7.0 ± 1.6%) than in non-tg mice (4.8 ± 0.8%) (Fig. 5B) whereas in the AA4– mature/recirculating B cell pool {lambda}1 B cells were greatly increased in ppc1-5H mice (25.1 ± 3.3%) but not in non-tg mice (5.3 ± 1.2%). The 1- to 2-fold increase in {lambda}1 B cells in the immature B compartment of tg mice as compared with non-tg mice is most likely due to preferential pairing of ppc1-5H with {lambda}1 L chain. However, the marked increase in {lambda}1 B cells in peripheral mature B cell compartment in ppc1-5H mice cannot be attributed to preferential pairing, rather, it suggests that {lambda}1 B cells are favorably selected during immature to mature B differentiation in the spleen.


Figure 5
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  		FIGURE 5. Percentages of {lambda}1 B cells in different B cell compartments in the spleen (A) and bone marrow (B) of ppc1-5H sd-tg and non-tg B6 mice. The populations of immature B (IMB), FO (FO), MZ (MZ), and mature B (MB) cells are shown in the boxes. The numbers indicate mean percentages of 5–10 mice ± SD of {lambda}1+ B cells within each population.

 
V{kappa} gene usage by ppc1-5H/{kappa}+ B cells

To determine whether {lambda}1 bias is due to a restriction in V{kappa} usage, we sequenced V{kappa} genes of 30 ppc1-5H/{kappa}+ hybridomas derived from the spleen of two ppc1-5H/B6 mice. Fig. 6A shows that a large variety of different V{kappa}s are able to combine with the ppc1-5 H chain. Furthermore, all four functional J{kappa} gene segments are equally used by these hybridomas (Fig. 6B).


Figure 6
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  		FIGURE 6. V{kappa} (A) and J{kappa} (B) gene usage by {kappa}+ hybridomas derived from ppc1-5H sd-tg mice. V{kappa} and J{kappa} genes were determined by RT-PCR and subsequent sequencing as described in Materials and Methods. Thirty hybridomas from two mice were examined.

 
The phenotype of ppc1-5H/{lambda}1 B cells

Splenic B cells from ppc1-5H sd-tg and non-tg mice were analyzed for expression of various surface markers. In comparison with non-tg and ppc1-5H/{kappa} B cells, the ppc1-5H/{lambda}1 B cells had an intermediate IgM level, a decreased IgD level, and an increased CD23 level (Fig. 7A). The majority of the {lambda}1 B cells were small-sized, similar to the bulk of the non-tg and ppc1-5H/{kappa} B cells. The ppc1-5H/{lambda}1 B cells did not express CD5 or CD43, markers characteristic of B1 cells (Fig. 7B). Therefore, the majority of the ppc1-5H/{lambda}1 NAA B cells are not B1 cells, and they exhibit an unusual phenotype, IgMintIgDlowCD23brightCD21int, which is different from that FO (IgMlowIgDhighCD23highCD21int) or MZ (IgMhighIgDlowCD23lowCD21high) B cells.


Figure 7
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  		FIGURE 7. Phenotypic analyses of ppc1-5H/{lambda}1+, ppc1-5H/{kappa}+, and non-tg B cells. A, Levels of surface marker expression shown as histogram. Solid line, ppc1-5H/{lambda}1; dotted line, ppc1-5H/{kappa}; gray histogram, non-tg B cells. B, Expression of surface markers shown as contour plots. At least five mice from each group were examined and gave similar results; plots shown are typical.

 
The decreased level of surface IgD on ppc1-5H/{lambda}1 B cells raises the possibility that they may be immature B cells. We examined the expression of maturation markers AA4.1, CD24, CD22, and CD62L. The ppc1-5H/{lambda}1 B cells, like the bulk of ppc1-5H/{kappa} and non-tg splenic B cells, were AA4.1–CD24lowCD22highCD62Lhigh (Fig. 7A), a phenotype consistent with mature B cells (52, 54, 55, 56). A panel of activation markers, including MHC class II, CD69, B7.1, B7.2, CD44, CD25, CD95, and CD79b were also examined. The ppc1-5/{lambda}1 B cells exhibited increased levels of MHC class II, CD69, and B7.2, and decreased level of CD79b (Ig-beta chain of the BCR) (Fig. 7A), indicating BCR engagement/activation (57, 58, 59, 60). The rest of the markers tested show comparable expression levels among the three types of B cells (data not shown).

The ppc1-5H/{lambda}1 B cells respond well to anti-BCR and mitogen stimulation

To further characterize the functional status of ppc1-5/{lambda}1 B cells, CD19+ splenic B cells from ppc1-5H and non-tg mice were labeled with CFSE, and placed in culture medium in the presence of LPS, anti-IgM F(ab')2, or anti-IgM F(ab')2 plus anti-CD40. The ppc1-5H/{lambda}1 B cells showed a good proliferative response to BCR cross-linking in the presence or absence of anti-CD40 as demonstrated by the reduction of CFSE fluorescence (Fig. 8A). This is in contrast to B1 cells, which fail to enter cell cycle in response to BCR cross-linking (61). Stimulation with LPS also triggered a proliferation of ppc1-5H/{lambda}1 B cells, but the response was slightly weaker than that of the ppc1-5H/{kappa} or non-tg B cells. This may be due to their prior BCR engagement (62).


Figure 8
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  		FIGURE 8. In vitro proliferation, Ab production, and survival of ppc1-5H B cells. A, CD19+ B cells from ppc1-5H and non-tg mice were stained with CFSE and cultured in the presence of anti-IgM F(ab')2 (20 µg/ml), anti-IgM F(ab')2 (10 µg/ml) plus anti-CD40 (5 µg/ml), or LPS (2 µg/ml) for 72 h. Numbers on top of each histogram represent number of divisions. Solid line, ppc1-5H/{lambda}1; dotted line, ppc1-5H/{kappa}; gray histogram, non-tg B cells. B, CD19+ B cells from ppc1-5H mice were cultured with LPS, and Abs in the supernatants were measured by ELISA at the indicated days. Supernatants were titrated in a series of 1/2 dilutions. C, B cells from ppc1-5H and non-tg mice were cultured in the medium for 8 days, and the numbers of viable cells were counted at various time points and expressed as percentage of starting cells. Results in A are representative of four separate experiments, and results in B and C are shown as the mean of four mice ± SD.

 
To examine the Ab production ability of the ppc1-5H/{lambda}1 B cells, CD19+ B cells were cultured with LPS and the Ab levels in the culture supernatants were measured at various time points. The production of IgM/{lambda} Abs paralleled that of IgM/{kappa} (Fig. 8B): they reached a significant level at day 4 and continued to increase until day 8 (the end point). The slightly lower level of IgM/{lambda} than IgM/{kappa} may be attributed to the lower proportion (25–30%) of {lambda} B cells in the culture. Comparison of IgG/{kappa} and IgG/{lambda} Abs revealed a difference: the level of IgG/{kappa} increased rapidly between days 2 and 4, but IgG/{lambda} Abs were not detected until day 6 and their level was relatively low (Fig. 8B). These results indicate that ppc1-5/{lambda}1 B cells are capable of Ab production upon LPS stimulation, but they may be partially resistant to LPS-induced isotype switching.

In vitro survival of ppc1-5H/{lambda}1 B cells

Different B cell populations may have different in vitro survival capabilities. For example, B1 cells have more extended survival than B2 cells (63). To examine this property, CD19+ B cells from ppc1-5H and non-tg mice were placed in culture medium without stimuli, and live cells were counted and examined by flow cytometry at various time points. After a 2-day delay, both ppc1-5/{kappa} and non-tg B cells died rapidly with <50% viable cells left at day 4 and <5% at day 6 (Fig. 8C). Similarly, over 90% of the ppc1-5H/{lambda}1 B cells died by day 6 although a slight delay at day 4 was observed. This pattern is similar to that of B2 cells (63).

B cell turnover in ppc1-5H sd-tg mice.

To determine the lifespan of ppc1-5H/{lambda}1 B cells, we performed a BrdU-labeling experiment. BrdU is a thymidine analog that is incorporated into DNA when cells divide. In rapidly dividing cells such as bone marrow immature B cells, BrdU uptake is a measurement of cell proliferation; in nondividing cells such as the bulk of splenic mature B cells (64), it is a measurement of cell turnover or renewal. The turnover rate inversely correlates with the lifespan of the B cells (54, 64, 65, 66). The ppc1-5H/{lambda}1 splenic B cells had similar BrdU uptake to that of the ppc1-5H/{kappa} and non-tg B cells over the course of 21 days (Fig. 9). The percentages of BrdU labeling are consistent with those of the previously published data in normal adult mice (54, 64). Therefore, the ppc1-5H/{lambda}1 NAA B cells have a lifespan comparable to that of mature peripheral B cells.


Figure 9
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  		FIGURE 9. BrdU labeling of B cells from ppc1-5H sd-tg mouse. A, A representative dot plot of BrdU labeling of B220+IgMa+ B cells at day 14 of BrdU feeding. B, Kinetics of BrdU labeling of ppc1-5H/{kappa}, ppc1-5H/{lambda}, and non-tg B cells. The results are shown as the mean of three to five mice ± SD.

 
Ontogeny of the ppc1-5/{lambda}1 B cells

To investigate the ontogeny of ppc1-5H/{lambda}1 NAA B cells, B cells from the liver and spleen of 1- and 7-day-old ppc1-5H mice were analyzed by flow cytometry. As in adult sd-tg mice, nearly all B cells in the liver and spleen of young mice expressed the H chain transgene (data not shown). In newborn mice, the percentages of IgM+ B cells were higher in sd-tg mice than in non-tg mice (Fig. 10), indicating that the H chain transgene accelerates B cell development early in life. By day 7, however, both sd-tg and non-tg mice had comparable percentages of IgM+IgD– immature B and IgM+IgD+ mature B cells. The percentages of {lambda}1 B cells in the newborn liver were 2-fold higher in ppc1-5H than in non-tg mice, a finding similar to that seen in adult bone marrow immature B cell compartment (see above). In the spleen, however, the percentages of {lambda}1 B cells in tg mice were >4-fold higher than in non-tg mice, indicating that selection of ppc1-5/{lambda}1 B cells occurred early in ontogeny.


Figure 10
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  		FIGURE 10. Ontogeny of ppc1-5H B cells. Lymphocytes were isolated from the liver and spleen of 1-day-old mice and from the spleen of 7-day-old mice. The analyses were done on gated B220+ cells (A) or IgM+ cells (B). The percentages of {lambda}1+ cells as the mean of three mice ± SD are as follows: ppc1-5H liver, 8.0 ± 1.9; non-tg liver, 3.9 ± 1.3; ppc1-5H 1-day spleen, 14.9 ± 1.7; non-tg 1-day spleen, 4.0 ± 0.8; ppc1-5H 7-day spleen, 29.4 ± 3.2; non-tg 7-day spleen, 6.3 ± 1.6.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
We have generated a new sd-tg mouse model in which a prototypic polyreactive natural autoantibody, ppc1-5H/{lambda}1, is expressed. The ppc1-5H/{lambda}1 NAA is produced in the sd-tg mice in large quantity without deliberate immunization, confirming that this Ab is naturally occurring. We have further validated this model by demonstrating that the serum ppc1-5H/{lambda}1 NAA sustains the same polyreactive/autoreactive binding profile as the original ppc1-5 mAb. Our initial studies of this model have revealed important new information regarding the origin and selection of NAA.

The cellular origin of NAA

Given that the ppc1-5 NAA is derived from the newborn liver where B1 cells originate, and that it is encoded by a {lambda} L chain which is overexpressed in the B1 cell repertoire (67), we expected the ppc1-5H/{lambda}1 B cells to be B1 cells. To our surprise, these B cells exhibit a phenotype that is different from that of B1: they are small in size and are negative for CD5 and CD43. In addition, they are not concentrated in the peritoneal cavity but rather are mainly located in the follicles of the spleen and lymph nodes, and they are part of the circulating B cells in the peripheral blood. These are features of FO B cells. However, the ppc1-5H/{lambda}1 B cells are distinguished from the bulk of FO cells by their decreased surface IgD and increased CD23 levels. It is not clear whether ppc1-5H/{lambda}1 NAA B cells represent a distinct B cell population or are a subset of FO B cells with a distinct activation status. Our finding that ppc1-5 B cells express activation-associated markers appears to support the latter notion.

The finding that ppc1-5/{lambda}1 NAA is not produced by B1 cells appears to contradict the general belief that B1 cells are the major source of NAAs (20, 22, 23). However, numerous studies on polyreactive NAAs and B1-derived Abs have revealed differences between these Abs. Although many polyreactive NAAs bind a large variety of foreign and self-Ags (5, 9, 68), B1-derived Abs have more restricted specificities and often bind cell membrane or bacterial wall components (22). In addition, polyreactive NAAs use a broad spectrum of Ig genes similar to those used by conventional Abs (9, 16, 17, 18) whereas B1 Abs are encoded by a limited set of VH/VL genes (69, 70, 71). In humans, up to 20% of peripheral blood B cells and 30% of splenic B cells are polyreactive, and many of these are not B1 cells (72). There are reports indicating that NAA can be produced by MZ or B2 cells in mice (24, 73). Bone marrow reconstitution studies have shown that B1 cells are not essential for NAA regeneration (74). These studies indicate that B cells other than B1 can contribute to the production of NAAs. Our findings are consistent with this notion.

Previous studies of Ig tg mice have demonstrated that segregation of B cell populations is dictated by BCR specificity in that B cells expressing B1-derived BCR develop into B1 cells whereas B cells expressing B2-derived BCR become B2 cells (39, 45, 63, 75). However, recent studies have shown that BCR signal strength plays an important role in deciding B cell fate. B cells expressing a typical B1 BCR can be directed to MZ differentiation when Ag dose is reduced (43). Likewise, mutations that impair BCR signaling prevent B1 and FO but favor MZ B cell development, and mutations that enhance BCR signaling are associated with increased FO and decreased MZ B cells (26, 76). Based on this signal strength model, NAA precursor B cells may differentiate into more than one B cell population as a result of differences in the type and amount of self-Ags they encounter. In other words, the phenotypic fate of ppc1-5 B cells may be determined not only by their Ag-binding specificity but also by the strength of signals mediated by their polyreactive BCR.

It has been shown that tolerant B cells often express markers associated with Ag activation (77, 78). This raises the possibility that the ppc1-5H/{lambda}1 B cells may be tolerant or anergic B cells. However, several lines of evidence argue against this idea: 1) ppc1-5H/{lambda}1 B cells are not functionally "anergic" because they produce large amounts of Abs in vivo, and they respond well to anti-IgM and LPS stimulation in vitro; 2) they have a lifespan similar to that of normal mature B cells whereas anergic B cells are often short-lived (66, 79, 80); 3) they are phenotypically mature and located within the B cell follicles whereas anergic B cells are usually immature and excluded from the follicles (80, 81). Therefore, ppc1-5H/{lambda}1 NAA B cells have the characteristics of mature, functional B cells.

Positive selection of ppc1-5/{lambda}1 NAA B cells

The ppc1-5 sd-tg mice have a large population of {lambda}1 B cells, constituting 25–40% of the mature B cell repertoire. This is unexpected given the small number of {lambda}1 B cells (3–5%) in non-tg mice. Could this be the result of overexpression of the transgene? Although the ppc1-5 H chain is expressed by >90% of the B cells in the H-chain only sd-tg mice, this does not necessarily lead to {lambda}1 predominance because the {lambda}1 present is native and not a transgene. It is merely 1 of the ~100 endogenous L-chain genes that can potentially be expressed. Given a normal probability distribution of L-chain expression, the {lambda}1 frequency should be low in ppc1-5H tg mice, as seen in non-tg mice. Another possible cause of increased {lambda}1 B cells in ppc1-5H mice is preferential H/L pairing because {lambda}1 is the original partner of ppc1-5 H chain. Indeed, this may account for the up to 2-fold increase in {lambda}1 B cells in the immature B cell compartment in the spleen, bone marrow, and fetal liver, assuming minimal Ag interaction at this early stage. However, the further and more striking increase of {lambda}1 B cells in the mature splenic B compartment is unlikely to be caused by preferential H/L pairing because gene rearrangement and Ab assembly have ceased at this stage. In addition, B cell turnover studies indicate that such increase is not due to prolonged survival of ppc1-5H/{lambda}1+ B cells. Instead, the findings suggest a selection at the immature to mature B cell transition: either a positive selection of {lambda}1 B cells or a negative selection of {kappa} B cells. The latter would predict a restricted V{kappa} gene usage and a biased J{kappa} gene usage (29, 82). However, {kappa}+ hybridomas recovered from the ppc1-5H mice use a variety of V{kappa} genes, and there is no bias toward downstream J{kappa} gene segments. Therefore, we favor a positive selection of ppc1-5H/{lambda}1 B cells. This is consistent with a previous report by Levine et al. (34) in which they find a significant skewing of VL repertoire in H-chain-only tg mice when immature B cells enter the mature B compartment in the spleen, which those authors ascribe to positive selection for a few VH/VL pairs rather than negative selection against nearly all others.

Assuming the positive selection of ppc1-5 NAA B cells, it is not clear whether this process would be self-Ag dependent or due to ligand-independent tonic signaling (83). It has been shown that production of B1-derived anti-Thy-1 autoantibody is dependent on the presence of Thy-1 self-Ag (46). More recently, Julien et al. (73) have reported that mouse B cells expressing a tg human polyreactive Ab specific for ssDNA and human Ig are enriched in the spleen in the presence of human IgFc. The ppc1-5H/{lambda}1 NAA has a broad reactivity to multiple self-constituents and it is therefore difficult to prove Ag-dependent selection by removing the self-Ags. However, because the ppc1-5H/{lambda}1 B cells are specifically chosen from a large repertoire of heterogeneous B cells at a relatively late developmental stage, it is most likely that their selection is a self-Ag-driven process. The activated phenotype of ppc1-5H/{lambda}1 B cells is consistent with this notion.

How NAA B cells escape negative selection is unknown. One possibility is that their Ag-binding affinity may be too low to trigger a tolerogenic signal, yet is high enough to induce a positive selection signal (44). Alternatively, their polyreactivity may facilitate interaction with T cells, dendritic cells, or TLRs, which deliver a second signal for their activation (84). Our model provides an opportunity to examine these possibilities.


   Acknowledgments
 
We thank Dr. Martin Weigert for continuing support; Dr. M. Baetscher for help with construction of sd-tg mice; Drs. M. Fields and J. Erikson for help with the immunohistochemistry study; C. Fredman for figure preparation; Drs. M. B. Rittenberg, W. Rodgers, and J. F. Kearney for insightful discussion; and Dr. P. Ulrich for critical review of the manuscript.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by grants (to C.C.) from National Institutes of Health AI-50818, the PEW Charitable Trust, and the Cancer Research Institute. Back

2 Q.T. and M.B. contributed equally to this work. Back

3 Address correspondence and reprint requests to Dr. Ching Chen, Department of Pathology L113, Oregon Health and Science University, Portland, OR 97239. E-mail address: chenq{at}ohsu.edu Back

4 Abbreviations used in this paper: NAA, natural autoantibody; FO, follicular; MZ, marginal zone; tg, transgenic; sd-tg, site-directed tg; KLH, keyhole limpet hemocyanin; ES, embryonic stem. Back

Received for publication February 23, 2006. Accepted for publication June 2, 2006.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
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A. Matejuk, M. Beardall, Y. Xu, Q. Tian, D. Phillips, B. Alabyev, K. Mannoor, and C. Chen
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