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The Journal of Immunology, 2006, 176: 939-948.
Copyright © 2006 by The American Association of Immunologists

Split Tolerance in Peripheral B Cell Subsets in Mice Expressing a Low Level of Ig{kappa}-Reactive Ligand1

Djemel Aït-Azzouzene2,*, Laurent Verkoczy*, Bao Duong*,{dagger}, Patrick Skog*, Amanda L. Gavin* and David Nemazee*

* Department of Immunology, and {dagger} Kellogg School of Science and Technology Doctoral Program in Chemical and Biological Sciences, The Scripps Research Institute, La Jolla, CA 92037.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Peripheral B cell tolerance differs from central tolerance in anatomic location, in the stage of B cell development, and in the diversity of Ag-responsive cells. B cells in secondary lymphoid organs are heterogeneous, including numerous subtypes such as B-1, marginal zone, transitional, and follicular B cells, which likely respond differently from one another to ligand encounter. We showed recently that central B cell tolerance mediated by receptor editing was induced in mice carrying high levels of a ubiquitously expressed {kappa}-macroself Ag, a synthetic superantigen reactive to Ig{kappa}. In this study, we characterize a new transgenic line that has a distinctly lower expression pattern from those described previously; the B cell tolerance phenotype of these mice is characterized by the presence of significant numbers of immature {kappa}+ B cells in the spleen, the loss of mature follicular and marginal zone B cells, the persistence of {kappa}+ B-1 cells in the peritoneal cavity, and significant levels of serum IgM,{kappa}. These findings suggest distinct signaling thresholds for tolerance among peripheral B cell subsets reactive with an identical ligand.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Although it has been established for many years that B cell tolerance is an active process (1), the parameters that permit persistence of autoreactive cells and eventual Ab secretion when tolerance is broken are poorly understood. At early stages of B cell development, tolerance mechanisms include developmental arrest, receptor editing, and apoptosis of cells that fail to effectively edit (2). In contrast, Ags that are unavailable in the bone marrow or that have low valence or affinity to the BCR fail to induce central tolerance; instead such Ags may promote tolerance in the periphery by the induction of anergy and clonal elimination in cells at a later developmental stage (3, 4, 5, 6).

Analysis of tolerance in peripheral B cells is complicated because of the heterogeneity of these cells. Major subsets include newly formed "transitional" B cells, follicular B cells, marginal zone (MZ)3 B cells, B-1 cells, and memory B cells. Transitional B cells include the immediate bone marrow emigrants, T-1 cells, and their developmental successors, T-2 cells (7, 8). Most transitional B cells turn over rapidly, but some are recruited into a long-lived pool that includes follicular B cells, splenic MZ B cells, and other subtypes. B-1 cells, which are mainly, but not exclusively, derived from fetal proB cells, preferentially populate the peritoneal cavity and lamina propria and are found in low numbers in other lymphoid tissues (9, 10). It is believed that B-1 and MZ B cells have self-renewal capacity, whereas follicular B cells, although long-lived, must be replenished from sIg– bone marrow progenitors (11, 12).

B cell subsets may also differ from one another with respect to reactivity and tolerance to self-Ags. Transitional B cells, particularly the T-1 subset, are believed to be highly tolerance susceptible (13, 14). B-1 and MZ B cells are believed to retain low-affinity self-reactivity and perhaps require it for survival or differentiation (15, 16, 17, 18, 19, 20, 21), whereas the evidence for such self-ligand-dependent positive selection among normal follicular cells is speculative or indirect (22, 23, 24). In contrast, in some autoantibody transgenic models, self-reactive MZ B cells are eliminated by levels of autoantigen that render follicular B cells anergic but viable (25); other studies also suggest that MZ B cells are particularly sensitive to BCR-mediated apoptosis (26, 27). B-1 cells seem to require continual, weak BCR signaling for development and persistence (10, 16, 28). Indeed, the repertoire of BCRs that promote B-1 development is largely nonoverlapping with the repertoire that promotes follicular B cell development (29, 30), probably because higher levels of BCR signaling are required for differentiation to B-1 vs follicular B cell phenotype (10, 16, 19, 28, 31, 32, 33). It would be desirable to develop an experimental system in which tolerance in different B lymphocyte subsets could be evaluated using a ligand common to all of these diverse subtypes.

We recently introduced a new model of B cell tolerance involving custom-designed superantigens, called macroself Ags, that allows analysis of tolerance in a normal, polyclonal immune system (34). In an initial study, a ubiquitously expressed, membrane-tethered, Ig{kappa}-reactive macroself Ag induced profound self-tolerance by receptor editing. In this study, results are presented from a transgenic mouse line expressing very low levels of the same macroself Ag in which tolerance by peripheral deletion of B-2 and MZ B cells predominates, whereas B-1 cells are spared. The results provide new insight into the relative tolerance sensitivity of B cell subsets.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

Young adult mice (6–12 wk of age) were analyzed in these studies. All mice were bred and maintained in The Scripps Research Institute Animal Resources facility according to Institutional Animal Care and Use Committee guidelines. C57BL6/J (B6) and B6.CD45.1 mice were obtained from The Jackson Laboratory.

Generation of {kappa}-macroself transgenes and production of transgenic mice

The construction of the {kappa}-macroself coding sequences, the pUli{kappa} transgene, and methods used to generate pUIi{kappa} transgenic mice have been described previously (34). Mice analyzed were backcrossed at least four times to the B6.CD45.1 background.

Bone marrow chimeras

Recipient mice were {kappa}-macroself transgenics or littermate controls; all carried the CD45.1 allele. Recipients received 950 rad of {gamma} radiation on the day of transfer. Bone marrow donors were all of the CD45.2 allotype. Bone marrow cell suspensions were depleted of T cells by treatment with biotinylated anti-Thy1.2 Abs and anti-biotin magnetic beads (Miltenyi Biotec) according to the suggested protocol. Five million cells were transferred i.v. per recipient. After 6 wk, recipients were killed, and their lymphoid tissues were analyzed by flow cytometry. Only chimeras in which ≥98% of cells in bone marrow and spleen were donor derived were included in the analysis.

Flow cytometric analysis

Nucleated cell suspensions were prepared from bone marrow, spleen, mesenteric lymph nodes, and peritoneal cavity. Erythrocytes were eliminated from the spleen and bone marrow preparations by ammonium chloride treatment. Cells were stained in a solution of PBS, 1% BSA, and 0.2% azide containing appropriately diluted combinations of the following mAbs: biotin-coupled mouse anti-rat IgG1 developed with streptavidin-PE; PE and biotin rat anti-mouse Ig{kappa} (187.1); biotin anti-Ig{lambda}1–3, followed by PE- or allophycocyanin-streptavidin; PerCP-coupled or FITC-coupled anti-CD45R/B220 (RA3–6B2); Cy5-coupled anti-IgM (331.12); PE-coupled anti-CD45.1 (eBioscience); FITC-coupled anti-CD45.2 (eBioscience); FITC-coupled anti-CD21; PE-coupled anti-CD23; PE-coupled anti-CD24 (HSA); and FITC-coupled anti-CD5. Abs were purchased from BD Pharmingen unless specified. Biotin-coupled Abs were revealed with streptavidin-PE (BD Pharmingen). Cells were gated on the basis of forward and side scatter criteria to avoid contamination by dead cells or debris. Stained cells were analyzed on a FACSCaliber flow cytometer (BD Biosciences) using the FlowJo analysis program.

RNA analysis of transgene expression

Total RNA was extracted from the spleen, bone marrow, liver, kidney, muscles, lungs, and heart of nontransgenic littermate, pUli{kappa}low transgenic, and pUli{kappa}high transgenic mice with TRIzol (Invitrogen). cDNA was generated from total RNA using the SuperScript III First-Strand Synthesis System for RT-PCR kit according to the manufacturer’s instructions (Invitrogen). For {kappa}-macroself Ag expression, PCRs were done in a final volume of 50 µl using the Platinum Taq polymerase system (Invitrogen) with the following primers: 5'-gtcatgaaccatcactttacaatctggg-3'; 5'-cagcggaggcggtgggtcggg-3'. Samples were amplified for 25 or 30 cycles: 1 min at 94°C, 1 min at 60°C, 1 min at 72°C. PCR products were electrophoresed in 1% agarose gel containing 5 µg/ml ethidium bromide. PCR products were visualized on gel exposure to UV light using an AlphaImage apparatus (Alpha Innotech). A 960-bp PCR product was amplified from tissues expressing the {kappa}-macroself Ag.

Ig gene rearrangement PCR assays

Nontransgenic littermate and pUli{kappa}low spleen B cells were first stained for B220 and Ig{kappa}. Ig{kappa}-positive cells were sorted in 1x PBS, 1 mM EDTA, 25 mM HEPES (pH 7.0), and 1% FCS using a FACS Vantage cell sorter (BD Biosciences). Ig{lambda} spleen B cells from pUli{kappa}high transgenic mice were isolated using the anti-B220 magnetic bead cell purification system (Miltenyi Biotec). The purity of these preparations was >95% in all cases. Genomic DNA was extracted with the QIAamp DNA Mini kit (Qiagen) according to the manufacturer’s protocol. PCRs were done in a final volume of 50 µl containing 100, 25, or 12 ng of B cell genomic DNA. V{lambda}1-to-J{lambda}1 excision product and V{kappa}1-to-J{kappa}1 and RS-to-IRS DNA rearrangements were detected and using the primers and the PCR conditions described previously (35, 36, 37). PCR products were electrophoresed in 1.5% agarose gels, blotted on nylon membranes, and probed with DNA probes as described previously (35, 36, 37).

BrdU labeling

pUli{kappa}low transgenic and nontransgenic littermate mice were given daily injections of 1 mg of BrdU in the peritoneum for 3 to 7 days. For BrdU uptake studies, surface-stained spleen and peritoneum cells were fixed, permeabilized, DNase treated, and stained with FITC anti-BrdU Ab using a BrdU Flow kit (BD Biosciences) according to the manufacturer’s instructions.

Serum Ig determinations

Polyvinylchloride plastic microplates (Falcon) were coated with rat mAbs specific for IgG2a + 2b, IgA, IgG1 (BD Pharmingen), and IgM (331.12). After washing and blocking, sera (diluted in PBS supplemented with 1% BSA) were incubated 3 h at room temperature. Bound Ig was detected using biotinylated anti-mouse Ig{lambda}1–3, anti-mouse Ig{kappa}, or HRP-conjugated anti-mouse Ig{kappa} (187.1). Biotinylated Abs were revealed using streptavidin-peroxidase (Sigma-Aldrich), followed by the addition of the chromogenic substrate TMB (3,3'-tetramethylbenzidine; Pierce). The reaction was stopped with the addition of a 2 M H2SO4 solution. Absorbance (450 nm) was measured on a Spectra MAX250 model plate reader (Molecular Devices). Standard curves were obtained using the following mouse Abs: IgM,{kappa} (G 155-228); IgM,{lambda} (11E10; Southern Biotechnology Associates); IgG2b,{kappa}; IgG2b,{lambda} (S23); IgG1,{kappa}; and IgG1,{lambda} anti-NIP.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
pUIi{kappa}low transgenic mice

B cell tolerance was studied in transgenic mice expressing the pUli{kappa} gene construct, which encodes a membrane-tethered, Ig{kappa}-reactive single-chain Ab under the control of the ubiquitin C promoter (Fig. 1A). A novel, low-expressing {kappa}-macroself Ag transgenic line (pUli{kappa}low) was generated and compared with the previously described pUli{kappa} transgenic line 2 (34). For the sake of simplicity, we will refer in this report to the pUli{kappa} line 2 strain as pUIi{kappa}high, although it expresses a level of {kappa}-macroself Ag that is intermediate among our panel of transgenics. Ig{kappa}+ cells in pUIi{kappa}high mice were shown previously to undergo central B cell tolerance by developmental arrest in the bone marrow and extensive receptor editing (34). Similar results were obtained with 11 of 12 pUli{kappa} transgenic lines. As we show below, the pUli{kappa}low mouse line described here, which appears to have the lowest transgene expression levels, had a distinct B cell tolerance phenotype. The pUli{kappa}low mouse expresses very low levels of the {kappa}-macroself Ag on the bone marrow and spleen cells compared with pUIi{kappa}high mice (Fig. 1B). The pUliklow mouse expresses low levels of soluble {kappa}-macroself Ag in the serum (<50 ng/ml), as determined by ELISA (data not shown). RT-PCR analysis revealed that transgene expression could be detected in most tissues of pUli{kappa}low mice (Fig. 1C), but, in contrast to pUIi{kappa}high tissues, at lower numbers of amplification cycles, many tissues scored negative. This expression difference provided an opportunity to study how low levels of self-Ag influence tolerance induction.



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  		FIGURE 1. A transgenic mouse line expressing low levels of Ig{kappa}-reactive macroself Ag. A, Schematic representation of a DNA construct used for microinjection to generate transgenic mice. A single-chain Fv generated from the anti-{kappa} hybridoma 187 is linked to the hinge and membrane proximal domains of rat IgG1, followed by transmembrane and cytoplasmic tail regions (Tm/Cy) of H-2Kb. The elements shown are approximately to scale. UTR, Untranslated region. B, Flow cytometric analysis of {kappa}-macroself Ag expression in the bone marrow and spleen of the indicated transgenic mice as detected using an anti-rat IgG1Fc Ab (dotted lines, nontransgenic cells; solid lines, pUli{kappa} transgenic cells). C, top panels, RT-PCR analysis of {kappa}-macroself Ag expression in the spleen (S), bone marrow (B), liver (Li), kidney (K), muscle (M), lung (Lu), and heart (H) of the indicated mice. Bottom panels, RT-PCR analysis of actin expression is used as a cDNA loading control. +, {kappa}-macroself transgenic tail DNA; –, nontransgenic tail DNA. Amplification reactions were performed for the indicated numbers of cycles. Tg, Transgenic.

 
Tolerance phenotype of Ig{kappa}+ cells in pUli{kappa}low mice

Lymphoid organs of pUIi{kappa}high and pUli{kappa}low mice were compared using flow cytometry for B cell numbers and phenotype (Figs. 2 and 3). B cells of pUli{kappa}low mice differed from B cells of pUIi{kappa}high and nontransgenic littermates in several ways. First, in bone marrows of pUli{kappa}low transgenic mice, the population of B220high/Ig{kappa}+ recirculating B cells was absent, whereas the fraction of newly formed B220int/Ig{kappa}+ cells was similar to nontransgenic littermate controls both in terms of cell numbers and sIg{kappa} density (Fig. 2A, cf left top and bottom rows). In contrast, newly formed {kappa}+ cells in pUIi{kappa}high bone marrow largely internalized sIg{kappa} (Fig. 2A, left middle panel; Ref.34). Furthermore, whereas in pUIi{kappa}high mice essentially no Ig{kappa}+ B cells were present in the periphery (Fig. 2A, middle row), pUli{kappa}low mice had significant, although reduced, numbers of Ig{kappa}+ B cells in the spleen and lymph nodes (top row). Compared with nontransgenic mice, pUli{kappa}low mice had a striking reduction in the percentages of sIg{kappa}+ B cells in the spleen and lymph nodes to one-third and one-tenth of normal, respectively (Fig. 3), which, when corrected to take account of their lower cell numbers, corresponds to 8 and 2% of normal absolute numbers, respectively. These results indicate significant but partial clonal elimination of Ig{kappa}+ B cells in the periphery of pUli{kappa}low mice.



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  		FIGURE 2. Flow cytometric analysis of Ig{kappa} B cells in lymphoid tissues of {kappa}-macroself Ag transgenic mice pUli{kappa}low and pUli{kappa}high. A and B, Two color flow cytometry plots of the indicated bone marrow, spleen, or lymph node tissues of pUli{kappa}low mice (top rows), pUli{kappa}high mice (middle rows), and littermate control mice (bottom rows). A, Costaining with B220 and Ig{kappa} Abs. In plots of bone marrow cells (left panels), the lower boxes identifying B220int/Ig{kappa}+ cells indicate putative newly formed B cells, whereas the upper boxes mark recirculating Ig{kappa}+ cells. Analysis of spleens and lymph nodes shows percentages of positive cells in the marked quadrants indicated. Note reduced numbers and B220 density of splenic and lymph node Ig{kappa}+ cells in transgenic mice. B, Costaining with Ig{lambda} and Ig{kappa} Abs. non Tg, nontransgenic.

 


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  		FIGURE 3. Statistical analysis of the observed frequencies of {kappa}+, {lambda}+, and B220+ cells in the indicated tissues of {kappa}-macroself Ag transgenic and nontransgenic mice given with overall viable cell numbers (see Fig. 2). In all experiments, bone marrow samples were analyzed using a lymphocyte gate to exclude most myeloid lineage cells. BM, Bone marrow; LN, lymph node; Non-Tg, nontransgenic.

 
Several findings suggested that {kappa}-to-{lambda} editing was not significantly elevated in pUli{kappa}low mice. One line of evidence came from an analysis of {lambda}+ B cell frequencies in lymphoid tissues. Although in pUli{kappa}low spleen and lymph nodes the percentages of Ig{lambda}+ cells were 3- to 5-fold increased compared with controls (Figs. 2B and 3), this corresponded to only a modest numerical increase in the spleen and lymph nodes of 2-fold and 20%, respectively. In bone marrow of pUli{kappa}low mice, numbers of newly formed B220int/{lambda}+ cells were also modestly increased relative to nontransgenic littermates (Fig. 4, A and C), but this difference was not statistically significant. In contrast, and as expected (34), the pUIi{kappa}high transgenic line had virtually no Ig{kappa}+ cells in the periphery, a 7-fold increase in {lambda}+ cells in the spleen and lymph nodes, and a 300–400% increase in newly formed {lambda}+ cells (Figs. 3 and 4C). A second line of evidence came from PCR quantitation of DNA rearrangements associated with {kappa}-to-{lambda} editing, including RS recombination to the {kappa} locus J-C intronic sites and V{lambda}-to-J{lambda} excision products (Fig. 4, B and D). In these experiments, sIg{kappa}+ splenic B cells from pUli{kappa}low mice did not have significant elevations of such recombination products compared with littermate control cells. B220+ cells from pUIi{kappa}high mice, which served as positive controls in these experiments, had significantly elevated levels of RS recombination and {lambda} excision products. We conclude that, in contrast to pUIi{kappa}high mice, in pUli{kappa}low mice tolerance induction occurs to a significant extent at a post-bone marrow stage of development because of poor {kappa}-macroself Ag expression in bone marrow.



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  		FIGURE 4. Quantification of receptor editing in {kappa}-macroself transgenic mice. A, Bone marrow cells from nontransgenic littermate and pUli{kappa}low and pUli{kappa}high macroself transgenic mice were stained for B220 and Ig{lambda}1–3 and analyzed by flow cytometry. The B220int/{lambda}+ population is found in the lower analysis box, whereas the upper boxes identify the recirculating, mature {lambda}+ cells. B, PCR analysis of DNA rearrangements in splenic B cells. Genomic DNA was isolated from sorted Ig{kappa}+ B cells from pUli{kappa}low transgenic mice and their nontransgenic littermates and from magnetic bead-purified B220+ pUli{kappa}high splenocytes. Genomic DNAs were used as PCR templates over a 4-fold dilution series as indicated. Amplified PCR products were detected by Southern blot using specific probes. The results from a single nontransgenic sample and two independent pUli{kappa}low and pUli{kappa}high samples are shown. Control PCR amplified a DNA stretch 3' to the RS element (control). C, Summary of quantification of newly formed bone marrow {lambda}+ cells as shown in A. D, Relative quantification of V{kappa}1-J{kappa}1, RS-to-IRS1, and V{lambda}1-J{lambda}1 excision product rearrangements detected by PCR as indicated in B. Non-Tg, Nontransgenic.

 
Ig{kappa} B cells in the spleens of pUli{kappa}low {kappa}-macroself transgenic mice have an immature phenotype

Because Ig{kappa}+ cells were readily detected in spleens of pUli{kappa}low transgenic mice, their cell-surface phenotype was assessed by flow cytometry. Several lines of evidence suggested that the {kappa} cells in pUli{kappa}low transgenic spleens were mainly phenotypically immature (Fig. 5). A large fraction had an IgMhigh/IgDlow phenotype, and few had a high level of IgD (Fig. 5A). Compared with nontransgenic {kappa}+ cells, pUli{kappa}low {kappa}+ B cells clearly had fewer CD23+ cells, reduced levels of B220 and CD21, and elevated levels of HSA (Fig. 5B). AA4.1+ cells (transitional type 1 cells) were proportionately more frequent than in controls (Fig. 5C). The pUli{kappa}low transgenic mice also appeared to have peripheral Ig{kappa}+ B cells with an IgMlow/IgDint phenotype that carried markers of immaturity, such as high HSA. Interestingly, MZ phenotype CD21high/CD23– cells appeared to be specifically depleted of Ig{kappa}+ cells in pUli{kappa}low mice, whereas Ig{lambda}+ cells were abundant in this compartment (Fig. 5D, bottom panels, and data not shown). Although the fraction of transitional B cells is preserved in the pUli{kappa}low transgenic mice, MZ B cells were increased in frequency relative to other B cell subsets, but not in overall numbers (Fig. 5E). We were unable to detect {kappa}+ cells of a B-1 phenotype in the spleens of pUli{kappa}low mice (data not shown). The results suggest that in the periphery of pUli{kappa}low transgenic mice, {kappa}+ B cells undergo developmental arrest or fail to survive long enough to differentiate normally.



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  		FIGURE 5. Immature phenotype of peripheral Ig{kappa}+ cells in {kappa}-macroself Ag transgenic mice. A, pUli{kappa}low spleen cells gated on sIg{kappa} were compared for the expression levels of IgM and IgD. B, Comparison of the levels of the indicated markers on splenic {kappa}+ cells of pUli{kappa}low transgenic mice (unfilled, bold lines) with littermate control mice (filled). C, AA4.1 vs B220 staining of Ig{kappa}+-gated splenic cells of pUli{kappa}low transgenic compared with control mice. D, Selective enrichment of {lambda}+ cells in MZ compartment of pUli{kappa}low transgenic mice as assessed with CD21 and CD23 Abs. MZ B cells are enriched in the CD21+/CD23– fraction indicated by the lower right boxes within the plots. The comparison of {kappa} and {lambda} cells of pUli{kappa}low spleens (bottom panels) with littermate control (top left) and pUli{kappa}high (top right) is shown. E, Summary of analysis in D. Values were as defined in the analysis boxes in D, with the CD21–/CD23– box enumerating T1 cells, the CD23–/CD21high box defining MZ B cells, and the CD23+/CD21low box including both T2 and follicular (F0) type B cells. non Tg or Non-Tg, Nontransgenic.

 
Persistence of Ig{kappa}+ B-1 phenotype B cells in peritonea of {kappa}-macroself transgenic mice

Tolerance in the B-1 compartment was assessed simply by evaluating the sIgL phenotype of peritoneal B cells. Interestingly, pUli{kappa}low mice had significant numbers of sIg{kappa}+ cells in the peritoneal cavity, whereas pUIi{kappa}high transgenic mice lacked all peripheral sIg{kappa}+ cells (Fig. 6, A and C). To determine the B cell subset type of sIg{kappa}+ in the peritonea of pUli{kappa}low mice, gated Ig{kappa}+ cells were evaluated for expression of the B-1 cell markers CD43, CD5, and Mac-1/CD11b (Fig. 6B). The sIg{kappa}+ cells of pUli{kappa}low mice clearly had a B-1a phenotype (CD43+CD5+CD11b+) and also had the characteristically larger size of B-1 cells as measured by forward light scatter. In contrast, B-2 type cells in the peritonea of pUli{kappa}low mice were devoid of sIg{kappa}+. One interpretation of these results is that B-1 cells have a higher threshold for immune tolerance than do MZ and follicular B cells (see Discussion).



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  		FIGURE 6. Presence of sIg{kappa}+ B-1 cells in the peritoneal cavity of pUli{kappa}low {kappa}-macroself transgenic mice. A, Flow cytometric analysis of sIg{kappa}+ and {lambda}+ cells in the peritonea of transgenic and littermate mice. B, Gated Ig{kappa}+ peritoneal cells were analyzed for other markers characteristic of B-1 type cells, including increased forward light scatter (FSC) and expression of CD5, CD43, and Mac-1/CD11b. Bold lines indicate pUli{kappa}low transgenic {kappa}+ cells, and light lines with shaded fill indicate wild-type control {kappa}+ peritoneal cells. C, Statistical analysis of the frequencies of {kappa} and {lambda} B cells in peritoneal cavities of pUli{kappa}low and pUli{kappa}high mice compared with nontransgenic controls. Non-Tg, Nontransgenic.

 
BrdU uptake studies

To assess the turnover of Ig{kappa} B cells in the spleen and peritoneal cavity, mice were given BrdU for 3 or 7 days, and the uptake into DNA was determined using a flow cytometry assay. As shown in Fig. 7, first panel, ~80% of Ig{kappa}+ cells in the spleens of pUli{kappa}low mice were labeled by day 7, whereas only ~20% were labeled in nontransgenic littermates. Furthermore, labeling of Ig{lambda}+ spleen cells was similar in pUli{kappa}low mice and nontransgenic littermates (~20% BrdU+ at day 7) (Fig. 7, second panel). Combined with the evidence of reduced Ig{kappa}+ B cell numbers and immature phenotype in the spleens of pUli{kappa}low mice, we conclude that Ig{kappa}+ splenic B cells of pUli{kappa}low mice undergo tolerance-induced clonal deletion.



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  		FIGURE 7. Immunofluorescence analysis of BrdU uptake in the spleen and peritoneal cavity of pUli{kappa}low macroself Ag transgenic and nontransgenic littermate mice. BrdU uptake with the time of labeling is shown for Ig{kappa}+ B cells and Ig{lambda}+ B cells of transgenic mice ({diamondsuit}) or littermates ({diamond}) in the indicated tissues. In the peritoneal cavity, the frequencies of BrdU+, Igk+, CD11b+ and BrdU+, Ig{lambda}+, CD11b+ B1 cells are shown. The number of mice analyzed from each time point and genotype are indicated on the first left histogram. Tg, Transgenic; WT, wild type.

 
In striking contrast, peritoneal Ig{kappa}+ B cells of the same pUli{kappa}low mice did not turn over rapidly, as determined by their low (~10%) level of BrdU uptake after 7 days of labeling (Fig. 7, third and fourth panels). This result supports the notion that peritoneal Ig{kappa}+ B cells of pUli{kappa}low mice are long-lived and distinct from the splenic Ig{kappa}+ B cell population. We conclude that the residual peritoneal Ig{kappa}+ B cells of pUli{kappa}low mice are long-lived B-1a cells that are relatively insensitive to tolerance-induced deletion by the {kappa}-macroself Ag.

Use of macroself transgenic mice as recipients in radiation chimeras

To assess {kappa}-macroself Ag-induced tolerance in a context in which B cells themselves cannot express macroself Ag, a bone marrow radiation chimera approach was adopted (Fig. 8). Wild-type bone marrow was transferred into lethally irradiated pUli{kappa}low, pUli{kappa}high, and nontransgenic mice and recipients analyzed 6 wk after reconstitution. As shown in Fig. 8, pUli{kappa}low recipients had reduced sIg{kappa}+ B cell numbers in the spleen and lymph nodes but not in bone marrow, with a modest increase in {lambda}+ B cells. In contrast, the peripheral lymphoid organs of pUli{kappa}high recipients had few {kappa}+ B cells and many {lambda}+ B cells (Fig. 8, A and C). Costaining for sIg{kappa} and IgM gave an independent measure of Ag receptor levels independent of L chain usage (Fig. 8B). Overall, pUli{kappa}low and pUli{kappa}high recipients appeared to recapitulate the tolerance phenotypes of intact transgenic mice. However, the extent of deletion in pUli{kappa}low recipients was less pronouced than that in intact pUli{kappa}low mice, possibly because of the presence of fewer superantigen-expressing cells. The results further suggested that central tolerance and receptor editing predominate in pUli{kappa}high recipients, whereas in pUli{kappa}low recipients, a peripheral, rather than central, B cell tolerance phenotype is seen.



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  		FIGURE 8. Flow cytometric analysis comparing Ig{kappa}+ B cells in pUli{kappa}low and pUli{kappa}high transgenic mice reconstituted with wild-type bone marrow. A, sIg{lambda} vs sIg{kappa} two-color analysis of the indicated tissues. Note that pUli{kappa}low mice had significant numbers of peripheral Ig{kappa}+ B cells, whereas pUli{kappa}high transgenic mice did not. B, sIg{kappa} vs IgM analysis. Donor and recipient genotypes are indicated on the left side of the flow cytometry histograms. C, Statistical analysis of the frequencies of {kappa} and {lambda} B cells along with total cells in the bone marrow (B. Marrow), spleen, and lymph nodes (L. Nodes) of pUli{kappa}low ({cjs2108}) and pUli{kappa}high ({blacksquare}) chimeras compared with nontransgenic recipient controls ({square}). The numbers in parentheses indicate the number of chimeras analyzed. WT, Wild type; Tg, transgenic.

 
Serum Ab levels in {kappa}-macroself transgenic mice

Levels of serum Ig{kappa} and Ig{lambda} in pUli{kappa}low transgenic mice were assessed to determine whether tolerance suppressed spontaneous Ig{kappa} Ab secretion and to determine the Ig H chain isotype profile of the Abs (Table I). A substantial but incomplete reduction in serum IgM,{kappa} levels was found in pUli{kappa}low transgenic mice, whereas levels of IgA,{kappa} and IgG,{kappa} were reduced nearly to background levels. In contrast, essentially no Ig{kappa} Ig of any kind was found in pUli{kappa}high transgenic sera (34). A reciprocal increase in serum Ig{lambda} levels was found in the pUli{kappa}low transgenic mice (Table II). We conclude that in pUli{kappa}low transgenic mice, partial peripheral B cell tolerance occurs in mouse Ig{kappa}+ cells but that considerable numbers of Ig{kappa}+ immature B cells and B-1 cells are retained, leading to the spontaneous formation of significant levels of serum IgM,{kappa}.


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  		Table I. Serum Ig{kappa} levels in pUli{kappa}low transgenic and littermate micea

 

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  		Table II. Serum Ig{lambda} levels in pUli{kappa}low transgenic and littermate mice

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
A novel model system to study peripheral B cell tolerance

We describe here a novel transgenic mouse line carrying Ig{kappa}-reactive macroself Ag that induces peripheral B cell tolerance in a polyclonal immune system. Tolerance in pUli{kappa}low mice results in a striking reduction in {kappa}+ cells of MZ and follicular phenotype, a net decrease in B cells, and an increased rate of {kappa}+ B cell turnover. These features are all consistent with tolerance-induced peripheral deletion. Although in pUli{kappa}low mice {lambda}+ B cells are somewhat elevated in numbers, particularly in the spleen, we have uncovered little evidence of {kappa}-macroself Ag-induced receptor editing. In pUli{kappa}low mice, a partial reduction in, but not elimination of, {kappa}+ cells was also noted in the B-1 compartment of the peritoneal cavity. A previous study assessed transgenic lines expressing the same {kappa}-macroself cell-surface protein at higher levels in the bone marrow, including pUli{kappa}high mice, in which a central tolerance phenotype was seen, characterized by a near total absence of peripheral {kappa}+ cells and extensive receptor editing (34). Because the pUli{kappa}low transgenic mice described here carry the same transgene and encode exactly the same protein product as the pUli{kappa}high mice, the altered phenotype described here is solely explained by the level of transgene expression. Furthermore, because the {kappa}-macroself ligand reacts identically with the Ag receptors of all {kappa}-expressing B cell subsets, without regard to fine specificity, it provides a relatively unbiased way to compare tolerance thresholds of different peripheral B cell subsets within the same animal.

The phenotype of the pUli{kappa}low mice, although unique among the panel of transgenics carrying the pUli{kappa} transgene, was similar to that found in a second {kappa}-macroself transgenic line carrying a modified transgene driven by the MHC class I Kb promoter and lacking the leader intron (data not shown). Therefore, it is unlikely that genetic alterations to the transgene explain the phenotype of pUli{kappa}low mice. Rather, we suspect that in pUli{kappa}low mice, and in the second line with a similar phenotype, the low level of macroself Ag in the bone marrow fails to induce significant receptor editing, leading to the migration of Ig{kappa}+ cells to the periphery, where the prolonged duration of low-level signaling promotes peripheral tolerance. It is difficult to say from the data available whether the lack of {kappa}+ B-2 and MZ B cells in pUli{kappa}low mice is dependent on the stage of development of the cells at the time of significant Ag encounter or the microenvironment in which Ag is encountered. Previous studies have provided evidence supporting both of these possibilities (4, 5, 35, 36, 38, 39, 40). In any case, the signaling induced by low-level {kappa}-macroself ligand in both mice is such that Ig{kappa}+ B-1 cells are spared, whereas other B cell subsets are counterselected.

Differential effects on B cell subsets

The relationship between peripheral B cell subset type and sensitivity to B cell tolerance or autoantibody formation is poorly understood. The present studies are significant because they are the first to directly assess the sensitivity of phenotypically and functionally distinct B cell compartments to the same Ag. The pUli{kappa}low transgenic mice had striking losses of Ig{kappa}+ cells in follicular and MZ B cell compartments, whereas clonal elimination was limited in the B-1 compartment. Remarkably, {kappa}+ cells constituted a major fraction (approximately one-third) of the peritoneal B-1 cell compartment of pUli{kappa}low mice, whereas {kappa}+ B-2 phenotype cells were absent in the same cell preparations. Peritoneal B-1 cells are known to be tolerance susceptible after acute administration of ligands (41) but are likely also dependent on self-Ag ligands for survival, development, or expansion (16, 21, 28, 42, 43). It may be that the relative paucity of Ig{kappa}+ follicular and MZ B cells in pUli{kappa}low transgenic mice is the result of the elimination or developmental block of their precursors at the immature B cell stage. The fetal precursors of B-1a cells are distinct from those of follicular and MZ B cells, which are bone marrow derived. Thus, it is possible that the tolerance thresholds of these precursors differ, or that they encounter different levels of macroself Ag. However, it is also possible that maturing MZ and follicular B cells have a lower threshold for tolerance induction than do B-1 lineage cells. In any case, mature B-1 cells appear to persist in an antigenic environment in which MZ and B-2 cells are counterselected.

It is unclear whether the signal strength of the BCR/{kappa}-macroself Ag drives the differentiation of some cells to the B-1 phenotype in pUli{kappa}low mice or whether B-1 development is independently regulated by the Ag receptor combining sites. It has been proposed based on work with endoplasmic reticulum-targeted hen egg lysozyme transgenic mice that Ags that promote B-1 development may preferentially be intracellular self-Ags (21). According to this model, intracellular Ags are stimulatory because exposure to them is limited to situations in which cell death occurs, a setting that would deliver TLR costimulation promoting B-1 development. However, in that study, low levels of hen egg lysozyme were apparent on the cell surface. Furthermore, Hayakawa et al. (28) found that one class of B-1 cells sees, and is selected by, the familiar Thy-1 molecule, a cell surface Ag. Although difficult to rule out, because all Ags must be expressed intracellularly at some level, we have no evidence that the {kappa}-macroself Ag used here, with its Ig leader peptide, MHC class I-derived transmembrane and cytoplasmic tail, is selectively retained inside cells. A more straightforward interpretation for our results would be that the low levels of accessible, membrane-displayed {kappa}-macroself Ag present in pUli{kappa}low mice fail to achieve a critical threshold of signaling needed for tolerance in the B-1 compartment, while failing to disrupt or actively induce B-1-positive selection.

It is interesting that the {kappa}+ cells in the peritonea of pUli{kappa}low mice solely had the phenotype of fetal derived B-1a B cells and lacked the bone marrow-derived B-1b or B-2 phenotypes. Our finding of low BrdU uptake in these cells suggests that {kappa}+ cells of pUli{kappa}low mice are long-lived and therefore resistant to clonal elimination by the macroself Ag, whereas bone marrow-derived B-1b cells fail to develop in the presence of the identical self-ligands. This result is reminiscent of those of Hayakawa et al. (28) in the anti-Thy B1 model. In addition, in that study, Ab reactive with self-Ag (Thy1, CD90) was present and actively induced by autoantigen. Similar to the {kappa}-macroself Ag expression profile in the pUliklow transgenic mice, the Thy1 autoantigen is also broadly distributed in mouse tissues (44). However, because of the normally high levels of IgM,{kappa} in serum, it is unclear whether the IgM,{kappa} Ab in pUli{kappa}low mice is actively induced by {kappa}-macroself Ag or rather is partly counterselected by it. Additional experiments will be needed to address this issue.

The present study underscores the observation that autoreactive B cells failing to see Ag in the bone marrow may be tolerized in the periphery by deletion or an anergy-associated reduction in lifespan (5, 6, 45, 46, 47, 48). The finding that MZ B cells apparently are eliminated in this model is consistent with studies with experimental tolerogens that this compartment may be particularly tolerance sensitive (25, 26, 27) but is inconsistent with another body of data suggesting that the MZ compartment is enriched in cells with self-reactive specificities (18, 42, 49). One possible explanation for this discrepancy is that, in contrast to the macroself Ag used here, certain self-Ags may promote B cell survival by delivering associated Tlr ligands, such as nucleic acids (50, 51).


   Acknowledgments
 
We gratefully acknowledge Drs. N. R. Klinman, M. G. McHeyzer-Williams, D. H. Kono, and A. J. Feeney of The Scripps Research Institute for their critiques of this manuscript and Dr. Glen Nemerow for the use of the ELISA plate reader.


   Disclosures
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 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 in part by research and training grants from the National Institutes of Health (RO1AI59714, RO1GM44809, T32AI07244, T32HL07195, and T32AI07606). Back

2 Address correspondence and reprint requests to Dr. Djemel Aït-Azzouzene, The Scripps Research Institute, 10550 North Torrey Pines Road, Mail Drop IM-29, La Jolla, CA 92037. E-mail address: djamait{at}scripps.edu Back

3 Abbreviations used in this paper: MZ, marginal zone; HSA, heat stable Ag (CD24). Back

Received for publication August 22, 2005. Accepted for publication October 27, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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