Predominance of a novel splenic B cell population in mice expressing a transgene that encodes multireactive antibodies: support for additional heterogeneity of the B cell compartment
Kathleen M. Tumas-Brundage1,,
Evangelia Notidis1,,
Lynn Heltemes1,,
Xianghua Zhang,
Lawrence J. Wysocki and
Tim Manser1,
Department of Immunology, National Jewish Medical and Research Center and University of Colorado School of Medicine, Denver, CO 80262, USA
1 Kimmel Cancer Institute and the Department of Microbiology and Immunology, Jefferson Medical College, Philadelphia, PA 19107, USA
Correspondence to:
T. Manser
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Abstract
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We generated IgHµ
transgenic mice using a VH gene that in A/J mice encodes multireactive BCR in the preimmune B cell compartment and is predominantly expressed by a memory B cell subpopulation. Most primary splenic B cells in these mice have a size, cell-surface phenotype and in vitro response profile distinct from mature follicular (B2), marginal zone (MZ) or B1 B cells, but are long-lived and appear to be slowly cycling. They reside in conventional B cell areas of the spleen and mount robust foreign antigen-driven germinal center responses, but do not efficiently differentiate to secretory phenotype. We propose that these qualities result from ongoing, low-avidity BCRself-ligand interactions and promote entry into the memory pathway. Given these data, and the enormous diversity and characteristic multireactivity of the preimmune antibody repertoire, we also suggest that it may be more appropriate to view the primary B cell compartment as a continuum of functional and phenotypic `layers', rather than as a group of discrete B1, B2 and MZ subsets.
Keywords: B cell, development, memory, subsets, transgenic mice
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Introduction
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B cell development takes place in a ordered series of steps, beginning in the fetal liver or adult bone marrow and culminating in peripheral lymphoid organs. First, the pre-BCR and subsequently the BCR play key roles in regulating progression in this developmental pathway (13). Engagement of self-ligands by the BCR can have profound inhibitory effects on development. Depending on the concentration and nature of the ligand, these may include death, receptor editing, developmental arrest or anergy (4). However, inability to express a BCR precludes B cell survival in both the bone marrow (BM) and the periphery (5,6). Moreover, there is mounting evidence that self-ligand engagement by the BCR can result in positive clonal selection, as well as influence the microenvironmental locale and peripheral B cell subset in which a clone resides (714).
One view of the primary B cell compartment is that its peripheral subsets collectively create a `layered' immune system, with each subset specialized to perform particular functions (15). In this context, the role of B1 cells seems to be to constitutively secrete IgM with specificities that allow binding to common pathogen structures, promotion of clearance of dead or damaged cells, immunoregulation and priming of the memory B cell response (1619). The marginal zone (MZ) B cell compartment is thought to be necessary for rapid responses to thymus-independent antigens encountered in the general circulation (20). Finally, follicular (B2) cells are viewed as the precursors of thymus-dependent responses leading to formation of the germinal center reaction and the memory compartment (21,22). Many primary B cells that do not fulfill the requirements for assignment to the B1, B2 or MZ subsets may well be in transit from the central lymphoid organs to one of these peripheral compartments, or from one peripheral compartment to another. Alternatively, some may represent more stable compartments that constitute yet additional functional `layers' of the immune system (5,23,24).
During the anti-p-azophenylarsonate (Ars) response in A/J mice a particular B cell clonotype that is a minor participant in the primary response comes to dominate anamnestic responses. The BCR expressed by this clonotype is encoded by a single combination of VH, D, JH, V
and J
gene segments (25,26). We term this clonotype and the antibodies it expresses `canonical'. Canonical antibodies expressed by primary B cells are multireactive, binding both Ars and a variety of self-ligands, including DNA, with low avidity (27). While, by definition, primary canonical B cells are precursors to memory B cells, the peripheral B cell compartment in which they reside has not been well studied due to their extremely low precursor frequency (28). For this reason we have generated and analyzed transgenic mice using a heavy chain gene representative of those expressed by canonical clonotypes prior to the Ars response.
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Methods
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Mice
The two transgene constructs used are shown schematically in Fig. 1(A)
. The µ
construct contains the heavy chain VDJ coding and promoter regions from the A/J canonical anti-Ars hybridoma 36-65, with the heavy chain intronic enhancer and the BALB/c µ and
constant region genes and substantial amounts of 3' flanking DNA. The 36-65 VDJ lacks somatic mutations. The HS1234 construct (29) contains four 3'
heavy chain enhancer elements (including the 1, 2, 3b and 4 DNase I hypersensitive regions) and was included to increase the probability that the transgenic heavy chain V gene was subjected to normal hypermutation. This issue is currently being investigated and will be the subject of a future report. Transgene inserts were purified and injected in equimolar amounts into the male pronuclei of fertilized FVB/nJ eggs. Initial PCR and Southern blotting screens revealed six founders with the µ
transgene out of which five also had the HS1234 transgene. Two founder lines (Ars20 and Ars30) were obtained that consistently passed both transgene constructs to their progeny. The Ars20 line contains four or five copies of the µ
construct, while the Ars30 line contains one copy. Founder mice were backcrossed to the A/J strain. Litters were screened for the presence of µ
and HS1234 transgenes as well as for the presence of A/J IgH alleles at the endogenous heavy chain loci. The screening for the A/J IgH alleles was done by Southern blot analysis probing for a restriction fragment length polymorphism near the µ switch region. Most mice used in the reported studies were maintained on a mixed A/JxFVB background. All A/J mice used were 812 weeks old and obtained from Jackson Laboratory (Bar Harbor, ME). All mice were housed under specific pathogen-free conditions, and given autoclaved food and water. The use of mice in these studies was conducted in compliance with Institute guidelines and all protocols using animals were approved by the Institutional Animal Care and Use Committee.

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Fig. 1. Transgene structure and allelic exclusion in the Ars20 B cell compartment. (A) The general structure of the µ and HS1234 constructs used to generate transgenic mice. The figure is drawn only approximately to scale. (B) The results of flow cytometric analysis of sIgMa (transgene allotype) and sIgDe (endogenous allotype) expression by spleen cells in an Ars20 transgenic mouse and a control littermate. Allelic exclusion was assessed using this approach since a mAb specific for the IgMe heavy chain is not available.
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Flow cytometry
Cells were isolated from lymphoid organs of 10- to 20-week-old, naive transgenic, age-matched transgene-negative littermates or normal A/J mice. Staining of cells was done using rat whole IgG and mouse whole IgG to block non-specific binding. One or several of the following reagents were used: polyclonal rat anti-mouse IgDbiotin (Fisher Scientific Pittsburgh, PA), anti-mouse IgMphycoerythrin (PE) (clone 1B4B1; Southern Biotechnology Associates, Birmingham, AL), donkey anti-mouse IgMFITC (Jackson ImmunoResearch, West Grove, PA), anti-IgDebiotin (clone AF4-73.3), anti-CD43biotin (clone S7), anti-I-Akbiotin (clone 11-5.2b), anti-CD69biotin (clone H1.2F3), anti-CD95biotin (Fas clone Jo2), anti-CD80biotin, rat anti-mouse IgDbiotin (clone 11-26; Southern Biotechnology Associates), PE and FITCanti-B220 (CD45R clone RA3-6B2), PEanti-CD1d (clone 1B1), FITCanti-IgMa (clone DS-1), FITCanti-CD3 (clone 145-2C11), FITCanti-CD24 (HSA clone M1/69), FITCanti-CD40 (clone HM40-3), FITCanti-CD21/CD35 (clone 7G6), FITCanti-CD86 (clone GL-1), allophycocyaninanti-CD62L (L-selectin clone Mel-14), FITCanti-CD23 (clone B3B4), FITCstreptavidin, PEstreptavidin, and R670streptavidin (Gibco/BRL, Rockville, MD), and analyzed immediately or fixed in 1% paraformaldehyde. In some experiments PEanti-CD5 (clone 53-7), anti-CD24biotin (clone 30-F1), FITCanti-BP-1 or PEanti-BP-1 were used (all kind gifts of Dr R. R. Hardy). The anti-idiotypic antibodies 107 and E4 were purified from ascites and biotinylated using standard methods. Unless indicated otherwise, all antibodies were obtained from PharMingen (San Diego, CA). Analyses were done on a Coulter Epics Elite or on a Becton Dickinson FACStar plus using either the WinMDI 2.7/2.8 programs from the Scripps Institute (San Diego, CA) website or FlowJo (Tree Star, San Carlos, CA) software.
Immunohistochemistry
Spleens were isolated from naive or immunized mice, frozen and cryosections prepared as previously described (30). Sections were stained with one of the following sets of reagents: (i) biotin107 or biotinE4, branched streptavidinalkaline phosphatase (Dako, Glostrup, Denmark) and horseradish peroxidase (HRP)peanut agglutinin (PNA) (EY Laboratories, San Mateo, CA); (ii) biotinPNA (EY Laboratories), branched streptavidinalkaline phosphatase and HRP(Fab')2 fragment of donkey anti-mouse IgM (Jackson ImmunoResearch); or (iii) anti-B220 ascites (clone RA3-6B2), streptavidinalkaline phosphatase-conjugated (Fab')2 fragment mouse anti-rat Ig (Jackson ImmunoResearch) and HRPanti-CD4 (clone GK1.5 coupled to HRP using standard methods).
Hybridomas
Hybridomas were generated from lipopolysaccharide (LPS)- and dextran sulfate-stimulated spleens cells from naive Ars20 line mice (10 weeks of age) as previously described (31). Twenty randomly chosen wells from each fusion were subcloned by limiting dilution. Supernatants from the subclones were subjected to quantitative Ars, E4, 107, anti-mouse
, and single- and double-stranded DNA ELISAs as well as antinuclear antibody assays as previously described (31). Prior to addition to the ELISA plates, samples were treated with 100 mM ß-mercaptoethanol for 30 min at room temperature to reduce IgM to a monomeric form, thus reducing the non-specific background in the assays (32). In the evaluation of Ars and DNA binding activity, the 36-65 and 3H9 mAb were used respectively as ELISA standards. Values obtained in Ars, and single- and double-stranded DNA binding assays were normalized to those obtained in anti-
assays. Only those mAb displaying normalized binding values
10% produced by the positive control mAb standard were considered `positive'.
V
amplification and sequencing
Total RNA was prepared from 107+ hybridomas as previously described (33). RNA was used for RT-PCR using the 5' primer univ 5LK which has been shown to amplify all known V
genes (34) and the 3' primer SalI CK 5'-GCAGCTGTGGATGGTGGGAAG-3'. Since the hybridoma fusion partner, Sp2/0, expresses a sterile V
mRNA (35), we modified the RT-PCR protocol as follows: after reverse transcription, only five cycles of PCR were done followed by digestion of the products with the restriction enzyme TatI (MBI Fermentas, Amherst, NY) per the manufacturer's instructions. TatI cleaves the PCR product generated from the Sp2/0 fusion partner into small fragments that cannot be amplified in subsequent PCR reactions. Of all known mouse V
gene segments, the TatI restriction site is found in central regions of only 20 out of 109 segments (36). After a 1 h digestion with TatI, samples were ethanol precipitated. Precipitated products were pelleted, and washed twice with 70% ethanol. Pellets were resuspended in PCR buffer containing univ 5LK and SalI CK primers (see above) and 30 cycles of PCR were performed. PCR products were directly sequenced using an internal C
primer.
In vitro proliferation assays
B cells were purified from the spleens of mice as previously described (37). Cells were plated in 96-well dishes at 12x106 cells/ml and stimulated with LPS (Difco; Detroit, MI), goat anti-mouse IgM(Fab')2 (Pierce, Rockford, IL) or anti-mouse CD40 (clone FGK45, a gift of Dr Anton Rolink) as previously described (37). After 48 h in culture, cells were pulsed for 1216 h with [3H]thymidine (New England Nuclear Life Sciences, Boston, MA), harvested and 3H incorporation evaluated.
Serology and ELISAspot assays
ELISA assays were performed as previously described (31). ELISAspot assays were performed using Multiscreen 96-well plates (Millipore, Bedford, MA). Wells were coated overnight at 4°C with 10 µg/ml of goat anti-mouse Ig or ArsBSA. After extensive washing the plates were blocked with 10% FCS/RPMI 1640 and dilutions of cells then added in the same medium. After incubation at 37°C for 6 h, the plates were washed with PBS/0.5% Tween 20 and dilutions of biotinylated 107, E4 or rat anti-mouse
mAb were then added in PBS/1% BSA. After incubation overnight at 4°C, the plates were washed with TBS/0.5% Tween 20 and dilutions of streptavidinalkaline phosphatase were added in TBS. After incubation for 2 h at room temperature, the plates were washed with TBS/0.5% Tween 20 and the spots developed by the addition of Fastblue BB. After washing and drying the spots were scored at x510 magnification using a stereomicroscope.
Cell cycle and BrdU-labeling analyses
Splenic B cells were isolated from mice as described above. Ethanol-fixed and RNase-treated cells were stained with propidium iodide (PI) immediately after isolation, and 24, 48 and 72 h after culture with LPS (25 µg/ml), anti-mouse IgM [F(ab')2, 10 µg/ml], anti-CD40 (FGK45, 10 µg/ml) or media alone as previously described (37), and analyzed using a Coulter Epics Profile II and Elite software.
Ars20 transgene-positive and control littermates were injected i.p. with 0.6 mg BrdU (Sigma) in 0.2 ml PBS at 12-h intervals for 11 days. Three mice from each group, plus an unlabeled mouse from each group, were sacrificed on days 4, 8 and 11. The cells obtained from each mouse were stained for surface markers as described above and washed in cold PBS. Incorporated BrdU was analyzed by flow cytometry according to Allman et al. (38).
Adoptive transfer experiments
Donor B cells were isolated from the pooled spleens of two to four Ars20 transgene-positive mice as described above. Following a modified protocol of Garside et al. (39), 2.53.0x106 Ars20 splenic B cells were injected i.v. into one unirradiated (A/JxFVB/nJ) F1 mouse. The day prior to administration of the cells, recipient mice were bled to obtain background serum antibody levels. The day after cells were administered, some mice were injected i.p. with 100 µg Arskeyhole limpet hemocyanin (KLH) in either complete Freund's adjuvant as an alum precipitate. For serological analysis, these mice were bled at 1-week intervals. For immunohistochemistry analysis, these mice were bled at day 8 or 12 after initial immunization and their spleens frozen for cryosectioning. Unimmunized mice were sacrificed at various times after cell transfer (up to 3 weeks) and their spleens frozen for histology.
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Results
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Lines of transgenic mice were generated using the µ
and HS1234 constructs shown in Fig. 1(A)
(see Methods for details). The transgene constant regions are Igha allotype and the transgene(s) were back crossed to A/J mice (Ighe allotype). In adult mice in which both endogenous IgH loci were of A/J origin, transgene expression and allelic exclusion were evaluated using IgMa- and IgDe-specific mAb. As shown in Fig. 1(B),
IgDe+, IgMa spleen cells were absent in a mouse of the Ars20 transgenic line, but a large population of IgMa+ cells was detected only a very minor fraction (<2%) of which were also weakly IgDe+, indicating extremely good transgene exclusion of endogenous IgH expression. Analogous results were obtained using several independent lines of mice and other lymphoid organs (data not shown).
The frequency of B cells expressing canonical BCR is substantially increased in transgenic mice
The anti-idiotypic mAb 107 is specific for a subset of canonical antibodies, including the 36-65 (transgene heavy chain donor) mAb, containing rare VD and DJ junctional amino acids (40,41). Figure 2
(upper panels) demonstrates that the vast majority of Ars20 B220+ cells are 107+. The few 107 B cells presumably express light chains that preclude idiotope expression. Similar results were obtained from mice of other transgenic lines. In subsequent analyses we focused on the Ars20 line, in which uniform genetic transmission of both transgene constructs was always observed, suggesting they had co-integrated. A second anti-idiotypic antibody, E4, which is rather specific for V regions composed of the canonical heavy and light chain combination (42), stained subpopulations of splenic and lymph node Ars20 B cells (Fig. 2
, bottom panels). Such B cells are extremely rare in A/J mice prior to Ars immunization.

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Fig. 2. Surface transgene idiotype expression by Ars20 B cells. The panels show the results of flow cytometric analysis of 107+ and E4+ BCR (see text for details) expression by Ars20 and A/J splenic B cells. The dots exactly on the diagonal in the spleen analyses result from autofluorescent cells.
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Serum antibody levels in Ars20 mice
Total serum Ig levels were found to be ~75% lower in naive Ars20 mice as compared to non-transgenic littermates. More strikingly, these mice had very low (<10 µg/ml) serum titers of 107+ antibodies and undetectable levels of E4+ antibodies (data not shown). These results stood in marked contrast to the data presented in Fig. 2
, showing that most splenic and lymph node B cells express 107+ BCR and a subpopulation of these also expresses E4+ BCR.
Peripheral B and T cell compartments in Ars20 mice
Quantitative flow cytometric analysis of B and T cell populations demonstrated a somewhat reduced number of total B (B220+ or IgM+) cells in the spleen (average 35% reduction) and a more substantial reduction in the lymph nodes (>50% reduction) in Ars20 mice compared to non-transgenic littermates. Visual examination of Peyer's patches revealed on average only two or three Peyer's patches per Ars20 mouse, as compared to 810 in control littermates. Analysis of the T cell compartment in spleen and lymph nodes with a variety of mAb specific for activation and differentiation revealed no numerical or phenotypic abnormalities (data not shown).
The majority of splenic Ars20 B cells do not have a follicular (B2) phenotype
In the spleen and lymph nodes of adult Ars20 mice, the mature, resting B cell population was substantially reduced. When Percoll gradients were done on either whole or T-depleted splenocytes, >30% of B cells in naive A/J mice or non-transgenic littermates pelleted through a
= 1.079 density layer (i.e. small, resting B cells). However, on average 3-fold fewer splenic B cells from Ars20 mice located to this position in the gradients, with most banding at the interface of the
= 1.079 and 1.066 layers. Flow cytometry also revealed a deficiency of splenic B cells with low forward and side light scatter values in Ars20 mice (data not shown).
Figure 3
shows that the majority of Ars20 splenic B cells express elevated levels of sIgM and sIgD characteristic of a subpopulation of splenic B cells present in littermates. Moreover, major subpopulations of either 107+ or E4+ cells in the spleen express elevated levels of CD21/35, CD24 and CD1d, but low levels of CD23, also characteristic of subpopulations present in littermates. In littermates, CD21/35highCD1dhighCD23low, IgMhighCD21/35high and IgMhighCD24high subpopulations account for 1015% of splenic B cells. In contrast, among both 107+ and E4+ B cells in adult Ars20 mice these subpopulations make up 5075%. In addition, Ars20 splenic B cells in general express elevated levels of CD21/35, CD1d and CD24, and reduced levels of CD23, as compared to the major subpopulation of littermate splenic B cells. Ars20 splenic B cells expressing 107+ or E4+ BCR are also deficient in a subpopulation of IgMhighCD21/35low cells that appears to correspond to B1 cells (R. R. Hardy, pers. commun.). Additional staining analyses revealed generally reduced levels of CD62L (L-selectin), and generally increased levels of MHC class II and CD40 on Ars20 splenic B cells (data not shown). While levels of CD69, CD86 (B7-2), CD43 (S7), CD95 (Fas) and B220 also appeared to be slightly elevated on these cells; this may result from their increased size.

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Fig. 3. Cell-surface phenotype of Ars20 transgenic B cells. Spleen, lymph node and peritoneal cells from three Ars20 mice and three littermates were isolated, pooled, stained with various combinations of mAb fluorescent conjugates and analyzed by flow cytometry, all as described in Methods. In these analyses data obtained from the live lymphocyte gate of littermate cells was compared with data obtained from both the live lymphocyte and either 107+ or E4+ gates of Ars20 cells. In each panel the intersection of the cross-hairs marks the center of the distribution of the major stained population of cells in the corresponding littermate samples, to allow better visualization of the differences or similarities observed in the Ars20 samples. There were too few E4+ cells in the lymph node and peritoneal samples for accurate gating and so these data are not shown.
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In contrast to splenic B cells, Fig. 3
shows that 107+ cells present in the lymph nodes of Ars20 mice display a phenotype consistent with mature follicular cells, in that the majority are IgMlowCD21/35lowCD24lowCD1dintCD23high (the levels of sIgD on these cells is also similar to littermate lymph node B cells, data not shown). In the peritoneum, Ars20 107+ B cells reveal staining profiles consistent with the presence of mainly IgMhigh cells. However, these cells are deficient in CD21/35lowCD23low cells (Fig. 3
), indicating a deficiency in conventional B1 cells as was observed in the spleen. In addition, while 107+ cells are abundant in the peritoneum and clearly present in the lymph nodes, E4+ cells are rare in both these locales.
In vivo life span and cell cycle activity of Ars20 splenic B cells
Neither the IgD+ or 107+ splenic compartments, or the E4+ subpopulation, appeared to have substantially different intermediate-term in vivo BrdU labeling rates as compared to the IgD+ splenic compartment in transgene-negative littermates (Fig. 4A
). PI cell cycle analyses revealed that the percentage of splenic B cells in the S/G2/M region was elevated 3- to 10-fold in Ars20 mice as compared to littermates (Fig. 4B
). In combination with the BrdU analyses, these data suggest that a major subpopulation of Ars20 splenic B cells is slowly cycling in vivo. Consistent with this conclusion, ~2-fold more splenic B cells (IgD+, B220+) incorporated BrdU over a 4-h pulse-labeling period in Ars20 mice as compared to littermates (data not shown).

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Fig. 4. Life span and cell cycle status of Ars20 splenic B cells. (A) The results of an intermediate term in vivo BrdU-labeling study of Ars20 and littermate B220+ spleen cells. Each point was derived from the independent analysis of the spleen cells of three mice (see Methods). Ars20 cells were analyzed for the percentage of E4+, 107+ or IgD+ cells that also stained with the anti-BrdU mAb. Littermate cells were analyzed for the percent of IgD+ cells that also stained with the anti-BrdU mAb. (B) The results of PI cell cycle analysis performed on the T cell-depleted and Percoll gradient-purified B cells of Ars20 and littermate spleens (pooled from three mice each). Immediately after purification, B cells were fixed, stained with PI and analyzed by flow cytometry. The percent of cells in the G2/S/M region of the histograms for the Ars20 (bold line) and littermate (light line) samples are indicated above the interval marker. Such PI data obtained from the splenic B cells of three Ars20 mice gave an average percentage of cells in the G2/S/M region of 7.6 (SD = 4.3).
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Ars20 splenic B cell responses to in vitro stimulation
In response to LPS stimulation, splenic B cells from Ars20 mice were consistently hyper-responsive as compared to B cells from either A/J mice (Fig. 5A
) or non-transgenic littermates (data not shown), particularly at high concentrations of this mitogen. In contrast, Ars20 splenic B cells proliferated to comparable extents as compared to control B cells in response to agonistic anti-CD40 treatment (Fig. 5B
) and were hypo-responsive to stimulation via cross-linking of their BCR with anti-µ (Fig. 5C
). These data were corroborated using PI cell cycle analyses (data not shown).

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Fig. 5. In vitro proliferative responses of Ars20 splenic B cells. Splenic B cells were purified from three Ars20 mice and three littermates or three A/J mice. Pooled cells from each type of mouse were then stimulated with the indicated concentrations of (A) LPS, (B) agonistic anti-CD40 mAb or (C) goat anti-mouse IgM F(ab')2 fragments in vitro for 48 h, pulsed with [3H]thymidine for 1218 h and 3H incorporated into DNA measured, all as described in Methods. Ars20 splenic B cells were consistently hyperproliferative to high concentrations of LPS as compared to either littermate or A/J splenic B cells, but depending on the batch of LPS used they sometimes displayed similar proliferation levels to low (<1 µg/ml) concentrations of this mitogen. (A) The results of one experiment, where A/J splenic B cells were used as a control, in which hyperproliferation was observed at both high and low concentrations of LPS. In this experiment, fewer c.p.m. were incorporated into DNA than in the anti-IgM and anti-CD40 experiments since less [3H]thymidine had been added to the cultures.
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ELISAspot analyses of secretion of anti-Ars, 107+ and E4+ antibodies were also performed on Ars20 and control A/J splenic B cell cultures stimulated for 35 days with LPS. An ~5-fold increase in Ars+ ELISAspots was obtained from Ars20 B cells as compared to controls. The frequency of E4+ ELISAspots was also increased (to ~0.4% of input B cells) among Ars20 B cells. 107+ antibody forming cells (AFC) were undetectable in littermate cultures, but were detected at a frequency of ~12% of total input B cells in Ars20 cultures. However, these frequencies of AFC obtained from transgenic B cells were extremely low given the precursor frequencies indicated in Fig. 2
and the average size of the spots the Ars20 AFC produced was small (data not shown).
Ars20 transgenic B cells home to the follicles of the spleen
Figure 6
shows the results of immunohistochemical staining of three parallel spleen sections from a representative naive Ars20 mouse and a non-transgenic littermate. In the transgenic sections, 107 stained the vast majority of cells in the follicles and MZ. Staining of parallel sections with B cell (anti-B220, blue)- and T cell (anti-CD4, red)-specific reagents showed that the overall lymphoid microenvironmental organization of the spleen was normal in the Ars20 mouse (Fig. 6
, bottom panels). B cell location was also assessed using anti-IgM (red staining; Fig. 6
, middle panels). The overall anti-IgM staining pattern in the Ars20 spleen is analogous to the staining pattern seen in the non-transgenic mouse. Staining of parallel sections with E4 revealed that canonical BCR-expressing B cells were present throughout the follicles and MZ. Identical results were obtained for several other Ars20 mice (data not shown).

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Fig. 6. Normal splenic lymphoid microarchitecture and location of transgene-expressing B cells in Ars20 mice. Three adjacent spleen sections from a naive Ars20 mouse and a naive littermate were stained with combinations of PNA and antibodies specific for the indicated markers and developed, all as described in Methods. In the top two panels, PNA staining appears red and 107 staining appears blue. In the middle two panels, PNA staining appears blue and IgM staining appears red. In the bottom two panels, B220 staining appears blue and CD4 staining appears red. Note that in the Ars20 section 107+ cells are distributed throughout the follicular and MZ regions, and are rare in the red pulp and the T cell zones. Original magnification of images was x100. These data are representative of those obtained from several Ars20 spleens.
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Ars20 splenic B cells efficiently participate in germinal center reactions
Small numbers of Ars20 B cells were injected into syngeneic, unirradiated A/JxFVB/nJ mice, followed by immunization of the chimeric mice with ArsKLH. At various times thereafter mice were bled and spleens taken for histological analysis. Figure 7(C)
shows that, as expected from the in vitro studies presented above, only low levels of 107+ serum antibodies were produced by the chimeric mice and only at late stages of the primary response. In contrast, the immunohistochemistry analysis revealed abundant germinal centers that stained with 107 or both 107 and E4 at both days 8 and 12 after immunization (a day 12 example is shown in Fig.7A and B
). At neither of these time points were substantial numbers of 107+ or E4+ cells observed outside of germinal centers. If chimeric mice generated in this way were not immunized with ArsKLH, 107+ and E4+ cells could be observed throughout the MZ and follicular regions of the white pulp, and the number of these cells remained stable for 3 weeks after transfer (the latest time point examined, data not shown).

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Fig. 7. Ars20 107+ and E4+ splenic B cells efficiently participate in the germinal center reaction but produce only low levels of serum antibody during an Ars response. Purified and pooled splenic B cells from three Ars20 mice were injected into syngeneic, non-irradiated recipients that were immunized with ArsKLH 1 day later (see Methods). At various times after immunization, chimeric mice were either bled or sacrificed and their spleens processed for histology. (A) The results of 107 (blue) and PNA (red) staining of a spleen section from such a chimeric mouse sacrificed at day 12 after immunization. (B) The results of staining of an adjacent spleen section with PNA (red) and E4 (blue). Original magnification of the images was x100. Note that numerous 107+ and E4+ cells are observed in all three germinal centers shown, and few such cells are observed outside of germinal centers. These data are representative of those obtained in two independent experiments. In both experiments, numerous germinal centers containing 107+ and E4+ cells were consistently observed in individual spleen sections obtained in the day 1214 after immunization time frame. (C) Results of an analysis of 107+ (transgene encoded) serum antibody production in pooled sera obtained from two chimeric mice at the indicated times after immunization. Note that only low levels (<30 µg/ml) of transgene-encoded serum antibody were produced even at late times after immunization.
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A large percentage of Ars20 B cells have multireactive BCR
Hybridomas were generated from the LPS and dextran sulfate stimulated spleen cells of two Ars20 mice. Of 28 randomly selected 107+ hybridomas, nine produced anti-Ars antibodies. All of these also bound single- and double-stranded DNA. In total, 19 of the hybridomas produced mAb that bound single-stranded DNA, double-stranded DNA or both. Most of the Ars-binding mAb bound Ars less well than did the 36-65 mAb, and most of the DNA-binding mAb bound this ligand less well than the 3H9 mAb (43), attesting to the low avidity of these binding reactions. Four of the mAb (two with unknown antigen specificities, one with a specificity for single- and double-stranded DNA, and one with a specificity for Ars, and single- and double-stranded DNA) were analyzed in an ANA assay. All of the mAb uniformly stained the cytoplasm and also stained nuclei in a punctate pattern (data not shown).
Analysis of V
family usage in the hybridomas demonstrated that all hybridomas analyzed appeared to co-express single light chain genes with the transgene encoded heavy chain. These genes were members of seven of the 18 known V
families. The predominant V
family was the V
9A/B family, found to be expressed in one-third of the hybridomas analyzed, and there was a predominance of J
5 gene segment usage by all hybridomas. However, the expression of a particular V
or V
J
did not correlate with a particular antibody specificity.
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Discussion
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Most splenic Ars20 B cells, including canonical clonotypes, are functionally and phenotypically distinct from mature follicular (B2) B cells and B1 cells. Comparison of the data we have obtained with previously published data on splenic B cells subsets (20,4447) indicates that Ars20 splenic B cells bear the strongest resemblance to MZ B cells. They are sIgMhighCD21/35highCD23low and express high levels of the CD1d non-classical MHC antigen. They also display increased size, appear to be slowly cycling and have a heightened response to LPS but are hyporesponsive to anti-IgM. In addition, Ars20 B cells are in general inefficient at seeding the lymph nodes and Peyer's patches. However, they efficiently colonize both the splenic follicles and MZ, properties reminiscent of the `T2' transitional population recently defined by Carsetti et al. (44). Indeed, the in vitro response characteristics of these cells might well be explained by developmental `immaturity'.
Nonetheless, Ars20 splenic B cells have unique characteristics as well. They do not produce high levels of secreted antibodies, either spontaneously, or after in vitro or in vivo stimulation in contrast to MZ B cells (20). In addition, elevated levels of sIgD is a characteristic of most Ars20 B cells. This is not a general property of T2, MZ or B1 B cells (20,44). While CD1dhigh follicular B cells express high levels of sIgD, they are sIgMlowCD23high (45,46). These elevated levels of IgD do not appear to result from the fact that the Ars20 transgene is present in multiple copies (four or five) since B cells in the Ars30 line, which carry only one copy of the µ
transgene, display this same property (L. Heltemes and T. Manser, unpublished observations). Also, the B cells present in the lymph nodes of Ars20 mice express normal levels of sIgD and the levels of sIgD expression on most Ars20 splenic B cells is similar to a subpopulation of splenic B cells observed in littermates. Since most Ars20 splenic B cells have a decreased buoyant density, appear to be slowly cycling and express levels of other markers consistent with a `partially activated' phenotype, we favor the interpretation that the elevated levels of sIgD result from ongoing, low-avidity engagement of self-ligands. These elevated sIgD levels may also inhibit tolerance induction, as has previously been suggested to be the case for follicular B cells (48).
Ars20 B cells also display intriguing phenotypic parallels with B cells present in a VH81X Ig heavy chain transgenic line of mice previously characterized by Kearney et al. (49) and a line of VH `knockin' mice expressing the T15 VH gene generated by Rajewsky et al. (50). The VH81X transgenic B cells have specificities for multiple intracellular autoantigens and display a phenotype consistent with self-antigen-mediated activation (51). Interestingly, the most autoreactive subpopulation of these B cells preferentially resides in the splenic MZ, and has a morphologic and cell-surface phenotype similar to that of MZ B cells in normal mice (13). The VHT15 `knockin' B cells display some of the phenotypic qualities of `T2' B cells (50), and the T15 VH gene confers specificity for the common self-ligand phosphatidylcholine. The in vitro reactivity of many of the Ars20 mAb we isolated makes intracellular nucleic acid-rich components one of the likely candidates for a group of self-ligands that might influence the developmental fate of many Ars20 B cells in vivo.
When small numbers of Ars20 splenic B cells were transferred to non-irradiated, syngeneic recipients, immunization with ArsKLH resulted in the widespread participation of the 107+, and 107+E4+ (canonical) subpopulations in the germinal center response, but not in the AFC response. This indicates that Ars-specific Ars20 B cells, including canonical clonotypes, are efficient precursors to the memory B cell pathway. We have previously shown that canonical clonotypes do not give rise to a periarteriolar lymphoid sheath, or red pulp AFC `focus' response, but are first observed in germinal centers during the anti-Ars response of A/J mice (30). Differentiation to AFC phenotype appears to require substantially higher levels and probably qualitatively distinct types of T cell `help' as compared to differentiation via the germinal center pathway to memory phenotype (52,53). The self-reactivity of most Ars20 B cells may shunt them into the germinal center/memory B cell pathway, by inducing a state of differentiation that precludes the receipt of the T cell help requisite for development into antibody-secreting cells. Once in the germinal center microenvironment, BCR somatic hypermutation and phenotypic selection processes may result in such clonotypes losing their autoreactivity, and gaining increased affinity for Ars. We have previously argued that this is how canonical clonotypes come to be the predominant contributors to the anti-Ars anamnestic serum antibody response (31).
Collectively, our data are consistent with the idea that the quality and quantity of BCR self-ligand engagement dramatically influence primary B cell developmental fate in the periphery (11,54). Since BCR cannot be monospecific and, in fact, the primary B cell compartment is characterized by the expression of multireactive BCR (16,55,56), it is likely that many stable and functional members of this compartment are subjected to such self-antigen-mediated `regulation'. Given the enormous diversity of the preimmune antibody repertoire, this reasoning suggests that it may be more appropriate to view this compartment as a continuum of functional and phenotypic `layers', rather than as a group of discrete B1, B2 and MZ subsets.
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Acknowledgments
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We thank Drs Mark Groudine and Linda Madisen for providing the HS1234 construct, the Kimmel Cancer Center Transgenic and Flow Cytometry Facilities (supported in part by a grant from the NIH, CA56036), Dr Dave Allman for alerting us to the studies on transitional B cells by Carsetti et al. and for discussions on transitional B cells in general, and all members of the Manser laboratory for their indirect contributions to this work. This study was supported by grants to L. W. (AI39563) and T. M. (AI23739, AI38965) from the NIH. E. N. and L. H. were supported by NIH training grants AI-07492 and CA-72318 respectively.
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Abbreviations
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AFC Antibody forming cells |
Ars p-azophenylarsonate |
BM bone marrow |
HRP horseradish peroxidase |
KLH keyhole limpet hemocyanin |
LPS lipopolysaccharide |
MZ marginal zone |
PE phycoerythrin |
PI propidium iodide |
PNA peanut agglutinin |
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Notes
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The first two authors contributed equally to this work
Transmitting editor: J. Kearney
Received 29 October 2000,
accepted 21 December 2000.
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