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The Journal of Immunology, 2007, 178: 2192-2203.
Copyright © 2007 by The American Association of Immunologists, Inc.

AID–/–µs–/– Mice Are Agammaglobulinemic and Fail to Maintain B220–CD138+ Plasma Cells1

Kaori Kumazaki*, Boaz Tirosh*, René Maehr{dagger}, Marianne Boes{ddagger}, Tasuku Honjo§ and Hidde L. Ploegh2,*

* Whitehead Institute for Biomedical Research, Cambridge, MA 02142; {dagger} Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138; {ddagger} Department of Dermatology, Brigham and Women’s Hospital, Harvard Institute of Medicine, Boston, MA 02115; and § Department of Immunology and Genomic Medicine, Graduate School of Medicine, Kyoto University, Kyoto, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The terminal stage of B cell differentiation culminates in the formation of plasma cells (PC), which secrete large quantities of Igs. Despite recent progress in understanding the molecular aspect of PC differentiation and maintenance, the requirement for the synthesis of secretory Igs as a contributing factor has not been explored. To address this issue, we generated activation-induced cytidine deaminase (AID)/secretory µ-chain (µs) double-knockout mice, in which a normally diverse repertoire of B cell receptors is retained, yet B cells are unable to synthesize secretory Igs. These mice possess polyclonal B cells but have no serum Igs. Following immunization in vivo, PCs, identified by CD138 expression and loss of the B220 marker, were starkly reduced in number in spleen and bone marrow of AID–/–µs–/– agammaglobulinemic mice compared with wild-type mice. Upon mitogenic stimulation in vitro, AID–/–µs–/– B cells differentiated into plasmablasts to some extent, but showed reduced survival compared with wild-type B cells. We found no evidence that this reduced survival was attributable to accumulation of membrane IgM. Our results indicate that the synthesis of secretory Igs is a requirement for maintenance of B220–CD138+ PCs.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Plasma cells (PC)3 are terminally differentiated secretory cells that play a critical role in humoral immunity by producing copious amounts of soluble Abs. Upon encounter with Ags, B cells either differentiate into short-lived PCs that secrete low-affinity IgM, or participate in a germinal center (GC) reaction accompanied by class switch recombination (CSR) and somatic hypermutation (SHM), to yield PCs that secrete high-affinity IgG or IgA (1, 2). Terminal differentiation into PCs requires excessive remodeling of the endoplasmic reticulum (ER) to accommodate the large quantities of newly synthesized Igs, and to control the quality of assembled multimeric Igs in preparation for secretion (3, 4, 5). The molecular mechanisms involved in the regulation of terminal PC differentiation are being deciphered at the level of the transcription factors responsible, but little is known about the significance of the secretory Igs themselves as a factor that contributes to terminal differentiation and maintenance of PCs.

Several transcriptional regulators have been implicated in the control of PC differentiation (1), of which XBP-1 was the first shown to be essential (6). There is a close correlation between high rates of Ig secretion and the activation of XBP-1 (7). Expression of the active form of XBP-1 requires engagement of the IRE-1 protein, whose activation is believed to be triggered by the accumulation of misfolded polypeptides, although the agents commonly used to activate IRE-1 are rather toxic compounds such as tunicamycin, DTT, or thapsigargin (8). In the absence of XBP-1, B cells fail to differentiate into PCs (6). The inability to deal with an onslaught of newly ER-inserted proteins and the misfolded proteins that are the inevitable byproduct of protein synthesis might propel such XBP-1 deficient B cells toward programmed cell death. However, we have shown that XBP-1 deficient B cells contain comparable levels of µ-chain mRNA, yet are far less efficient at translating this mRNA than their wild-type counterparts (7). These observations suggest that the relationship between PC differentiation and high rates of Ig secretion is more complex than envisioned originally.

Even though the transcripts that encode the membrane and secreted forms of Igs are part of the same transcription unit, polyclonal activation of B cells incapable of IgM secretion does not result in a measurable increase in the synthesis of membrane IgM (7). The decision between the synthesis of membrane vs secreted Igs is made by choice of polyadenylation site (9), but exactly what regulates the levels of these different mRNAs is not known. Clearly, the inability to generate an mRNA that encodes the secreted µ-chain is not automatically linked to an increase in the synthesis of the membrane bound product, notwithstanding seemingly adequate secretory capacity (7). These observations, too, suggest a hitherto unappreciated relationship between Ig secretion by B cells and their ability to differentiate into PCs.

To explore the relationship between secretory Ig and PC differentiation more directly, we generated mice that are unable to synthesize any secretory Ig in significant quantities, yet retain a normally diverse repertoire of BCRs. We used µs–/– mice, unable to secrete IgM because of the elimination of elements that enable synthesis of the secretory form of IgM (10). These mice retain the capacity to engage other Ig constant regions by CSR, and so possess normal serum levels of all Ig isotypes except IgM. Crossing the µs–/– animals onto an AID-deficient background eliminates both the possibility of SHM and CSR (11) and consequently such animals should lack circulating serum Igs, as is indeed the case (see Results). Although agammaglobulinemic animals have been produced previously through introduction of a membrane IgM-encoding transgene on a genetic background incapable of executing VDJ recombination of the H chain locus (12, 13), the B cells in such animals lack the normally diverse repertoire of BCRs. The rearranged Ig transgene is not embedded in the usual chromosomal environment that may contribute to its regulated expression, and so might impinge on the terminal stages of B cell differentiation. Moreover, the issue of PC generation has not been previously examined in the context of the B cell’s ability to secrete Igs. Upon stimulation of secretory Ig-deficient, yet otherwise normal B cells, can PC equivalents be generated and maintained?

In the present study, we compared in detail the B cell differentiation pathway of agammaglobulinemic AID–/–µs–/– mice with wild type and each of the single knockout strains. We show that Ag-independent B cell development/differentiation is close to normal in AID–/–µs–/– mice. However, following T-dependent Ag immunization in vivo, B220–CD138+ stage PCs were starkly reduced in number in the spleen and bone marrow of AID–/–µs–/– agammaglobulinemic mice. Upon mitogenic stimulation in vitro, AID–/–µs–/– B cells proliferated and differentiated into CD138+ plasmablasts to some extent, but showed reduced survival compared with B cells from wild-type mice. Our results suggest that the synthesis of secretory Igs is required to maintain PCs.


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

AID–/– mice (11) were obtained from the animal facility of Dr. F. Alt (Harvard Medical School, Boston, MA). The AID–/– mice had been backcrossed to C57BL/6J for six generations in Dr. Alt’s laboratory. The µs–/– mice (10) were provided by Dr. J. Chen (Massachusetts Institute for Technology, Cambridge, MA) and Dr. M. Carroll (Harvard Medical School). The µs–/– mice used had been backcrossed to C57BL/6J for 10 generations in Dr. Chen’s laboratory. AID–/–µs–/– double-knockout mice were generated by crossing AID–/– and µs–/– mice. C57BL/6J mice were used as wild-type controls, and were originally purchased from The Jackson Laboratory. All mice were bred and maintained under pathogen-free environment.

Age- (8–11 wk) and sex- matched mice from each group, which were raised at the same time in the same environment at the Harvard Medical School animal facility, were used for serum analyses and B cell characterization by flow cytometry. Two to four mice from each group were analyzed per experiment, and each experiment was repeated four times. Flow cytometry analyses were performed using the same reagents and instruments for each experiment, and the results were pooled for statistical analysis. For in vitro experiments, spleens from age- (12–14 wk) and sex-matched mice from each group that were raised in the same environment at the Whitehead Institute for Biomedical Research animal facility (Cambridge, MA) were used. All the studies were performed according to institutional guidelines for animal use and care.

Immunization

For each group, age- (9–11 wk) and sex- matched mice were immunized with nitrophenyl-conjugated chicken gammaglobulin (NP-CG; Biosearch Technologies) or OVA (grade V; Sigma-Aldrich) to elicit a response to TD-Ags. For NP-CG immunization, 100 µg of NP-CG emulsified in alum was injected i.p., and analyses were performed 9 days and 2 wk after the immunization. For immunization with OVA, mice were injected i.p. with 100 µg of OVA emulsified in CFA (Sigma-Aldrich) on day 0, then boosted with the same dose of OVA emulsified in IFA (Sigma-Aldrich) on days 7, 14, and 21 by i.p. injection. At day 24, blood was taken from each group of mice for assessment of specific Ab production by ELISA. A significant increase in anti-OVA Abs was detected in the sera of wild-type (both IgG1 and IgM), AID–/– mice (IgM), and µs–/– mice (IgG1) (data not shown), as evidence of successful immunization. Mice were sacrificed on day 28 for analysis of bone marrow and spleen.

Serum analysis

Serum samples were assayed for Ig isotype levels by ELISA using goat anti-mouse Ig, HRP-conjugated goat Abs specific for each mouse Ig isotype (Southern Biotechnology Associates), and a 3,3', 5,5'-tetramethylbenzidine-based developing reagent (Sigma-Aldrich). Ig concentrations were calculated using ELISA software (Softmax Pro; Molecular Devices) from a standard curve produced from serial 2-fold dilutions of mouse Ig standards (Southern Biotechnology Associates).

Immunoblotting of serum Ig was conducted using HRP-conjugated goat Abs specific for IgM (µ), IgG1 ({gamma}1), IgA ({alpha}), {kappa}-chain, and {lambda}-chain (Southern Biotechnology Associates). The level of total protein in each serum sample was measured before SDS-PAGE, and the same amount of total serum protein was loaded in each lane in each immunoblotting.

Cell preparation and flow cytometry

Spleens, lymph nodes, and Peyer’s patches were dissociated into single-cell suspensions by mechanical disruption in ice-cold RPMI 1640 medium (Invitrogen Life Technologies) containing 10% FCS. Peritoneal cells were obtained by lavage with 10 ml of the same medium. Bone marrow cells were obtained by flushing femurs and tibias of the mice. Erythrocytes in spleens and bone marrows were lysed with 0.14 M NH4Cl and 17 mM Tris-Cl (pH7.4). Cells were surface-stained on ice with combination of FITC-, PE-, PerCP-, allophycocyanin, or Alexa 647-conjugated mAbs in PBS, 2% FCS, and 0.1% sodium azide. Staining with biotinylated mAbs was followed by a secondary staining with streptavidin-PE or streptavidin-allophycocyanin (BD Biosciences). After staining, the samples were washed and resuspended in the same medium. Stained cells were examined by cytofluorometry on a FACSCalibur (BD Biosciences), and data were analyzed using CellQuest software (BD Biosciences). Commercially obtained mAbs include anti-CD4 (L3T4), -CD5 (53-7.3), -CD8{alpha} (53-6.7), -CD11b (Mac-1{alpha}), -CD19 (1D3), -CD21/CD35 (7G6), -CD23 (B3B4), -CD24 (HSA, M1/69), -CD38 (90), -CD43 (S7), -CD138 (Syndecan1, 281-2), -BP-1 (6C3), -CD1d (1B1), -CD45R/B220 (RA3-6B2), -IgM (IB4B1), -IgD (11-26C.2a), and -Fas (Jo2) from BD Biosciences, anti-C1qRp (AA4.1) from eBioscience, and anti-mouse IgM (µ) -Alexa 647 from Molecular Probes. FITC-conjugated peanut agglutinin (PNA) was purchased from Vector Laboratories.

Histological and immunofluorescence analyses

For histological examination, organs were fixed with 4% paraformaldehyde, embedded in paraffin, and stained with H&E. For immunofluorescence staining, tissues were embedded in Tissue Tek OCT compound (Sakura Fintek) and snap-frozen, then cryostat sections (6-µm thick) were thawed, air-dried and fixed in ice-cold acetone for 10 min. For detection of GC B cells, sections were incubated with biotinylated PNA (Vector Laboratories) and rat anti-mouse CD19 (BD Biosciences), followed by incubation with Alexa 568-conjugated streptavidin and Alexa 488-conjugated goat anti-rat Ab (Molecular Probes). For detection of PCs or plasmablasts, sections were incubated with PE-conjugated anti-mouse CD138 and FITC-conjugated anti-mouse CD45R/B220 (BD Biosciences).

Cell culture and in vitro stimulation

The conditions for in vitro B cell stimulation were as described (7). In brief, B cells were purified from mouse splenocytes by magnetic depletion with anti-CD43 beads (Miltenyi Biotec), then cells were plated at 106 cells/ml in complete medium containing RPMI 1640 supplemented with 10% FBS, 2 mM glutamine, 50 U/ml penicillin, 50 µg/ml streptomycin, 50 µM 2-ME, 1x nonessential amino acids, and 1 mM sodium pyruvate. For proliferation assays, cells were labeled with CFSE (Molecular Probes) according to the manufacturer’s instructions.

Cells were stimulated with 100 nM CpG (1826-CPG; TIB Molbiol) followed by 20 µg/ml LPS (Sigma-Aldrich) added after 24 h of culture. For conditions that promote in vitro class switching, cells were incubated with 1 µg/ml anti-mouse CD40 (HM40-3; BD Biosciences) and 25 ng/ml recombinant murine IL-4 (R&D System). Flow cytometry was performed every 24 h. Dead cells were identified by labeling with TOPRO-3 iodide (Molecular Probes). Annexin V-FITC (BD Biosciences) staining was performed according to the manufacturer’s instruction.

RT-PCR analysis

Total RNA was isolated using TRIzol (Invitrogen Life Technologies). RNAs were used for first-strand synthesis with the Superscript reverse transcriptase (Invitrogen Life Technologies). PCR primers 5'-ACACGCTTGGGAATGGACAC-3' and 5'-CCATGGGAAGATGTTATGGG-3' encompassing the missing sequences in XBP-1 were used for the PCR amplification with Platinum PCR Supermix (Invitrogen Life Technologies). Cycling conditions were as follows: 95°C for 3 min and 58°C for 40 s, 35 cycles of 72°C for 45 s, 95°C for 45 s. A PCR for GAPDH was performed to validate cDNA synthesis. We separated PCR products by electrophoresis in 11% PAGE gel and visualized them by ethidium bromide staining. Semiquantitative PCR for µm was performed as described (6, 7).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Lack of serum IgM, IgG, and IgA in AID–/–µs–/– mice

To determine whether AID–/–µs–/– mice are deficient in circulating Igs, we performed immunoblotting and ELISA analyses using the sera from AID–/–µs–/– mice as well as the sera from wild-type, AID–/–, and µs–/–mice. As expected from the previous reports (10, 11), IgG1 ({gamma}1 chain) and IgA ({alpha}-chain) were not detected in the sera of AID–/– mice, and IgM (µ chain) was not detected in the sera of µs–/– mice by immunoblotting (Fig. 1A). AID–/– mice had more IgM than wild-type mice, consistent with the "hyperIgM" syndrome that also characterizes AID-deficient humans (14). B cells of neither the AID–/– nor the µs–/– mice have an intrinsic defect in Ig secretion.


Figure 1
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  		FIGURE 1. Absence of Ig in the sera of AID–/–µs–/– mice. A, The presence of serum Igs was examined by Western blotting using Abs for {gamma}1, µ-, {alpha}-, {kappa}-chain, and {lambda}-chain. Neither class of Ig was detected in the sera of AID–/–µs–/– mice. B, Serum Ig levels by ELISA. Sera from wild-type (•), AID–/– ({circ}), µs–/– ({square}), and AID–/–µs–/– ({triangleup}) mice were collected and their Ig levels were determined by ELISA. Ig levels <0.01 µg/ml were plotted below the baseline. Each point represents an individual mouse.

 
No µ, {gamma}1, {alpha}, {kappa}, or {lambda} polypeptides were detected in the sera of AID–/–µs–/– mice by immunoblotting (Fig. 1A). ELISA analysis confirmed the absence of IgG1 ({gamma}1 chain), IgG2a ({gamma}2{alpha} chain), IgG2b ({gamma}2beta chain), IgG3 ({gamma}3 chain), and IgA ({alpha}-chain) in the sera of AID–/–µs–/– mice (Fig. 1B). In the sera of AID–/–µs–/– and µs–/– mice, very low levels of IgM (µ chain) were detected by ELISA (Fig. 1B). The ELISA does not distinguish between intact IgM and shed IgM-derived fragments that retain immunoreactivity. The absence of intact µ-chain by immunoblotting, even for overdeveloped blots, suggests that the levels of intact µ-chain are negligible, if present at all. Because L chains are synthesized in molar excess over H chains, even B cells that produce only membrane IgM will still release some L chain. This accounts for the low, but detectable level of {kappa} and {lambda} chain in AID–/–µs–/– mice as measured by ELISA (data not shown).

Flow cytometric analyses demonstrated the presence of membrane IgM (Fig. 2C) and the absence of membrane IgG (data not shown) in the B cells freshly harvested from AID–/–µs–/– mice.


Figure 2
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  		FIGURE 2. B cell development and differentiation in AID–/–µs–/– mice by flow cytometric analyses. Representative results from at least four separate experiments are shown. A, Bone marrow cells from each group of mice were stained with B220, CD43 (S7), and IgM. Dot plots are gated on B220+S7– B cells. Fraction (Fr.) D (B220+(dull)S7–IgM–) corresponds to pre-B cells, Fr. E (B220+(dull)S7–IgM+) corresponds to newly generated immature B cells, and Fr. F (B220+(hi)S7–IgM+) corresponds to recirculating mature B cells. The numbers indicate the percentages of gated cells in total bone marrow cells. B and C, Splenocytes from four groups of mice were stained with B220, AA4.1, CD21, and CD23 (B) or with B220, AA4.1, IgM, and IgD (C). Dot plots are gated on B220+AA4.1– mature B cells. CD21highCD23– cells were defined as MZ B cells, and CD21intCD23+ cells were defined as follicular (FO) B cells (B). Numbers indicate the percentages of gated cells in total splenocytes. D, PNAhighFashigh GC B cells were analyzed in the splenocytes of four groups of mice 2 wk after immunization with NP-CG. Dot plots are gated on B220+ B cells. Numbers indicate the percentages of gated cells in total splenocytes.

 
Early B cell development in the bone marrow in AID–/–µs–/– mice

To characterize B cell development and differentiation in AID–/–µs–/– mice, we performed flow cytometric analyses using the cells collected from bone marrow, spleen, Peyer’s patches, and peritoneal cavity of each of the four groups of mice.

There was no difference in total number of bone marrow cells among the four groups (Table I). The different populations of bone marrow B cells were classified according to the scheme proposed by Hardy et al. (15, 16), by the surface expression of S7 (leukosialin, CD43), BP-1, 30F1 (heat stable Ag, CD24), B220, and IgM. There was no significant difference in the proportions of pre pro-B, early pro-B, late pro-B, and pre-B cells (fraction (Fr.) A, B, C, and D, respectively) among the four groups.


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  		Table I. Number and frequency of cells in different organsa

 
The percentage of newly generated immature B cells (Fr. E, B220+dullS7–IgM+) was higher in AID–/–µs–/– and µs–/– mice than in wild-type mice (Fig. 2A). The percentage of recirculating mature B cells (Fr. F, B220++brightS7–IgM+) was lower in µs–/– but not in AID–/– or AID–/–µs–/– mice, which might reflect the decreased percentage of B cells in the spleen of µs–/– mice (Table II, differences that are significant at p < 0.05 are indicated in bold).


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  		Table II. Numbers and frequencies of splenic B and T cell populationsa

 
Thus, we observed only minor changes in the B cell compartment in the bone marrow of AID–/–µs–/– mice, and these changes did not appear to be affecting early B cell development. We conclude that at this level of analysis, early development of B cells in AID–/–µs–/– is close to normal.

Splenic B cell and T cell compartment in AID–/–µs–/– mice

There was no significant difference in total number of splenocytes and percentage of B cells between AID–/–µs–/– and wild-type mice, whereas the total number of splenocytes was smaller in AID–/– mice, and the percentage of B cells was lower in µs–/– mice than in wild type (Table II). The ratio of surface {kappa}+ B cells/{lambda}+ B cells was somewhat lower in AID–/–µs–/– and µs–/– mice than in wild type (Table II), suggesting that the absence of µs may affect the type of B cell ({kappa}+ or {lambda}+) that survive.

The percentages of CD4+ T cells were equivalent for the four groups. We observed decreases in the percentage of CD8+ T cells in AID–/–µs–/– and AID–/– mice compared with wild type, and an increase in the percentage of CD8+ T cells in µs–/– mice. Although statistically significant, these differences were not radical and therefore considered unlikely to have a major effect on development and homeostasis of the T cell compartment.

Marginal zone (MZ) and follicular B cells in the spleen of AID–/–µs–/– mice

Mature B cells in the spleen can be classified into MZ and follicular B cells on the basis of CD21 and CD23 expression (17, 18). The percentage of MZ (B220+AA4.1–CD21highCD23–) B cells was 4- to 5-fold higher in AID–/–µs–/– mice compared with wild-type mice (Table II, Fig. 2B). We also examined the expression of CD1d, which is more highly expressed on MZ than on follicular B cells (17, 19, 20). The percentage of CD1dhighCD23– MZ B cells in AID–/–µs–/– showed a 4-fold increase compared with wild-type mice (data not shown). The percentages of follicular (B220+AA4.1–CD21intCD23+) B cells in AID–/–µs–/– did not differ compared with wild-type mice. MZ B cells express high levels of surface IgM and low levels of surface IgD, while follicular B cells express low levels of surface IgM and high levels ofsurface IgD (21). The percentage of AA4.1–IgMhighIgDlow mature B cells was much higher in AID–/–µs–/– than in wild-type mice (Fig. 2C), again indicative of an increased percentage of MZ B cells in AID–/–µs–/– mice. Because PCs are very few in the spleen of AID–/–µs–/– mice, as will be described below, the increase in MZ B cell number could be related to the reduction in number of PCs. If correct, this could be evidence for a role for PCs in limiting the number of MZ B cells.

Germinal center reaction in AID–/–µs–/– mice

The total number and percentage of PNAhighFashigh GC B cells was significantly higher in the spleen of AID–/–µs–/– mice than in wild-type mice, both before and after immunization (Table II, Fig. 2D). This observation fits the phenotype of AID–/– mice (11). The total numbers and percentages of GC cells were higher in the Peyer’s patches of AID–/–µs–/– and AID–/– mice than in those of wild-type mice (Table I). Immunofluorescence analysis also showed enhancement of GC B cell formation in the spleen of AID–/–µs–/– and AID–/– mice compared with wild-type mice (Fig. 3C). Thus, the features of enhanced GC reaction in AID–/–µs–/– mice were fully consistent with those seen in AID–/– mice.


Figure 3
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  		FIGURE 3. Histological analysis of lymphoid tissue. A, Spleens from each group of naive mice were stained with H & E. White pulp (WP) and red pulp (RP) are less organized in the spleen of AID–/–µs–/– mice compared with other groups of mice. Images are shown at x40 magnification. B, Peyer’s patches from four groups of mice were stained with H & E. Intestinal glands (IG) are largely included in the sections of wild-type and µs–/– mice. B cell follicles (B-Fo) are highly developed, and Peyer’s patches are markedly enlarged in AID–/–µs–/– and AID–/– mice. Images are shown at x40 magnification. C, GC B cells in the spleens by immunofluorescence. Two weeks after immunization with NP-CG, cryosections of spleens from each group of mice were stained for PNA (Alexa 568, red) and CD19 (Alexa 488, green). GC formation (coexpression, yellow) is enhanced in the spleens of AID–/–µs–/– and AID–/– mice. Images are shown at x100 magnification.

 
B-1B cell population in AID–/–µs–/– mice

Because µs–/– mice show an increase in the number of B-1 cells (10), we also examined the population of B-1a cells in the spleen and peritoneal cavity in each group of mice. The percentages of splenic CD5+IgM+ B-1a B cells were much higher in both AID–/– µs–/– and µs–/– mice than in wild-type mice (Table II). In peritoneal lavage cells, the total numbers of both B220+Mac-1+CD5+ B-1a B cells and B220+Mac-1+CD5– B-1b B cells were much higher in both AID–/–µs–/– mice and µs–/– mice than in wild-type mice (Table I). The total number of B220+Mac-1– B-2 B cells was also higher in AID–/–µs–/– mice than in wild-type mice, although the percentage of B-2 B cells was similar (Table I). Thus, the feature of increased B-1 B cells in the spleen and peritoneal cavity in AID–/–µs–/– mice was consistent with that observed for µs–/– mice.

We conclude that the changes in B cell differentiation in naive AID–/–µs–/– mice, as assessed by cytofluorometry, are modest, and the perturbations in the B cell compartment have minimal effects on the CD4 and CD8 T cell populations. Therefore Ag-independent B cell development/differentiation and T cell homeostasis are close to normal in AID–/–µs–/– mice.

Morphological features of the spleens and Peyer’s patches of AID–/–µs–/– mice

We conducted histological examinations of spleen, thymus, peripheral lymph nodes, and GALT. The spleens of AID–/–µs–/– mice were enlarged compared with the other three groups by macroscopic observation (data not shown). H&E-stained sections of the spleens of AID–/–µs–/– mice revealed less-structured white pulp in comparison with the other groups of mice (Fig. 3A). Peyer’s patches of AID–/–µs–/– and AID–/– mice were obviously enlarged compared with wild-type mice, by both macroscopic inspection and microscopic examination (Fig. 3B). Mesenteric lymph nodes of AID–/–µs–/– and AID–/– mice were also enlarged compared with wild-type mice (data not shown). Because increased GC formation was evident in both AID–/–µs–/– mice and AID–/– mice by flow cytometry (Tables I and II, Fig. 2D) and immunofluorescence microscopy (Fig. 3C), the enlargement of GALT in these mice may be due to the enhanced GC reactions in response to intestinal microbial Ags. There were no obvious histological differences in H&E-stained sections of thymus and peripheral lymph nodes among the four groups of mice (data not shown).

The enlargement of GALT in AID–/–µs–/– mice as well as in AID–/– mice can be caused by hyperreaction of GC. However, the disorganization of white pulp that characterizes the spleen of the AID–/–µs–/– mice, is likely caused by the unique features of this strain, i.e., impaired secretion of any class of Ig, or impaired PC maintenance, as will be described below.

Decreased PCs in the spleen and bone marrow of AID–/–µs–/– mice

To determine whether PCs are maintained normally in AID–/– µs–/– mice, we examined the population of B220–CD138+ PCs in the spleen and bone marrow. To focus on terminally differentiated PCs, we excluded from our analysis the B220+CD138+ population, which includes B220int-lowCD138+ plasmablasts (22, 23, 24). There was no significant difference in the percentage of B220int-lowCD138+ plasmablasts among the four groups of mice (Table II). Following immunization with OVA (Fig. 4A) and NP-CG (Fig. 4B), both the percentage and total number of B220–CD138+ PCs were significantly lower in the spleen of AID–/–µs–/– mice compared with wild-type mice (Table II, Fig. 4). In the bone marrow of AID–/–µs–/– mice, the percentages of B220–CD138+ PCs were significantly lower, both before and after immunization (Table I, Fig. 4A). The total number of PCs in the bone marrow was also much lower in AID–/–µs–/– mice than in wild-type mice after immunization.


Figure 4
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  		FIGURE 4. PCs are nearly absent in AID–/–µs–/– mice. A, After repeated immunization with OVA, bone marrow cells, and splenocytes from each group of mice were stained with B220, CD5, CD138, and IgM for flow cytometric analyses. Dot plots are gated on B220–CD5– cells. Numbers indicate the percentages of gated cells in total bone marrow cells or in total splenocytes. The percentage of B220–CD138+ PCs was markedly decreased in AID–/–µs–/– mice. In the analyses of wild-type, AID–/–, and µs–/– mice, there was a clear distinction between IgM– and IgM+ PCs. B, Nine days after immunization with NP-CG, splenocytes from each group of mice were stained with B220, CD138, IgM, and IgG1. Dot plots are gated on B220– cells, and numbers indicate the percentages of gated cells in total splenocytes. Pink dots represent IgM+B220–CD138+ PCs and green dots represent IgM–B220–CD138+ PCs. Majority of IgM–B220–CD138+ PCs express detectable levels of IgG1, whereas most of IgM+B220–CD138+ PCs are negative for IgG1. Representative results from at least three separate experiments are shown.

 
Although PCs are generally considered to express little or no surface Ig (2, 25), there are some recent reports that PCs can express Ig on their surface (26, 27, 28). We could divide B220–CD138+ PCs of wild type and each of the single knockout mice into two groups, according to a clear distinction in surface IgM expression (Fig. 4). After immunization of normal mice with NP-CG, most of the splenic IgM+ PCs were negative for IgG1, and the majority of IgM– PCs expressed a detectable level of IgG1 (Fig. 4B). PCs occur in at least two types: short-lived PCs that produce low-affinity IgM Abs, and class-switched PCs that produce high-affinity IgG or IgA Abs. It is commonly held that PCs that produce isotypes other than IgM are class-switched PCs (1, 2). Therefore, we shall refer to IgM+ PCs as "unswitched" PCs and to IgM– PCs as "switched" PCs. In wild-type mice, we detected both IgM+ unswitched and IgM– switched PCs in spleen and bone marrow. In AID–/– mice, most of B220–CD138+ PCs were IgM+ PCs, whereas most of B220–CD138+ PCs were IgM– switched PCs in µs–/– mice. In AID–/–µs–/– mice, both types of PCs were essentially undetectable in spleen and bone marrow (Fig. 4). These results indicate that the maintenance of terminally differentiated PCs is impaired in AID–/–µs–/– mice in vivo.

XBP-1 splicing is not inhibited in AID–/–µs–/– B cells upon mitogenic stimulation in vitro

Splicing of XBP-1 is imperative for differentiation of B cells into PCs (4, 6). Because PCs were not detected in AID–/–µs–/– mice in vivo, a plausible explanation might be the failure to evoke splicing of XBP-1. Such failure might derive from the absence of secretory Ig and misfolded byproducts, usually held responsible for induction of an unfolded protein response (UPR), which starts with activation of IRE-1 and is followed by IRE-1 degradation and splicing of XBP-1 mRNA. To address this possibility, we examined by RT-PCR the splicing of XBP-1 mRNA in response to mitogenic stimulation of isolated splenic B cells with CpG/LPS. Similar to wild-type controls, spliced XBP-1 mRNA was readily seen for AID–/–µs–/– at day 1, and was continuously generated until day 3 (Fig. 5A). Spliced XBP-1 mRNA was also observed for AID–/– and µs–/– mice from days to day3 (data not shown). The absence of secretory Ig does not prevent the activation of IRE1 and splicing of XBP-1 mRNA in the course of polyclonal B cell activation, nor do we observe an excessive level of spliced XBP-1 mRNA in AID–/–µs–/– mice compared with wild type.


Figure 5
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  		FIGURE 5. A, XBP-1 splicing in splenic B cells from AID–/–µs–/– mice under in vitro stimulation. B cells were purified from the spleens of each group of mice, and stimulated with CpG and LPS. RNA was extracted at the indicated times, and splicing of XBP-1 mRNA was analyzed by RT-PCR. Spliced XBP-1 mRNA was observed for AID–/–µs–/– mice similar to wild type. B, semiquantitative RT-PCR analysis of cDNA prepared from RNA of CpG/LPS-treated B cells. Three-fold dilution series of cDNA were used as input material for the PCR primers specific for µm or GAPDH as reference. There was no significant difference in the levels of µm mRNA in wild-type and AID–/–µs–/– B cells.

 
µm mRNA is not up-regulated in AID–/–µs–/– B cells upon mitogenic stimulation in vitro

We considered a possibility that overproduction of membrane IgM due to the increase in membrane µ (µm) mRNA, which would normally be channeled into µs mRNA in the course of PC differentiation, may exert toxicity and result in failure of PC maintenance in AID–/–µs–/– mice. To address this possibility, we compared the levels of µm mRNA by semiquantitative RT-PCR in CpG/LPS treated B cells. We found similar levels of µm mRNA in wild-type and AID–/–µs–/– mice on days 2 and 3 (Fig. 5B). Therefore, we consider the presence of toxic level of µm unlikely. However, we cannot formally exclude the possibility that AID–/– µs–/– B cells/plasmablasts that express elevated µm transcript levels rapidly die and are lost to analysis, and therefore µm mRNA levels were similar among the groups. This explanation would require selective death of those cells that express enhanced levels of µm mRNA, such that the residual population of survivors would express the identical mRNA levels as seen in wild type, not withstanding the fact that these survivors are still AID–/–µs–/–.

Alternatively, because the µs–/– mouse strain retains a neomycin phosphotransferase (neo)-gene insertion, the failure of generating PCs in AID–/–µs–/– mice might be attributed to increased synthesis of the Neo cassette in the course of PC differentiation. We consider this possibility unlikely, because normal differentiation into GFP+ PCs is seen in IgHGFP/+ mice, in which GFP is inserted into IgH locus with a Neo cassette (29).

Differentiation and survival of AID–/–µs–/– plasmablasts in vitro

Many aspects of B cell differentiation into Ig-secreting plasmablasts can be recapitulated in vitro by exposing splenic B cells to polyclonal B cell activators such as CpG and LPS. We purified B cells from spleens of each group of mice and stimulated them with CpG and LPS to induce differentiation of B cells into B220+CD138+ plasmablasts. We plated identical numbers of B cells from each group, and performed flow cytometry every 24 h. Dead cells were identified by labeling with TOPRO-3.

The formation of CD138+ plasmablasts was observed for all four groups of mice. We assessed TOPRO3–CD138+ cells, i.e., live plasmablasts, by the following criteria: 1) number of live CD138+ plasmablasts relative to total cell number (Fig. 6A), 2) total number of live CD138+ plasmablasts in each culture (Fig. 6B), 3) number of live CD138+ plasmablasts relative to total (live and dead) CD138+ plasmablasts (data not shown). By all of these criteria, the numbers of live CD138+ plasmablasts were lower for the AID–/–µs–/– and µs–/– mice compared with wild type. For the 48–72 h interval, we observed significantly lower numbers of live plasmablasts for the AID–/–µs–/– and µs–/– mice compared with wild type (Fig. 6B). Our results indicate that B cells from AID–/–µs–/– mice can differentiate in vitro at least to the stage of CD138+ plasmablasts; but survival of these cells appears to be compromised.


Figure 6
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  		FIGURE 6. In vitro stimulation of splenic B cells from AID–/–µs–/– mice. B cells were purified from the spleens of each group of mice and stimulated with CpG followed by stimulation with LPS after 24 h. For the proliferation assay (D), cells were stained with CFSE before the stimulation. Dead cells were identified by TOPRO-3 staining. A and B, CD138+ plasmablasts were detectable in the culture of all four groups of mice, however, the number and percentage of live plasmablasts (TOPRO3–CD138+ cells) were lower in the culture of AID–/–µs–/– and µs–/– mice compared with wild type (*, p < 0.05 compared with wild). Data represent mean ± SD from three separate experiments. C, Annexin V/TOPRO3 costaining of stimulated B cells on day 2. Dot plots are gated on CD138+ plasmablasts. Annexin V+,TOPRO3– cells are early apoptotic cells, and Annexin V+,TOPRO3+ cells are dead cells that include both late apoptotic cells and necrotic cells. There was no significant difference in the ratio of early apoptotic plasmablasts to dead plasmablasts between AID–/–µs–/– and wild type. D, Flow cytometry on day 3 showed B cell proliferation in all four groups, although distribution of CFSE fluorescence was heterodisperse in the analyses for AID–/–µs–/– and µs–/– mice. Histograms (left and middle panels) are shown for TOPRO3– live cells, and dot plots (right panels) are shown for all the cells in each culture.

 
To see whether impaired survival of AID–/–µs–/– plasmablasts is due to apoptotic censoring, we performed annexin V/TOPRO3 costaining using cultured B cells stimulated by CpG/LPS. Although the percentage of total Annexin V+ plasmablasts was much higher in AID–/–µs–/– culture compared with wild type, the proportion of early apoptotic plasmablasts (annexin V+, TOPRO3–) relative to total dead plasmablasts (annexin V+, TOPRO3+) was similar between AID–/–µs–/– and wild type (Fig. 6C). Therefore, we cannot conclude that impaired survival of AID–/–µs–/– plasmablasts is simply due to apoptosis.

Because only IgM– switched PCs were detected in the spleen of µs–/– mice in vivo (Fig. 4), we assumed that only PCs that undergo CSR and acquire the capacity to secrete isotype switched Igs are maintained in µs–/– mice. Therefore, to examine whether signals that trigger in vitro CSR improve the survival of µs–/– plasmablasts, we induced class switching by inclusion of anti-CD40 and IL4. We observed 25–30% of IgG+ cells in cultures from wild-type and µs–/– mice (data not shown). For the 72–96 h interval, the ratio of CD138+ live plasmablasts tended to be higher in the culture from µs–/– mice compared with AID–/–µs–/– mice (data not shown), although this difference was not statistically significant.

To determine whether AID–/–µs–/– B cells proliferate normally upon stimulation in vitro, we labeled purified B cells with CFSE and then incubated them with CpG and LPS. Flow cytometric analysis on day3 revealed B cell proliferation in all four groups (Fig. 6D). The distribution of CFSE fluorescence was heterodisperse with less discrete peaks for µs–/– and AID–/–µs–/– mice. For the proliferating cells, no differences in cell size were apparent. We concluded that B cells from AID–/–µs–/– mice can proliferate and differentiate to the stage of plasmablasts in vitro, but survive less well than wild-type plasmablasts.

Absence of PCs in the spleen of AID–/–µs–/– mice by immunohistochemistry after immunization with TD-Ag

After four sequential immunizations with OVA, we performed immunohistochemical analysis on spleens from each group of mice. We noted the presence of B220–CD138+ PCs in wild-type, AID–/– and µs–/– mice, but not in AID–/–µs–/– mice (Fig. 7). Immunohistochemistry also confirmed the presence of B220+CD138+ plasmablasts in the spleen of AID–/–µs–/– mice.


Figure 7
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  		FIGURE 7. Immunofluorescence for PCs in the spleen of AID–/–µs–/– mice. After sequential immunization with OVA, cryosections of spleens from each group of mice were stained with FITC-B220 and PE-CD138. Clusters of B220–CD138+ PCs (red) were observed in the extrafollicular space in wild-type, AID–/–, and µs–/– mice but not in the spleen of AID–/–µs–/– mice, where only B220+CD138+ plasmablasts (orange) were detectable. Images are shown at x100 magnification. Higher magnification micrograph (x200) is shown for the indicated area in AID–/–µs–/– spleen.

 
This result is consistent with, and extends the in vitro data, and strongly suggests that in AID–/–µs–/– mice plasmablasts can arise, but PCs, if they arise, do not persist.


   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
We observed a near complete absence of PCs in the AID–/–µs–/– agammaglobulinemic mice. Some of the other features in B cell differentiation in AID–/–µs–/– mice were compatible with the observations in either of single knockout mice (10, 11). An increase of the B-1 B cell population was reported in µs–/– mice, and a physiologic role of secretory IgM was inferred as a component of a feedback loop that contributes to B-1 cell differentiation and/or maintenance (10). AID–/–µs–/– mice show an enhanced GC reaction, observed also in AID–/– mice. Upon encounter with Ag and Th cells, some follicular B cells migrate into the primary follicle and expand within the GC, where B cells undergo proliferation accompanied by SHM and CSR (1, 30). The inhibition of this proliferation may be provoked by SHM or CSR, so that activated B cells are given the opportunity to differentiate (11). Because IgM– PCs were few in both AID–/– and AID–/–µs–/– mice, IgM– switched splenic PCs that should have undergone SHM and CSR may also be required under normal circumstances for feedback regulation of B cell proliferation in the GC (Fig. 8). The increase in numbers of MZ B cells in AID–/–µs–/– mice can also be explained by impaired negative feedback regulation due to a lack of IgM+ unswitched splenic PCs (Fig. 8). The disorganized structure of white and red pulp in H&E staining of AID–/–µs–/– spleen may be due to the accumulation of MZ B cells. These changes in B the cell compartment are not radical, and therefore we conclude that in AID–/–µs–/– mice, B cell development and differentiation are close to normal, at least before the terminal stage of PC formation.


Figure 8
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  		FIGURE 8. Schematic summary of B cell and PC differentiation in AID–/–µs–/– agammaglobulinemic mice. Switched PCs are nearly absent because of the defects in SHM and CSR. Enhanced GC reaction may be caused by impaired feedback regulation due to the lack of SHM and CSR. B cells develop and differentiate into unswitched PC to some extent. However, unswitched PCs are not maintained normally; failure in production of secretory IgM (sIgM) may cause early death of unswitched PCs. Accumulation of MZ B cells may be caused by impaired feedback regulation due to the lack of unswitched PCs and/or sIgM. FoB, follicular B cell; PB, plasmablasts; {kappa}, {kappa}-chain; mIgM, membrane IgM.

 
Although PC differentiation is characterized by complex pathways and heterogeneity in marker expression (31, 32, 33), we could classify B220–CD138+ PCs into two groups, according to surface IgM expression. In AID–/– mice, the absence of IgM– switched PCs is consistent with a lack of SHM and CSR. IgM+ unswitched PCs were observed not only in the spleen but also in the bone marrow of AID–/– mice, and of wild-type mice at lower frequency. Class-switched PCs that find survival niches become long-lived and migrate into bone marrow, which is their preferred location (1, 34). The IgM+ PCs detected in the bone marrow of AID–/– mice cannot have undergone CSR or SHM (i.e., unswitched and "short-lived" in general), and therefore certain populations of "short-lived" unswitched PCs may also recirculate through the bone marrow, although the lifespan of these cells is not clear.

In the spleen and bone marrow of µs–/– mice, we observed only IgM– switched PCs, whereas both types of PCs were very few in AID–/–µs–/– mice. The absence of switched PCs in AID–/–µs–/– mice, as well as in AID–/– mice, must be due to the defect in CSR. However, in AID–/–µs–/– and µs–/– mice, it was unclear whether B cells fail to differentiate into unswitched PCs, or whether unswitched PCs survive less well than in wild-type mice. To address this question, we performed in vitro stimulation using CpG and LPS, agents that rapidly induce differentiation of MZ B cells into plasmablasts, the precursors of unswitched PCs (17, 35). Because we detected CD138+ cells in cultures from both AID–/– µs–/– and µs–/– mice, MZ B cells in these strains can differentiate at least to the stage CD138+ plasmablasts by T-independent Ag stimulation in vitro. The detection of B220+CD138+ cells in both AID–/–µs–/– and µs–/– mice after TD-Ag immunization in vivo also demonstrates the potential of follicular B cells to differentiate into plasmablasts. The decrease in number of "live" CD138+ cells in AID–/–µs–/– and µs–/– cultures in the course of stimulation with CpG/LPS indicates impaired survival of plasmablasts/PCs. The tendency toward improved survival of µs–/– CD138+ cells by induction in vitro of CSR suggests that, in µs–/– mice, death of plasmablasts/PCs can in part be rescued by CSR.

The shortened survival of unswitched PCs in AID–/–µs–/– and µs–/– mice is likely attributable to the shared feature of these two strains, i.e., abolished secretion of IgM. We might consider the possibility that plasmablasts/PCs of AID–/–µs–/– and µs–/– mice are unable to accommodate excess L chain, not all of which is necessarily secreted if it failed to assemble with µs chain. Plasmablasts/PCs might therefore die as they ramp up L chain synthesis. Under this assumption, plasmablasts/PCs in µs–/– mice can survive only by CSR, as L chain can be secreted with {gamma}-, {alpha}-, or {epsilon}-chains. Although we observed {kappa}-chain secretion in AID–/– µs–/– cells as well as in wild type upon in vitro stimulation (data not shown), our qualitative analysis cannot directly exclude this possibility.

XBP-1 is required for PC differentiation (6), and is a key element of the UPR, activated during ER stress imposed by treatment with drugs such as tunicamycin, thapsigargin, or with DTT (8). The UPR is required a properly functioning ER (36, 37). Iwakoshi et al. (4, 38) established the relationship between PC differentiation, the UPR, and XBP-1. Treatment with tunicamycin, thapsigargin, and DTT usually result in robust splicing of XBP-1 mRNA. However, the physiological factors that trigger the UPR in the course of PC differentiation are not at all well defined. Mature naive B cells synthesize equal amounts of µm and µs (39). In the course of PC differentiation, B cells shift from synthesis of the µm to predominantly µs (3). The steep increase in µs synthesis, perhaps to a level that exceeds the folding capacity of the ER of the developing PCs, might indeed entail ER stress, as evident from splicing of XBP-1 mRNA. Treatment of B cells with low concentrations of cycloheximide abrogates XBP-1 splicing, suggesting that at least de novo protein synthesis is required for induction of the UPR (4). Additionally, Iwakoshi and colleagues (7) demonstrated that Cre-mediated removal of a floxed µ H chain locus dramatically reduces the levels of XBP-1s generated in LPS-treated B cells. Finally, we have shown that XBP-1 is required to maintain high levels of synthesis of µs chains, even though µ mRNA levels hardly differ for wild-type and XBP-1–/– B cells. Taken together, these data suggest a feed-forward loop that connects the synthesis of µs with the generation of XBP-1s. Against expectations, we observed that in the absence of µs, XBP-1 mRNA is spliced in response to CpG/LPS treatment. This suggests that factors other than induction of Ig synthesis contribute to the activation of IRE1 upon stimulation. Indeed, careful analysis of the kinetics of µ-chain synthesis and the appearance of XBP-1s in CH12 B cells subjected to LPS treatment showed that splicing of XBP-1 preceded the up-regulation of µ chain synthesis (40). We therefore propose that the initial activation of IRE1 does not necessarily rely on de novo synthesis of µ-chains.

The intrinsic survival of B cells ex vivo is limited to 3–4 days. This confounds a quantitative assessment of the contribution of individual factors to the viability of B cells in culture. Nonetheless, we observed reduced survival of AID–/–µs–/– and µs–/– in comparison to wild-type or AID–/– B cells. This indicates that synthesis of µs protects B cells from cell death. The µs glycoprotein contains 5 N-linked glycosylation sites and many disulfide bonds. In the course of its folding, µs interacts directly and indirectly with a multitude of chaperones, including Bip, PDI, EroI, ERp44 and others (41). It is conceivable that the differentiation program that leads to PC prepares the ER in advance of the barrage of newly synthesized µs molecules. Because proper amounts of chaperones are required for successful folding, assembly and secretion of IgM, the complete absence of µs perturbs ER homeostasis, and might so provoke cell death. The molecular mechanism(s) engaged in this process remain to be identified.

There exist several mouse models that lack serum Igs (12, 13, 42, 43, 44). To our knowledge, our AID–/–µs–/– mouse is the first agammaglobulinemic mouse model that retains a normally diverse BCR repertoire. The inability to secrete IgM (µs–/–) paired with the absence of CSR (AID–/–) yields the expected agammaglobulinemic mice. Shlomchik and coworkers (12, 13) generated a transgenic mouse that lacks circulating Abs but with B cells that retain membrane IgM. Their mouse lacks the JH segments required for VDJ recombination and B cell development, and monoclonal B cell development is rescued by introduction of a VH IgM transgene that specifies membrane IgM. In their elegant mouse model, B cell responses can be explored by immunization, but only with the Ag for which the transgenic IgM is specific. Their mouse model has been used successfully to investigate the role of secreted Abs in memory B cell kinetics, in response to specific Ag (45). In contrast, our mouse model, with a fully diverse BCR repertoire, can be challenged with multiple types of Ags. AID–/–µs–/– mice should facilitate further studies of the roles of serum Igs in immune responses against diverse infectious agents.

In conclusion, we have shown that PCs are nearly absent in the spleen and bone marrow of AID–/–µs–/– mice that cannot synthesize any class of secretory Ig. Synthesis of secretory Ig by PCs thus appears to be required for their maintenance.


   Acknowledgments
 
We thank Amy Mcquay for her excellent technical assistance. We also thank Dr. Klaus Rajewsky for discussion of the results.


   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 grant from the National Institutes of Health (to H.L.P.). B.T. was supported by a Dorot Foundation fellowship. Back

2 Address correspondence and reprint requests to Dr. Hidde L. Ploegh, Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, MA 02142. E-mail address: ploegh{at}wi.mit.edu Back

3 Abbreviations used in this paper: PC, plasma cell; GC, germinal center; CSR, class switch recombination; SHM, somatic hypermutation; ER, endoplasmic reticulum; NP-CG, nitrophenyl-conjugated chicken gammaglobulin; UPR, unfolded protein response; AID, activation-induced cytidine deaminase; µs, secretory µ; µm, membrane µ; XBP-1, X-box binding protein 1; IRE, inositol-requiring enzyme; PNA, peanut agglutinin; MZ, marginal zone. Back

Received for publication August 1, 2006. Accepted for publication December 1, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Shapiro-Shelef, M., K. Calame. 2005. Regulation of plasma-cell development. Nat. Rev. Immunol. 5: 230-242. [Medline]
  2. Calame, K. L., K. I. Lin, C. Tunyaplin. 2003. Regulatory mechanisms that determine the development and function of plasma cells. Annu. Rev. Immunol. 21: 205-230. [Medline]
  3. van Anken, E., E. P. Romijn, C. Maggioni, A. Mezghrani, R. Sitia, I. Braakman, A. J. Heck. 2003. Sequential waves of functionally related proteins are expressed when B cells prepare for antibody secretion. Immunity 18: 243-253. [Medline]
  4. Iwakoshi, N. N., A. H. Lee, P. Vallabhajosyula, K. L. Otipoby, K. Rajewsky, L. H. Glimcher. 2003. Plasma cell differentiation and the unfolded protein response intersect at the transcription factor XBP-1. Nat. Immunol. 4: 321-329. [Medline]
  5. Brewer, J. W., L. M. Hendershot. 2005. Building an antibody factory: a job for the unfolded protein response. Nat. Immunol. 6: 23-29. [Medline]
  6. Reimold, A. M., N. N. Iwakoshi, J. Manis, P. Vallabhajosyula, E. Szomolanyi-Tsuda, E. M. Gravallese, D. Friend, M. J. Grusby, F. Alt, L. H. Glimcher. 2001. Plasma cell differentiation requires the transcription factor XBP-1. Nature 412: 300-307. [Medline]
  7. Tirosh, B., N. N. Iwakoshi, L. H. Glimcher, H. L. Ploegh. 2005. XBP-1 specifically promotes IgM synthesis and secretion, but is dispensable for degradation of glycoproteins in primary B cells. J. Exp. Med. 202: 505-516. [Abstract/Free Full Text]
  8. Zhang, K., R. J. Kaufman. 2006. Protein folding in the endoplasmic reticulum and the unfolded protein response. Handb. Exp. Pharmacol. 172: 69-91. [Medline]
  9. Edwalds-Gilbert, G., C. Milcarek. 1995. Regulation of poly(A) site use during mouse B-cell development involves a change in the binding of a general polyadenylation factor in a B-cell stage-specific manner. Mol. Cell Biol. 15: 6420-6429. [Abstract]
  10. Boes, M., C. Esau, M. B. Fischer, T. Schmidt, M. Carroll, J. Chen. 1998. Enhanced B-1 cell development, but impaired IgG antibody responses in mice deficient in secreted IgM. J. Immunol. 160: 4776-4787. [Abstract/Free Full Text]
  11. Muramatsu, M., K. Kinoshita, S. Fagarasan, S. Yamada, Y. Shinkai, T. Honjo. 2000. Class switch recombination and hypermutation require activation-induced cytidine deaminase (AID), a potential RNA editing enzyme. Cell 102: 553-563. [Medline]
  12. Chan, O. T., L. G. Hannum, A. M. Haberman, M. P. Madaio, M. J. Shlomchik. 1999. A novel mouse with B cells but lacking serum antibody reveals an antibody-independent role for B cells in murine lupus. J. Exp. Med. 189: 1639-1648. [Abstract/Free Full Text]
  13. Hannum, L. G., A. M. Haberman, S. M. Anderson, M. J. Shlomchik. 2000. Germinal center initiation, variable gene region hypermutation, and mutant B cell selection without detectable immune complexes on follicular dendritic cells. J. Exp. Med. 192: 931-942. [Abstract/Free Full Text]
  14. Revy, P., T. Muto, Y. Levy, F. Geissmann, A. Plebani, O. Sanal, N. Catalan, M. Forveille, R. Dufourcq-Labelouse, A. Gennery, et al 2000. Activation-induced cytidine deaminase (AID) deficiency causes the autosomal recessive form of the hyper-IgM syndrome (HIGM2). Cell 102: 565-575. [Medline]
  15. Hardy, R. R., C. E. Carmack, S. A. Shinton, J. D. Kemp, K. Hayakawa. 1991. Resolution and characterization of pro-B and pre-pro-B cell stages in normal mouse bone marrow. J. Exp. Med. 173: 1213-1225. [Abstract/Free Full Text]
  16. Hardy, R. R., K. Hayakawa. 2001. B cell development pathways. Annu. Rev. Immunol. 19: 595-621. [Medline]
  17. Oliver, A. M., F. Martin, G. L. Gartland, R. H. Carter, J. F. Kearney. 1997. Marginal zone B cells exhibit unique activation, proliferative and immunoglobulin secretory responses. Eur. J. Immunol. 27: 2366-2374. [Medline]
  18. Maehr, R., M. Kraus, H. L. Ploegh. 2004. Mice deficient in invariant-chain and MHC class II exhibit a normal mature B2 cell compartment. Eur. J. Immunol. 34: 2230-2236. [Medline]
  19. Roark, J. H., S. H. Park, J. Jayawardena, U. Kavita, M. Shannon, A. Bendelac. 1998. CD1.1 expression by mouse antigen-presenting cells and marginal zone B cells. J. Immunol. 160: 3121-3127. [Abstract/Free Full Text]
  20. Amano, M., N. Baumgarth, M. D. Dick, L. Brossay, M. Kronenberg, L. A. Herzenberg, S. Strober. 1998. CD1 expression defines subsets of follicular and marginal zone B cells in the spleen: beta2-microglobulin-dependent and independent forms. J. Immunol. 161: 1710-1717. [Abstract/Free Full Text]
  21. Pillai, S., A. Cariappa, S. T. Moran. 2005. Marginal zone B cells. Annu. Rev. Immunol. 23: 161-196. [Medline]
  22. William, J., C. Euler, M. J. Shlomchik. 2005. Short-lived plasmablasts dominate the early spontaneous rheumatoid factor response: differentiation pathways, hypermutating cell types, and affinity maturation outside the germinal center. J. Immunol. 174: 6879-6887. [Abstract/Free Full Text]
  23. Ardavin, C., P. Martin, I. Ferrero, I. Azcoitia, F. Anjuere, H. Diggelmann, F. Luthi, S. Luther, H. Acha-Orbea. 1999. B cell response after MMTV infection: extrafollicular plasmablasts represent the main infected population and can transmit viral infection. J. Immunol. 162: 2538-2545. [Abstract/Free Full Text]
  24. Finke, D., H. Acha-Orbea. 2001. Differential migration of in vivo primed B and T lymphocytes to lymphoid and non-lymphoid organs. Eur. J. Immunol. 31: 2603-2611. [Medline]
  25. MacLennan, I. C., K. M. Toellner, A. F. Cunningham, K. Serre, D. M. Sze, E. Zuniga, M. C. Cook, C. G. Vinuesa. 2003. Extrafollicular antibody responses. Immunol. Rev. 194: 8-18. [Medline]
  26. Fagarasan, S., K. Kinoshita, M. Muramatsu, K. Ikuta, T. Honjo. 2001. In situ class switching and differentiation to IgA-producing cells in the gut lamina propria. Nature 413: 639-643. [Medline]
  27. Kamata, T., F. Nogaki, S. Fagarasan, T. Sakiyama, I. Kobayashi, S. Miyawaki, K. Ikuta, E. Muso, H. Yoshida, S. Sasayama, T. Honjo. 2000. Increased frequency of surface IgA-positive plasma cells in the intestinal lamina propria and decreased IgA excretion in hyper IgA (HIGA) mice, a murine model of IgA nephropathy with hyperserum IgA. J. Immunol. 165: 1387-1394. [Abstract/Free Full Text]
  28. Smith, K. G., T. D. Hewitson, G. J. Nossal, D. M. Tarlinton. 1996. The phenotype and fate of the antibody-forming cells of the splenic foci. Eur. J. Immunol. 26: 444-448. [Medline]
  29. Delogu, A., A. Schebesta, Q. Sun, K. Aschenbrenner, T. Perlot, M. Busslinger. 2006. Gene repression by Pax5 in B cells is essential for blood cell homeostasis and is reversed in plasma cells. Immunity 24: 269-281. [Medline]
  30. McHeyzer-Williams, M. G.. 2003. B cells as effectors. Curr. Opin. Immunol. 15: 354-361. [Medline]
  31. Underhill, G. H., K. P. Kolli, G. S. Kansas. 2003. Complexity within the plasma cell compartment of mice deficient in both E- and P-selectin: implications for plasma cell differentiation. Blood 102: 4076-4083. [Abstract/Free Full Text]
  32. Medina, F., C. Segundo, A. Campos-Caro, I. Gonzalez-Garcia, J. A. Brieva. 2002. The heterogeneity shown by human plasma cells from tonsil, blood, and bone marrow reveals graded stages of increasing maturity, but local profiles of adhesion molecule expression. Blood 99: 2154-2161.
  33. Kallies, A., J. Hasbold, D. M. Tarlinton, W. Dietrich, L. M. Corcoran, P. D. Hodgkin, S. L. Nutt. 2004. Plasma cell ontogeny defined by quantitative changes in blimp-1 expression. J. Exp. Med. 200: 967-977. [Abstract/Free Full Text]
  34. Manz, R. A., A. Thiel, A. Radbruch. 1997. Lifetime of plasma cells in the bone marrow. Nature 388: 133-134. [Medline]
  35. Martin, F., A. M. Oliver, J. F. Kearney. 2001. Marginal zone and B1 B cells unite in the early response against T-independent blood-borne particulate antigens. Immunity 14: 617-729. [Medline]
  36. Lee, A. H., N. N. Iwakoshi, L. H. Glimcher. 2003. XBP-1 regulates a subset of endoplasmic reticulum resident chaperone genes in the unfolded protein response. Mol. Cell Biol. 23: 7448-7459. [Abstract/Free Full Text]
  37. Harding, H. P., M. Calfon, F. Urano, I. Novoa, D. Ron. 2002. Transcriptional and translational control in the mammalian unfolded protein response. Annu. Rev. Cell. Dev. Biol. 18: 575-599. [Medline]
  38. Iwakoshi, N. N., A. H. Lee, L. H. Glimcher. 2003. The X-box binding protein-1 transcription factor is required for plasma cell differentiation and the unfolded protein response. Immunol. Rev. 194: 29-38. [Medline]
  39. Mason, J. O., G. T. Williams, M. S. Neuberger. 1988. The half-life of immunoglobulin mRNA increases during B-cell differentiation: a possible role for targeting to membrane-bound polysomes. Genes Dev. 2: 1003-1011. [Abstract/Free Full Text]
  40. Gass, J. N., N. M. Gifford, J. W. Brewer. 2002. Activation of an unfolded protein response during differentiation of antibody-secreting B cells. J. Biol. Chem. 277: 49047[Abstract/Free Full Text]
  41. Sitia, R., I. Braakman. 2003. Quality control in the endoplasmic reticulum protein factory. Nature 426: 891-894. [Medline]
  42. Shlomchik, M. J., M. P. Madaio, D. Ni, M. Trounstein, D. Huszar. 1994. The role of B cells in lpr/lpr-induced autoimmunity. J. Exp. Med. 180: 1295-1306. [Abstract/Free Full Text]
  43. Chen, J., M. Trounstine, F. W. Alt, F. Young, C. Kurahara, J. F. Loring, D. Huszar. 1993. Immunoglobulin gene rearrangement in B cell deficient mice generated by targeted deletion of the JH locus. Int. Immunol. 5: 647-656. [Abstract/Free Full Text]
  44. Kitamura, D., J. Roes, R. Kuhn, K. Rajewsky. 1991. A B cell-deficient mouse by targeted disruption of the membrane exon of the immunoglobulin µ chain gene. Nature 350: 423-426. [Medline]
  45. Anderson, S. M., L. G. Hannum, M. J. Shlomchik. 2006. Cutting edge: memory B cell survival and function in the absence of secreted antibody and immune complexes on follicular dendritic cells. J. Immunol. 176: 4515-4519. [Abstract/Free Full Text]



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