The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Bergqvist, P.
Right arrow Articles by Lycke, N. Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Bergqvist, P.
Right arrow Articles by Lycke, N. Y.
The Journal of Immunology, 2006, 177: 7772-7783.
Copyright © 2006 by The American Association of Immunologists, Inc.

Gut IgA Class Switch Recombination in the Absence of CD40 Does Not Occur in the Lamina Propria and Is Independent of Germinal Centers1

Peter Bergqvist, Eva Gärdby, Anneli Stensson, Mats Bemark2 and Nils Y. Lycke2,3

Department of Microbiology and Immunology, Mucosal Immunobiology and Vaccine Research Center, Institute of Biomedicine, Göteborg University, Göteborg, Sweden


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Conflicting findings have recently been presented as to the sites and sources of B cells that undergo class switch recombination (CSR) to IgA in the gut. In this study we provide compelling evidence in CD40–/– mice demonstrating that IgA CSR can be independent of CD40 signaling and germinal center formation and does not occur in the gut lamina propria (LP) itself. We found that CD40–/– mice had near normal levels of gut total IgA despite lacking germinal centers and completely failing to raise specific responses against the T cell-dependent Ags cholera toxin and keyhole limpet hemocyanin. The Peyer’s patches in CD40–/– mice expressed unexpectedly high levels of activation-induced cytidine deaminase mRNA and germline {alpha} transcripts, but few postswitch circular DNA transcripts, arguing against significant IgA CSR. Moreover and more surprisingly, wild-type mice exhibited no to low IgA CSR in mesenteric lymph nodes or isolated lymphoid follicles. Importantly, both strains failed to demonstrate any of the molecular markers for IgA CSR in the gut LP itself. Whereas all of the classical sites for IgA CSR in the GALT in CD40–/– mice appeared severely compromised for IgA CSR, B cells in the peritoneal cavity demonstrated the expression of activation-induced cytidine deaminase mRNA comparable to that of wild-type mice. However, peritoneal cavity B cells in both strains expressed intermediate levels of the germinal center marker GL7 and exhibited no germline {alpha} transcripts, and only three of 51 mice analyzed showed the presence of postswitch circular DNA transcripts. Taken together, these findings strongly argue for alternative inductive sites for gut IgA CSR against T cell-independent Ags outside of the GALT and the nonorganized LP.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
In recent years elegant studies using normal and genetically deficient mice have clearly documented two pathways for IgA formation, both of which could contribute greatly to the total production of IgA in the gut lamina propria (LP)4 (1, 2, 3). It is thought that these pathways operate in parallel, but the relative importance of each and how they are regulated are unknown at present (4, 5). The classical pathway for T cell-dependent (TD) IgA B cell differentiation is closely associated with the Peyer’s patches (PP), which are predominantly populated with naive IgMdull/IgDbright B220+ B2 cells (6) By contrast, T cell-independent (TI) Ags, derived from the gut commensal flora, are thought to drive differentiation of IgA B cells also at sites outside of the PP (7, 8). This IgA production appears to be generated to a large extent from IgMbright/IgDdull B1 cells that are primarily present in the peritoneal cavity (PerC), but B2 cells in the PP may also contribute (9, 10, 11, 12). Germfree animals exhibit poorly developed GALT and produce little IgA, supporting the notion that gut IgA plays a role in an evolutionary mutualism with the bacteria colonizing the intestine (13). Whether this IgA production is completely independent of T cells is still not quite clear. On the one hand, bacterial colonization of TCRbeta{gamma}-deficient mice resulted in substantial IgA production, suggesting that T cells were not required. On the other, the transfer of B1 cells without T cells to SCID mice failed to produce significant gut IgA, arguing in favor of T cell involvement (7, 12, 14). Such involvement may, however, not require cognate T-B cell interactions but may still depend on CD40L expression or other factors surface expressed or released by activated T cells (15, 16). Another site of potential interest for IgA B cell development is the large number of isolated lymphoid follicles (ILF) that are scattered along the antimesenteric border of the small intestine (8, 17). These ILF resemble the PP in many ways, as they consist of clusters of naive B2-cells and also often contain germinal centers (GCs) (17). The ILF are lined by a follicle-associated epithelium that includes M cells, which allow for Ag to be taken up into the follicle and activate B cells (8, 17, 18, 19). Yet, limited information is available as to the specific role of these ILF in the overall production of gut IgA, albeit PP-null mice appear to have functioning ILF IgA inductive sites (17).

A well-documented gut IgA precursor population is found in the PerC, which hosts predominantly self-replenishing B1 cells. These cells may carry germline {alpha} transcripts, indicative of a potential to undergo IgA class switch recombination (CSR) (20). The anatomical site and factors required for the PerC B1 cells to become committed to IgA are still to be determined. It has been suggested that some IgA CSR could take place in the mesenteric lymph nodes (MLN) (19, 21). However, lymphotoxin {alpha} receptor-deficient mice, lacking both PP and MLN and reconstituted with normal bone marrow, produced near normal gut IgA levels, arguing against PP or MLN as necessary sites for IgA B cell development (22). Still, because dendritic cells (DC) can take-up luminal content through the epithelial barrier and thus transport bacteria to the MLN, they could serve as IgA inductive sites independently of the PP (23). Also, the LP itself has been proposed to be a possible primary site for IgA CSR for TI Ags (24). This is, though, a controversial finding, and two recent independent reports have failed to support the notion that naive B cells can undergo IgA CSR in the nonorganized LP (25, 26).

CSR and somatic hypermutation (SHM) are important processes for refining and shaping the function of humoral immunity. Subsequent to Ag exposure, these events take place in the organized secondary lymphoid tissues and are linked to the formation of the GC (27, 28, 29). Whereas CSR to IgG subclasses, except for IgG3 in the mouse, are known to depend on GC formations, less is known about the role of GC in IgA B cell differentiation (2). We and others have reported a number of different gene knockout or transgenic mouse models with a poor correlation between GC in the GALT and the presence of gut IgA and ability to respond to oral immunization (7, 30). For example, IL-4–/–, CD19–/–, or CTLA4-Hg1 transgenic mice respond poorly to oral immunizations with cholera toxin (CT) but demonstrate normal GC reactions in PP and have near normal levels of gut IgA (31, 32). By contrast, CD28–/– or TNFRI–/– mice have no GC in PP but produce near normal levels of gut IgA (7, 30). Thus, the presence of GC in PP does not constitute a requirement for IgA production in the gut LP. In fact, PP-null mice were shown to still have high numbers of IgA plasma cells in the gut LP, suggesting alternative inductive sites for IgA CSR (21).

CSR is a complex process and requires the interplay of a number of different enzymes and genes that are not yet completely understood. Recently, an enzyme called activation-induced cytidine deaminase (AID), which is necessary for CSR and SHM, was discovered (33). Mouse and humans lacking AID fail to produce Abs other than IgM (34), and the mice suffer from hyperplasia of ILF in the LP (35). The role that AID has in CSR is still debated. There are two main hypotheses, disputing whether RNA or DNA is the substrate for AID (36). However, the end result of AID activity is recombination of Ig genes and the production of switch circles that are looped out of the chromosomal DNA (37). These looped-out postswitch circles are present for a short time after CSR, and during this period spliced RNA transcripts between germline I promoters and Cµ can also be detected. Thus, the presence of the postswitch circles and the postswitch circular DNA transcripts represents our best marker for a just completed CSR (37). Together with the expression of germline {alpha} transcripts and AID mRNA transcripts, we have very sensitive molecular tools for the detection of CSR IgA. Using these tools, evidence for active CSR was reported to occur in the LP itself (24), but these results have been disputed. The controversy relates to whether or not the isolated cells could have been derived from ILF rather than the LP (24).

Notwithstanding this controversy, there is ample documentation that naive IgM B cells can undergo IgA CSR under the influence of certain factors acting alone or together. In most species, TGFbeta has been shown to induce IgA CSR, and deletions of the TGFbeta-1 or the TGFbeta receptor type II genes result in severe loss of IgA in mice (38, 39). Yet, TGFbeta alone is insufficient to drive IgA CSR in B cells in vitro but requires a combination with, e.g., LPS or CD40 to provide mitogenic stimulation (40). However, recent reports have documented the possibility that IgA CSR could be stimulated by B cell-activating factor (BAFF) and a proliferation-inducing ligand (APRIL) acting through TACI (transmembrane activator, calcium modulator, and cyclophilin ligand interactor) alone or together with cytokines such as IL-10, and, as it appears, even independently of CD40-signaling (41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51). These factors, produced from stromal cells, macrophages, or DC in the tissue, would then act directly on naive IgM B cells but may still not be produced completely independent of gut T cells, as suggested by Casola et al. (16). Because CD40-CD40L interactions control the expression of costimulatory molecules, the lack of CD40 may also indirectly influence such noncognate T cell functions. Furthermore, whereas CD40L is expressed not only by activated T cells but also by activated DC, the lack of CD40 could potentially have an impact on overall IgA formation. Even TI Ag-driven IgA CSR may, in this way, depend on CD40 signaling (52). Thus, both TD and TI Ag-stimulated gut IgA production pathways may require CD40 signaling to be effective.

In the present study we have used wild-type (WT) and CD40–/– mice to analyze to what extent gut IgA CSR is dependent on CD40 signaling and to address the compartments of the GALT from which IgA CSR may emanate. In particular, we have addressed whether IgA CSR occurs in the nonorganized gut LP. We have also investigated the relationship between GC reactions, gut IgA formation, and the expression of the molecular markers for IgA CSR as well as germline {alpha} transcripts, AID mRNA and postswitch circular transcripts in the GALT, LP and PerC.


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

CD40–/– and WT mice (both on a C57BL/6 background) were bred and housed at the experimental biomedicine animal facility at Göteborg University (Göteborg, Sweden) under specific pathogen-free conditions. For comparison, we also used IgA–/– and double-deficient CD28/ICOS mice (53). The mice were sacrificed at 6–10 wk of age and used for experiments.

Immunohistochemistry

Naive CD40–/– and C57BL/6 mice were sacrificed and the intestine, PP, or MLN were removed, embedded in TissueTek OCT compound, and snap frozen in liquid nitrogen. Frozen sections (7 µm) of the small intestine were prepared on microslides using a cryostat (Leica), and the sections were stored at –70°C until used. The slides were fixed in 50% acetone for 30 s and in 100% acetone for 5 min at 4°C and air dried at room temperature. The tissue was rehydrated in PBS followed by blocking with normal horse serum in PBS (5%) for 15 min in a humidifying chamber. For detection of germinal centers, the sections were stained with FITC-conjugated anti-GL7 mAb diluted 1/100 (BD Pharmingen) and biotinylated anti-B220 diluted 1/100 (BD Pharmingen) and were stained for 30–60 min. The B220 was visualized by adding streptavidin-conjugated Texas Red (DakoCytomation) 1/100 dilution. Consecutive sections were stained with stained with B220 together with FITC-conjugated anti-IgA diluted 1/100 (BD Pharmingen) and FITC-conjugated anti-Ki67 diluted 1/5 (BD Pharmingen). The sections were washed in PBS and mounted with DakoCytomation fluorescent mounting medium and visualized using a Leica LSC microscope.

For visualization and counting of ILF, intestines were divided into four parts, rolled up on a toothpick, embedded in TissueTek OCT compound, snap frozen in liquid nitrogen, and sectioned as described above. The sections were blocked with 0.3% H2O2 in PBS for 5 min, rinsed in PBS, and then blocked with normal horse serum in PBS (5%) for 15 min in a humidifying chamber. To block endogenous biotin, sections were incubated with a avidin/biotin blocking kit (Vector Laboratories) followed by incubation with primary biotinylated anti-B220 for 30–60 min. Following a brief rinse in PBS, peroxidase-conjugated avidin/biotin complexes were prepared and added to the sections according to the manufacturer’s instructions, incubated for 45 min, washed with PBS, and visualized by adding 3-amino-9-ethylcarbazole (Vector Laboratories). The sections were counterstained with hematoxylin, mounted with Faramount (DakoCytomation), and visualized using a Leica LSC microscope.

Cell turnover assessment

BrdU (Sigma-Aldrich) was administered orally by feeding the mice once with 500 µl of BrdU (1 mg/ml) in H2O and simultaneously injecting i.p. 500 µl of BrdU (4 mg/ml) in PBS. Twenty-four hours later the mice were given another BrdU pulse orally. Seventeen hours, 3 days, 1 wk, or 3 wk after BrdU treatment the mice were sacrificed, and the intestine was removed and frozen as described above. Before immunofluorescent staining the sections were treated with 2 M HCl for 30 min at 37°C, neutralized in 0.1 M Na2B4O7 for 5 min. The sections were then stained and mounted as described above using FITC-conjugated anti-BrdU (1/5) and anti-IgA-TXRD (1/100). The number of BrdU-labeled cells in the lamina propria was determined within a fixed area in all sections and visualized in a diagram.

Lamina propria lymphocytes were prepared and BrdU incorporation was visualized using a BrdU flow kit (BD Biosciences) and double stained with anti-IgA according to the instructions and analyzed by FACS. Naive intestines from CD40–/– mice and WT mice were removed and washed two times with PBS. The tissue was snap frozen and sectioned as described above. To visualize apoptosis we used the ApopTag in situ TUNEL apoptosis detection kit (Chemicon). The sections were counterstained with Texas Red-conjugated anti-IgA (Southern Biotechnology Associates) diluted 1/100 for 30 min, washed in PBS, and mounted with DakoCytomation fluorescent mounting medium.

ELISPOT

A 96-well MultiScreen plate (Millipore) was washed in PBS and coated with 5 g/ml purified rat-anti-mouse-IgA Abs (BD Pharmingen) or 3 nmol/ml ganglioside GM1 followed by 3 µg/ml CT or 100 µg/ml keyhole limpet hemocyanin (KLH) at 4°C overnight. The plates were washed in PBS, blocked with 0.2% BSA in PBS, and 10,000 purified lamina propria lymphocytes, prepared as previously described (30), were added in duplicate and diluted 1/3 in four serial dilutions directly on the plate and incubated at 37°C for 3 h. The plate was then washed twice with PBS plus 0.05% Tween 20 and twice with PBS, followed by incubation with alkaline phosphate-conjugated goat anti-mouse-IgA Abs (Southern Biotechnology Associates) overnight at 4°C. The next day the plates were washed twice with PBS plus 0.05% Tween 20 and twice with PBS. The spots were visualized by the addition of one 5-bromo-4-chloro-3-indolylphosphate tablet (Sigma-Aldrich) dissolved in 10 ml of H2O, and 100 µl was added to each well and incubated for 10 min in room temperature. The plate was then washed twice with PBS plus 0.05% Tween 20, twice in PBS, dried, and analyzed using the ImmunoSpot ELISPOT system (Cellular Technology).

ELISA

Ninety-six-well plates (Nunc) were coated with 100 µg/ml KLH (100 µl/well), 0.5 nM/ml GM1 followed by 0.5 µg/ml CT overnight at 4°C. For total Ig titers, the 96-well plates were conjugated with 5 µg/ml anti-mouse IgG1, IgG2a, IgA, or IgM (Southern Biotech) and incubated at 4°C overnight. The plates were washed three times with PBS followed by blocking in PBS plus 0.1% BSA for 30 min at 37°C. The PBS/BSA was discarded, serum samples were diluted 1/100 and added to the plates, and a 3-fold serial dilution was made. The gut lavage was diluted 1/10 and a 2-fold serial dilution was made. The samples were incubated at 4°C overnight and washed three times in PBS-Tween 20. To the CT- and KLH-specific ELISA HRP-conjugated rat anti-mouse Abs (DakoCytomation) were added at a dilution of 1/200 in PBS plus 0.1% BSA and incubated for 2 h at room temperature. The plates were washed with PBS-Tween 20 and developed using o-phenylenediamine dihydrochloride tablets (Sigma-Aldrich) according to the manufacturer’s instructions. The plate was analyzed at 450 nm. For total Ig measurements the plates were incubated with alkaline phosphate-conjugated rabbit anti-mouse Ig (Southern Biotechnology Associates) diluted 1/500 in PBS plus 0.1% BSA for 2 h at RT. The plates were washed with PBS-Tween and developed using Sigma 104 phosphatase tablets (Sigma-Aldrich) according to instructions. The plates were analyzed at 405 nm.

Flow cytometry and cell sorting

Lymphocytes from PP and MLN were prepared by excising the PP from the intestine and passing them through a cell strainer of 100-µm nylon mesh (BD Falcon). Peritoneal cells were isolated by carefully peeling off the skin from the mouse peritoneum, and 8 ml of ice-cold PBS was injected into the peritoneal cavity. After 5 min the peritoneum was cut open and the fluid was retrieved using a transfer pipette. The cells were suspended in PBS supplemented with 0.1% BSA and 0.02% EDTA and stained with anti-B220-PE (BD Pharmingen) and GC-specific anti-GL7-FITC Abs (54) (BD Pharmingen) or anti-IgA-FITC (Southern Biotechnology Associates). For intracellular Ki67 staining the cells were fixed and permeabilized using the FoxP3 staining set (eBioscience), but the anti Ki67-FITC Ab (BD Pharmingen) was used instead of the anti-Foxp3 Ab. To ensure single cell suspensions, the cells were filtered through a MACS preseparation filter (Miltenyi Biotec). The cells were sorted in a FACSAria device (BD Biosciences) in PBS supplemented with 0.1% BSA and 0.02% EDTA. Sorted cells were spun down at 2000 rpm at 4°C followed by RNA extraction using the RNeasy micro kit (Qiagen). The data were analyzed using the FlowJo software (Tree Star).

RNA preparation and RT-PCR

The small intestines were removed and punch biopsies were made from the PP using a 2-mm diameter dermal puncher, isolated, and stored in TRIzol (Invitrogen Life Technologies). RNA was prepared according to the TRIzol protocol, dissolved in 50 µl of UltraPure distilled water (Invitrogen Life Technologies), and stored at –80°C. The RNA concentration was measured and 4 µg of RNA was used for cDNA synthesis using oligo(dT) RETROscript primers (Ambion) and SuperScript III RT polymerase (Invitrogen Life Technologies). MLN were isolated from naive mice and stored in TRIzol, and RNA was prepared as described above.

ILF-free sections of gut LP

To ensure analysis of ILF-free sections, 2- to 3-cm pieces of the small intestine were frozen and sectioned as described (55). Every fifth section was placed on a slide and the sections in between were stored in RLT buffer (RNeasy lysis buffer; Qiagen) at –70°C. The sections on the slide were stained with Texas Red-conjugated anti-IgM (Southern Biotechnology Associates) and FITC-conjugated anti-IgA (BD Pharmingen) to localize ILF. If two consecutive sections on the slide were ILF-negative, the sections in between were stored in RLT buffer and considered to be ILF free. RNA was prepared from these sections using the RNeasy micro kit (Qiagen).

Peritoneal washouts

Mice were sacrificed and the cells were isolated as described above. The cells were spun down at 2000 rpm at 4°C and RNA was extracted using the RNeasy mini kit (Qiagen).

Primers

To detect germline {alpha} transcripts the primers I{alpha}F (5'-CCA GGC ATG GTT GAG ATA GAG ATA G-3') and C{alpha}R (5'-GAG CTG GTG GGA GTG TCA GTG-3') were used. Cycling was performed under the following conditions: denaturation at 95°C for 5 min followed by 30 cycles of PCR (95°C for 1 min, 62°C for 1 min, and 72°C for 1 min) followed by a final extension at 72°C for 10 min. For AID transcripts the following primers were used: mAID119 (5'-GAG GGA GTC AAG AAA GTC ACG CTG G-3'), mAIDR1 (5'-CCA GGC TTT GAA AGT TCT TTC ACG-3'), and, as B cell loading control, CD79a forward (5'-AAC ACA GGG GCT TGT ACT GG-3') and CD79a reverse (5'-ATC TCC AAT GTG GAG GTT GC-3'). The cycling was performed as described above with 25 cycles and an annealing temperature of 59°C. Circular transcripts were detected by a nested PCR using the outer primers Iµ4 (5'-ACC CTG GAT GAC TTC AGT GT-3') and I{alpha}up4 (5'-CAT CTG GAC TCC TCT GCT CA-3') and the inner primers I{alpha}F (5'-CCA GGC ATG GTT GAG ATA GAG ATA G-3') and CµR (5'-AAT GGT GCT GGG CAG GAA GT-3'). Each PCR was performed as described above with 30 cycles each and an annealing temperature of 56°C for the first PCR and of 62°C for the second one.

Southern blot

The AID and CD79 PCR products were separated on a 2% agarose gel and the gel was photographed to visualize AID and CD79. To confirm the AID expression, the fragments was transferred to a Hybond+ membrane (Amersham Biosciences) by passive diffusion using 0.4 M NaOH overnight. The membrane was dried and cross-linked by UV light and then incubated for 1 h in blocking solution at 45°C. An AID-specific probe, AIDhyb_1 (5'-ATA TGG ACA GCC TTC TGA TGA AGC AA AGA AGT TTC-3'), was labeled by mixing 5 pmol of AIDhyb_1 probe, 5 µl of 10x T4 PNK buffer (Promega), 1 µl of T4 polynucleotide kinase (Promega), 5 µl of Redivue adenosine 5'-[{gamma}-32P]triphosphate, triethylammonium salt (Amersham Biosciences), and water to 50 µl. The mixture was incubated for 1 h at 37°C, and free nucleotides were removed using a ProbeQuant G-50 micro column (Amersham Biosciences). The labeled probe was added to the membrane and incubated at 45°C overnight. Hybridization was detected on 32P-sensitive film (Kodak).

One-step PCR

RNA prepared from sorted cells, cells from sectioning, and peritoneal washouts was analyzed using one-step RT-PCR (Invitrogen Life Technologies). The RNA was purified as described above and 2 µl of RNA was used in the RT-PCR. For the cDNA synthesis, gene-specific primers were added and the cDNA synthesis temperature was the same as the annealing temperature for the PCR except during the detection of germline {alpha} transcripts, when the cDNA synthesis temperature was 59°C and PCR annealing temperature was 62°C.

Statistical analysis

Data were compared using Student’s t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
CD40–/– mice show near normal formation of gut IgA

Although it is well established that cognate T cell-B cell interactions are required for systemic IgG responses, less is known about CSR to IgA and to mucosal IgA in particular (4, 5). CSR is classically thought to be restricted to GC, which do not develop in the absence of CD40 (56). Therefore, we predicted a dramatically impaired CSR and mucosal IgA formation in CD40–/– mice. However, quite in contrast, we found substantial IgA production in the gut of CD40–/– mice. In fact, labeling frozen sections of the small intestine with anti-IgA revealed almost normal numbers of IgA plasma cells in gut villi (Fig. 1A). Moreover, total IgA levels in gut lavage were comparable with those in WT mice, and the frequency of IgA-producing, spot-forming cells (SFC) in freshly isolated LP cells was not significantly reduced in CD40–/– mice (Fig. 1, B and C). Whereas IgM was increased 3-fold and IgA reduced to 50% of normal serum levels, IgG1 and IgG2a Abs were below detection limits in serum from CD40–/– mice (Fig. 1D)


Figure 1
View larger version (38K):
[in this window]
[in a new window]
  		FIGURE 1. Significant IgA production in the small intestine of CD40–/– mice. Naive CD40–/– and WT mice small intestines were analyzed for IgA production. A, Cryosections from rolled up small intestines were stained with anti-IgA (green) and anti-IgM (red). Each section contains one ILF cluster brightly staining for IgM+ cells. B, Levels of secreted IgA in gut lavage as determined by ELISA are shown. C, Relative level of gut IgA producing cells as determined by ELISPOT is shown. The diagram shows one representative experiment of three. D, Serum Ab levels of the indicated Ig subclasses were determined by ELISA. Mean values ± SD of three individual experiments are shown. *, p < 0.05; **, p < 0.01; n.s., no significance.

 
Next we addressed whether the substantial IgA production observed in the LP of CD40–/– mice readily reflected an ability to respond to TD Ags in the gut. To this end, oral immunizations with KLH supplemented with CT adjuvant were performed (57). We found no IgA or IgG responses to these Ags in CD40–/– mice, whereas WT mice demonstrated strong specific serum IgA and IgG and gut LP IgA production (Fig. 2, A–D). None of the CD40–/– mice responded with specific IgM Abs against CT, and only 5 of 15 responded against KLH, whereas WT mice responded strongly with IgM Abs against both Ags (Fig. 2, C and D) Furthermore, a similar result was obtained in serum after parenteral immunizations with KLH plus CT adjuvant with no specific IgG or IgA production in CD40–/– mice, whereas IgM Abs against KLH, but not CT, were detected in some mice (Fig. 2, E and F). Of note, parenteral immunizations also failed to stimulate specific IgA responses in serum in WT mice (Fig. 2, E and F). Taken together, our data indicated that CSR to IgA, as opposed to IgG-subclasses (except IgG3), can occur independently of CD40. However, as expected, CD40-CD40L interactions are clearly required to mount specific IgA and IgG responses to orally or parenterally administered TD Ags (58).


Figure 2
View larger version (15K):
[in this window]
[in a new window]
  		FIGURE 2. Lack of specific responses to TD Ags in CD40–/– mice. CD40–/– and WT mice were immunized three times orally or i.p. with KLH using CT as an adjuvant, and 6–8 days after the final immunization the specific IgA response in the LP was analyzed by ELISPOT and the level of serum Abs was measured by ELISA. A, LP KLH-specific spot forming cells after oral immunization are shown. B, LP CT-specific SFC after oral immunization are shown. C, Log10 titers of KLH-specific Abs in serum after oral immunization are shown. D, Log10 titers of CT-specific Abs in serum after oral immunization are shown. E, Log10 titers of KLH-specific Abs in serum after i.p. immunization are shown. F, Log10 titers of CT-specific Abs in serum after i.p. immunization are shown. Shown is one representative experiment of three. Mean values ± SD are shown (p < 0.01). ND, Not detected.

 
Lack of IgA inductive sites in the GALT of CD40–/– mice

Given that CD40–/– mice have poor CSR to IgG and an impaired ability to respond to orally administered TD Ags but show near normal levels of total gut IgA, we analyzed various compartments of the GALT for possible inductive sites for IgA CSR. Frozen sections were investigated for the presence of GC, IgA production, and proliferating cells using the Ki67 Ag as a marker. Whereas the PP of WT mice contained distinct GC areas with aggregates of cells brightly staining with GL7-, IgA- and Ki67-specific Abs, PP from CD40–/– mice failed to label with any of these markers (Fig. 3A). FACS analysis of PP from CD40–/– mice showed <1% of the B220+ cells expressing GL7, whereas in WT mice an average of 6% PP B cells were GL7+ (Fig. 3B). In WT PP B cells, roughly 5% were stained with an anti-IgA mAb, whereas in CD40–/– mice <1% of the cells were labeled (Fig. 3B). For comparison, we used the low/background levels of IgA- and GL7-labeling B cells in PP from IgA–/– or CD28/ICOS double-deficient mice (no GC-reactions), respectively, as negative controls (53).


Figure 3
View larger version (54K):
[in this window]
[in a new window]
  		FIGURE 3. Germinal center formation in PP. A, Immunohistochemical analysis of PP are shown. Naive PP from CD40–/– and WT mice were excised, snap frozen, and sectioned followed by staining with anti-B220 (red) and anti-GL7, anti-Ki67, and anti-IgA (green) as indicated. B, Flow cytometric analysis of PP from naive mice. PP were excised and pooled from two mice and stained with the indicated Abs. To measure the background level of IgA staining, IgA knockout mice were used (IgA–/–) and, as a measurement of background GL7 staining, CD28 and ICOS double-knockout mice were used (CD28/ICOS–/–). The figure shows one representative experiment of five.

 
Another potential site for IgA-switching is ILF along the antimesenteric lining of the small intestine (Fig. 1A). The frequency and distribution of ILF in CD40–/– mice were comparable to that in WT mice with increasing numbers toward the distal part of the small intestine (Fig. 4A). A thorough analysis of the ILF revealed a lack of IgA- and Ki67-positive cells in CD40–/– mice (Fig. 4B). ILF from WT mice were also largely devoid of IgA+ cells, and no GL7- or Ki67-labeling areas were found (Fig. 4B), suggesting that these structures do not contribute much to the overall production of IgA, even in the normal gut. In young mice (3 wk of age) from both strains we were unable to detect any ILF, but IgA SFC numbers in gut LP were significant and comparable in CD40–/– and WT mice (Fig. 4, C and D), again supporting the notion that IgA formation in the gut LP of CD40–/– and WT mice does not rely on the presence of ILF but appear to be recruited from other inductive sites. Furthermore, the gut draining MLN in both CD40–/– and WT mice appeared to be poor sites for IgA CSR. Although none of the MLN from CD40–/– mice stained with any of the three markers, a few small and scattered GC were found in WT MLN, failing to support the notion that MLN is an important site for CSR to IgA (19) (Fig. 4E). FACS analysis demonstrated low levels of GL7-expressing cells in WT and CD40–/– mice, not exceeding the frequency seen in PP of CD40–/– mice (Fig. 4E). Thus, we were unable to find a site in the GALT with ongoing GC formation and CSR to IgA in CD40–/– mice, and even in WT mice the ability to develop such a site appeared to be poor outside of the PP.


Figure 4
View larger version (70K):
[in this window]
[in a new window]
  		FIGURE 4. ILF frequency and distribution are comparable in WT and CD40–/– mice. A, The diagram illustrates the distribution of ILF determined by consecutive sectioning along rolled up intestines. The intestine was divided into four parts, rolled up, and snap frozen, followed by an analysis of the presence of ILF by staining sections with anti-B220. Mean values ± SD of three individual experiments are shown. B, The illustration shows three consecutive sections of a typical ILF from CD40–/– and WT mice stained with anti-B220 (red) and anti-GL7, anti-Ki67, and anti-IgA (green). C, Small intestines from 3-wk-old and adult mice (8 wk) were rolled up and stained with anti-B220 and counterstained with hematoxylin. Arrowheads in the photographs of the adult intestines indicate the presence of ILF. D, The relative frequency of IgA-producing cells in the LP of 3-wk-old mice is shown. The diagram shows one representative experiment of three. E, Immunohistochemical analysis of MLN is shown. Naive MLN were excised, snap frozen, and sectioned, followed by staining with anti-B220 (red) and anti-GL7, anti-Ki67, and anti-IgA (green). F, Representative FACS analysis of GL7 expression in MLN from naive CD40–/– and WT mice are shown. n.s., No significance.

 
A simple explanation for the large number of IgA plasma cells in the LP, despite the impaired ability to respond to TD Ags, could be altered gut plasma cell dynamics in the CD40–/– mice. We failed, however, to identify any difference in plasma cell turnover in the gut LP of CD40–/– mice as compared with that in WT mice, as evidenced by pulse-chase BrdU treatment experiments (Fig. 5A). Following BrdU loading of gut plasma cells we observed no difference in the decay of labeled cells in the LP in the two strains over time (Fig. 5, A–C). A careful assessment of BrdU-labeled cells in the LP of both strains on days 1, 3, 7, and 21 after treatment failed to reveal an altered IgA+ cell turnover in CD40–/– as compared with WT mice (Fig. 5C). Furthermore, a FACS analysis of the BrdU-treated animals showed that both strains had ~1% of IgA+BrdU+ cells in the small intestine 1 wk after BrdU labeling (data not shown). Moreover, the levels of IgA+ cells undergoing apoptosis within the gut mucosa of both strains were comparable, arguing against a difference in IgA plasma cell turnover between the strains (Fig. 5D). These data suggested that IgA plasma cells do not exhibit altered dynamics in gut LP of CD40–/– as compared with WT mice.


Figure 5
View larger version (26K):
[in this window]
[in a new window]
  		FIGURE 5. Unaltered gut plasma cell turnover in CD40–/– mice. A, Mice were given BrdU by simultaneously feeding it orally for 2 days and injecting it i.p., and the mice were then sacrificed at the indicated time points after challenge. The intestines were removed and stained with anti-IgA (red) and anti-BrdU (green). B, High magnification view of stained sections taken one week after BrdU treatment. C, BrdU-positive cells in the lamina propria at the different time points indicated. Numbers are average BrdU-positive cells from three different sections within one visual field in the microscope. D, Detection of apoptotic cells in the small intestine using a TUNEL assay is shown. Intestines from naive mice were stained for apoptotic cells (green) and IgA-positive cells (red).

 
Molecular screening for IgA CSR in the GALT of CD40–/– mice

To increase the sensitivity of the analysis for detection of IgA CSR in the GALT of CD40–/– mice, we used RT-PCR for the presence of germline {alpha} transcripts, AID mRNA, and postswitch circular DNA transcripts (24, 37). With this technique we confirmed that PP in WT mice are strong sites for IgA CSR with frequent and high mRNA expression of AID and germline {alpha} transcripts as well as the presence of postswitch circular DNA transcripts (Fig. 6A). By contrast, PP from CD40–/– mice showed the expression of germline {alpha} transcripts and AID mRNA, albeit weaker, but few postswitch DNA-transcripts were found, arguing against PP being a major site for IgA CSR in these mice (Fig. 6A). In fact, we detected postswitch DNA transcripts in <25% of 45 PP isolated from 10 different CD40–/– mice, whereas in 47 PP from 10 WT mice >85% demonstrated presence of these transcripts. Of note, given that no GC reactions (GL7- and Ki67-negative) were detected in PP of CD40–/– mice (Fig. 3), it was surprising to find relatively prominent AID mRNA expression (Fig. 6A). An extended analysis of sorted GL7-high, -intermediate, and -negative cells from both strains revealed readily detectable AID mRNA expression in the GL7-intermediate and -high populations, but not in the negative population. Thus, substantial AID mRNA-expression was evident in the GL7-intermediate population, albeit not nearly as strong as in the GL7-high population. (Fig. 6B). Moreover, in agreement with the histological analysis, the MLN of both CD40–/– and WT mice expressed very low levels of any of the molecular markers for IgA CSR (Fig. 6C). Of 21 MLN samples from nine CD40–/– mice analyzed, we detected AID mRNA in 20% and postswitch circular DNA-transcripts in <5%. However, MLN from WT mice also appeared to be poor sites for IgA CSR when compared with PP from the same mice, as we detected weak expression of AID mRNA and postswitch circular DNA transcripts in only 14% of the MLN samples. Although an unlikely site for IgA CSR, we also included the spleen in our analysis but found no germline {alpha} transcripts, weak or no AID mRNA-expression, and no postswitch DNA transcripts in this tissue in neither CD40–/– or WT mice (Fig. 6D).


Figure 6
View larger version (33K):
[in this window]
[in a new window]
  		FIGURE 6. Analysis of molecular markers for IgA CSR. RNA was prepared and analyzed by RT-PCR. The figure shows the expression of germline {alpha} transcripts ({alpha}-GT) measured by RT-PCR, AID transcripts measured by RT-PCR followed by Southern blot analysis and postswitch circle transcripts measured by nested RT-PCR. A, PP from naive CD40–/– and WT mice were analyzed. The CD79 PCR was performed in the same tube as the AID PCR and used as a B cell-specific loading control, and beta2m PCR was used as a RNA loading control. Samples without RT added (–RT) and samples from µmt mice were used as negative controls, and a PP sample from a WT mouse was used as a positive control. The different sizes in the postswitch circle transcripts correspond to different splice variants of the transcript. B, FACS sorting of PP cells from WT and CD40–/– mice was performed. PP lymphocytes from five individual mice were isolated and pooled and stained with anti-B220, anti-GL7, and anti-CD3 and were then sorted into GL7-negative (GL7–), GL7-intermediate (GL7 int), and GL7-high (GL7 hi) cells. The gates show the different sorted fractions before and after sorting. RNA was isolated from the different fractions and AID expression was analyzed by one-step RT-PCR amplification followed by Southern bloting. CD79 was used as a B cell-specific loading control. C, Expression of germline {alpha} transcripts, AID transcripts, and postswitch circle transcripts in MLN of naive CD40–/– and WT mice was performed as described above. D, Expression of germline {alpha} transcripts ({alpha}-GT), AID transcripts, and postswitch circle transcripts in the spleens of naive CD40–/– and WT mice was performed as described above.

 
The gut LP is not a site for IgA CSR

Previous work has indicated that the gut LP could be a site for TI IgA CSR (24). Because we had failed to explain the relative abundance of IgA in the gut LP of CD40–/– mice, we therefore turned to investigating the LP for evidence of IgA CSR. Gut LP cells showed no postswitch circular DNA transcripts or AID mRNA expression in either WT or CD40–/– mice, clearly rejecting the notion that IgA CSR normally occurs in this nonorganized tissue of the mucosal immune system (Fig. 7). We analyzed 12 individual mice from each strain and two different samples from each intestine and never found AID mRNA expression or postswitch circular DNA transcripts, whereas germline {alpha} transcripts were clearly detected. Of note, to insure that gut LP samples were devoid of ILF we used the method previously described by Stoel, M. et al. (55) (see Materials and Methods). Importantly, specimens containing ILF occasionally demonstrated AID mRNA expression (not shown).


Figure 7
View larger version (14K):
[in this window]
[in a new window]
  		FIGURE 7. Expression of molecular markers for ongoing CSR in the lamina propria. RNA from ILF-free lamina propria from CD40–/– and WT mice was isolated according to Materials and Methods. Expression levels of germline {alpha} transcripts ({alpha}-GT), AID transcripts, and postswitch circle transcripts was visualized by one-step RT-PCR followed by Southern blot (AID) and nested PCR (postswitch circle transcripts). The CD79 PCR was performed in the same tube as the AID PCR and used as a B cell-specific loading control, and the beta2m PCR was used as a RNA loading control.

 
Intact AID mRNA expression in peritoneal cavity B cells in CD40–/– mice

In the absence of data pointing to a probable site for IgA CSR in the CD40–/– mice, we analyzed B cells taken out of the PerC. This is a classical site for B1 cells, which have been suggested as seeding the gut LP with IgA plasma cells (9, 11). The question of PerC B cells express AID mRNA and postswitch DNA transcripts has, to our knowledge, not been previously investigated, although germline {alpha} transcripts and postswitch {alpha} transcripts have been reported (20). Thus, to this end we isolated PerC B cells and extracted RNA for subsequent RT-PCR analysis for IgA CSR. We found that PerC B cells from the two strains demonstrated high and comparable expression of AID mRNA, but very few samples expressed germline {alpha} transcripts or postswitch circular DNA-transcripts in either CD40–/– or WT mice (Fig. 8A). In total, 56 mice were analyzed and only three of these (one WT and two CD40–/–) indeed expressed postswitch circular DNA transcripts. FACS sorting of PerC B cells demonstrated that AID mRNA expression was largely restricted to the GL7-intermediate population, in particular in the CD40–/– mice (Fig. 8B). Furthermore, we could detect proliferating Ki67+IgMbright cells in the PerC of both strains (Fig. 8C). However we could not detect any difference in the frequency of Ki67-labeled cells in the PerC of the two strains, albeit there was a 3- to 4-fold lower level of proliferating Ki67+B220+ cells in the PP in CD40–/– mice as compared with that in WT mice (Fig. 8C).


Figure 8
View larger version (27K):
[in this window]
[in a new window]
  		FIGURE 8. Peritoneal cavity cells express AID but very few postswitch circular transcripts. A, Peritoneal cavities from naive CD40–/– and WT mice were washed out with PBS, and RNA was isolated from the collected cells. The expression of germline {alpha} transcripts ({alpha}-GT), AID transcripts, and postswitch circle transcripts was visualized by RT-PCR ({alpha}-GT), RT-PCR followed by Southern blotting (AID), and nested PCR (postswitch circle transcripts). The CD79 PCR was performed in the same tube as the AID PCR and used as a B cell-specific loading control, and the beta2m PCR was used as a RNA loading control. B, A FACS analysis of PerC cells is shown. The gates show the populations before and after sorting. RNA was prepared from the sorted fractions and analyzed for AID expression as in A. RNA prepared from a PP of a WT mouse was used as a positive control. GL7–, GL7-negative cells; GL7 int, GL7-intermediate cells; and GL7 hi, GL7-high cells. C, An analysis of the presence of proliferating cells in the PerC and the PP of CD40–/– and WT mice is shown. The cells were stained with anti-IgM or anti-B220, permeabilized, fixed, and stained with anti-Ki67. Mean values of IgM+Ki67+ cells were 7.4 ± 2.5% for CD40–/– (n = 7) and 8.0 ± 3.6% for WT (n = 7) in the PerC.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The present study of CD40–/– mice corroborate observations of normal to high IgA levels in patients with impaired CD40-signaling (59). Here we report near normal levels of gut IgA and a 50% reduction in serum IgA, whereas IgG1 and IgG2a were undetectable in CD40–/– mice. Thus, significant IgA CSR can occur in the complete absence of CD40 and GC reactions. Furthermore, we found that CD40–/– mice completely failed to respond to oral (as well as parenteral) immunizations with the TD Ags, CT, and KLH while, in fact, demonstrating near normal levels of total IgA plasma cells in their gut LP. In the search for a site where naive B cells would undergo IgA CSR in these mice, we were unsuccessful in documenting microscopic or molecular data to suggest that any of the traditional locations within the GALT or the spleen are strong sources for IgA CSR. Compared with WT mice, the CD40–/– mice exhibited dramatically reduced levels of IgA CSR in PP and MLN, whereas the ILF and LP were poor sites for IgA class switching in both strains. B cells in the PerC demonstrated significant levels of AID mRNA expression in both strains, and an equal but low frequency of postswitch circular DNA transcripts were detected. These findings are consistent with the notion that IgA in CD40–/– mice is produced against TI Ags and that B1 cells are potentially the main source of this IgA (10, 60). The question of whether it is the B1 PerC population or the strongly impaired IgA CSR in PP or MLN (in B1 or B2 cells) that contributes to most of the LP IgA plasma cells in CD40–/– mice, and to what extent, cannot be answered by the present study. However, we can conclude that the gut LP is unlikely to be a primary site for IgA CSR in either CD40–/– or WT mice because of a complete lack of AID mRNA expression and no evidence of postswitch circular DNA transcripts.

We made some remarkable observations regarding IgA CSR in the CD40–/– mice. A close interrelationship between expression of AID mRNA, germline {alpha} transcripts, and postswitch circular DNA transcripts was clearly found in WT PP, denoting this site a true place for IgA CSR. This finding was also supported by strong labeling with specific Abs for GC and IgA in tissue sections of PP from WT mice. By contrast, CD40–/– mice had PP that failed to label for GC or IgA and exhibited substantial levels of germline {alpha} transcripts and AID mRNA but substantially reduced postswitch circle DNA transcripts. Therefore, we believe that PP may not be an important site for IgA CSR in CD40–/– mice unless activated IgA CSR-committed B cells migrate out of the PP to complete the CSR. Alternatively, only 25% of normal CSR-activity in PP, as in the CD40–/– mice, suffices to provide WT levels of gut LP IgA plasma cells. Interestingly, despite the lack of GC in CD40–/– mice, the AID mRNA expression in PP B cells was prominent. AID is normally thought of in the context of GL7high B cells in the GC and is critically required for CSR and SHM (33, 61). Recent studies have also documented AID-expression in B cell tumors and extrafollicular large proliferating normal B cells (62, 63, 64). However, contrary to our finding, these latter cells showed evidence of CD40 signaling, were Ki67+, and resulted from TD Ag activation (62). Of note, CD40 signaling is one pathway that can induce AID expression as can IL-4 and TLR4 signaling or pathways that affect intracellular levels of protein kinase A (65, 66, 67, 68).

The B cell population that transcribed AID in the PP of CD40–/– animals expressed an intermediate level of GL7 undetectable by histology, whereas the GL7-negative B cells were also AID mRNA-negative. To our knowledge, no previous study has reported AID mRNA transcription in relation to GL7 membrane expression. We found that microscopically GL7-negative tissues can, indeed, host substantial AID mRNA expression. Both in PP and PerC B cells AID mRNA expression was found in GL7-intermediate cells, indicating that these cells have the potential to undergo CSR. It is also possible that these cells undergo IgA CSR, although this process would have to be nondeletional, as seen, e.g., in hairy leukemic cells, which also fail to produce circle DNA transcripts (63). Rather, we believe that IgA CSR in the activated B cells in PP or PerC is completed at some other not yet defined location before seeding the gut LP. This location does not appear to be the spleen or MLN. However, the significance of extrafollicular AID mRNA expression for CSR and SHM is still controversial (62). Therefore, it remains to be seen to what extent GL7-intermediate B cells are responsible for IgA CSR in response to TI Ags in normal individuals. Nevertheless, in this study we provide compelling evidence that CD40–/– mice have near normal total gut IgA plasma cell levels and yet exhibit only extrafollicular sites of AID mRNA expressing B cells. In addition, preliminary studies of SHM in IgA VH genes from gut LP cells from CD40–/– mice surprisingly revealed many mutated clones even compared with those observed in WT gut IgA cells, suggesting that even this normally GC-restricted quality could be provided by extrafollicular sites (P. Bergqvist, unpublished observation).

Evidence that LP would be a primary site for IgA CSR was first reported by Fagarasan et al. (24). The background to the study was that AID–/– mice had increased levels of B220+IgM+ cells in the LP, and the authors hypothesized that that these cells could undergo IgA CSR in the LP itself. (24). However, their data have recently been challenged by at least two studies that cannot confirm the notion that gut LP would be a primary site for IgA CSR (25, 26). The ILF compartment is grossly enlarged in AID–/– mice, and it is probable that the nonorganized LP is diminished in these mice in keeping with the interpretation that the purified gut cells were likely to contain cells from ILF and not exclusively nonorganized LP in AID–/– mice (24). In the present study we found that AID mRNA expression was detected in ILF-containing biopsies but not in the biopsies of normal nonorganized small intestinal LP. Shikina et al. (26) also used careful dissection of LP tissue to exclude ILF and, in agreement with our study, failed to detect AID mRNA and postswitch circle DNA transcripts. In human LP tissue, Boursier et al. (25) failed to detect the molecular markers for IgA CSR, including a lack of AID mRNA. Furthermore, clonal analysis of somatic mutations in the rearranged IgVH5-gene segment in IgA+ cells isolated from distant sites in the nonorganized LP showed greater similarity than when compared with mutations in neighboring sites, favoring a model of dissemination of precursor cells derived from a common source within the organized GALT (25). At variance with our study and that of Boursier et al., Shikina et al. did not detect germline {alpha} transcripts in LP (25, 26). The reason for this is unknown. Nevertheless, because expression of these germline {alpha} transcripts is necessary and precedes CSR but cannot initiate CSR in the absence of AID expression, these transcripts merely indicate the potential of B cells in the gut LP to undergo CSR (69, 70). We believe that the lack of AID mRNA and postswitch circle DNA transcripts as well as failure to demonstrate B cell proliferation, i.e., Ki67-labeling cells (P. Bergqvist, unpublished observation), disqualify gut LP as a primary site for IgA CSR. For these reasons we cannot ascribe IgA CSR to the PP or, indeed, all of the GALT, including MLN, in CD40–/– mice, as these sites fail to exhibit the signifying factors required for an active IgA CSR. If CSR is initiated in the GALT of these mice at all, cells must leave, e.g., the PP, before undergoing full deletional CSR to IgA. We believe that this site or sites are to be found.

In agreement with previous studies we found that CD40–/– mice lack GC completely (56). A common notion is that the presence of GC in the PP of WT mice constitute a source of gut LP IgA plasma cell precursors (6, 71). These GC B cells are predominantly B2 cells that appear to respond not only to TD Ags, but to a significant degree also to TI Ags associated with the commensal flora (13). Hence, the PP GC formations seen in CD19–/– mice, which largely lack B1 cells, could be B2 cells reacting to the commensal flora, whereas these mice are unable to respond to oral immunizations with TD Ags (31). In fact, GC formations in PP may not even be dependent on BCR recognition but result from the presence of luminal bacteria and T cells (16). Because genetically deficient mice such as CD19–/– or TNFRI–/– mice differ with regard to the presence of GC in their PP and still make good levels of IgA, it must be concluded that there is no direct relationship between GC in GALT and IgA CSR in either B1 or B2 cells (7, 31).

Two tissues were surprisingly poor sites for IgA CSR also in WT mice. Considering the paucity of detectable molecular markers for IgA CSR in both MLN and the ILF relative to that in PP, we must conclude that they appear to be subordinate for the total gut IgA formation. Because previous reports have demonstrated active IgA CSR in ILF in particular, we analyzed this site in detail as a source for the progeny of LP IgA plasma cells (8). However, we found almost no ILF in young (3-wk-old) mice, but substantial IgA SFC were detected in gut LP in both CD40–/– and WT mice. Although the ILF were formed independent of CD40 and appeared to develop in response to the luminal flora of bacteria, they appear to have little to do with populating the gut LP with IgA plasma cells. The ILF in CD40–/– mice were comparable in numbers and distribution to those in WT mice and hosted naive IgM+ cells. In both WT and CD40–/– mice IgA CSR in ILF were not prominent, as determined by the lack of postswitch circle DNA transcripts and poor correlation to gut IgA SFC (P. Bergqvist, unpublished observation). Furthermore, MLN has been found to be critical for processing luminal bacteria carried to the node by DC migrating from PP and the gut LP (72). These and other findings have focused attention on MLN as a possible site for IgA CSR in the GALT. Our study does not support this idea. Rather, it is the phagocytic and eliminating activity against the transported bacteria that is critically located at the MLN, and the MLN is not a site for IgA CSR of the gut LP IgA plasma cells.

Previous reports have suggested that PerC B-1 cells could contribute to as much as half of the total IgA production in the mouse small intestine (11, 55). The PerC B-1 cells in both WT and CD40–/– did not express IgA (P.Bergqvist, unpublished results) but, interestingly, were strong in AID mRNA transcription. This report is, to our knowledge, the first to document AID mRNA and postswitch circle DNA transcripts in PerC B cells. However, the frequency of postswitch circle DNA transcripts was very low, and relatively few cells were Ki67-positive as compared with PP, suggesting little ongoing proliferation. At variance with de Waard et al. (20), we failed to detect germline {alpha} transcripts in the PerC B cells. Taken together, this raises the question of whether these cells were already committed or were on their way to initiate or complete IgA CSR at some other location. AID mRNA expression by itself is not sufficient for CSR; additional cofactors are required (73). Of note, compared with PP B2 cells in the PP of WT mice, the frequency of postswitch circle DNA expression was very low in PerC B1 cells. Moreover, none of the earlier reports have established where, indeed, PerC B-1 cells are exposed to Ag or where the cells undergo IgA CSR. It has been suggested that PerC B1 cells can undergo IgA CSR in MLN, but our results in CD40–/– mice largely fail to support this theory because postswitch circular DNA transcripts were hardly found (72, 74). Future studies using germfree and recently colonized mice will explore these possibilities.


   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 study was supported by the Swedish Research Council, the Swedish Cancer Foundation, the Sahlgrenska University Hospital Foundation, the Swedish Foundation for Strategic Research, the Mucosal Immunobiology and Vaccine Center, and European Union Grants QLK2-CT-2001-01702, QLK2-CT-199-00228, and LSHP-CT- 2003-503240. Back

2 M.B. and N.Y.L. share senior authorship. Back

3 Address correspondence and reprint requests to Dr. Nils Lycke, Department of Microbiology and Immunology, University of Göteborg, 405 30 Göteborg, Sweden. E-mail address: nils.lycke{at}microbio.gu.se Back

4 Abbreviations used in this paper: LP, lamina propria; AID, activation-induced cytidine deaminase; APRIL, a proliferation-inducing ligand; BAFF, B cell-activating factor; CSR, class switch recombination; CT, cholera toxin; DC, dendritic cell; GC, germinal center; ILF, isolated lymphoid follicle; KLH, keyhole limpet hemocyanin; MLN, mesenteric lymph node; PerC, peritoneal cavity; PP, Peyer’s patch; SFC, spot forming cell; SHM, somatic hypermutation; TD, T cell dependent; TI, T cell independent; WT, wild type. Back

Received for publication May 24, 2006. Accepted for publication September 13, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Macpherson, A. J., L. Hunziker, K. McCoy, A. Lamarre. 2001. IgA responses in the intestinal mucosa against pathogenic and non-pathogenic microorganisms. Microbes Infect. 3: 1021-1035. [Medline]
  2. Strober, W., S. Fagarasan, N. Lycke. 2005. IgA B cell development. J. Mestecky, and M. E. Lamm, and W. Strober, and J. Bienenstock, and J. R. McGhee, and L. Mayer, eds. Mucosal immunology 3rd Ed.583-616. Academic Press, Boston.
  3. Fagarasan, S., T. Honjo. 2003. Intestinal IgA synthesis: regulation of front-line body defences. Nat. Rev. Immunol. 3: 63-72. [Medline]
  4. Fagarasan, S., T. Honjo. 2004. Regulation of IgA synthesis at mucosal surfaces. Curr. Opin. Immunol. 16: 277-283. [Medline]
  5. Brandtzaeg, P., F. E. Johansen. 2005. Mucosal B cells: phenotypic characteristics, transcriptional regulation, and homing properties. Immunol. Rev. 206: 32-63. [Medline]
  6. Husband, A. J., J. L. Gowans. 1978. The origin and antigen-dependent distribution of IgA-containing cells in the intestine. J. Exp. Med. 148: 1146-1160. [Abstract/Free Full Text]
  7. Macpherson, A. J., D. Gatto, E. Sainsbury, G. R. Harriman, H. Hengartner, R. M. Zinkernagel. 2000. A primitive T cell-independent mechanism of intestinal mucosal IgA responses to commensal bacteria. Science 288: 2222-2226. [Abstract/Free Full Text]
  8. Lorenz, R. G., R. D. Newberry. 2004. Isolated lymphoid follicles can function as sites for induction of mucosal immune responses. Ann. NY Acad. Sci. 1029: 44-57. [Medline]
  9. Kroese, F. G., E. C. Butcher, A. M. Stall, L. A. Herzenberg. 1989. A major peritoneal reservoir of precursors for intestinal IgA plasma cells. Immunol. Invest. 18: 47-58. [Medline]
  10. Kroese, F. G., W. A. Ammerlaan, A. B. Kantor. 1993. Evidence that intestinal IgA plasma cells in µ, {kappa} transgenic mice are derived from B-1 (Ly-1 B) cells. Int. Immunol. 5: 1317-1327. [Abstract/Free Full Text]
  11. Kroese, F. G., E. C. Butcher, A. M. Stall, P. A. Lalor, S. Adams, L. A. Herzenberg. 1989. Many of the IgA producing plasma cells in murine gut are derived from self-replenishing precursors in the peritoneal cavity. Int. Immunol. 1: 75-84. [Abstract/Free Full Text]
  12. Bos, N. A., J. J. Cebra, F. G. Kroese. 2000. B-1 cells and the intestinal microflora. Curr. Top. Microbiol. Immunol. 252: 211-220. [Medline]
  13. Macpherson, A. J., M. B. Geuking, K. D. McCoy. 2005. Immune responses that adapt the intestinal mucosa to commensal intestinal bacteria. Immunology 115: 153-162. [Medline]
  14. Bos, N. A., H. Q. Jiang, J. J. Cebra. 2001. T cell control of the gut IgA response against commensal bacteria. Gut 48: 762-764. [Free Full Text]
  15. Chirmule, N., J. Tazelaar, J. M. Wilson. 2000. Th2-dependent B cell responses in the absence of CD40-CD40 ligand interactions. J. Immunol. 164: 248-255. [Abstract/Free Full Text]
  16. Casola, S., K. L. Otipoby, M. Alimzhanov, S. Humme, N. Uyttersprot, J. L. Kutok, M. C. Carroll, K. Rajewsky. 2004. B cell receptor signal strength determines B cell fate. Nat. Immunol. 5: 317-327. [Medline]
  17. Hamada, H., T. Hiroi, Y. Nishiyama, H. Takahashi, Y. Masunaga, S. Hachimura, S. Kaminogawa, H. Takahashi-Iwanaga, T. Iwanaga, H. Kiyono, et al 2002. Identification of multiple isolated lymphoid follicles on the antimesenteric wall of the mouse small intestine. J. Immunol. 168: 57-64. [Abstract/Free Full Text]
  18. Lorenz, R. G., D. D. Chaplin, K. G. McDonald, J. S. McDonough, R. D. Newberry. 2003. Isolated lymphoid follicle formation is inducible and dependent upon lymphotoxin-sufficient B lymphocytes, lymphotoxin beta receptor, and TNF receptor I function. J. Immunol. 170: 5475-5482. [Abstract/Free Full Text]
  19. Yamamoto, M., M. N. Kweon, P. D. Rennert, T. Hiroi, K. Fujihashi, J. R. McGhee, H. Kiyono. 2004. Role of gut-associated lymphoreticular tissues in antigen-specific intestinal IgA immunity. J. Immunol. 173: 762-769. [Abstract/Free Full Text]
  20. de Waard, R., P. M. Dammers, J. W. Tung, A. B. Kantor, J. A. Wilshire, N. A. Bos, L. A. Herzenberg, F. G. Kroese. 1998. Presence of germline and full-length IgA RNA transcripts among peritoneal B-1 cells. Dev. Immunol. 6: 81-87. [Medline]
  21. Yamamoto, M., P. Rennert, J. R. McGhee, M. N. Kweon, S. Yamamoto, T. Dohi, S. Otake, H. Bluethmann, K. Fujihashi, H. Kiyono. 2000. Alternate mucosal immune system: organized Peyer’s patches are not required for IgA responses in the gastrointestinal tract. J. Immunol. 164: 5184-5191. [Abstract/Free Full Text]
  22. Kang, H. S., R. K. Chin, Y. Wang, P. Yu, J. Wang, K. A. Newell, Y. X. Fu. 2002. Signaling via LTbetaR on the lamina propria stromal cells of the gut is required for IgA production. Nat. Immunol. 3: 576-582. [Medline]
  23. Rescigno, M., M. Urbano, B. Valzasina, M. Francolini, G. Rotta, R. Bonasio, F. Granucci, J. P. Kraehenbuhl, P. Ricciardi-Castagnoli. 2001. Dendritic cells express tight junction proteins and penetrate gut epithelial monolayers to sample bacteria. Nat. Immunol. 2: 361-367. [Medline]
  24. 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]
  25. Boursier, L., J. N. Gordon, S. Thiagamoorthy, J. D. Edgeworth, J. Spencer. 2005. Human intestinal IgA response is generated in the organized gut-associated lymphoid tissue but not in the lamina propria. Gastroenterology 128: 1879-1889. [Medline]
  26. Shikina, T., T. Hiroi, K. Iwatani, M. H. Jang, S. Fukuyama, M. Tamura, T. Kubo, H. Ishikawa, H. Kiyono. 2004. IgA class switch occurs in the organized nasopharynx- and gut-associated lymphoid tissue, but not in the diffuse lamina propria of airways and gut. J. Immunol. 172: 6259-6264. [Abstract/Free Full Text]
  27. Butcher, E. C., R. V. Rouse, R. L. Coffman, C. N. Nottenburg, R. R. Hardy, I. L. Weissman. 1982. Surface phenotype of Peyer’s patch germinal center cells: implications for the role of germinal centers in B cell differentiation. J. Immunol. 129: 2698-2707. [Abstract]
  28. Klaus, G. G., A. Kunkl. 1982. The role of T cells in B cell priming and germinal centre development. Adv. Exp. Med. Biol. 149: 743-749. [Medline]
  29. Berek, C., A. Berger, M. Apel. 1991. Maturation of the immune response in germinal centers. Cell 67: 1121-1129. [Medline]
  30. Gardby, E., J. Wrammert, K. Schon, L. Ekman, T. Leanderson, N. Lycke. 2003. Strong differential regulation of serum and mucosal IgA responses as revealed in CD28-deficient mice using cholera toxin adjuvant. J. Immunol. 170: 55-63. [Abstract/Free Full Text]
  31. Gardby, E., N. Y. Lycke. 2000. CD19-deficient mice exhibit poor responsiveness to oral immunization despite evidence of unaltered total IgA levels, germinal centers and IgA-isotype switching in Peyer’s patches. Eur. J. Immunol. 30: 1861-1871. [Medline]
  32. Gardby, E., P. Lane, N. Y. Lycke. 1998. Requirements for B7-CD28 costimulation in mucosal IgA responses: paradoxes observed in CTLA4-H {gamma} 1 transgenic mice. J. Immunol. 161: 49-59. [Abstract/Free Full Text]
  33. 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]
  34. 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]
  35. Fagarasan, S., M. Muramatsu, K. Suzuki, H. Nagaoka, H. Hiai, T. Honjo. 2002. Critical roles of activation-induced cytidine deaminase in the homeostasis of gut flora. Science 298: 1424-1427. [Abstract/Free Full Text]
  36. Honjo, T., H. Nagaoka, R. Shinkura, M. Muramatsu. 2005. AID to overcome the limitations of genomic information. Nat. Immunol. 6: 655-661. [Medline]
  37. Kinoshita, K., M. Harigai, S. Fagarasan, M. Muramatsu, T. Honjo. 2001. A hallmark of active class switch recombination: transcripts directed by I promoters on looped-out circular DNAs. Proc. Natl. Acad. Sci. USA 98: 12620-12623. [Abstract/Free Full Text]
  38. Cazac, B. B., J. Roes. 2000. TGF-beta receptor controls B cell responsiveness and induction of IgA in vivo. Immunity 13: 443-451. [Medline]
  39. van Ginkel, F. W., S. M. Wahl, J. F. Kearney, M. N. Kweon, K. Fujihashi, P. D. Burrows, H. Kiyono, J. R. McGhee. 1999. Partial IgA-deficiency with increased Th2-type cytokines in TGF-beta 1 knockout mice. J. Immunol. 163: 1951-1957. [Abstract/Free Full Text]
  40. McIntyre, T. M., M. R. Kehry, C. M. Snapper. 1995. Novel in vitro model for high-rate IgA class switching. J. Immunol. 154: 3156-3161. [Abstract]
  41. Zan, H., A. Cerutti, P. Dramitinos, A. Schaffer, P. Casali. 1998. CD40 engagement triggers switching to IgA1 and IgA2 in human B cells through induction of endogenous TGF-beta: evidence for TGF-beta but not IL-10-dependent direct Sµ->S{alpha} and sequential Sµ->S{gamma}, S{gamma}->S{alpha} DNA recombination. J. Immunol. 161: 5217-5225. [Abstract/Free Full Text]
  42. Han, S., B. Zheng, Y. Takahashi, G. Kelsoe. 1997. Distinctive characteristics of germinal center B cells. Semin. Immunol. 9: 255-260. [Medline]
  43. van Kooten, C., J. Banchereau. 2000. CD40-CD40 ligand. J. Leukocyte Biol. 67: 2-17. [Abstract]
  44. Castigli, E., S. Scott, F. Dedeoglu, P. Bryce, H. Jabara, A. K. Bhan, E. Mizoguchi, R. S. Geha. 2004. Impaired IgA class switching in APRIL-deficient mice. Proc. Natl. Acad. Sci. USA 101: 3903-3908. [Abstract/Free Full Text]
  45. Litinskiy, M. B., B. Nardelli, D. M. Hilbert, B. He, A. Schaffer, P. Casali, A. Cerutti. 2002. DCs induce CD40-independent immunoglobulin class switching through BLyS and APRIL. Nat. Immunol. 3: 822-829. [Medline]
  46. He, B., N. Raab-Traub, P. Casali, A. Cerutti. 2003. EBV-encoded latent membrane protein 1 cooperates with BAFF/BLyS and APRIL to induce T cell-independent Ig heavy chain class switching. J. Immunol. 171: 5215-5224. [Abstract/Free Full Text]
  47. Schneider, P.. 2005. The role of APRIL and BAFF in lymphocyte activation. Curr. Opin. Immunol. 17: 282-289. [Medline]
  48. Mackay, F., P. Schneider, P. Rennert, J. Browning. 2003. BAFF and APRIL: a tutorial on B cell survival. Annu. Rev. Immunol. 21: 231-264. [Medline]
  49. Austin, A. S., K. M. Haas, S. M. Naugler, A. A. Bajer, D. Garcia-Tapia, D. M. Estes. 2003. Identification and characterization of a novel regulatory factor: IgA-inducing protein. J. Immunol. 171: 1336-1342. [Abstract/Free Full Text]
  50. Moon, B. G., S. Takaki, K. Miyake, K. Takatsu. 2004. The role of IL-5 for mature B-1 cells in homeostatic proliferation, cell survival, and Ig production. J. Immunol. 172: 6020-6029. [Abstract/Free Full Text]
  51. Sangster, M. Y., J. M. Riberdy, M. Gonzalez, D. J. Topham, N. Baumgarth, P. C. Doherty. 2003. An early CD4+ T cell-dependent immunoglobulin A response to influenza infection in the absence of key cognate T-B interactions. J. Exp. Med. 198: 1011-1021. [Abstract/Free Full Text]
  52. Yoneyama, H., K. Matsuno, E. Toda, T. Nishiwaki, N. Matsuo, A. Nakano, S. Narumi, B. Lu, C. Gerard, S. Ishikawa, K. Matsushima. 2005. Plasmacytoid DCs help lymph node DCs to induce anti-HSV CTLs. J. Exp. Med. 202: 425-435. [Abstract/Free Full Text]
  53. Suh, W. K., A. Tafuri, N. N. Berg-Brown, A. Shahinian, S. Plyte, G. S. Duncan, H. Okada, A. Wakeham, B. Odermatt, P. S. Ohashi, T. W. Mak. 2004. The inducible costimulator plays the major costimulatory role in humoral immune responses in the absence of CD28. J. Immunol. 172: 5917-5923. [Abstract/Free Full Text]
  54. Han, S., B. Zheng, D. G. Schatz, E. Spanopoulou, G. Kelsoe. 1996. Neoteny in lymphocytes: Rag1 and Rag2 expression in germinal center B cells. Science 274: 2094-2097. [Abstract/Free Full Text]
  55. Stoel, M., H. Q. Jiang, C. C. van Diemen, J. C. Bun, P. M. Dammers, M. C. Thurnheer, F. G. Kroese, J. J. Cebra, N. A. Bos. 2005. Restricted IgA repertoire in both B-1 and B-2 cell-derived gut plasmablasts. J. Immunol. 174: 1046-1054. [Abstract/Free Full Text]
  56. Castigli, E., F. W. Alt, L. Davidson, A. Bottaro, E. Mizoguchi, A. K. Bhan, R. S. Geha. 1994. CD40-deficient mice generated by recombination-activating gene-2-deficient blastocyst complementation. Proc. Natl. Acad. Sci. USA 91: 12135-12139. [Abstract/Free Full Text]
  57. Hornquist, E., N. Lycke. 1993. Cholera toxin adjuvant greatly promotes antigen priming of T cells. Eur. J. Immunol. 23: 2136-2143. [Medline]
  58. Kawabe, T., T. Naka, K. Yoshida, T. Tanaka, H. Fujiwara, S. Suematsu, N. Yoshida, T. Kishimoto, H. Kikutani. 1994. The immune responses in CD40-deficient mice: impaired immunoglobulin class switching and germinal center formation. Immunity 1: 167-178. [Medline]
  59. Jain, A., C. A. Ma, E. Lopez-Granados, G. Means, W. Brady, J. S. Orange, S. Liu, S. Holland, J. M. Derry. 2004. Specific NEMO mutations impair CD40-mediated c-Rel activation and B cell terminal differentiation. J. Clin. Invest. 114: 1593-1602. [Medline]
  60. Snider, D. P., H. Liang, I. Switzer, B. J. Underdown. 1999. IgA production in MHC class II-deficient mice is primarily a function of B-1a cells. Int. Immunol. 11: 191-198. [Abstract/Free Full Text]
  61. Muramatsu, M., V. S. Sankaranand, S. Anant, M. Sugai, K. Kinoshita, N. O. Davidson, T. Honjo. 1999. Specific expression of activation-induced cytidine deaminase (AID), a novel member of the RNA-editing deaminase family in germinal center B cells. J. Biol. Chem. 274: 18470-18476. [Abstract/Free Full Text]
  62. Cattoretti, G., M. Buettner, R. Shaknovich, E. Kremmer, B. Alobeid, G. Niedobitek. 2006. Nuclear and cytoplasmic AID in extrafollicular and germinal center B cells. Blood 107: 3967-3975. [Abstract/Free Full Text]
  63. Forconi, F., S. S. Sahota, D. Raspadori, M. Ippoliti, G. Babbage, F. Lauria, F. K. Stevenson. 2004. Hairy cell leukemia: at the crossroad of somatic mutation and isotype switch. Blood 104: 3312-3317. [Abstract/Free Full Text]
  64. Moldenhauer, G., S. W. Popov, B. Wotschke, S. Bruderlein, P. Riedl, N. Fissolo, R. Schirmbeck, O. Ritz, P. Moller, F. Leithauser. 2006. AID expression identifies interfollicular large B cells as putative precursors of mature B-cell malignancies. Blood 107: 2470-2473. [Abstract/Free Full Text]
  65. Zhou, C., A. Saxon, K. Zhang. 2003. Human activation-induced cytidine deaminase is induced by IL-4 and negatively regulated by CD45: implication of CD45 as a Janus kinase phosphatase in antibody diversification. J. Immunol. 170: 1887-1893. [Abstract/Free Full Text]
  66. Dedeoglu, F., B. Horwitz, J. Chaudhuri, F. W. Alt, R. S. Geha. 2004. Induction of activation-induced cytidine deaminase gene expression by IL-4 and CD40 ligation is dependent on STAT6 and NF{kappa}B. Int. Immunol. 16: 395-404. [Abstract/Free Full Text]
  67. Basu, U., J. Chaudhuri, C. Alpert, S. Dutt, S. Ranganath, G. Li, J. P. Schrum, J. P. Manis, F. W. Alt. 2005. The AID antibody diversification enzyme is regulated by protein kinase A phosphorylation. Nature 438: 508-511. [Medline]
  68. Pasqualucci, L., Y. Kitaura, H. Gu, R. Dalla-Favera. 2006. PKA-mediated phosphorylation regulates the function of activation-induced deaminase (AID) in B cells. Proc. Natl. Acad. Sci. USA 103: 395-400. [Abstract/Free Full Text]
  69. Coffman, R. L., D. A. Lebman, P. Rothman. 1993. Mechanism and regulation of immunoglobulin isotype switching. Adv. Immunol. 54: 229-270. [Medline]
  70. Yu, K., F. Chedin, C. L. Hsieh, T. E. Wilson, M. R. Lieber. 2003. R-loops at immunoglobulin class switch regions in the chromosomes of stimulated B cells. Nat. Immunol. 4: 442-451. [Medline]
  71. Tseng, J.. 1981. Transfer of lymphocytes of Peyer’s patches between immunoglobulin allotype congenic mice: repopulation of the IgA plasma cells in the gut lamina propria. J. Immunol. 127: 2039-2043. [Abstract]
  72. Macpherson, A. J., T. Uhr. 2004. Induction of protective IgA by intestinal dendritic cells carrying commensal bacteria. Science 303: 1662-1665. [Abstract/Free Full Text]
  73. Ta, V. T., H. Nagaoka, N. Catalan, A. Durandy, A. Fischer, K. Imai, S. Nonoyama, J. Tashiro, M. Ikegawa, S. Ito, et al 2003. AID mutant analyses indicate requirement for class-switch-specific cofactors. Nat. Immunol. 4: 843-848. [Medline]
  74. Macpherson, A. J., T. Uhr. 2004. Compartmentalization of the mucosal immune responses to commensal intestinal bacteria. Ann. NY Acad. Sci. 1029: 36-43. [Medline]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
S. Yuvaraj, G. Dijkstra, J. G. M. Burgerhof, P. M. Dammers, M. Stoel, A. Visser, F. G. M. Kroese, and N. A. Bos
Evidence for Local Expansion of IgA Plasma Cell Precursors in Human Ileum
J. Immunol., October 15, 2009; 183(8): 4871 - 4878.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
J. Bessa, A. Jegerlehner, H. J. Hinton, P. Pumpens, P. Saudan, P. Schneider, and M. F. Bachmann
Alveolar Macrophages and Lung Dendritic Cells Sense RNA and Drive Mucosal IgA Responses
J. Immunol., September 15, 2009; 183(6): 3788 - 3799.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
N. S. Longo, P. L. Lugar, S. Yavuz, W. Zhang, P. H. L. Krijger, D. E. Russ, D. D. Jima, S. S. Dave, A. C. Grammer, and P. E. Lipsky
Analysis of somatic hypermutation in X-linked hyper-IgM syndrome shows specific deficiencies in mutational targeting
Blood, April 16, 2009; 113(16): 3706 - 3715.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
O. Morteau, C. Gerard, B. Lu, S. Ghiran, M. Rits, Y. Fujiwara, Y. Law, K. Distelhorst, E. M. Nielsen, E. D. Hill, et al.
An Indispensable Role for the Chemokine Receptor CCR10 in IgA Antibody-Secreting Cell Accumulation
J. Immunol., November 1, 2008; 181(9): 6309 - 6315.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
W. Xu, P. A. Santini, A. J. Matthews, A. Chiu, A. Plebani, B. He, K. Chen, and A. Cerutti
Viral Double-Stranded RNA Triggers Ig Class Switching by Activating Upper Respiratory Mucosa B Cells through an Innate TLR3 Pathway Involving BAFF
J. Immunol., July 1, 2008; 181(1): 276 - 287.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Bergqvist, P.
Right arrow Articles by Lycke, N. Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Bergqvist, P.
Right arrow Articles by Lycke, N. Y.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS