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Gut IgA Class Switch Recombination in the Absence of CD40 Does Not Occur in the Lamina Propria and Is Independent of Germinal Centers This information is current as of September 28, 2021. Peter Bergqvist, Eva Gärdby, Anneli Stensson, Mats Bemark and Nils Y. Lycke J Immunol 2006; 177:7772-7783; ; doi: 10.4049/jimmunol.177.11.7772 http://www.jimmunol.org/content/177/11/7772 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2006 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

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 Ga¨rdby, Anneli Stensson, Mats Bemark,2 and Nils Y. Lycke2,3

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 ␣ transcripts, but few posts- Downloaded from witch 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 transcripts, and only three of 51 http://www.jimmunol.org/ 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. The Journal of Immunology, 2006, 177: 7772–7783.

n recent years elegant studies using normal and genetically (7, 8). This IgA production appears to be generated to a large deficient mice have clearly documented two pathways for extent from IgMbright/IgDdull B1 cells that are primarily present in IgA formation, both of which could contribute greatly to the the peritoneal cavity (PerC), but B2 cells in the PP may also con- I 4 total production of IgA in the gut lamina propria (LP) (1–3). It is tribute (9–12). Germfree animals exhibit poorly developed GALT thought that these pathways operate in parallel, but the relative and produce little IgA, supporting the notion that gut IgA plays a by guest on September 28, 2021 importance of each and how they are regulated are unknown at role in an evolutionary mutualism with the bacteria colonizing the present (4, 5). The classical pathway for T cell-dependent (TD) intestine (13). Whether this IgA production is completely indepen- IgA B cell differentiation is closely associated with the Peyer’s dent of T cells is still not quite clear. On the one hand, bacterial patches (PP), which are predominantly populated with naive colonization of TCR␤␥-deficient mice resulted in substantial IgA IgMdull/IgDbright B220ϩ B2 cells (6) By contrast, T cell-independent production, suggesting that T cells were not required. On the other, (TI) Ags, derived from the gut commensal flora, are thought to the transfer of B1 cells without T cells to SCID mice failed to drive differentiation of IgA B cells also at sites outside of the PP 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 Department of Microbiology and Immunology, Mucosal Immunobiology and Vaccine other factors surface expressed or released by activated T cells (15, Research Center, Institute of Biomedicine, Go¨teborg University, Go¨teborg, Sweden 16). Another site of potential interest for IgA B cell development Received for publication May 24, 2006. Accepted for publication September is the large number of isolated lymphoid follicles (ILF) that are 13, 2006. scattered along the antimesenteric border of the small intestine (8, 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 17). These ILF resemble the PP in many ways, as they consist of with 18 U.S.C. Section 1734 solely to indicate this fact. clusters of naive B2-cells and also often contain germinal centers 1 This study was supported by the Swedish Research Council, the Swedish Cancer (GCs) (17). The ILF are lined by a follicle-associated epithelium Foundation, the Sahlgrenska University Hospital Foundation, the Swedish Foundation that includes M cells, which allow for Ag to be taken up into the for Strategic Research, the Mucosal Immunobiology and Vaccine Center, and Euro- pean Union Grants QLK2-CT-2001-01702, QLK2-CT-199-00228, and LSHP-CT- follicle and activate B cells (8, 17–19). Yet, limited information is 2003-503240. available as to the specific role of these ILF in the overall produc- 2 M.B. and N.Y.L. share senior authorship. tion of gut IgA, albeit PP-null mice appear to have functioning ILF 3 Address correspondence and reprint requests to Dr. Nils Lycke, Department of IgA inductive sites (17). Microbiology and Immunology, University of Go¨teborg, 405 30 Go¨teborg, Sweden. A well-documented gut IgA precursor population is found in the E-mail address: [email protected] PerC, which hosts predominantly self-replenishing B1 cells. These 4 Abbreviations used in this paper: LP, lamina propria; AID, activation-induced cy- cells may carry germline ␣ transcripts, indicative of a potential to tidine deaminase; APRIL, a proliferation-inducing ligand; BAFF, B cell-activating factor; CSR, class switch recombination; CT, cholera toxin; DC, dendritic cell; GC, undergo IgA class switch recombination (CSR) (20). The anatom- germinal center; ILF, isolated lymphoid follicle; KLH, keyhole limpet hemocyanin; ical site and factors required for the PerC B1 cells to become 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 inde- committed to IgA are still to be determined. It has been suggested pendent; WT, wild type. that some IgA CSR could take place in the mesenteric lymph

nodes (MLN) (19, 21). However, lymphotoxin ␣ receptor-deficient ligand (APRIL) acting through TACI (transmembrane activator, mice, lacking both PP and MLN and reconstituted with normal calcium modulator, and cyclophilin ligand interactor) alone or to- bone marrow, produced near normal gut IgA levels, arguing gether with cytokines such as IL-10, and, as it appears, even in- against PP or MLN as necessary sites for IgA B cell development dependently of CD40-signaling (41–51). These factors, produced (22). Still, because dendritic cells (DC) can take-up luminal con- from stromal cells, macrophages, or DC in the tissue, would then tent through the epithelial barrier and thus transport bacteria to the act directly on naive IgM B cells but may still not be produced MLN, they could serve as IgA inductive sites independently of the completely independent of gut T cells, as suggested by Casola et PP (23). Also, the LP itself has been proposed to be a possible al. (16). Because CD40-CD40L interactions control the expression primary site for IgA CSR for TI Ags (24). This is, though, a con- of costimulatory molecules, the lack of CD40 may also indirectly troversial finding, and two recent independent reports have failed influence such noncognate T cell functions. Furthermore, whereas to support the notion that naive B cells can undergo IgA CSR in CD40L is expressed not only by activated T cells but also by the nonorganized LP (25, 26). activated DC, the lack of CD40 could potentially have an impact CSR and somatic hypermutation (SHM) are important processes on overall IgA formation. Even TI Ag-driven IgA CSR may, in for refining and shaping the function of humoral immunity. Sub- this way, depend on CD40 signaling (52). Thus, both TD and TI sequent to Ag exposure, these events take place in the organized Ag-stimulated gut IgA production pathways may require CD40 secondary lymphoid tissues and are linked to the formation of the signaling to be effective. GC (27–29). Whereas CSR to IgG subclasses, except for IgG3 in In the present study we have used wild-type (WT) and CD40Ϫ/Ϫ the mouse, are known to depend on GC formations, less is known mice to analyze to what extent gut IgA CSR is dependent on CD40

about the role of GC in IgA B cell differentiation (2). We and signaling and to address the compartments of the GALT from Downloaded from others have reported a number of different gene knockout or trans- which IgA CSR may emanate. In particular, we have addressed genic mouse models with a poor correlation between GC in the whether IgA CSR occurs in the nonorganized gut LP. We have GALT and the presence of gut IgA and ability to respond to oral also investigated the relationship between GC reactions, gut IgA immunization (7, 30). For example, IL-4Ϫ/Ϫ, CD19Ϫ/Ϫ,or formation, and the expression of the molecular markers for IgA CTLA4-Hg1 transgenic mice respond poorly to oral immuniza- CSR as well as germline ␣ transcripts, AID mRNA and postswitch

tions with cholera toxin (CT) but demonstrate normal GC reactions circular transcripts in the GALT, LP and PerC. http://www.jimmunol.org/ in PP and have near normal levels of gut IgA (31, 32). By contrast, CD28Ϫ/Ϫ or TNFRIϪ/Ϫ mice have no GC in PP but produce near Materials and Methods normal levels of gut IgA (7, 30). Thus, the presence of GC in PP Mice does not constitute a requirement for IgA production in the gut LP. CD40Ϫ/Ϫ and WT mice (both on a C57BL/6 background) were bred and In fact, PP-null mice were shown to still have high numbers of IgA housed at the experimental biomedicine animal facility at Go¨teborg Uni- plasma cells in the gut LP, suggesting alternative inductive sites versity (Go¨teborg, Sweden) under specific pathogen-free conditions. For Ϫ/Ϫ for IgA CSR (21). 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. CSR is a complex process and requires the interplay of a number of different enzymes and genes that are not yet completely under- Immunohistochemistry by guest on September 28, 2021 stood. Recently, an enzyme called activation-induced cytidine Naive CD40Ϫ/Ϫ and C57BL/6 mice were sacrificed and the intestine, PP, deaminase (AID), which is necessary for CSR and SHM, was dis- or MLN were removed, embedded in TissueTek OCT compound, and snap covered (33). Mouse and humans lacking AID fail to produce Abs frozen in liquid nitrogen. Frozen sections (7 ␮m) of the small intestine other than IgM (34), and the mice suffer from hyperplasia of ILF were prepared on microslides using a cryostat (Leica), and the sections were stored at Ϫ70°C until used. The slides were fixed in 50% acetone for in the LP (35). The role that AID has in CSR is still debated. There 30 s and in 100% acetone for 5 min at 4°C and air dried at room temper- are two main hypotheses, disputing whether RNA or DNA is the ature. The tissue was rehydrated in PBS followed by blocking with normal substrate for AID (36). However, the end result of AID activity is horse serum in PBS (5%) for 15 min in a humidifying chamber. For de- recombination of Ig genes and the production of switch circles that tection of germinal centers, the sections were stained with FITC-conjugated anti-GL7 mAb diluted 1/100 (BD Pharmingen) and biotinylated anti- are looped out of the chromosomal DNA (37). These looped-out B220 diluted 1/100 (BD Pharmingen) and were stained for 30–60 min. postswitch circles are present for a short time after CSR, and dur- The B220 was visualized by adding streptavidin-conjugated Texas Red ing this period spliced RNA transcripts between germline I pro- (DakoCytomation) 1/100 dilution. Consecutive sections were stained with moters and C␮ can also be detected. Thus, the presence of the stained with B220 together with FITC-conjugated anti-IgA diluted 1/100 postswitch circles and the postswitch circular DNA transcripts rep- (BD Pharmingen) and FITC-conjugated anti-Ki67 diluted 1/5 (BD Pharm- ingen). The sections were washed in PBS and mounted with DakoCyto- resents our best marker for a just completed CSR (37). Together mation fluorescent mounting medium and visualized using a Leica LSC with the expression of germline ␣ transcripts and AID mRNA microscope. transcripts, we have very sensitive molecular tools for the detec- For visualization and counting of ILF, intestines were divided into four tion of CSR IgA. Using these tools, evidence for active CSR was parts, rolled up on a toothpick, embedded in TissueTek OCT compound, snap frozen in liquid nitrogen, and sectioned as described above. The sec- reported to occur in the LP itself (24), but these results have been tions were blocked with 0.3% H2O2 in PBS for 5 min, rinsed in PBS, and disputed. The controversy relates to whether or not the isolated then blocked with normal horse serum in PBS (5%) for 15 min in a hu- cells could have been derived from ILF rather than the LP (24). midifying chamber. To block endogenous biotin, sections were incubated Notwithstanding this controversy, there is ample documentation with a avidin/biotin blocking kit (Vector Laboratories) followed by incu- that naive IgM B cells can undergo IgA CSR under the influence bation with primary biotinylated anti-B220 for 30–60 min. Following a ␤ brief rinse in PBS, peroxidase-conjugated avidin/biotin complexes were of certain factors acting alone or together. In most species, TGF prepared and added to the sections according to the manufacturer’s instruc- has been shown to induce IgA CSR, and deletions of the TGF␤-1 tions, incubated for 45 min, washed with PBS, and visualized by adding or the TGF␤ receptor type II genes result in severe loss of IgA in 3-amino-9-ethylcarbazole (Vector Laboratories). The sections were coun- mice (38, 39). Yet, TGF␤ alone is insufficient to drive IgA CSR in terstained with hematoxylin, mounted with Faramount (DakoCytomation), and visualized using a Leica LSC microscope. B cells in vitro but requires a combination with, e.g., LPS or CD40 to provide mitogenic stimulation (40). However, recent reports Cell turnover assessment have documented the possibility that IgA CSR could be stimulated BrdU (Sigma-Aldrich) was administered orally by feeding the mice once ␮ by B cell-activating factor (BAFF) and a proliferation-inducing with 500 l of BrdU (1 mg/ml) in H2O and simultaneously injecting i.p. 7774 IgA B CELL DIFFERENTIATION IN CD40-DEFICIENT MICE

500 ␮l of BrdU (4 mg/ml) in PBS. Twenty-four hours later the mice were (Miltenyi Biotec). The cells were sorted in a FACSAria device (BD Bio- given another BrdU pulse orally. Seventeen hours, 3 days, 1 wk, or 3 wk sciences) in PBS supplemented with 0.1% BSA and 0.02% EDTA. Sorted after BrdU treatment the mice were sacriﬁced, and the intestine was re- cells were spun down at 2000 rpm at 4°C followed by RNA extraction moved and frozen as described above. Before immunoﬂuorescent staining using the RNeasy micro kit (Qiagen). The data were analyzed using the the sections were treated with 2 M HCl for 30 min at 37°C, neutralized in FlowJo software (Tree Star).

0.1MNa2B4O7 for 5 min. The sections were then stained and mounted as described above using FITC-conjugated anti-BrdU (1/5) and anti- RNA preparation and RT-PCR IgA-TXRD (1/100). The number of BrdU-labeled cells in the lamina pro- The small intestines were removed and punch biopsies were made from the pria was determined within a fixed area in all sections and visualized in a PP using a 2-mm diameter dermal puncher, isolated, and stored in TRIzol diagram. (Invitrogen Life Technologies). RNA was prepared according to the Lamina propria lymphocytes were prepared and BrdU incorporation was TRIzol protocol, dissolved in 50 ␮l of UltraPure distilled water (Invitrogen visualized using a BrdU flow kit (BD Biosciences) and double stained with Life Technologies), and stored at Ϫ80°C. The RNA concentration was anti-IgA according to the instructions and analyzed by FACS. Naive in- Ϫ Ϫ measured and 4 ␮g of RNA was used for cDNA synthesis using oligo(dT) testines from CD40 / mice and WT mice were removed and washed two RETROscript primers (Ambion) and SuperScript III RT polymerase (In- times with PBS. The tissue was snap frozen and sectioned as described vitrogen Life Technologies). MLN were isolated from naive mice and above. To visualize apoptosis we used the ApopTag in situ TUNEL apo- stored in TRIzol, and RNA was prepared as described above. ptosis detection kit (Chemicon). The sections were counterstained with Texas Red-conjugated anti-IgA (Southern Biotechnology Associates) di- ILF-free sections of gut LP luted 1/100 for 30 min, washed in PBS, and mounted with DakoCytoma- tion fluorescent mounting medium. 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 ELISPOT was placed on a slide and the sections in between were stored in RLT buffer

Ϫ Downloaded from A 96-well MultiScreen plate (Millipore) was washed in PBS and coated (RNeasy lysis buffer; Qiagen) at 70°C. The sections on the slide were with 5 g/ml purified rat-anti-mouse-IgA Abs (BD Pharmingen) or 3 stained with Texas Red-conjugated anti-IgM (Southern Biotechnology As- nmol/ml ganglioside GM1 followed by 3 ␮g/ml CT or 100 ␮g/ml keyhole sociates) and FITC-conjugated anti-IgA (BD Pharmingen) to localize ILF. limpet hemocyanin (KLH) at 4°C overnight. The plates were washed in If two consecutive sections on the slide were ILF-negative, the sections in PBS, blocked with 0.2% BSA in PBS, and 10,000 purified lamina propria between were stored in RLT buffer and considered to be ILF free. RNA lymphocytes, prepared as previously described (30), were added in dupli- was prepared from these sections using the RNeasy micro kit (Qiagen). cate and diluted 1/3 in four serial dilutions directly on the plate and incu- Peritoneal washouts bated at 37°C for 3 h. The plate was then washed twice with PBS plus http://www.jimmunol.org/ 0.05% Tween 20 and twice with PBS, followed by incubation with alkaline Mice were sacrificed and the cells were isolated as described above. The phosphate-conjugated goat anti-mouse-IgA Abs (Southern Biotechnology cells were spun down at 2000 rpm at 4°C and RNA was extracted using the Associates) overnight at 4°C. The next day the plates were washed twice RNeasy mini kit (Qiagen). 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 Primers tablet (Sigma-Aldrich) dissolved in 10 ml of H O, and 100 ␮l was added 2 ␣ ␣ Ј to each well and incubated for 10 min in room temperature. The plate was To detect germline transcripts the primers I F(5-CCA GGC ATG GTT Ј ␣ Ј then washed twice with PBS plus 0.05% Tween 20, twice in PBS, dried, GAG ATA GAG ATA G-3 ) and C R(5-GAG CTG GTG GGA GTG Ј and analyzed using the ImmunoSpot ELISPOT system (Cellular TCA GTG-3 ) were used. Cycling was performed under the following Technology). 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 by guest on September 28, 2021 ELISA extension at 72°C for 10 min. For AID transcripts the following primers were used: mAID119 (5Ј-GAG GGA GTC AAG AAA GTC ACG CTG ␮ ␮ Ninety-six-well plates (Nunc) were coated with 100 g/ml KLH (100 l/ G-3Ј), mAIDR1 (5Ј-CCA GGC TTT GAA AGT TCT TTC ACG-3Ј), and, ␮ well), 0.5 nM/ml GM1 followed by 0.5 g/ml CT overnight at 4°C. For as B cell loading control, CD79a forward (5Ј-AAC ACA GGG GCT TGT ␮ total Ig titers, the 96-well plates were conjugated with 5 g/ml anti-mouse ACT GG-3Ј) and CD79a reverse (5Ј-ATC TCC AAT GTG GAG GTT IgG1, IgG2a, IgA, or IgM (Southern Biotech) and incubated at 4°C over- GC-3Ј). The cycling was performed as described above with 25 cycles and night. The plates were washed three times with PBS followed by blocking an annealing temperature of 59°C. Circular transcripts were detected by a in PBS plus 0.1% BSA for 30 min at 37°C. The PBS/BSA was discarded, nested PCR using the outer primers I␮4(5Ј-ACC CTG GAT GAC TTC serum samples were diluted 1/100 and added to the plates, and a 3-fold AGT GT-3Ј) and I␣up4 (5Ј-CAT CTG GAC TCC TCT GCT CA-3Ј) and serial dilution was made. The gut lavage was diluted 1/10 and a 2-fold the inner primers I␣F(5Ј-CCA GGC ATG GTT GAG ATA GAG ATA serial dilution was made. The samples were incubated at 4°C overnight and G-3Ј) and C␮R(5Ј-AAT GGT GCT GGG CAG GAA GT-3Ј). Each PCR washed three times in PBS-Tween 20. To the CT- and KLH-specific was performed as described above with 30 cycles each and an annealing ELISA HRP-conjugated rat anti-mouse Abs (DakoCytomation) were temperature of 56°C for the first PCR and of 62°C for the second one. 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 de- Southern blot veloped using o-phenylenediamine dihydrochloride tablets (Sigma-Aldrich) according to the manufacturer’s instructions. The plate was analyzed at 450 The AID and CD79 PCR products were separated on a 2% agarose gel and nm. For total Ig measurements the plates were incubated with alkaline phos- the gel was photographed to visualize AID and CD79. To confirm the AID ϩ phate-conjugated rabbit anti-mouse Ig (Southern Biotechnology Associates) expression, the fragments was transferred to a Hybond membrane (Am- diluted 1/500 in PBS plus 0.1% BSA for2hatRT.Theplates were washed ersham Biosciences) by passive diffusion using 0.4 M NaOH overnight. with PBS-Tween and developed using Sigma 104 phosphatase tablets (Sigma- The membrane was dried and cross-linked by UV light and then incubated Aldrich) according to instructions. The plates were analyzed at 405 nm. for1hinblocking solution at 45°C. An AID-specific probe, AIDhyb_1 (5Ј-ATA TGG ACA GCC TTC TGA TGA AGC AA AGA AGT TTC-3Ј), Flow cytometry and cell sorting was labeled by mixing 5 pmol of AIDhyb_1 probe, 5 ␮lof10ϫ T4 PNK buffer (Promega), 1 ␮l of T4 polynucleotide kinase (Promega), 5 ␮lof Lymphocytes from PP and MLN were prepared by excising the PP from Redivue adenosine 5Ј-[␥-32P]triphosphate, triethylammonium salt (Amer- the intestine and passing them through a cell strainer of 100-␮m nylon sham Biosciences), and water to 50 ␮l. The mixture was incubated for 1 h mesh (BD Falcon). Peritoneal cells were isolated by carefully peeling off at 37°C, and free nucleotides were removed using a ProbeQuant G-50 the skin from the mouse peritoneum, and 8 ml of ice-cold PBS was injected micro column (Amersham Biosciences). The labeled probe was added to into the peritoneal cavity. After 5 min the peritoneum was cut open and the the membrane and incubated at 45°C overnight. Hybridization was de- fluid was retrieved using a transfer pipette. The cells were suspended in tected on 32P-sensitive film (Kodak). 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) One-step PCR (BD Pharmingen) or anti-IgA-FITC (Southern Biotechnology Associates). For intracellular Ki67 staining the cells were fixed and permeabilized using RNA prepared from sorted cells, cells from sectioning, and peritoneal the FoxP3 staining set (eBioscience), but the anti Ki67-FITC Ab (BD washouts was analyzed using one-step RT-PCR (Invitrogen Life Technol- Pharmingen) was used instead of the anti-Foxp3 Ab. To ensure single cell ogies). The RNA was purified as described above and 2 ␮l of RNA was suspensions, the cells were filtered through a MACS preseparation filter used in the RT-PCR. For the cDNA synthesis, gene-specific primers were The Journal of Immunology 7775 added and the cDNA synthesis temperature was the same as the annealing temperature for the PCR except during the detection of germline ␣ 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 CD40Ϫ/Ϫ 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 clas- sically 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 CD40Ϫ/Ϫ mice. How- ever, quite in contrast, we found substantial IgA production in the gut of CD40Ϫ/Ϫ mice. In fact, labeling frozen sections of the small

intestine with anti-IgA revealed almost normal numbers of IgA Downloaded from 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 CD40Ϫ/Ϫ 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 http://www.jimmunol.org/ limits in serum from CD40Ϫ/Ϫ mice (Fig. 1D) Next we addressed whether the substantial IgA production observed in the LP of CD40Ϫ/Ϫ mice readily reflected an ability to respond to TD Ags in the gut. To this end, oral immunizations with FIGURE 1. Significant IgA production in the small intestine of KLH supplemented with CT adjuvant were performed (57). We Ϫ/Ϫ Ϫ/Ϫ Ϫ/Ϫ CD40 mice. Naive CD40 and WT mice small intestines were ana- found no IgA or IgG responses to these Ags in CD40 mice, lyzed for IgA production. A, Cryosections from rolled up small intestines whereas WT mice demonstrated strong specific serum IgA and were stained with anti-IgA (green) and anti-IgM (red). Each section con- IgG and gut LP IgA production (Fig. 2, A–D). None of the tains one ILF cluster brightly staining for IgMϩ cells. B, Levels of secreted Ϫ Ϫ CD40 / mice responded with specific IgM Abs against CT, and IgA in gut lavage as determined by ELISA are shown. C, Relative level of by guest on September 28, 2021 only 5 of 15 responded against KLH, whereas WT mice responded gut IgA producing cells as determined by ELISPOT is shown. The diagram strongly with IgM Abs against both Ags (Fig. 2, C and D) Fur- shows one representative experiment of three. D, Serum Ab levels of the thermore, a similar result was obtained in serum after parenteral indicated Ig subclasses were determined by ELISA. Mean values Ϯ SD of Ͻ ءء Ͻ ء immunizations with KLH plus CT adjuvant with no specific IgG or three individual experiments are shown. , p 0.05; , p 0.01; n.s., no Ϫ Ϫ significance. 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 re- from IgAϪ/Ϫ or CD28/ICOS double-deficient mice (no GC-reac- sponses in serum in WT mice (Fig. 2, E and F). Taken together, tions), respectively, as negative controls (53). our data indicated that CSR to IgA, as opposed to IgG-subclasses Another potential site for IgA-switching is ILF along the anti- (except IgG3), can occur independently of CD40. However, as mesenteric lining of the small intestine (Fig. 1A). The frequency expected, CD40-CD40L interactions are clearly required to mount and distribution of ILF in CD40Ϫ/Ϫ mice were comparable to that specific IgA and IgG responses to orally or parenterally adminis- in WT mice with increasing numbers toward the distal part of the tered TD Ags (58). small intestine (Fig. 4A). A thorough analysis of the ILF revealed Ϫ/Ϫ Ϫ/Ϫ a lack of IgA- and Ki67-positive cells in CD40 mice (Fig. 4B). Lack of IgA inductive sites in the GALT of CD40 mice ILF from WT mice were also largely devoid of IgAϩ cells, and no Given that CD40Ϫ/Ϫ mice have poor CSR to IgG and an impaired GL7- or Ki67-labeling areas were found (Fig. 4B), suggesting that ability to respond to orally administered TD Ags but show near these structures do not contribute much to the overall production of normal levels of total gut IgA, we analyzed various compartments IgA, even in the normal gut. In young mice (3 wk of age) from of the GALT for possible inductive sites for IgA CSR. Frozen both strains we were unable to detect any ILF, but IgA SFC num- sections were investigated for the presence of GC, IgA production, bers in gut LP were significant and comparable in CD40Ϫ/Ϫ and and proliferating cells using the Ki67 Ag as a marker. Whereas the WT mice (Fig. 4, C and D), again supporting the notion that IgA PP of WT mice contained distinct GC areas with aggregates of formation in the gut LP of CD40Ϫ/Ϫ and WT mice does not rely cells brightly staining with GL7-, IgA- and Ki67-specific Abs, PP on the presence of ILF but appear to be recruited from other in- from CD40Ϫ/Ϫ mice failed to label with any of these markers (Fig. ductive sites. Furthermore, the gut draining MLN in both 3A). FACS analysis of PP from CD40Ϫ/Ϫ mice showed Ͻ1% of CD40Ϫ/Ϫ and WT mice appeared to be poor sites for IgA CSR. the B220ϩ cells expressing GL7, whereas in WT mice an average Although none of the MLN from CD40Ϫ/Ϫ mice stained with any of 6% PP B cells were GL7ϩ (Fig. 3B). In WT PP B cells, roughly of the three markers, a few small and scattered GC were found in 5% were stained with an anti-IgA mAb, whereas in CD40Ϫ/Ϫ mice WT MLN, failing to support the notion that MLN is an important Ͻ1% of the cells were labeled (Fig. 3B). For comparison, we used site for CSR to IgA (19) (Fig. 4E). FACS analysis demonstrated the low/background levels of IgA- and GL7-labeling B cells in PP low levels of GL7-expressing cells in WT and CD40Ϫ/Ϫ mice, not 7776 IgA B CELL DIFFERENTIATION IN CD40-DEFICIENT MICE Downloaded from http://www.jimmunol.org/

FIGURE 3. Germinal center formation in PP. A, Immunohistochemical analysis of PP are shown. Naive PP from CD40Ϫ/Ϫ and WT mice were

excised, snap frozen, and sectioned followed by staining with anti-B220 by guest on September 28, 2021 Ϫ/Ϫ FIGURE 2. Lack of specific responses to TD Ags in CD40 mice. (red) and anti-GL7, anti-Ki67, and anti-IgA (green) as indicated. B, Flow Ϫ/Ϫ CD40 and WT mice were immunized three times orally or i.p. with cytometric analysis of PP from naive mice. PP were excised and pooled KLH using CT as an adjuvant, and 6–8 days after the final immunization from two mice and stained with the indicated Abs. To measure the back- the specific IgA response in the LP was analyzed by ELISPOT and the ground level of IgA staining, IgA knockout mice were used (IgAϪ/Ϫ) and, level of serum Abs was measured by ELISA. A, LP KLH-specific spot as a measurement of background GL7 staining, CD28 and ICOS double- forming cells after oral immunization are shown. B, LP CT-specific SFC knockout mice were used (CD28/ICOSϪ/Ϫ). The figure shows one repre- after oral immunization are shown. C, Log10 titers of KLH-specific Abs in sentative experiment of five. 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, Log titers of 10 the BrdU-treated animals showed that both strains had ϳ1% of CT-specific Abs in serum after i.p. immunization are shown. Shown is one ϩ ϩ IgA BrdU cells in the small intestine 1 wk after BrdU labeling representative experiment of three. Mean values Ϯ SD are shown (p Ͻ ϩ 0.01). ND, Not detected. (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 exceeding the frequency seen in PP of CD40Ϫ/Ϫ mice (Fig. 4E). the strains (Fig. 5D). These data suggested that IgA plasma cells Ϫ Ϫ Thus, we were unable to find a site in the GALT with ongoing GC do not exhibit altered dynamics in gut LP of CD40 / as com- formation and CSR to IgA in CD40Ϫ/Ϫ mice, and even in WT pared with WT mice. mice the ability to develop such a site appeared to be poor outside Molecular screening for IgA CSR in the GALT of the PP. Ϫ/Ϫ A simple explanation for the large number of IgA plasma cells of CD40 mice in the LP, despite the impaired ability to respond to TD Ags, could To increase the sensitivity of the analysis for detection of IgA CSR be altered gut plasma cell dynamics in the CD40Ϫ/Ϫ mice. We in the GALT of CD40Ϫ/Ϫ mice, we used RT-PCR for the presence failed, however, to identify any difference in plasma cell turnover of germline ␣ transcripts, AID mRNA, and postswitch circular in the gut LP of CD40Ϫ/Ϫ mice as compared with that in WT mice, DNA transcripts (24, 37). With this technique we confirmed that as evidenced by pulse-chase BrdU treatment experiments (Fig. PP in WT mice are strong sites for IgA CSR with frequent and 5A). Following BrdU loading of gut plasma cells we observed no high mRNA expression of AID and germline ␣ transcripts as well difference in the decay of labeled cells in the LP in the two strains as the presence of postswitch circular DNA transcripts (Fig. 6A). over time (Fig. 5, A–C). A careful assessment of BrdU-labeled By contrast, PP from CD40Ϫ/Ϫ mice showed the expression of cells in the LP of both strains on days 1, 3, 7, and 21 after treatment germline ␣ transcripts and AID mRNA, albeit weaker, but few failed to reveal an altered IgAϩ cell turnover in CD40Ϫ/Ϫ as com- postswitch DNA-transcripts were found, arguing against PP being pared with WT mice (Fig. 5C). Furthermore, a FACS analysis of a major site for IgA CSR in these mice (Fig. 6A). In fact, we The Journal of Immunology 7777 Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 4. ILF frequency and distribution are comparable in WT and CD40Ϫ/Ϫ 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 Ϯ SD of three individual experiments are shown. B, The illustration shows three consecutive sections of a typical ILF from CD40Ϫ/Ϫ 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 CD40Ϫ/Ϫ and WT mice are shown. n.s., No signiﬁcance. 7778 IgA B CELL DIFFERENTIATION IN CD40-DEFICIENT MICE

detected postswitch DNA transcripts in Ͻ25% of 45 PP isolated from 10 different CD40Ϫ/Ϫ mice, whereas in 47 PP from 10 WT mice Ͼ85% demonstrated presence of these transcripts. Of note, given that no GC reactions (GL7- and Ki67-negative) were detected in PP of CD40Ϫ/Ϫ mice (Fig. 3), it was surprising to ﬁnd relatively prominent AID mRNA expression (Fig. 6A). An ex- tended 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). More- over, in agreement with the histological analysis, the MLN of both CD40Ϫ/Ϫ and WT mice expressed very low levels of any of the molecular markers for IgA CSR (Fig. 6C). Of 21 MLN samples from nine CD40Ϫ/Ϫ mice analyzed, we detected AID mRNA in 20% and postswitch circular DNA-transcripts in Ͻ5%. 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 Downloaded from 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 ␣ transcripts, weak or no AID mRNA-expression, and no postswitch DNA transcripts in this tissue in neither CD40Ϫ/Ϫ or

WT mice (Fig. 6D). http://www.jimmunol.org/

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 CD40Ϫ/Ϫ 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 CD40Ϫ/Ϫ mice, clearly re- jecting the notion that IgA CSR normally occurs in this nonor- by guest on September 28, 2021 ganized 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 ␣ 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).

Intact AID mRNA expression in peritoneal cavity B cells in CD40Ϫ/Ϫ mice In the absence of data pointing to a probable site for IgA CSR in the CD40Ϫ/Ϫ 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

FIGURE 5. Unaltered gut plasma cell turnover in CD40Ϫ/Ϫ 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). The Journal of Immunology 7779 Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 6. Analysis of molecular markers for IgA CSR. RNA was prepared and analyzed by RT-PCR. The figure shows the expression of germline ␣ transcripts (␣-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 CD40Ϫ/Ϫ 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 ␤2m PCR was used as a RNA loading control. Samples without RT added (ϪRT) and samples from ␮mt 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 CD40Ϫ/Ϫ 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 (GL7Ϫ), 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 ␣ transcripts, AID transcripts, and postswitch circle transcripts in MLN of naive CD40Ϫ/Ϫ and WT mice was performed as described above. D, Expression of germline ␣ transcripts (␣-GT), AID transcripts, and postswitch circle transcripts in the spleens of naive CD40Ϫ/Ϫ and WT mice was performed as described above. 7780 IgA B CELL DIFFERENTIATION IN CD40-DEFICIENT MICE Downloaded from FIGURE 7. Expression of molecular markers for ongoing CSR in the lamina propria. RNA from ILF-free lamina propria from CD40Ϫ/Ϫ and WT mice was isolated according to Materials and Methods. Expression levels of germline ␣ transcripts (␣-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 ␤2m PCR was used as a RNA loading control.

Ϫ/Ϫ

has, to our knowledge, not been previously investigated, although were analyzed and only three of these (one WT and two CD40 ) http://www.jimmunol.org/ germline ␣ transcripts and postswitch ␣ transcripts have been re- indeed expressed postswitch circular DNA transcripts. FACS sort- ported (20). Thus, to this end we isolated PerC B cells and ex- ing of PerC B cells demonstrated that AID mRNA expression was tracted RNA for subsequent RT-PCR analysis for IgA CSR. We largely restricted to the GL7-intermediate population, in particular found that PerC B cells from the two strains demonstrated high and in the CD40Ϫ/Ϫ mice (Fig. 8B). Furthermore, we could detect pro- comparable expression of AID mRNA, but very few samples ex- liferating Ki67ϩIgMbright cells in the PerC of both strains (Fig. pressed germline ␣ transcripts or postswitch circular DNA-tran- 8C). However we could not detect any difference in the frequency scripts in either CD40Ϫ/Ϫ or WT mice (Fig. 8A). In total, 56 mice of Ki67-labeled cells in the PerC of the two strains, albeit there by guest on September 28, 2021

FIGURE 8. Peritoneal cavity cells express AID but very few postswitch circular transcripts. A, Peritoneal cavities from naive CD40Ϫ/Ϫ and WT mice were washed out with PBS, and RNA was isolated from the collected cells. The expression of germline ␣ transcripts (␣-GT), AID transcripts, and postswitch circle transcripts was visualized by RT-PCR (␣-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 ␤2m 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. GL7Ϫ, 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 CD40Ϫ/Ϫ 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 IgMϩKi67ϩ cells were 7.4 Ϯ 2.5% for CD40Ϫ/Ϫ (n ϭ 7) and 8.0 Ϯ 3.6% for WT (n ϭ 7) in the PerC. The Journal of Immunology 7781 was a 3- to 4-fold lower level of proliferating Ki67ϩB220ϩ cells reported AID mRNA transcription in relation to GL7 membrane in the PP in CD40Ϫ/Ϫ mice as compared with that in WT mice expression. We found that microscopically GL7-negative tissues (Fig. 8C). can, indeed, host substantial AID mRNA expression. Both in PP and PerC B cells AID mRNA expression was found in GL7-in- Discussion termediate cells, indicating that these cells have the potential to Ϫ Ϫ The present study of CD40 / mice corroborate observations of undergo CSR. It is also possible that these cells undergo IgA CSR, normal to high IgA levels in patients with impaired CD40-signal- although this process would have to be nondeletional, as seen, e.g., ing (59). Here we report near normal levels of gut IgA and a 50% in hairy leukemic cells, which also fail to produce circle DNA reduction in serum IgA, whereas IgG1 and IgG2a were undetect- transcripts (63). Rather, we believe that IgA CSR in the activated Ϫ Ϫ able in CD40 / mice. Thus, significant IgA CSR can occur in the B cells in PP or PerC is completed at some other not yet defined complete absence of CD40 and GC reactions. Furthermore, we location before seeding the gut LP. This location does not appear Ϫ/Ϫ found that CD40 mice completely failed to respond to oral (as to be the spleen or MLN. However, the significance of extrafol- well as parenteral) immunizations with the TD Ags, CT, and KLH licular AID mRNA expression for CSR and SHM is still contro- while, in fact, demonstrating near normal levels of total IgA versial (62). Therefore, it remains to be seen to what extent GL7- plasma cells in their gut LP. In the search for a site where naive B intermediate B cells are responsible for IgA CSR in response to TI cells would undergo IgA CSR in these mice, we were unsuccessful Ags in normal individuals. Nevertheless, in this study we provide in documenting microscopic or molecular data to suggest that any compelling evidence that CD40Ϫ/Ϫ mice have near normal total of the traditional locations within the GALT or the spleen are gut IgA plasma cell levels and yet exhibit only extrafollicular sites strong sources for IgA CSR. Compared with WT mice, the of AID mRNA expressing B cells. In addition, preliminary studies Downloaded from Ϫ/Ϫ CD40 mice exhibited dramatically reduced levels of IgA CSR Ϫ/Ϫ of SHM in IgA VH genes from gut LP cells from CD40 mice in PP and MLN, whereas the ILF and LP were poor sites for IgA surprisingly revealed many mutated clones even compared with class switching in both strains. B cells in the PerC demonstrated those observed in WT gut IgA cells, suggesting that even this significant levels of AID mRNA expression in both strains, and an normally GC-restricted quality could be provided by extrafollicu- equal but low frequency of postswitch circular DNA transcripts lar sites (P. Bergqvist, unpublished observation). were detected. These findings are consistent with the notion that Evidence that LP would be a primary site for IgA CSR was first http://www.jimmunol.org/ IgA in CD40Ϫ/Ϫ mice is produced against TI Ags and that B1 cells reported by Fagarasan et al. (24). The background to the study was are potentially the main source of this IgA (10, 60). The question Ϫ Ϫ ϩ ϩ that AID / mice had increased levels of B220 IgM cells in the of whether it is the B1 PerC population or the strongly impaired LP, and the authors hypothesized that that these cells could un- IgA CSR in PP or MLN (in B1 or B2 cells) that contributes to most dergo IgA CSR in the LP itself. (24). However, their data have of the LP IgA plasma cells in CD40Ϫ/Ϫ mice, and to what extent, recently been challenged by at least two studies that cannot con- cannot be answered by the present study. However, we can confirm the notion that gut LP would be a primary site for IgA CSR clude that the gut LP is unlikely to be a primary site for IgA CSR Ϫ/Ϫ Ϫ Ϫ (25, 26). The ILF compartment is grossly enlarged in AID in either CD40 / or WT mice because of a complete lack of AID

mice, and it is probable that the nonorganized LP is diminished in by guest on September 28, 2021 mRNA expression and no evidence of postswitch circular DNA these mice in keeping with the interpretation that the purified gut transcripts. cells were likely to contain cells from ILF and not exclusively We made some remarkable observations regarding IgA CSR in Ϫ/Ϫ Ϫ Ϫ nonorganized LP in AID mice (24). In the present study we the CD40 / mice. A close interrelationship between expression of AID mRNA, germline ␣ transcripts, and postswitch circular found that AID mRNA expression was detected in ILF-containing DNA transcripts was clearly found in WT PP, denoting this site a biopsies but not in the biopsies of normal nonorganized small in- true place for IgA CSR. This finding was also supported by strong testinal LP. Shikina et al. (26) also used careful dissection of LP labeling with specific Abs for GC and IgA in tissue sections of PP tissue to exclude ILF and, in agreement with our study, failed to from WT mice. By contrast, CD40Ϫ/Ϫ mice had PP that failed to detect AID mRNA and postswitch circle DNA transcripts. In hu- label for GC or IgA and exhibited substantial levels of germline ␣ man LP tissue, Boursier et al. (25) failed to detect the molecular transcripts and AID mRNA but substantially reduced postswitch markers for IgA CSR, including a lack of AID mRNA. Further- more, clonal analysis of somatic mutations in the rearranged circle DNA transcripts. Therefore, we believe that PP may not be ϩ Ϫ Ϫ an important site for IgA CSR in CD40 / mice unless activated IgVH5-gene segment in IgA cells isolated from distant sites in IgA CSR-committed B cells migrate out of the PP to complete the the nonorganized LP showed greater similarity than when com- CSR. Alternatively, only 25% of normal CSR-activity in PP, as in pared with mutations in neighboring sites, favoring a model of the CD40Ϫ/Ϫ mice, suffices to provide WT levels of gut LP IgA dissemination of precursor cells derived from a common source plasma cells. Interestingly, despite the lack of GC in CD40Ϫ/Ϫ within the organized GALT (25). At variance with our study and mice, the AID mRNA expression in PP B cells was prominent. that of Boursier et al., Shikina et al. did not detect germline ␣ AID is normally thought of in the context of GL7high B cells in the transcripts in LP (25, 26). The reason for this is unknown. Nev- GC and is critically required for CSR and SHM (33, 61). Recent ertheless, because expression of these germline ␣ transcripts is studies have also documented AID-expression in B cell tumors and necessary and precedes CSR but cannot initiate CSR in the ab- extrafollicular large proliferating normal B cells (62–64). How- sence of AID expression, these transcripts merely indicate the po- ever, contrary to our finding, these latter cells showed evidence of tential of B cells in the gut LP to undergo CSR (69, 70). We CD40 signaling, were Ki67ϩ, and resulted from TD Ag activation believe that the lack of AID mRNA and postswitch circle DNA (62). Of note, CD40 signaling is one pathway that can induce AID transcripts as well as failure to demonstrate B cell proliferation, expression as can IL-4 and TLR4 signaling or pathways that affect i.e., Ki67-labeling cells (P. Bergqvist, unpublished observation), intracellular levels of protein kinase A (65–68). disqualify gut LP as a primary site for IgA CSR. For these reasons The B cell population that transcribed AID in the PP of we cannot ascribe IgA CSR to the PP or, indeed, all of the GALT, CD40Ϫ/Ϫ animals expressed an intermediate level of GL7 unde- including MLN, in CD40Ϫ/Ϫ mice, as these sites fail to exhibit the tectable by histology, whereas the GL7-negative B cells were also signifying factors required for an active IgA CSR. If CSR is ini- AID mRNA-negative. To our knowledge, no previous study has tiated in the GALT of these mice at all, cells must leave, e.g., the 7782 IgA B CELL DIFFERENTIATION IN CD40-DEFICIENT MICE

PP, before undergoing full deletional CSR to IgA. We believe that B1 cells can undergo IgA CSR in MLN, but our results in this site or sites are to be found. CD40Ϫ/Ϫ mice largely fail to support this theory because post- In agreement with previous studies we found that CD40Ϫ/Ϫ switch circular DNA transcripts were hardly found (72, 74). Future mice lack GC completely (56). A common notion is that the studies using germfree and recently colonized mice will explore presence of GC in the PP of WT mice constitute a source of gut these possibilities. LP IgA plasma cell precursors (6, 71). These GC B cells are predominantly B2 cells that appear to respond not only to TD Disclosures Ags, but to a significant degree also to TI Ags associated with The authors have no financial conflict of interest. the commensal flora (13). Hence, the PP GC formations seen in CD19Ϫ/Ϫ mice, which largely lack B1 cells, could be B2 cells References reacting to the commensal flora, whereas these mice are unable 1. Macpherson, A. J., L. Hunziker, K. McCoy, and A. Lamarre. 2001. IgA responses to respond to oral immunizations with TD Ags (31). In fact, GC in the intestinal mucosa against pathogenic and non-pathogenic microorganisms. Microbes Infect. 3: 1021–1035. formations in PP may not even be dependent on BCR recogni- 2. Strober, W., S. Fagarasan, and N. Lycke. 2005. IgA B cell development. In tion but result from the presence of luminal bacteria and T cells Mucosal immunology, 3rd Ed. J. Mestecky, M. E. Lamm, W. Strober, (16). Because genetically deficient mice such as CD19Ϫ/Ϫ or J. Bienenstock, J. R. McGhee, and L. Mayer, eds. Academic Press, Boston. P. Ϫ Ϫ 583–616. TNFRI / mice differ with regard to the presence of GC in 3. Fagarasan, S., and T. Honjo. 2003. Intestinal IgA synthesis: regulation of front- their PP and still make good levels of IgA, it must be concluded line body defences. Nat. Rev. Immunol. 3: 63–72. 4. Fagarasan, S., and T. Honjo. 2004. Regulation of IgA synthesis at mucosal sur- that there is no direct relationship between GC in GALT and faces. Curr. Opin. Immunol. 16: 277–283.

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