of March 25, 2018.This information is current as

Center Formationand Occurs Prior to Manifest GerminalRecombination Is Restricted to the GALT T Cell-Independent IgA Class Switch

BemarkPeter Bergqvist, Anneli Stensson, Nils Y. Lycke and Mats

ol.0901895http://www.jimmunol.org/content/early/2010/03/05/jimmun

published online 5 March 2010J Immunol 

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The Journal of Immunology

T Cell-Independent IgA Class Switch Recombination IsRestricted to the GALT and Occurs Prior to ManifestGerminal Center Formation

Peter Bergqvist, Anneli Stensson, Nils Y. Lycke, and Mats Bemark

Recently, we reported that CD402/2 mice, exhibiting exclusively T cell-independent IgA class switch recombination (CSR),

demonstrated near normal levels of IgA plasma cells in the gut lamina propria (LP), despite the complete lack of germinal

centers (GCs). In this study, we have extended our analysis focusing on how to reconcile these findings using flow cytometry and

molecular markers for IgA CSR. In agreement with our previous results with small intestinal LP, the colon LP was found to host

IgA CSR only when lymphoid follicles were present. Thus, no IgA CSR was observed in the nonorganized colon LP. By contrast,

the Peyer’s patch (PP) was the dominant IgA CSR site in both CD402/2 and wild type (WT) mice, and they both hosted similar

levels of mRNA expression for B cell activating factor of the TNF family, a proliferation inducing ligand, and inducible NO

synthase, potential switch-factors for IgA. Unexpectedly, we found that PP B cells undergoing IgA CSR were GL7-intermediate.

These cells had not undergone somatic hypermutations (SHMs), whereas GL7-high cells in WT PP, which exhibited GCs, were

heavily mutated. Moreover, IgA plasma cells in the LP of CD402/2 mice demonstrated few mutations in their Ig V regions,

whereas WT LP B cells from different sites showed extensive SHMs, which were also clonally related. Therefore, IgA CSR can

occur in PP at a stage preceding manifest GC (GL7-intermediate), whereas SHM require GC formations (GL7-high). These

findings reconcile that IgA CSR can occur in PP in the absence of GC with the fact that CD402/2 mice host near normal levels of

IgA plasma cells in the LP. The Journal of Immunology, 2010, 184: 000–000.

Aunique feature of the mucosal membrane is the largeproduction of IgA Abs, which are generated in responseto the commensal flora or invading pathogens (e.g., food

Ags) (1). Despite the importance of this defense system, our un-derstanding of the mechanisms responsible for IgA B cell differ-entiation and their microanatomic localization is still incomplete(2–5). Although oral immunizations with T cell-dependent Agscan elicit strong gut IgA responses, it is the IgA formation againstT cell-independent Ags that has attracted much attention in recentstudies (6–12). In particular, IgA Ab formation against the com-mensal flora has been studied intensely, and results from germ-free mice indicate that such Abs are a dominant proportion of thetotal IgA found in the gut (13–16). Whether IgA class switchrecombination (CSR) against T cell-independent and T cell-dependent Ags occurs at the same or different sites is currentlydebated (17). Conflicting reports have essentially described twoscenarios for where and how naive B cells undergo IgA CSR, with

the classical model ascribing IgA CSR to the organized GALT,whereas recent reports have identified the nonorganized laminapropria (LP) of the small and large intestine as potential sites forIgA CSR (10, 11, 18–20).Similar to CSR in peripheral lymph nodes or the spleen, the

former relies on the formation of germinal centers (GCs) and isdependent on CD40 and TGFb, whereas the latter occurs in theabsence of GC, is independent of CD40, and is supported byproduction in situ of inducible NO synthase (iNOS), B cell acti-vating factor of the TNF (BAFF), and a proliferation inducingligand (APRIL) by epithelial cells, dendritic cells (DCs), orstromal cells (21–26). Whereas IgA production is near normal inthe absence of CD40, it is dramatically impaired in APRIL- orBAFF-deficient mice (27–30). Moreover, the action of thesemolecules is independent of CD40 and TGFb (9, 11, 21, 25).iNOS-deficient mice were also found to be impaired in IgA CSR,a mechanism that probably was secondary to a defect in BAFFand/or APRIL produced by DCs in the GALT and nonorganizedLP of the intestine (22). Nevertheless, both pathways for IgA CSRrequire B cell proliferation and expression of activation-inducedcytidine deaminase (AID) (31–33). Our recent study in CD40-deficient mice, and also previous studies in ICOS- or CD28-deficient mice, supports the hypothesis that near normal IgA plasmacell levels can be found in the LP in the complete absence of GCs inPeyer’s patches (PPs) (8, 30). We proposed previously that suchIgA CSR could have occurred at alternative sites outside of theGALT. However, we failed to find evidence for IgA CSR in the no-organized LP of the small intestine or in the peritoneal cavity, twoof the reportedly strongest candidates for IgA CSR (20, 30, 34). Arecent study has, however, pointed to the nonorganized LP of thecolon as a possible site for IgA CSR (11). The present study is anextended analysis of alternative sites for IgA CSR in CD402/2

mice, focusing on the nonorganized colon LP for evidence ofmolecular markers for IgA CSR, which involved using sensitive

Department of Microbiology and Immunology, Mucosal Immunobiology and VaccineCenter, Institute of Biomedicine, University of Gothenburg, Gothenburg, Sweden

Received for publication June 16, 2009. Accepted for publication January 22, 2010.

This work was supported by the Swedish Research Council, the Swedish CancerFoundation, Sahlgrenska University Hospital LUA/ALF grants, European UnionGrants QLK2-CT-2001-01702 and LSHP-CT-2003-503240, and the Mucosal Immu-nobiology and Vaccine Center Swedish Foundation for Strategic Research.

Address correspondence and reprint requests to Dr. Mats Bemark, Department of Micro-biology and Immunology, Institute of Biomedicine, University of Gothenburg, P.O. Box435, SE 405 30 Gothenburg, Sweden. E-mail address: mats.bemark@immuno.gu.se

Abbreviations used in this paper: AID, activation-induced cytidine deaminase; APRIL,a proliferation-inducing ligand; BAFF, B cell activating factor of the TNF family; CSR,class switch recombination; CT, circle transcript; DC, dentritic cell; GC, germinal center;GL7int, GL7 intermediate; ILF, isolated lymphoid follicle; iNOS, inducible NO synthase;LP, lamina propria; MLN, mesenteric lymph node; PP, Peyer’s patch; RT, reverse tran-scriptase; SHM, somatic hypermutation; SI, small intestine; VH, variable H chain; WT,wild type.

Copyright� 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00

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quantitative RT-PCR for detection of a-germline transcripts andAID mRNA and a semiquantitative PCR for the detection of switcha-circle transcript (CT). Moreover, we analyzed in detail the PPs inCD402/2 and wild type (WT) mice and determined the relation-ships between GL7-expression and evidence for IgA CSR and so-matic hypermutations (SHMs) in these populations.

Materials and MethodsMice

CD402/2 and WT mice (both on a C57BL/6 background) were bred andhoused at the animal facility Experimentell Biomedicin at the Universityof Gothenburg under specific pathogen-free conditions.

RNA extraction and cDNA synthesis

ThePPs and 1-cmpieces of the colonwere isolated anddirectly submerged in350ml buffer RLT (Qiagen, Hilden, Germany). The tissuewas disrupted andhomogenized for 2 min using the tissue lyser (Qiagen) according to themanufacturer’s instructions, followed by RNA extraction using the RNeasyMini Kit (Qiagen). To isolate RNA from sorted cells or from tissue sections,we used the RNeasy Micro Kit (Qiagen) according to the manufacturer’sinstructions. The concentration of the RNAwas determined, and 4 mg RNAwas used for cDNA synthesis using oligo(dT)18 primers (Fermentas, Bur-lington, Ontario, Canada) and superscript III reverse transcriptase (RT)polymerase (Invitrogen, Carlsbad, CA). The cDNA synthesis was performedat 50˚C for 1 h followed by 15 min at 70˚C to inactivate the enzyme.

Real-time PCR

All real-time PCR reactions were performed on a Lightcycler 480 (Roche,Basel, Switzerland) using the probes master mix (Roche) according to theinstructions. Template (2 ml) was used in all reactions, and the reactionswere run in multiplex together with CD79a as a B cell loading controland as triplicates to minimize pipetting errors. To detect germlinea-transcripts, the following primers were used: germline_for_qpcr, 59-CCA GGC TAG ACA GAG GCA AG-39; germline_rev_qpcr, 59-CGGAAG GGA AGT AAT CGT GA-39. As probe, we used the Roche uni-versal library probe #27 (Roche). The following primers were used forAID transcripts: mAID_for_qpcr, 59-TCC TGC TCA CTG GAC TTC G-39; mAID_rev_qpcr, 59-GCG TAG GAA CAA CAA TTC CAC-39. Asprobe, we used the Roche universal library probe #71. The B cell loadingcontrol primers were: CD79a_for, 59-GAT GCC AGG GGG TCT AGAAG-39; CD79a_rev_qpcr, 59-GTT CAC CGT CAG GGA TGG-39. Asprobe, we used a Texas Red-labeled oligo 59-RED-CGT CAT GCC GACCCG CAG T-BHQ2-39. Cycling was performed under the followingconditions: denaturation at 95˚C for 5 min followed by 45 cycles of PCR(95˚C for 10 s, 60˚C for 15 s, and 72˚C for 1 s). The gene expression wascalculated by the software and normalized against the CD79a expressionin every reaction. To analyze BAFF, APRIL and iNOS we used preva-lidated custom RT2 quantitative PCR arrays (SABiosciences, Frederick,MD). The PCR plate contained the primers, and we added 2 ml templateto each well and SYBR Green Master Mix (Roche). The PCR reactionwas run for 40 cycles of 95˚C for 15 s, 60˚C for 30 s and 72˚C for 15 s.The samples were normalized to HPRT, and the relative expression wascalculated using the DDCT method with the efficiency set to 2 and WTexpression set to 1 as a reference.

One-step nested PCR

To analyze circular a-transcripts we used the one-step RT-PCR technique.The RNA was purified as described, and 2 ml RNA was used in the RT-PCR reaction. Gene-specific primers were added, and the cDNA synthesisand the PCR were performed in the same tube with the following primers:Im4, 59-ACC CTG GAT GAC TTC AGT GT-39; Iaup4, 59-CAT CTGGAC TCC TCT GCT CA-39. The PCR program was as follows: cDNAsynthesis at 56˚C for 30 min followed by an initial denaturation and in-activation of the RT enzyme at 94˚C for 2 min. The cycling conditionswere: 94˚C for 30 s, 56˚C for 30 s, 72˚C for 1 min, repeated 30 times,followed by a final extension at 72˚C for 10 min. The next round of PCRused 2 ml of the product from the first PCR as a template using the fol-lowing primers: IaF, 59-CCA GGC ATG GTT GAG ATA GAG ATA G-39;CmR, 59-AAT GGT GCT GGG CAG GAA GT-39. The cycling was per-formed under the following conditions: denaturation at 95˚C for 5 min and30 cycles of 95˚C for 1 min, 62˚C for 1 min, and 72˚C for 1 min. The finalstep in the PCR was an extension step at 72˚C for 10 min. Finally, theproducts were separated and analyzed on a 1.5% agarose gel.

Dilution PCR

To semiquantify the expression of circular a-transcripts, we used a dilutionseries assay approach in which RNA from PPs were diluted in a 1:2 di-lution series, and 2 ml from every dilution was subsequently used in a one-step nested PCR as described above. Every dilution giving a positive resultwas scored according to how diluted the sample was; for example, a pos-itive result when diluted 8 times was scored an 8, and a negative result wasscored as 0. All samples and dilutions were run in five replicates to min-imize jackpot effects and similar problems caused by the PCR.

Immunohistochemistry

Naive mice were sacrificed and the small intestine, colon, or the PP wereremoved, embedded in TissueTek OCT compound, and snap frozen in liquidnitrogen. Frozen sections (7 mm) of the small intestine were prepared onmicroslides using a cryostat (Leica, Wetzlar, Germany), and the sectionswere stored at 270˚C until used. The slides were fixed in 50% acetone for30 s and 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 horseserum in PBS (5%) for 15 min in a humidifying chamber. For detection ofgerminal centers, the sections were stained for 30–60 min with FITC con-jugated anti-GL7 mAb diluted 1:100 (BD Pharmingen, San Diego, CA) andbiotinylated anti-B220 diluted 1:500 (BD Pharmingen). The B220 was vi-sualized by adding streptavidin conjugated Texas Red (DakoCytomation,Glostrup, Denmark) 1:100 dilution. Consecutive sections were stained withB220 together with FITC conjugated anti-IgA diluted 1:100 (BD Pharmin-gen) or FITC conjugated anti-Ki67 diluted 1:5 (BD Pharmingen). The sec-tions were washed in PBS and mounted with DakoCytomation fluorescentmounting medium and visualized using a Leica DMLB microscope.

Flow cytometry and cell sorting

PPs were excised and transferred into ice cold PBS supplemented with 0.1%BSA. Single-cell suspensions were prepared by passing the tissue througha 100-mm nylon mesh cell strainer (BD Falcon). The cells were washedand stained with anti–B220-PeCy7 (BD Pharmingen) and anti–GL7-FITC(BD Pharmingen) at 1:300 dilution for 30 min on ice. The cells were thenwashed twice with PBS + 0.1% BSA and sorted or analyzed usinga FACSAria or a LSR II (BD Biosciences). Sorted cells were sorted intoPBS + 0.1% BSA and immediately centrifuged at 600 3 g and re-suspended in 350 ml buffer RLT (Qiagen) for subsequent RNA or DNAextraction using the RNeasy Micro Kit (Qiagen) or the Allprep DNA/RNAMini Kit (Qiagen).

Colony hybridization for GL7 mutational analysis

To analyze the mutational frequency of PP cells, we adopted a methodpreviously described (35). Briefly, we isolated DNA from GL7low, GL7intermediate (GL7int), and GL7high sorted cells from PPs and performeda genomic PCR, using the following primers: FR3J558, 59-GCC TGA CATCTG AGG ACT CTG C-39; and IgH_intron, 59-CCT CTC CAG TTT CGGCTG AAT CC-39. The program for the PCR was 98˚C for 1 min followedby 30 cycles of 98˚C for 10 s, 63˚C for 1 min, and 72˚C for 1 min. Theprogram ended with a final extension step at 72˚C for 10 min. To minimizemutations inserted by the PCR, we used the proofreading high-fidelityenzyme Phusion (Finnzymes, Espoo, Finland). The PCR generated blunt-ended products that were cloned directly using the Zero Blunt TOPO PCRCloning Kit (Invitrogen), and the plasmid was transformed into chemicallycompetent Escherichia coli and spread and selected using antibiotic re-sistance and blue/white screening. The selected colonies were patched ontoa selective plate, grown overnight, and lifted onto a nitrocellulose mem-brane (GE Healthcare, Piscataway, NJ). The colonies were then lysed byadding 10% SDS to the membrane, followed by a denaturing step in 1.5 MNaCl and 0.5 M NaOH. The membrane was then neutralized in 1 M Tris-HCl (pH 7.4) and 1.5 M NaCl and finally submerged in 23SSC solutionbefore the membrane was left to air dry and ultraviolet cross-linked using700 J/cm2. All steps were performed for 5 min each. The membrane wassubsequently blocked for 1 h at 42˚C in 15 ml blocking solution (0.5 M PO4

buffer [pH 7.2] containing 1 mMEDTA and 7% SDS) in a rotating chamber;5 pmol of the probe JH4: 59-TAT GCT ATG GAC TAC TGG-39 was ra-dioactively labeled by adding 5 ml ATP, [g-32P]–6000 Ci/mmol, 10 mCi/mlEasyTide Lead, 250 mCi (Perkin Elmer, Wellesley, MA), 5 ml 103 T4 PNKbuffer (Promega, Madison, WI), 1 ml T4 PNK (Promega) and up to 50 mlwater, followed by a 1 h incubation at 37˚C. Free nucleotides were removedby using a ProbeQuant G-50 micro column (GE Healthcare) and the labeledprobe was added to the membrane and incubated at 42˚C overnight. Hy-bridization was detected on a Pharos FX molecular imager (Bio-Rad,Hercules, CA), and positive colonies were picked and grown in liquid se-lectivemedia overnight followed by plasmid preparation usingGenElute HP

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PlasmidMiniprep Kit (Sigma-Aldrich, St. Louis,MO). Finally, the plasmidswere sent for sequencing (EurofinsMWGoperon, Ebersberg, Germany) andthe sequences were blasted against mouse Ab genes to identify mutationsusing IgBLAST at www.ncbi.nlm.nih.gov/igblast/.

Isolation of follicle free colon tissue

The mice were sacrificed and the colons were removed. To ensure analysisof follicle-free sections, 2–3-cm pieces of the colon were frozen andsectioned as described (30, 36). Every fifth section was placed on a slide,and the sections in between were stored in buffer RLT (Qiagen) at 270˚C.The sections on the slide were stained with Texas Red conjugated anti-IgM(SouthernBiotech, Birmingham, AL) and FITC conjugated anti-IgA (BDPharmingen) to localize the follicles. If two consecutive sections on theslide were positive or negative for follicles, the sections in between (storedin buffer RLT) were considered to also contain follicles or to be folliclefree. RNA was prepared from these sections using the RNeasy Micro Kit(Qiagen) and analyzed by PCR as described above.

Cloning and IgA sequencing

The mice were sacrificed and the small intestine was removed and dividedinto three pieces; one proximal, one middle, and one distal part. To ensureanalysis of isolated lymphoid follicle (ILF)-free tissue, the same procedureas for the colon was used. Subsequent cDNA synthesis was performed withAccuscript RT polymerase (Stratagene, La Jolla, CA) using an IgA-specificprimer (59-TGA CAT TGG TGG GTT TAC-39) followed by PCR using thefollowing primers: Vhdegen: 59-CTT CCG GAA TTC SAR GTN MAGCTG SAG SAG TC-39; IgArev: 59-TTC CTC GAG AGG GCA GGT GGGAAG TTT A-39. The PCR was performed using the high fidelity poly-merase PFU (Fermentas). The PCR product and the pBSK (Fermentas)were cleaved using XhoI and EcoRI (Fermentas) and purified on a gelusing the QIAquick Gel Extaction Kit (Qiagen). The fragments were li-gated into the plasmid, and the ligation was transformed into chemicallycompetent DH5a (Invitrogen) that was spread on selective plates for blue/white screening. White colonies were inoculated into 2 ml of selective LBmedium grown overnight at 37˚C, and the plasmid was purified using theGeneElute Plasmid Miniprep Kit (Sigma-Aldrich) followed by sequencing(Eurofins MWG Operon). The sequences were blasted against the IgBlast(NCBI) database to compare the sequences with the germline sequences,and aligned in Bioedit (www.mbio.ncsu.edu/bioedit/bioedit.html) using theClustalW algorithm for easy analysis of the constant IgA region and theCDR3 regions.

Statistical analysis

The statistical analysis was based on Mann-Whitney U test comparing twopopulations with a non-Gaussian distribution. Individual p values based ona 95% confidence interval are stated in every figure.

ResultsAn abundance of gut IgA plasma cells despite the complete lackof germinal centers in CD402/2 mice

In our previous study of CD402/2 mice, we searched for alter-native sites for IgA CSR in the GALT and in the nonorganized gutintestinal LP that could reconcile our conflicting findings of nearnormal levels of IgA plasma cells in the gut LP with a completelack of GCs in the PP (Fig. 1A, 1B) (30). We observed significantlevels of IgA CSR in the PP of CD402/2 mice, whereas all otherlocations, including the mesenteric lymph node (MLN), largelylacked IgA CSR activity (30). Because recent reports have pro-vided evidence of IgA CSR against T cell-independent Ags in theLP of the human colon, we analyzed this tissue in greater detail inthe CD402/2 mice (11). Similar to the small intestine, the colon ofboth CD402/2 and WT mice had comparable IgA plasma cellnumbers and few IgM+ B cells in the LP (Fig. 1B, 1C). A majorityof the latter, IgM+ B cells, were found in the organized lymphoidfollicles in the gut intestine (Fig. 1B, 1C). Unexpectedly, whereasWT and CD402/2 mice hosted similar numbers of small ILFs, theCD402/2 mice largely lacked bigger colonic patches and only oneto two were found per colon, compared with six to eight per colonin WT mice (Fig. 1C). Few IgM+ B cells that potentially couldundergo IgA CSR were observed in the nonorganized LP of thesmall intestine and colon of both CD402/2 and WT mice.

IgA class switch recombination does not occur in the laminapropria of the colon

To better assess the relative IgA CSR activity in various tissues wedeveloped highly sensitive quantitative PCR methods for detectionof a-germline transcripts and AID mRNA and a semiquantitativePCR for the detection of switch a-CT. The latter method proved todetect as few as four transcripts in a sample. We compared the IgACSR activity in samples from the PP, small intestine, and colon LPof CD402/2 and WT mice. In agreement with our earlier study,we found switch a-CT in the PPs of both WT and CD402/2 miceand occasionally in colon and small intestinal LP biopsies (Fig.2A) (30). In fact, only a small proportion of LP samples containedswitch a-CT; from 17 WT and 22 CD402/2 mice, 15% and 16%,respectively, of the colon samples contained switch a-CT (Fig.2B). To rule out the inclusion of organized lymphoid follicles(ILFs or colonic patches), we undertook extensive sectioning ofthe biopsies (Fig. 3A). After microscopical analysis for the pres-ence of organized lymphoid tissues, we isolated RNA from the

B

AB220 /GL7

WT PP

B220 /GL7

CD40-/- PP

WT Small Intestine CD40-/- Small Intestine

IgM/ IgAIgM/ IgA

WT colon CD40-/- colon

IgM/ IgAIgM/ IgA

C

FIGURE 1. Absence of GCs in the PP of CD402/2 mice. The GALT of

CD402/2 mice exhibited no GCs and LP host near normal numbers of IgA

plasma cells, but few IgM+ cells. A, The illustration shows frozen sections

from the PP of CD402/2 and WT mice labeled with anti-GL7 FITC

(green) and anti–B220-TxR Abs (red). B, Sections of the small intestine

were prepared and labeled with anti-IgA FITC (green) and anti-IgM TxR

(red) ABs. C, Similarly, sections of colon were prepared and labeled with

anti-IgA FITC (green) and anti-IgM TxR (red) Abs. White arrowheads

indicate colonic ILFs and red arrowheads indicate colonic patches in WT

mice, characterized by their larger sizes (left panel). CD402/2 mice had

reduced numbers of colonic patches. Insets shows higher magnification of

rare IgM+ cells in the nonorganized LP of the small intestine or colon (B,

C). Images are representative of at least 10 mice analyzed from each strain

Original magnification of the whole gut mounts 310 (insets 340).

The Journal of Immunology 3

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sections and analyzed these samples for the presence of IgA CSRmarkers (Fig. 3B). Consistently, we found that AID mRNA wasdetected only in samples that hosted lymphoid follicles, and thatswitch a-CTwere never found in follicle-free samples of colon LPfrom either WT or CD402/2 mice (Fig. 3C). However, in 22% and14%, respectively, of the sections that hosted lymphoid follicles,we also detected switch a-CTs as well as AID mRNA (Fig. 3C).Thus, using this refined method, IgA CSR activity was found to beexclusively restricted to the organized colonic lymphoid tissues,whereas no evidence for IgA CSR was found in the nonorganizedLP in either strain (Fig. 3C).

PPs are dominant sites for IgA CSR in both CD402/2 and WTmice

Because we failed to detect switch a-CT in the small intestinal andcolon LP, we analyzed the IgA CSR activity in the PP of CD402/2

mice in greater detail. To this end we used the refined quantitativeRT-PCR methods that we had developed. We found thata-germline RNA and AID mRNA expression were reduced in thePP of CD402/2 compared with those in WT mice (Fig. 4A, 4B).Moreover, only half of the PP samples from the CD402/2 micehad switch a-CT, whereas almost 100% of the PP containedswitch a-CT in WT mice (Fig. 4C). In fact, a semiquantitativePCR analysis of RNA samples using serial dilutions demonstratedthat samples from WT mice contained twice the amount of switcha-CT compared with PP samples from CD402/2 mice (Fig. 4D).Therefore, the estimated overall IgA CSR activity in PPs ofCD402/2 mice, lacking GC formations completely, was ∼25% ofthat in WT PPs. Thus, PP from CD402/2 mice hosted significantIgA CSR and, compared with the ILFs in which ∼80–85% of thesamples were negative for IgA CSR, it appeared that the PP wasthe dominant site for IgA CSR.

Unaltered presence of IgA switch factors in PPof CD402/2mice

BecauseTcell-independent IgACSRhasbeen reported todependonBAFF,APRIL and/or iNOSproduction,wenext investigatedmRNAlevels of these factors in PP fromCD402/2 andWTmice (9, 22, 26).We found comparable mRNA expression of all three factors in both

FIGURE 2. Switch a-CT expression in biopsies from the GALT and LP

of WT and CD402/2 mice. Random biopsies from the PP, small intestine

(SI) or colon from WT and CD402/2 mice showed a high frequency of

switch a-CT in PP, but few in the small intestine or colon. A, Examples of

gel analysis of switch a-CT in random biopsy specimens taken from the

PP, colon, or small intestine. The two differently sized bands correspond to

two different splice variants of the transcript (49). B, The compiled data

from a large number of random samples (indicated in the center of the pie

chart) from the colon of 17 WT and 22 CD402/2 mice, illustrating the

frequency of switch a-CT, which was found to be ∼15% in these biopsy

secimens from WT or CD402/2 mice.

WT/CD40-/-C

CD40-/-

IgM / IgA

WT

IgM / IgAB

Follicle+

+ -

18/21

5/6 13/15

+ -

4/3 1/3 0/1 13/14

-

12/15

0/0 12/15

0/0 0/0 0/0 12/15

AID mRNA

Switch α-CT+ -

+ -

+ - + -

Onto slide

Into RLTBuffer

16

1116212631

6µmsections

Colon

Biopsy

2345

789

10

} Tube 1

} Tube 2

12131415

} Tube 3

Etc.

RNA preparationfrom follicle free orfollicle containingtissue.

1

2

3

Stain sections with anti IgA and anti IgM antibodies to visualize follicles.

4

A

CD40-/-

IgM / IgA

WT

IgM / IgA

ILFILF

No ILFNo ILF

FIGURE 3. The nonorganizedLPof the colon fromCD402/2 orWTmice

lack expression of switch a-CT or AID mRNA. A detailed investigation of

molecular markers for IgA CSR in colon LP samples devoid of organized

lymphoid tissues was performed. After careful microscopic evaluations of

frozen sections from colon for the presence or absence of ILFs or colonic

patches, we undertook PCR analysis for detection of switch a-CT or AID

mRNA. A, A schematic representation of the procedure to isolate follicle-

free material from the colon LP for further analysis for switch a-CTor AID

mRNA is shown. Tissue sections were labeled with anti-IgMAbs to identify

organized lymphoid follicles. B, Representative examples of follicles (upper

panels, showing IgM+ B cells, red) or follicle-free colon LP (lower panel) in

CD402/2 or WT mice following labeling with anti-IgA FITC (green) and

anti-IgM TxR (red) Abs. Original magnification 320. C, An extensive

analysis was performed to assess the frequency of switch a-CT or AID

mRNA positive samples from nonorganized colon LP samples. The diagram

represents the compiled data frommultiple biopsies taken from 15WT (red)

and 15 CD402/2 (green) mice. First we assessed the presence (+) or absence

(2) of follicles and found that 18 of 30 samples inWTand 21 of 36 samples in

CD402/2mice hosted follicles. Of the samples hosting follicles 5 of 18 WT

and 6 of 21 CD402/2 scored positive for AID mRNA expression, whereas

none of the follicle-free samples from either WT or CD402/2 mice carried

AIDmRNA. Finally, expression of switcha-CTwas found in four offiveWT

and three of six CD402/2 samples that hosted follicles and AID mRNA,

whereas, of AID mRNA negative samples in these groups, only 1 of 15 ex-

pressed switch a-CT in CD402/2 and none in WT mice. Corroborating the

lack of IgA CSR in follicle-free nonorganized colon LP we failed to detect

switch a-CT in any of these samples (0 of 12 WT and 0 of 15 CD402/2).

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strains, indicating that the PP milieu in the CD402/2mice was wellequipped to support T cell-independent IgA CSR (Fig. 5). Note-worthy, the presence and level of expression of BAFF, APRIL andiNOS mRNA was clearly independent of GC formations as theywere found at similar levels in PP of CD402/2mice as inWTmice.

IgACSRoccurs at theGL7 intermediate stage prior tomanifestGCformation

The relationship between the IgA CSR that occurred in GC-freePPs of CD402/2 mice and that in GC-rich PPs of WT mice wasanalyzed next. We made the unexpected finding that a substantialB cell population expressed GL7int levels in both CD402/2 andWT mice, whereas only WT mice hosted a significant populationof GL7high cells, as analyzed by FACS (Fig. 6A). Because theGL7int PP B cells were nearly as frequent in CD402/2 as in WTmice, we hypothesized that these cells were responsible for IgACSR in the PPs of both strains. Because a prerequisite for CSR iscell division, we analyzed the PP B cells for expression of Ki67+,a marker for proliferating cells, and found significant numbers ofGL7int cells to be Ki67+ (Fig. 6B). Furthermore, AID mRNAexpression, necessary for CSR, was found in GL7int cells fromCD402/2 mice, albeit at lower levels than in WT mice (Fig. 6C).Of note, the strongest expression of AID mRNA by far was foundin GL7high PP B cells (not shown). Extending the analysis to in-clude switch a-CT expression, in six experiments we found thatGL7neg and GL7high populations failed to demonstrate switcha-CT (Fig. 6D). In contrast, in both CD402/2 and WT PP GL7int

B cells, we found significant expression of switch a-CT (Fig. 6D).In a total of 16 experiments, we found evidence of switch a-CTand IgA CSR in 50% of the experiments (not shown). Whencomparing the degree of SHM in the JH4 intron in GL7int andGL7high PP B cells from WT mice, we observed that GL7int cellswere mostly unmutated, similar to the GL7neg PP B cells, whereasGL7high cells were highly mutated (Fig. 6E). These results suggestthat T cell-independent IgA CSR in PP B cells occurs at the GL7int

stage, in the absence of GCs, whereas SHM follows later when theB cells are GL7high, express high levels of AID mRNA, and havemanifested GC formation.

The distribution of somatic hypermutations reflects clonalrelationships in gut IgA plasma cells

To test our hypothesis that IgA CSR occurred in GL7int PP B cells,we used the different levels of SHM in GL7int and GL7high cells asa marker for the origin of LP IgA plasma cells. If the hypothesiswas correct, CD402/2 mice, hosting predominantly GL7int cells,would be expected to have few mutations in LP IgA plasma cells.Therefore, we analyzed random V regions of the IgA cells fromspatially separated segments of the LP in CD402/2 and WT mice.We RT-PCR cloned the IgAV regions using a degenerate upstreamV region primer together with a downstream IgA C region primer.Importantly, this allowed us to determine the level of mutationsassociated with SHM, as opposed to mutations resulting from thecloning, because constant regions do not undergo SHM. In WTmice, 84% of the cloned V regions from LP cells carried muta-tions, with an average of 5.2 mutations per sequenced V region(Fig. 7A; Table I). In contrast, LP IgA cells in CD402/2 miceremained largely unmutated, supporting the finding that IgA CSRhad occurred in GL7int B cells before manifest GC in the PP, butthat efficient SHM required GC formations. Whereas ∼14% of theLP sequences from CD402/2 mice showed mutations, the averagenumber was only 0.3 mutations per sequenced V region. These Vregion mutations in CD402/2 mice, however, occurred at a higherfrequency than mutations restricted to the IgA constant regions(0.9 mutations per 1000 bp versus 0.1 mutations per 1000 bp),indicating that LP IgA cells probably undergo some SHM despitethe lack of GC in PP. Nevertheless, in a total of almost 75,000 bp

FIGURE 4. Substantial IgA CSR activity in the PP of CD402/2 mouse.

We undertook a quantitative analysis of molecular markers for IgA CSR in

the PP from CD402/2 and WT mice. A, Germline a transcripts were an-

alyzed using real time PCR. Each data point represents one PP, and the line

shows the mean expression. The total number of mice in the analysis was

14 WT and 18 CD402/2 mice. B, AID mRNA expression from the same

PPs that were analyzed in A. All samples in A and B were compared with

a calibrator containing a pool of WT PP cDNA that was set to 1. To no-

rmalize for the number of B cells in each sample, the germline a tran-

scripts and AID mRNA expression levels were normalized against the

B cell-specific CD79a mRNA expression. C, The diagram shows a detailed

analysis of switch a-CT from the same PPs as in A and B. Each data point

represents the frequency of switch a-CT positive PPs in individual mice

and 5 PPs analyzed per mouse. The line in the diagram shows the mean

frequency of positive PPs. D, A dilution series of PP cDNAwas performed

with samples from three individual mice and pooled material from 4 PPs.

Each black dot represents the sample dilution that gave a positive score for

switch a-CT expression. Gray circles indicate no detectable switch a-CT.

This is one representative experiment of three giving similar results.

p values of significance are given.

FIGURE 5. Similar expression of IgA switch factors in thePPofCD402/2

and WT mice. Real-time quantitative PCR analysis of potential IgA switch

factors in the PP revealed that the relative expression of BAFF, APRIL, and

iNOS mRNA was similar in CD402/2 and WT mice. The relative mRNA

expression level of the indicated genes was determined after normalizing

against HPRT mRNA. In each experiment, the mRNA level in pooled sam-

ples of PP from three individual CD402/2 mice was then expressed as

fold change relative to that found in PPs fromWTmice, whichwas set to 1.0.

These data are given as fold change against PP WT levels and represents

two separate experiments and shows the means6 SD.

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analyzed we found 19.6-fold higher SHM levels in V regionscloned from WT compared with IgA LP cells from CD402/2

mice. Thus, the largely unmutated V-regions of LP IgA plasmacells corresponded well with the lack of GCs in the PPs of CD402/

2 mice, whereas WT PPs, hosting manifest GC formations, pro-duced heavily mutated LP IgA plasma cells. Remarkably, in all sixmice, WT and CD402/2, we observed IgA V region sequenceswith identical CDR3s from the proximal, middle, or distal parts ofthe small intestine (Fig. 7B). This finding suggested that clonalexpansion of B cells had occurred before seeding the LP. In par-ticular, the clonal relationships between LP IgA cells from dis-tinctly separate sites of the intestine in WT mice were evidentwhen sequences were arranged into mutational trees (Fig. 7B, 7C).In addition, when we extended the analysis to include colonsamples, we found no significant differences in the number ofmutations between the small intestine and colon in WT mice (Fig.8A, Table I). We observed clonally related sequences with sharedmutations between the small intestine and the colon in WT mice,although this appeared to be less common compared with simi-larities between sequences taken from distant places within thesmall intestine or the colon (Fig. 8A, 8B). Thus, these data provide

FIGURE 6. IgA CSR in PPs from CD402/2 mice occurs in GL7int cells

before manifest GC formation. B cells with differential expression of

GL7 were sorted from PPs of CD402/2 and WT mice and analyzed for

IgA CSR and SHM. A, PP B cells pooled from three mice were labeled

with anti-B220 Abs and sorted by flow cytometry into GL7neg, GL7int,

and GL7high fractions. Few cells were found in the GL7high population in

CD402/2 mice compared with that in WT mice, where the GL7int PP B

cells were almost comparable in number between the two strains. B,

Evidence of cell proliferation in the PP B cell GL7-fractions was ob-

tained after labeling with anti-Ki67 Abs and FACS analysis. The GL7int

population hosted large numbers of proliferating cells. This representa-

tive experiment is one of three with similar results. C, The fold change 6SD in AID mRNA expression detected by a quantitative RT-PCR analysis

revealed AID expression in the GL7int fraction of both CD402/2 and WT

mice. The level of AID mRNA expression was calibrated against WT PP

AID mRNA expression, which was set to 1.0 and using CD79 mRNA as

the B cell-specific housekeeping gene. D, Subfractions of PP B cells from

WT mice were further processed for RNA isolation, and samples were

subjected to a one-step nested PCR analysis for switch a-CT, which was

found exclusively in GL7int PP B cells in six experiments. A total of 16

experiments were then performed with sorted GL7int PP B cells from WT

or CD402/2 mice. Switch a-CT were found in .50% of these experi-

ments. E, Mutational analysis of pooled and sorted GL7low, GL7int, and

GL7high PP B cells isolated from WT mice. DNA was isolated and ana-

lyzed for JH4 intron mutations, and the results were given as the number

of mutations per sequence. These date are compiled from three identical

experiments.

FIGURE 7. Clonally related IgV sequences are found inWTandCD402/2

small intestinal LP IgA+ cells, but few mutations in CD402/2 LP IgA+ cells.

Small intestinal LP biopsies from three CD402/2 or WT mice were isolated

and used to extract mRNA for further analysis of mutations and clonal rela-

tionships between IgH V–Ca regions. cDNA synthesis was performed with

a Ca downstream to specifically amplify Ca rearrangements. A, The pie

charts show themutation frequency inWTandCD402/2mice, where the size

of the segments shows the number of sequences containing the number of

mutations indicated in each segment. The total number of sequences analyzed

is given in the center of the chart. B, Analysis of small intestine clonally re-

lated IgV sequenceswith identicalCDR3 rearrangements from three spatially

separated segments. Mutations are shown with a vertical line and shared

mutations are indicated with either an asterisk (p) or pound sign (#). The

clonally identical V, D, and J segments are indicated by their name as found

using the IgBLAST (www.ncbi.nlm.nih.gov/igblast). The image shows two

examples in which all three sites in the small intestine share clonality. C, A

lineage tree analysis generated from the data from B. Gray cells represent

hypothetical intermediate stages.

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additional support for the hypothesis that IgA CSR had not oc-curred in the nonorganized LP, because clonally related sequenceswere observed at great distances in the intestine, suggesting thatLP IgA B cells undergo CSR and SHM in the GALT beforeseeding the intestinal mucosa.

DiscussionThis report unequivocallydemonstrates that IgACSRdoes not occurinnonorganized lymphoid tissues of themousegutLP.We found thatIgA CSR was restricted to gut samples containing B cell follicles,whereas we failed to detect any switch a-CT in 35 mice with LPsamples lacking follicles. Therefore, our study fails to confirmprevious reports on IgACSR in human and mouse non-rganized LP(11, 20). Furthermore, to reconcile that IgA CSR was restricted tothe GALTof CD402/2mice despite the complete lack of GCs in thePPs, we provide evidence that IgA CSR in PP B cells can occur ata stage beforemanifestGC (i.e., when the cells are at aGL7int stage).In contrast, high levels of SHM in PP were found only in GL7high

B cells that were associated with manifest GCs, and an extendedanalysis of mutations of IgAV-region sequences revealed that highlevels ofmutations occurred in LP IgA plasma cells inWTmice, butnot in CD402/2mice. Our data indicate that PP B cells in CD402/2

and WT mice undergo IgA CSR at a stage before manifest GC. Incontrast, efficient SHMrequiresGC formation. The observation thatclonally related sequences were found in LP at distant sites furthersupports the hypothesis that LP IgA plasma cell precursors undergoCSR, SHM and expansion before seeding the LP and also arguesagainst local IgACSR in the nonorganized gut LP being a dominantsource for these cells.Previous studies have suggested that the nonorganized LP is a site

for substantial IgACSR inmice (20).Amore recent studybyHeet al.(11) suggested that IgA CSR could be induced in the nonorganizedLP of the human colon in a process that involved recognition ofintestinal bacteria by epithelial cells, the release of thymic stromallymphopoietin, and production of APRIL by gut epithelial cells andDCs. CSR in the colon was not only from IgM to IgA, but also fromIgA1 to IgA2. However, other studies in mice and humans do notfind any evidence that the nonorganized LP is a prominent site forIgA CSR (30, 37, 38). One explanation for the discrepancies be-tween the studies could be that ILFs may have contaminated theanalysis inwhich theLPwas found to support IgACSR. In our study,we microscopically analyzed the gut tissue for lymphoid follicles topreclude that we were analyzing follicle-containing biopsies whenassessing IgA CSR in gut intestinal LP. Our data show that AIDmRNA expression and switch a-CT were restricted to follicle-containing biopsies and were never found in follicle-free LP. Pre-vious studies, in which the organized follicles have been separated

from the nonorganized LP, support the findings that the LP is nota site for IgACSR (30, 37, 38). All of these studies essentially failedto detect molecular evidence for ongoing IgA CSR in the non-organized LP. In addition, we found few IgM cells that could un-dergo IgA CSR in the nonorganized LP, whereas most IgM cellswere found in organized follicles or in the PP.These findings excludethe LP as a primary site for IgACSR, at least in the mouse. Missingevidence of significant B cell proliferation in the nonorganizedLP inhumans, or that APRIL can promote cell proliferation, alsoweakensthis interpretation for human tissues (21, 37). Moreover, furtherarguing against IgA CSR in the nonorganized LP is our finding thatclonal relationships existed between IgA plasma cells isolated atdistant sites in the gut intestine. Such a presence is best explained bythe expansion of B cells that have already undergone IgA CSR andSHM in organized lymphoid tissues before seeding the gut intestinalLP. Studies in humans corroborate this interpretation, in whichclosely related gene sequences were found at widely separated sitesin the gut intestinal LP (5, 37, 39). Our data favor a model in whichgut LP plasma cells originate from precursors in the GALT that cansupport both IgA CSR and SHM, such as the PP, before seeding thenonorganized small intestinal or colon LP. In CD402/2 mice, webelieve the dominant site for IgA CSR is the PP, rather than the ILFor the colonic patches, because these mice largely failed to carrycolonic patches and only 15–20% of biopsy specimens that hostedorganized lymphoid tissue expressed switch a-CT. This hypothesisis further supported by our previous observation that young 3-wk-old CD402/2 mice that have PP, but lack most ILFs in the earlyphase of bacterial colonization, still exhibit significant levels of IgAplasma cells in the LP of the gut intestine (30).In our previous study of CD402/2 mice, we reported low IgA

CSR-activity in the MLN (,5% of the samples); therefore, we donot consider this site an important contributor to T cell-independent IgA CSR in conventionally reared mice (30). Nota-bly, MLN has been found to be important for IgA CSR in micetreated in utero with LTbR-Ig fusion protein and lacking PP,suggesting a compensatory function under conditions of an

Table I. Summary of IgA VH sequence data

WT CD402/2

Total no. of sequences 84 79Total no. of base pairs 24,509 23,291No. of mutated sequences 73 11No. of mutations in VH regions 434 21VH mutation frequency per 1000 base pairs 17.71 0.90No. of mutations in the IgA constant region 0 3Average no. of mutations per VH sequence 5.17 0.27Average no. of mutations per VH distal SI 5.79 NDAverage no. of mutations per VH middle SI 3.86 NDAverage no. of mutations per VH proximal SI 5.47 NDAverage no. of mutations per VH colon 6.92 ND

A summary of the data obtained from the analysis of mutations in IgAV regionsexpressed from LP plasma cells isolated from the intestine of CD402/2 and WT mice.

VH, variable H chain.

FIGURE 8. Clonally related IgV sequences are found in the colon and

the small intestine in WT LP IgA+ cells. Colon and small intestine LP

biopsy specimens from two WT mice were obtained and used to extract

mRNA for further analysis of mutations and clonal relationships between

IgH V–Ca regions. cDNA synthesis was performed with a Ca downstream

to specifically amplify Ca rearrangements. A, The pie charts show the

mutation frequency in WT small intestine and colon, where the size of the

segments shows the number of sequences containing the number of mu-

tations indicated in each segment. The total number of sequences analyzed

is given in the center of the chart. B, Analysis of small intestine or colon

clonally related IgV sequences with identical CDR3 rearrangements from

WT mice. The image shows an example of clonality between sequences in

the colon and in the small intestine. Mutations are shown with a vertical

line, and shared mutations are indicated with a pound sign (#).

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aberrant mucosal barrier (40, 41). In addition, we observed en-hanced IgA CSR activity in the MLN of germ-free mice newlysubjected to bacterial colonization and lacking an intact IgA-barrier function (P. Bergqvist, unpublished observation). Further-more, we did not find evidence for IgA CSR in the spleen, bonemarrow, peritoneal cavity, or nonorganized LP of the small in-testine and, as documented in the current study, nor in the LP ofthe colon. Therefore, we propose that T cell-independent IgA CSRoccurs predominantly in the PP in CD402/2 mice. We found that∼50% of all PP samples from CD402/2 mice contained switcha-CT. The B cell population that expressed switch a-CT and AIDmRNA in the PPs was GL7int, undetectable by histologic analysisin CD402/2 mice, whereas the GL7high B cells, detectable byhistologic analysis in WT mice, failed to express switch a-CT, butwere strongly mutated and expressed high levels of AID mRNA.The higher expression of AID mRNA in WT GL7high cells agreeswith the finding that AID expression and CSR in general is di-vision linked and reflects possibly both CSR and SHM in themanifest GC (33). Although GL7 is widely used as a GC marker,no study has correlated the switch a-CT and AID mRNA tran-scription in relation to GL7 expression (42–45). We found thatGL7int B cells exhibited all features associated with successfulIgA CSR (i.e., a-germline RNA transcripts, AID mRNA, switcha-CT). The implication of our finding is that T cell-independentIgA CSR occurs in the PP at a stage before manifest GC. The factthat PP from CD402/2 mice hosted comparable or higher levels ofgene expression for the potentially important IgA switch-factors,BAFF and APRIL (and perhaps iNOS), is important because itindicates that the PP milieu is well equipped for supporting IgACSR, even in the absence of GC (22, 26). This information cou-pled with the evidence of cell proliferation in the GL7int pop-ulation is the basis for proposing that the PP is the site for IgACSR also against T cell-independent Ags.To what extent the GL7int B cells were located in clusters or

dispersed in the tissue close to or within the B cell follicles of thePP was not analyzed. However, as these cells hosted Ki67+ pro-liferating cells, detectable only by FACS and not seen micro-scopically, we can only speculate that they were dispersed in thetissue. Whether GL7int B cells were located at an extrafollicular orfollicular site in the PP is not known. In support of an inter-follicular site for IgA CSR in PP, AID mRNA expression ac-companied by switch a-CT has been found in secondary lymphoidorgans outside the GC, scattered in the T cell zone and within themantle zone (46–48). Nevertheless, in this study we providecompelling evidence that T cell-independent gut IgA CSR occursin the PP at a stage before manifest GC formations and that suchIgA cells carry largely unmutated V region sequences. We pro-pose that T cell-independent IgA CSR in the GALT normallyprecedes SHM at a GL7int stage, before manifest GC. Further-more, our study excludes that IgA CSR occurs in the non-organized LP of the small intestine or the colon. Clonalrelationships were found between IgA cells in the small intestineand colon, further supporting the notion of a common source forIgA CSR before seeding the intestinal LP. The PP appears to becentral for IgA CSR in WT and CD40-deficient mice, and it hostsa milieu rich in IgA switch-factors regardless of the presence orabsence of GCs.

AcknowledgmentsWe thank the Centre for Cellular Imaging at the University of Gothenburg

for the use of imaging equipment and for excellent support from the staff.

DisclosuresThe authors have no financial conflicts of interest.

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