seminario 7 t regs gut 2009 westendorf 211 9

CD4+Foxp3+ regulatory T cell expansion induced byantigen-driven interaction with intestinal epithelialcells independent of local dendritic cells

A M Westendorf,1 D Fleissner,1 L Groebe,2 S Jung,3 A D Gruber,4 W Hansen,1 J Buer1

c Additional methods andfigures are published online onlyat http://gut.bmj.com/content/vol58/issue2

1 Institute of MedicalMicrobiology, UniversityHospital, Essen, Germany;2 Department of MucosalImmunity, Helmholtz Centre forInfection Research,Braunschweig, Germany;3 Department of Immunology,Weizmann Institute of Science,Rehovot, Israel; 4 Department ofVeterinary Pathology, FreieUniversitat Berlin, Germany

Correspondence to:Professor A M Westendorf,Mucosal Immunity, Institute ofMedical Microbiology, UniversityHospital Essen, Hufelandstr. 55,D-45122 Essen, Germany;[email protected]

Revised 6 August 2008Accepted 8 August 2008Accepted for Online First2 October 2008

ABSTRACTBackground: Regulatory T cells (Tregs) have potentialanti-inflammatory effects and are likely to be important inthe pathogenesis of chronic inflammatory bowel disease(IBD). However, the induction and expansion of Tregs atsites of mucosal inflammation are not yet fully understoodand may involve antigen presentation by local dendriticcells (DCs) and/or intestinal epithelial cells (IECs).Methods: To determine the unique ways in which the gutinduces or expands Tregs, a transgenic mouse model thatis based on the specific expression of a modelautoantigen (influenza haemagglutinin (HA)) in theintestinal epithelium (VILLIN-HA) was used. Gut-asso-ciated DCs and IECs isolated from these mice werephenotypically and functionally characterised for thepotential to interact with HA-specific Tregs in vitro and invivo.Results: Intestinal self-antigen expression leads toperipheral expansion of antigen-specific CD4+Foxp3+ Tregs.Although gut-associated DCs can induce antigen-specificCD4+Foxp3+ T cell proliferation, in vivo depletion of DCsdid not preclude proliferation of these cells. Interestingly,antigen presentation by primary IECs is sufficient toexpand antigen-specific CD4+Foxp3+ Tregs efficiently. Thisis dependent on major histocompatibility complex class II,but, in contrast to DCs, is unlikely to require transforminggrowth factor b and retinoic acid.Conclusion: This study provides experimental evidencefor a new concept in mucosal immunity: in contrast tocurrent thinking, expansion of Tregs can be achievedindependently of local DCs through antigen-specific IEC–Tcell interactions.

Regulatory T cells (Tregs) are believed to contributeto the maintenance of intestinal homeostasis, andtheir absence may predispose to inflammatorybowel disease (IBD). For the control of intestinalinflammation by Tregs different populations ofthymically or peripherally induced Tregs,CD4+CD25+, CD4+CD45RBlow T cells, TR1, TH3cells or CD8+ suppressor T cells, have beendescribed.1–4

To date, little is known about the mechanismsby which regulatory cells are generated orexpanded in vivo in the intestinal mucosa. It hasbeen suggested that dendritic cells (DCs) located inthe intestine are able to induce Tregs.

5 Furthermore,Huang and colleagues6 demonstrated that a dis-tinct DC subset constitutively endocytoses andtransports apoptotic epithelial cells to T cell areasin the mesenteric lymph node (MLN). Their resultssuggest a role for these DCs in inducing andmaintaining peripheral self-tolerance. New studies

support this idea by showing that the catalysis ofvitamin A into retinoic acid (RA) in gut-associatedDCs enhances the transforming growth factor b

(TGFb)-dependent conversion of naıve T cells intoTregs and also directs Treg homing to the gut.7 8

However, intestinal epithelial cells (IECs) havealso been implicated in the regulation of uncon-trolled T cell responses.9 10 The ability of IECsconstitutively to express molecules required forantigen presentation including major histocompat-ibility complex (MHC) class I and II, and to takeup and process soluble antigens, suggests that theymight also act as antigen-presenting cells (APCs).10–

12 Lack of or weak expression of co-stimulatorymolecules on the surface of IECs could result inanergy of T cells and might therefore be importantfor maintaining immune homeostasis.

Recently we could show that self-antigenexpression by intestinal epithelial cells in a T cellreceptor (TCR) transgenic mouse model leads to amild form of chronic inflammation that is mostprobably controlled by the local induction ofanergy or by Tregs.

4 12 In the present study, wemake use of this TCR transgenic mouse model toaddress the role of DCs and IECs for the inductionand/or expansion of Tregs in the intestinal mucosain vivo.

METHODSMiceTCR-HA transgenic (6.5) mice expressing an a/b-TCR recognising the MHC class II (H-2Ed:HA110–

120)-restricted epitope of the haemagglutin (HA)protein have been described previously.13 VILLIN-HA transgenic mice express the HA proteinspecifically in the intestinal epithelium.4 12

Heterozygous VILLIN-HA and TCR-HA mice weremated to generate (VILLIN-HA/TCR-HA) doubletransgenic mice. INS-HA/RAG2–/– transgenic miceexpress the HA protein under the control of theinsulin promoter.14 CD11c-DTR/GFP transgenicmice express a diphtheria toxin receptor (DTR)15

and were crossed to VILLIN-HA transgenic mice.

Isolation of DCsMLNs were first cut into small pieces and thentreated with 1 mg/ml collagenase D (Roche,Mannheim, Germany) diluted in phosphate-buf-fered saline (PBS) with 2% fetal calf serum (FCS).Enzymatic digestion was performed for 45 min at37uC. The remaining tissue was mechanicallyminced and resulting cell suspensions were pooledand filtered through a 100 mm cell strainer. Cellswere washed in PBS containing 2% FCS and 2 mM

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EDTA. For isolation of lamina propria (LP) DCs, the smallintestine was cut into small pieces, followed by sequentialstirring in medium to remove mucus and the epithelial layer. LPcells were released by digestion at 37uC with collagenase. DCswere separated with the MoFlow cell sorter (Cytomation,Glostrup, Denmark) after staining with a-CD11c antibody.

Isolation of IECsIECs were isolated as described previously.12 16 For geneexpression profiling and cellular assays, a single cell suspensionwas prepared by shearing with a pipette tip, and cells werestained with a-CD45 antibody to label haematopoietic cells.IECs were sorted by size, granularity and negative selection forCD45 with the MoFlow cell sorter (Cytomation).

CD4+ T cell isolationFor proliferation experiments, CD4+ T cells were isolated fromTCR-HA and TCR-HA/RAG2–/– splenocytes using the MACSCD4 T cell isolation kit according to the manufacturer’srecommendations (Miltenyi Biotec, Bergisch Gladbach,Germany).

Adoptive transferFor transfer experiments into INS-HA/RAG2–/– mice, lympho-cytes from the MLNs of TCR-HA and VILLIN-HA/TCR-HAtransgenic mice were stained with a-6.5, a-CD4 and a-CD25,and separated by cell sorting. Purified cells were resuspended inPBS. A total of 2.56105 6.5+CD4+ T cells were injectedintravenously into INS-HA/RAG2–/– transgenic mice. For invivo inhibition, 2.56105 regulatory 6.5+CD4+CD25+ T cells fromTCR-HA transgenic mice or 2.56105 6.5+CD4+ T cells fromVILLIN-HA/TCR-HA transgenic mice were co-transferred with2.56105 naıve 6.5+CD4+ T cells from TCR-HA transgenic mice.For adoptive transfer into VILLIN-HA transgenic mice, CD4+ Tcells from the MLN or the spleen of TCR-HA and VILLIN-HA/TCR-HA transgenic mice were isolated. Cells were labelled withCFSE (carboxyfluorescein succinimidyl ester) and 16107 CD4+ Tcells were injected intravenously into wild-type BALB/c andVILLIN-HA recipient mice. DC depletion was performed byintraperitoneal injection of 9 ng/g of diphtheria toxins intoVILLIN-HA/CD11c-DTR transgenic mice 1 day before and 1day after adoptive transfer.

Diabetes monitoringBlood glucose concentrations in mice were monitored up to day20 post-transfer using a Haemo Glukotest 200-800R (Roche).Mice were considered diabetic when glycaemia was .200 mg/dlfor two consecutive measurements.

HistologySmall intestines and MLNs were immersion fixed with bufferedformalin, embedded in paraffin, sectioned at 4 mm thickness,and stained with H&E. Immunohistochemistry for GFP+ DCswas performed by a-GFP (green fluorescent protein) antibodyand the avidin–biotin complex (ABC) method with diamino-benzidine as chromogen. Immunohistochemistry sections werecounterstained with haematoxylin.

Proliferation assayFor DC stimulation experiments, 66104 DCs from the MLN andthe LP of VILLIN-HA and wild-type BALB/c mice were co-cultured with 36105 CFSE-labelled CD4+ T cells enriched fromTCR-HA splenocytes for 5 days. Proliferation was measured by

loss of CFSE dye. For IEC stimulation experiments, 16105 IECsfrom VILLIN-HA and wild-type BALB/c mice were co-culturedwith 36105 CFSE-labelled CD4+ T cells enriched from TCR-HAsplenocytes for 5 days. Proliferation was measured by loss ofCFSE dye. Where indicated, 50 ng/ml of a TGFb-blockingantibody or 1 mM RA antagonist LE135 (Tocris, Ellisville,Missouri, USA) or LE540 (Wako, Neuss, Germany) were addedto the co-culture. In some experiments, before co-culture, IECswere incubated with a purified blocking MHC class IImonoclonal antibody (14-4-4S) at 35 mg/ml for 1 h at 4uC.The cells were then washed twice with PBS and resuspended inmedium for co-culture.

Additional methodsAntibodies, flow cytometry, DNA microarrays, CFSE labellingof T cells and real-time reverse transcription-PCR (RT-PCR) aredescribed in the supplementary methods.

StatisticsData are expressed as the mean (SEM) for each group. Student ttest was used to compare groups; p values ,0.05 wereconsidered significant.

RESULTS

Chronic mucosal antigen exposure leads to the development ofFoxp3+ T cells in the peripheryWe recently demonstrated that in VILLIN-HA/TCR-HA trans-genic mice, incomplete thymic deletion of HA-specific (6.5+)CD4+ T cells occurred and that HA-specific CD4+ T cellsinfiltrated into the intestinal mucosa, but failed to induceclinically overt tissue damage.12 To analyse whether this is dueto the induction of Tregs, Foxp3 expression in 6.5+CD4+

transgenic T cells from the MLN of TCR-HA and VILLIN-HA/TCR-HA transgenic mice were analysed by intracellularFACS (fluorescence activated cell sorting) staining. Our dataclearly revealed a specific upregulation of Foxp3 expression in6.5+CD4+ T cells from MLNs of VILLIN-HA/TCR-HA doubletransgenic mice (fig 1A). To test whether these antigen-experienced T cells have suppressive functions in vivo, wemade use of an antigen-specific mouse model for autoimmunediabetes. INS-HA transgenic mice express HA under the controlof the rat insulin promoter. Adoptive transfer of low numbers ofnaive 6.5+CD4+ T cells from TCR-HA transgenic mice into INS-HA/RAG2–/– mice leads to the induction of diabetes within10 days.17 To estimate the suppressive capacity 6.5+CD4+

transgenic T cells from the MLNs of VILLIN-HA/TCR-HAand TCR-HA transgenic mice, cells were sorted by FACS andadoptively transferred into INS-HA/RAG2–/– mice. Adoptivetransfer of naıve 6.5+CD4+ T cells from TCR-HA transgenicmice led to the induction of diabetes as monitored by bloodglucose level. In contrast, the transfer of 6.5+CD4+ T cells fromVILLIN-HA/TCR-HA transgenic mice into INS-HA/RAG2–/–

mice failed to induce diabetes (table 1). To analyse whethernaturally occurring 6.5+CD4+CD25+ T cells from TCR-HAtransgenic mice and 6.5+CD4+ T cells from VILLIN-HA/TCR-HA transgenic mice are able to prevent the development ofdiabetes, co-transfer experiments were performed. Naıve6.5+CD4+ T cells from TCR-HA transgenic mice were trans-ferred either with regulatory 6.5+CD4+CD25+ T cells from TCR-HA transgenic mice or with 6.5+CD4+ cells from VILLIN-HA/TCR-HA transgenic mice intravenously into INS-HA/RAG2–/–

recipient mice. None of the co-transferred INS-HA/RAG2–/–

recipient mice developed diabetes within 20 days (table 1).


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To analyse whether 6.5+CD4+Foxp3+ Tregs developed in thethymus of VILLIN-HA/TCR-HA transgenic mice, or whetherthese cells were induced or expanded in the periphery,lymphocytes were isolated from the thymus, the MLN andthe LP, and stained for the expression of intracellular Foxp3.Interestingly, the increased expression of Foxp3 was onlypresent in 6.5+CD4+ T cells in the periphery of VILLIN-HA/TCR-HA transgenic mice but not in the thymus (fig 1B).

To circumvent further possible thymic development of6.5+CD4+Foxp3+ T cells, adoptive transfer experiments intoVILLIN-HA transgenic mice were performed. Thus, CFSE-labelled 6.5+CD4+ T cells from TCR-HA transgenic mice wereintravenously injected into VILLIN-HA recipient mice and non-transgenic littermates. At day 7 after transfer, cells from theMLN were isolated and proliferation of HA-specific 6.5+CD4+ T

Figure 1 High frequency of haemagglutinin (HA)-specific Foxp3+ T cells in the mesenteric lymph node (MLN) of VILLIN-HA/TCR-HA transgenic mice.(A) Lymphocytes from the MLNs of TCR-HA and VILLIN-HA/TCR-HA transgenic mice were stained for 6.5, CD4 and intracellular Foxp3 expression. Cellswere gated on 6.5 and CD4 expression, and analysed regarding the expression of Foxp3. The number of Foxp3+ T cells is indicated as a percentage. (B)Lymphocytes from the THY (thymus), MLN and LP (lamina propria) of TCR-HA and VILLIN-HA/TCR-HA transgenic mice were stained for 6.5, CD4 andintracellular Foxp3 expression. Cells were gated on 6.5 and CD4 expression, and analysed regarding the expression of Foxp3. The percentage of Foxp3+

T cells is indicated. One representative experiment out of three independent experiments with similar results is shown. Proliferation of adoptivelytransferred HA-specific T cells in the MLNs of VILLIN-HA recipient mice is shown. A total of 16107 carboxyfluorescein succinimidyl ester (CFSE)-labelled CD4+ T cells from TCR-HA transgenic mice were transferred into VILLIN-HA recipient mice and non-transgenic littermates. (C) Seven days later,lymphocytes from the MLN were stained for the expression of CD4. Histograms show the CFSE fluorescence intensity on gated CD4+CFSE+ T cells. (D)Small intestines were isolated and sections were stained with H&E. (E) Lymphocytes from the MLN were stained for the expression of CD4 and Foxp3.Histograms show the expression of Foxp3 vs CFSE fluorescence intensity on gated CD4+CFSE+ T cells. One representative experiment out of threeindependent experiments with similar results is shown.

Table 1 Transfer of haemagglutinin (HA)-specific CD4+ T cells fromTCR-HA and VILLIN-HA/TCR-HA transgenic mice into INS-HA/RAG2–/–

recipient mice

Donor mouse (cell type)No. of transferredcells (6105)

Diabetic recipients(.200 mmol/l*)

TCR-HA (6.5+CD4+) 2.5 9/9

VILLIN-HA/TCR-HA(6.5+CD4+)

2.5 0/3

TCR-HA (6.5+CD4+) + and 2.5 0/3

TCR-HA (6.5+CD4+CD25+) 2.5

TCR-HA (6.5+CD4+) and 2.5 0/3

VILLIN-HA/TCR-HA(6.5+CD4+)

2.5

6.5+CD4+ transgenic T cells from TCR-HA and/or VILLIN-HA/TCR-HA transgenic micewere adoptively transferred into INS-HA/RAG2–/– mice.*Diabetes was monitored by blood glucose measurement.


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cells was determined by loss of CFSE dye. In contrast to6.5+CD4+ T cells isolated from non-transgenic littermates,6.5+CD4+ T cells from VILLIN-HA recipient mice exhibitedstrong proliferation in vivo (fig 1C). Remarkably, no intestinaltissue damage occurred in the VILLIN-HA recipient mice(fig 1D). Analysing the phenotype of the in vivo proliferatingcells, we found that not only Foxp32 but also significantnumbers of Foxp3+ T cells were stimulated in vivo by intestinalepithelial antigen (fig 1E).

Gut-associated DCs induce proliferation of CD4+Foxp3+ T cellsTo examine the role of gut-associated DCs in VILLIN-HA/TCR-HA transgenic mice and VILLIN-HA recipient mice for theinduction or expansion of Foxp3+CD4+ T cells, DCs wereisolated from the MLN and the LP and stained for theexpression of MHC class II, CD80 and CD86. Around 74% ofCD11c+ DCs isolated from the MLN and the LP expressed MHCclass II, 29% of DCs isolated from the MLN and 41% isolatedfrom the LP expressed CD80, and 15% of MLN DCs and 35% ofLP DCs expressed CD86, thus exhibiting the phenotype of aprofessional APC (fig 2A). To investigate whether DCs isolatedfrom the MLN and the LP could present HA associated withapoptotic IECs or shed HA to T cells in vivo, isolated DCs fromVILLIN-HA mice were co-cultured with CFSE-labelled 6.5+CD4+

T cells from TCR-HA transgenic mice without the addition ofexternal HA peptide. In contrast to DCs isolated from wild-typeBALB/c mice, DCs from VILLIN-HA transgenic mice inducedsignificant 6.5+CD4+ T cell proliferation as demonstrated by theloss of CFSE dye after 5 days of co-culture (fig 2B).

As it was demonstrated that DCs which transport apoptoticintestinal epithelial cells into the MLN seem to be important forself-tolerance6 and that gut-associated DCs are able to induce

the differentiation of CD4+Foxp3+ T cells, stimulated 6.5+CD4+

T cells were analysed for Foxp3 expression. Gating onproliferating CD4+ T cells, the intracellular Foxp3 stainingrevealed about 40% Foxp3+ T cells after co-culture with DCsisolated from the MLN of VILLIN-HA mice and 35% Foxp3+ Tcells after co-culture with LP DCs (fig 2C).

Ablation of DCs does not inhibit the expansion of Foxp3+ T cellsin vivoTo investigate the in vivo effect of DC depletion on T cellresponses, we attempted to achieve DC depletion by usingCD11c-DTR mice, in which CD11c+ cells express GFP and theDTR.15 18 VILLIN-HA transgenic mice were crossed to CD11c-DTR transgenic mice and two intraperitonal injections ofdiphtheria toxin resulted in a robust depletion of CD11c+ DCsin the MLN and the LP, as demonstrated by immunohisto-chemistry (fig 3A). In addition, FACS staining for CD11cperformed with cells isolated from the MLN exhibited thedepletion of DCs after injection of diphtheria toxin (fig 3B).Interestingly, depletion of DCs from VILLIN-HA/CD11c-DTRmice did not prevent proliferation of adoptively transferred HA-specific CD4+ T cells in the MLN (fig 3C). In addition, the ratiobetween proliferating Foxp32 and Foxp3+ T cells was compar-able with the ratio detected after adoptive transfer into non-DC-depleted VILLIN-HA/CD11c-DTR mice (fig 3D). Thesedata corroborate that antigen-sampled DCs are not the onlyplayers for the induction or expansion of Foxp3+ T cells in theintestine.

IECs induce the proliferation of CD4+Foxp3+ T cellsIECs may also play an important role in the generation orexpansion of Tregs, because these cells are able to process and

Figure 2 Gut-associated dendritic celss (DCs) from VILLIN-HA transgenic mice stimulate haemagglutinin (HA)-specific T cell proliferation. (A) Cells fromthe mesenteric lymph node (MLN) and the lamina propria (LP) of VILLIN-HA transgenic mice were stained for the expression of CD11c, majorhistocompatibility complex (MHC) class II, CD80 and CD86. Dot plots represent the percentage of MHC class II+, CD80+ and CD86+ cells gated on CD11c+

DCs. (B) DCs from the MLN and the LP of wild-type BALB/c and VILLIN-HA transgenic mice were co-cultured with carboxyfluorescein succinimidyl ester(CFSE)-labelled HA-specific CD4+ T cells isolated from the spleen of TCR-HA transgenic mice for at least 5 days. Cells were harvested and stained for theexpression of CD4 and Foxp3. Histograms show the proliferation of gated CD4+ T cells by the loss of CFSE dye. (C) Dot plots demonstrate the expression ofFoxp3 vs CFSE fluorescence intensity. Gating on proliferating CD4+ T cells, the percentage of Foxp3+ T cells is demonstrated.


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present luminal antigens to T cells in the LP.19 To study theinteraction between IECs and CD4+ lymphocytes, we estab-lished a FACS-based protocol for the isolation of primary IECs(Supplementary fig 1). There was a similar viability betweenIECs isolated from wild-type BALB/c and VILLIN-HA trans-genic mice demonstrated by propidium iodide (data not shown).Affymetrix array analysis of FACS-sorted IECs from VILLIN-HA transgenic mice revealed the typical fingerprint of IECs—that is, strong expression of fatty acid-binding protein and villin(table 2).20 21 In contrast, expression signals of marker moleculesfor haematopoietic cells (CD45), macrophages (CD14) and DCs(CD11c) were defined as absent by Affymetrix software(table 2).

To determine the capacity of IECs to induce specific CD4+ Tcell proliferation, IECs were first stained for expression of MHCclass II and co-stimulatory molecules. In contrast to DCs fromthe MLN and LP, IECs from VILLIN-HA transgenic miceexpressed only low levels of MHC class II (16%), CD80 (9%)and CD86 (9%) (fig 4A). To clarify whether intestinal HAexpression in epithelial cells of VILLIN-HA transgenic mice isefficient to elicit a HA-specific T cell response, IECs from wild-type BALB/c and VILLIN-HA mice were co-cultured with CFSE-labelled 6.5+CD4+ T cells without adding external HA peptide.Surprisingly, we could clearly demonstrate that IECs fromVILLIN-HA but not from wild-type BALB/c mice induced aproliferative response of HA-specific T cells (fig 4B). After5 days of co-culture, 6.5+CD4+ T cells were analysed for Foxp3expression. Strikingly, IECs with low MHC class II, CD80 andCD86 expression were able to stimulate proliferation of bothFoxp32 and Foxp3+ T cells in vitro (fig 4C). The percentage ofFoxp3+ proliferating HA-specific CD4+ T cells was even higherwhen compared with the percentage of Foxp3+ T cells after co-culture with DCs isolated from the MLN and the LP (52% vs40% and 35%). To rule out that co-culture-contaminating DCsare responsible for the proliferation of 6.5+CD4+Foxp3+ T cells,we titrated FACS-sorted DCs from the LP and the spleen to theIEC-6.5+CD4+ T cell co-culture. As demonstrated in fig 4D,titration of DCs as professional APCs increased the proliferativeresponse of HA-specific CD4+ T cells. IECs from VILLIN-HA

mice alone stimulated the proliferation of CD4+Foxp3+ T cells(35%). In contrast, when LP and splenic DCs were included inaddition to the co-culture, the ratio between Foxp3+ and Foxp32

proliferating CD4+ T cells shifted towards proliferation ofFoxp32 and only 20% Foxp3+ T cells (fig 4D).

Proliferation of HA-specific CD4+Foxp3+ T cells is not influencedby TGFb and RATo probe further the mechanism underlying the ability of IECsto expand or induce Foxp3+ T cells efficiently, the function ofTGFb and the vitamin A metabolic RA was investigated.Therefore, the expression of TGFb, and Aldh1a1 and Aldh1a2,which encode retinal dehydrogenases responsible for theconversion of RA, was analysed in IECs in comparison withDCs from the MLN and LP of VILLIN-HA transgenic mice.TGFb expression was nearly absent in IECs in comparison withrobust expression in DCs isolated from the MLN and LP (fig 5A).In addition, inclusion of a blocking monoclonal antibodyagainst TGFb did not significantly influence the proliferationof Foxp3+ T cells in vitro (fig 5B), indicating a negligible impactof TGFb for the observed mechanism of IEC-mediatedCD4+Foxp3+ T cell proliferation.

Figure 3 Dendritic cell depletion doesnot influence haemagglutinin (HA)-specific CD4+ T cell proliferation in vivo. Atotal of 16107 carboxyfluoresceinsuccinimidyl ester (CFSE)-labelled CD4+ Tcells from TCR-HA transgenic mice weretransferred into VILLIN-HA/CD11c-DTRtransgenic recipient mice. One day beforeand 1 day after the transfer, mice wereinjected intraperitoneally with phosphate-buffered saline (+PBS) or diphtheria toxin(+DT). Ablation of CD11c+GFP+ cells wasinvestigated in the mesenteric lymphnode (MLN) and the lamina propria onday 3 after transfer byimmunohistochemistry (A) and FACS(fluorescence ativated cell sorting)staining for CD11c (B). At day 5 afteradoptive transfer, cells from the MLNwere stained for the expression of CD4and Foxp3. (C–D) Dot plots show theproliferation of CD4+Foxp3+ T cells by lossof CFSE dye. One representativeexperiment out of three independentexperiments is shown. GFP, greenfluorescent protein.

Table 2 Gene expression levels of mucosal control genes for the purityof isolated intestinal epithelial cells (IECs)

Cell type/geneControl expression VILLIN-HAArray1/Array2

Classified as present(P) or absent (A) *

Epithelial cells

Fatty acid-binding protein(Fabp2)

23 968/24 610 PP

Villin 8786/7544 PP

Immune cells

CD45 (Prprc) 9/54 AA

CD14 61/58 AA

CD11c (Itgax) 38/11 AA

Control genes of cell types after cell sorting of IECs. Represented is the signal intensityof the indicated gene on Affymetrix MG U74Av2 oligonucleotide arrays. Results arefrom pooled individual mice (n.4).*Present or absent is defined by the Affymetrix software logarithm.


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In contrast to Aldh1a1, which is only expressed in IECs, theexpression of Aldh1a2 was particularly expressed in gut-associated DCs (fig 5A). To determine further the function ofRA in IEC-mediated stimulation of CD4+Foxp3+ T cells, IECsfrom VILLIN-HA mice were co-cultured with CFSE-labelled6.5+CD4+ T cells in the presence of the RA receptor antagonistsLE135 and LE540. In contrast to data which demonstrate thatRA inhibition blocks the induction of CD4+Foxp3+ T cell bymucosal DCs, neither LE135 nor LE540 inhibits the proliferationof HA-specific CD4+Foxp3+ T cells after co-culture with IECsfrom VILLIN-HA transgenic mice (fig 5C). In addition, co-culture of IECs and CFSE-labelled 6.5+CD4+ T cells providedwith exogenous RA did not influence the proliferation of Foxp3+

T cells (Supplementary fig 2). Therefore, the mechanism bywhich IECs induce or expand Tregs is independent of RA.

Phenotype of proliferating HA-specific CD4+ T cellsTo characterise the phenotype of HA-specific CD4+ T cells afterIEC-mediated stimulation, IECs from VILLIN-HA mice were co-cultured with CFSE-labelled 6.5+CD4+ T cells. ProliferatingCD4+ T cells were stained for marker molecules specific forregulatory T cells (Foxp3, CD103, PD-1, LAG3 and GITR) and a

set of cytokines (interleukin 10 (IL10), interferon c (IFNc), IL17and IL2) (Supplementary fig 3). Whereas PD-1 and LAG3 arestrongly expressed by CD4+Foxp3- T cells, CD103 and GITRexpression is specifically upregulated by proliferatingCD4+Foxp3+ T cells (Supplementary fig 3B). After re-stimula-tion with PMA(phorbol 12-myristate 13-acetate)/ionomycin,CD4+Foxp3+ and CD4+Foxp32 proliferative T cells expressedvery low levels of IL10 and IL2 (Supplementary fig 3C). Incontrast, 7.7% of CD4+Foxp3+ T cells expressed IFNc, and even18.9% of CD4+Foxp32 T cells. Very interestingly, nearly noCD4+Fopx3+ T cells produced IL17 (0.7%), in contrast to 7.0% ofthe CD4+Fopx32 T cells (Supplementary fig 3C). The pheno-typic characterisation clearly revealed the proliferation of twodistinct T cell populations at the same time, CD4+Foxp32

effector T cells on the one hand and CD4+Foxp3+ Tregs on theother hand.

Stimulation of HA-specific CD4+ T cells is mediated by MHCclass IIThe prerequisite for the interaction of APCs and CD4+ T cells isthe expression of MHC class II molecules on the surface of APCs.To assess the contribution of MHC class II to IEC-mediated

Figure 4 Intestinal epithelial cells (IECs) from VILLIN-HA transgenic mice stimulate haemagglutinin (HA)-specific T cell proliferation (A) IECs from thesmall intestine of VILLIN-HA transgenic mice were stained for the expression of MHC class II, CD80 and CD86. Dot plots represent the percentage ofMHC class II+, CD80+ and CD86+ cells. (B) IECs from wild-type BALB/c and VILLIN-HA mice were co-cultured with carboxyfluorescein succinimidylester (CFSE)-labelled HA-specific CD4+ T cells for at least 5 days. Cells were stained for the expression of CD4 and Foxp3. Histograms showproliferation of gated CD4+ T cells by the loss of CFSE dye. (C) The dot plot demonstrates the expression of Foxp3 vs CFSE fluorescence intensity.Gating on proliferating CD4+ T cells, the percentage of Foxp3+ T cells is demonstrated. One representative experiment out of five independentexperiments with similar results is shown. (D) IECs were co-cultured with CFSE-labelled HA-specific CD4+ T cells in the absence or presence of theindicated amounts of lamina propria (LP) or splenic (SP) dendritic cells (DCs) for at least 5 days. Cells were harvested and stained for the expression ofCD4 and Foxp3. Dot plots demonstrate the proliferation of HA-specific CD4+ T cells. Histograms show the expression of Foxp3 gated on proliferatingcells. One representative experiment out of three independent experiments is shown.


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stimulation of CD4+ T cells, isolated IECs were preincubatedwith an MHC class II-blocking monoclonal antibody and co-cultured with CFSE-labelled 6.5+CD4+ T cells. Preincubation ofIECs with MHC class II-blocking antibody, but not with anisotype control, significantly decreased the proliferation of HA-specific CD4+ T cells (fig 6A). No changes in the ratio betweenFoxp32 and Foxp3+ T cells were detectable (data not shown).However, it is still unclear whether IEC–CD4+ T cell interac-tions in our model led to either induction or expansion ofFoxp3+ regulatory T cells. To clarify this issue, the IEC–CD4+ Tcell interaction experiments were repeated with 6.5+CD4+ Tcells from TCR-HA transgenic mice on a recombinant activatinggene 2-deficient background (TCR-HA/RAG2–/–) which do notcontain Foxp3+ T cells. IECs from wild-type BALB/c andVILLIN-HA transgenic mice were co-cultured with CFSE-labelled 6.5+CD4+ T cells from TCR-HA or TCR-HA/RAG2–/–

transgenic mice. As depicted in fig 6B, IECs from VILLIN-HAtransgenic mice are able to stimulate 6.5+CD4+ T cellsindependently of their origin. However, only co-culture ofIECs with 6.5+CD4+ T cells from TCR-HA transgenic mice andnot from TCR-HA/RAG2–/– mice leads to the proliferation ofFoxp3+ T cells, demonstrating the active expansion and not theperipheral induction of antigen-specific Tregs by IECs.

DISCUSSIONRecent data illustrate that antigen-specific Tregs play animportant role in downregulating antigen-specific T cellresponses.22 23 Different types of naturally occurring and inducedTregs

1–4 have been described as being responsible for controllingintestinal inflammation. In a previous study, we could showthat the expression of HA in IECs of VILLIN-HA mice and theconcomitant expression of an a/b-TCR which recognises anMHC class II-restricted epitope of HA, leads to a mild form ofchronic intestinal inflammation. In this model, inflammation ispartially controlled by regulatory mechanisms.12 In the presentstudy, intracellular FACS staining clearly demonstrated aspecific upregulation of Foxp3 in HA-specific CD4+ T cells fromVILLIN-HA/TCR-HA mice. Previous observations in severalTCR transgenic mouse autoreactivity models suggest that high-avidity interactions between autoreactive thymocytes andthymic radioresistant APCs expressing the agonist ligand canresult in both deletion of specific thymocytes and an increase inthe proportion of Foxp3+ Tregs expressing the transgenic TCR.24–

26 Interestingly, the increased expression of Foxp3 in theVILLIN-HA/TCR-HA transgenic mice was restricted to theperiphery and not detectable in the thymus. This stronglysupports the idea that chronic mucosal antigen exposure

Figure 5 Intestinal epithelial cells (IECs) induce the expansion of Foxp3+ T cells independently of transforming growth factor b (TGFb) and retinoicacid (RA). (A) CD11c+ cells were sorted from the mesenteric lymph node (MLN) and the lamina propria (LP), and IECs were sorted from the smallintestine of VILLIN-HA transgenic mice. TGFb, Aldh1a1 and Aldh1a2 expression was assayed by quantitative PCR and normalised relative to expressionof RPS9. Data shown are representative of three independent experiments. *p,0.05. IECs from wild-type BALB/c and VILLIN-HA transgenic mice wereco-cultured with carboxyfluorescein succinimidyl ester (CFSE)-labelled haemagglutinin (HA)-specific CD4+ T cells. Where indicated, wells wereadditionally supplemented with 50 ng/ml a-TGFb (B) or 1 mM of the RA receptor antagonist LE135 or LE540 (C). After 5 days of culture, cells wereharvested and stained for the expression of CD4 and Foxp3. For proliferation, dot plots are gated on CD4+ T cells. The percentage of Foxp3+ T cells isgated on proliferating cells. Data are representative for at least two independent experiments.


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may lead to the expansion of Tregs via peripheral activation invivo.

For proper T cell activation, antigen must be presented to theTCR complex in the context of MHC molecules and involvesthe interaction of co-stimulatory molecules such as CD80 andCD86 on the surface of APCs with their counter-receptorsCD28 and CLTA-4 on the T cells.27 Cross-priming by DCs isconsidered to play an important role in initiating MHC class II-restricted immune responses, including T cell activation andtolerance.28 Huang et al6 demonstrated that DCs couldendocytose and transport apoptotic IECs to the T cell area inthe MLN and present the antigen in the context of MHC class Ior II. These DCs were implicated in tolerance induction. Inaddition, very recently, Su et al and Coombes et al7 8 demon-strated that gut-associated DCs are able to induce Foxp3+ Tregs

via TGFb and RA. In line with these findings, DCs isolated fromVILLIN-HA transgenic mice were able to take up shed HAantigen or apoptotic IECs and migrate to the MLN as theystimulated HA-specific Foxp32 and Foxp3+ T cell proliferationwithout adding external peptide in vitro. However, in vivodepletion of DCs from VILLIN-HA transgenic mice did notprevent the proliferation of Foxp32 and Foxp3+ HA-specific Tcells in vivo, suggesting other components in the intestinal APCnetwork for the maintenance of immune tolerance in the gut.

IECs were initially believed to be exclusively involved in theabsorptive process of digestion. However, IECs are a major pointof contact for enteric antigens and may therefore play a moredirect role in mucosal immunity, particularly in regulating T cellimmunity. The ability of IECs constitutively to expressmolecules involved in antigen presentation, including MHCclass I and II, further emphasises this hypothesis.10 11 Lack of/orweak expression of co-stimulatory molecules on IECs couldresult in anergy of T cells. However, here we demonstrated astrong stimulatory capacity of primary IECs isolated fromVILLIN-HA transgenic mice when co-cultured with HA-specificCD4+ T cells.

It has been established for human CD8+ T cells that IECs areable to activate different subsets of CD8+ T cells.29 Inalloreactions with human IECs and CD8+ T cells, the prolifera-tion of CD8+CD28+ effector T cells and CD8+CD282 Tregs wasdetected. The interaction of CD4+ T cells and IECs has not yetbeen studied in detail. Using IECs from VILLIN-HA transgenicmice as APCs for the presentation of the HA self-antigen, weshowed that IECs induced the proliferation of both Foxp32 andFoxp3+ HA-specific T cells. In addition, the stimulation of CD4+

T cell proliferation by IECs was dependent on MHC class II, asblocking of this molecule leads to a reduced proliferation of HA-specific CD4+Foxp32 and Foxp3+ T cells. The expansion ofeffector cells and Tregs with the same antigen specificity by IECsat the same time might explain the permanent balance betweenimmunity and tolerance reflected by the continuous low-gradeinflammation as one hallmark of the intestinal mucosa. In IBDthis orchestrated balance is dysregulated due to an uncontrolledpathogenic CD4+ T cell effector response triggered by endogen-ous and exogenous factors. This is in line with very recentlypublished data, which demonstrated that IECs from patientssuffering from IBD preferentially stimulate CD4+ T cellproliferation with a high level of secretion of IFNc.30

Currently, it is becoming clear that nutrient status caninfluence intestinal homeostasis—that is, vitamin A, and inparticular its transcriptionally active metabolite, RA, has beenshown to enhance the expression of gut homing receptors on Tcells.31 Based on the abundance of RA-synthesising enzymes (ie,retinal dehydrogenase, Aldh1) present in IECs and certainresident DC populations, the gut is a significant producer ofRA.31 New studies support the idea that the catalysis of vitaminA into RA in gut-associated DCs enhances the TGFb-dependentconversion of naıve T cells into Tregs. Interestingly, wedemonstrate here that gut-associated DCs express the isoformAldh1a2, whereas IECs mainly express Aldh1a1. In addition, incomparison with gut-associated DCs, IECs express negligibleamounts of TGFb. Furthermore, blocking experiments with RA

Figure 6 Effect of major histocompatibility complex (MHC) class II blockade on intestinal epithelial cell (IEC)–T cell interaction. IECs from wild-typeBALB/c and VILLIN-HA mice were incubated with anti-MHC class II-blocking antibody or with an isotype control, washed, and co-cultured withcarboxyfluorescein succinimidyl ester (CFSE)-labelled haemagglutinin (HA)-specific CD4+ T cells from TCR-HA transgenic mice for at least 5 days. Cellswere stained for the expression of CD4. Proliferation is demonstrated as the loss of CFSE on gated CD4+ T cells. Data are representative for at least twoindependent experiments. (B) IECs induce the expansion of CD4+Foxp3+ T cells. IECs from wild-type BALB/c and VILLIN-HA mice were co-cultured withCFSE-labelled HA-specific CD4+ T cells from TCR-HA and TCR-HA/RAG2–/– transgenic mice for at least 5 days. Cells were harvested and stained for theexpression of CD4 and Foxp3. Data are representative for at least two independent experiments.


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antagonists and TGFb-blocking antibodies did not influence thestimulatory capacity of IECs for CD4+Foxp3+ T cell prolifera-tion. These differences in the expression pattern could explainthe fact that gut-associated DCs are able to convert Foxp32 Tcells into Foxp3+ T cells, whereas IECs are important for theexpansion of pre-existing CD4+Foxp3+ T cells.

In summary, the results of this study indicate for the first timethat IECs play a direct role in maintaining the normal state oftolerance in the mucosal immune system due to their abilityactively to expand regulatory CD4+Foxp3+ T cells. In the contextof chronic mucosal inflammation, this might prevent progressiontoward severe intestinal autoimmunity. These results suggestthat strategies that focus on the therapeutic expansion of Tregs inthe treatment of patients suffering from IBD should consider thedirect IEC–T cell interaction as a new target.

Acknowledgements: This work was supported by a grant from the DeutscheForschungsgemeinschaft to AMW (WE 4472/1-1).

Competing interests: None.

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doi: 10.1136/gut.2008.151720 2009 58: 211-219 originally published online October 2, 2008Gut

A M Westendorf, D Fleissner, L Groebe, et al. local dendritic cellsintestinal epithelial cells independent ofinduced by antigen-driven interaction with

regulatory T cell expansion+Foxp3+CD4

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