ava i l ab l e a t www.sc i enced i r ec t . com

C l i n i ca l Immuno logy

www.e l sev i e r . com / l o ca t e / y c l im

Clinical Immunology (2012) 144, 87–97

β7 integrin controls immunogenic and tolerogenicmucosal B cell responsesAngela Schippers a, Annika Kochut b, Oliver Pabst c, Ursula Frischmann b, d,Thomas Clahsen a, Klaus Tenbrock a, Werner Müller b, e, Norbert Wagner a,⁎

a Department of Pediatrics, Medical Faculty, RWTH Aachen University, Aachen D-52074, Germanyb Department of Experimental Immunology, Helmholtz Centre for Infection Research, Braunschweig D-38124, Germanyc Institute of Immunology, Hannover Medical School, Hannover D-30625, Germanyd ZIK Septomics, Faculty of Medicine, Jena University, Jena D-07743, Germanye Faculty of Life Sciences, University of Manchester, Manchester M13 9PT, UK

Received 23 March 2012; accepted with revision 17 May 2012

Abbreviations APC, antigen presentDTH, delayed type hypersensitivity;MAdCAM-1, mucosal addressin cell anitrophenyl-chickenglobulin; OVA, ovaadhesion molecule 1; WT, wildtype.⁎ Corresponding author at: Departme

8082492.E-mail address: nwagner@ukaachen

1521-6616/$ - see front matter © 2012doi:10.1016/j.clim.2012.05.008

KEYWORDSIgG;IgA;Lymphocyte migration;Intestine;Tolerance;β7 integrin

Abstract IgA production in the gut-associated lymphoid tissue represents a pivotal defensemechanism against luminal pathogens. The other important challenge for the GALT is theinduction of local and systemic hyporesponsiveness (tolerance) to dietary antigens and luminalbacterial flora to prevent allergies or deleterious immunologic reactions to food orenvironmental antigens. In this study we analyzed the impact of β7 integrin on immunogenicand tolerogenic B cell responses in the gastrointestinal tract.β7 integrin deficient mice failed to mount a normal intestinal IgA response to ovalbumin and choleratoxin,whereas the IgG responsewas unchanged in comparison to controlmice. Oral B cell tolerance to

ovalbumin, measured as the suppression of specific serum IgG responses, did not develop in theabsence of β7 integrin. After adoptive transfer of spleen cells from β7 integrin +/+ mice into RAG-2deficient or RAG-2/β7 integrin double deficientmice, only RAG-2 deficientmicewere able to developoral B cell tolerance. These observations suggest that β7 integrin expression on cells of the innateimmune system contributes to the critical role of β7 integrin in the process of B cell tolerance.© 2012 Elsevier Inc. All rights reserved.

ing cell(s); ASC, antibody secreGALT, gut-associated lymphoidhesion molecule-1; NK, natulbumin; PC, plasma cells; pD

nt of Pediatrics, RWTH Aachen

.de (N. Wagner).

Elsevier Inc. All rights reserve

ting cell(s); CT, cholera toxin; LP, lamina propria; DC, dendritic cell(s);d tissue; GIT, gastrointestinal tract; IEL, intraepithelial lymphocyte;ral killer cell(s); NP, 4-hydroxy-3-nitrophenyl; NP-CG, 4-hydroxy-3-C, plasmacytoid DC; Treg, regulatory T cell(s); VCAM-1, vascular cell

University, Pauwelsstrasse 30, D-52074 Aachen, Germany. Fax: +49 241

d.

88 A. Schippers et al.

1. Introduction

The epithelium of the gastrointestinal tract (GIT) is thelargest surface of the body and is continuously exposed topathogens as well as harmless nutritive antigens. Differen-tiation of harmful from harmless antigens, leading to theinitiation of either protective immunity or tolerogenicresponses is particularly important in the GIT. Disturbancesof immunological homeostasis in the gut, e.g. resulting fromexcessive immune reactions to innocuous antigens ordeficiency of IL-2, IL-10 or TGF-β will eventually lead toinflammatory bowel disease [1]. The specific induction oflocal and systemic hyporesponsiveness to soluble antigensthrough previous exposure to the respective antigen via theoral route is termed oral tolerance [2,3]. Intestinal toler-ance can also influence immune responses outside the gut,e.g. by preventing immune responses like DTH, lymphocyteproliferation and the production of antibodies [2,3]. It is amultifactorial process thought to involve mechanisms likeimmune deviation, anergy/deletion of effector T cells [4,5]as well as active suppression by regulatory T (Treg) cells [6].There is also evidence that antigen presenting cells (APC)like dendritic cells (DC) [7–9], plasmacytoid DC (pDC)[10,11] and macrophages [12] help to generate tolerancewhile mediating inflammatory responses against harmfulpathogens.

Directed migration of immune cells is a prerequisite formaintaining the functional integrity of gut-associated intes-tinal tissue (GALT) and establishing either immunogenic ortolerogenic immune responses [13]. This directed migrationis secured by the surface expression of a defined repertoireof adhesion molecules and chemokine receptors thatrecognize their respective counter receptors or chemokineson endothelial cells.

Integrins are heterodimeric transmembrane cell adhesionreceptors consisting of noncovalently associated α and βchains [14]. The β7 integrin chain associates with either theα4 or the αE (CD103) chain. α4β7 integrin is expressed atlow levels on naïve T and B cells, NK cells, stimulatedmonocytes, macrophages, DC, eosinophils, most CD8+ recentthymic emigrants, and at a high level on memory oractivated gut-homing CD4+ T cell subsets, and IgA-secretingplasma cells (PC) [15]. αEβ7 is expressed by most intrae-pithelial lymphocytes, but only a minority of lamina propria(LP) lymphocytes and circulating blood lymphocytes [16]. Itis also expressed on some CD4+ T cells (e.g. a population ofnatural Treg cells), CD8+ cells, mast cells, and a subset of DC[7,17,18]. α4β7 integrin directs the migration of lympho-cytes into the draining gut MLN and lamina propria (LP),mainly via interaction with its endothelial ligand MAdCAM-1(mucosal addressin cell adhesion molecule-1) [19], whileαEβ7 integrin facilitates retention of lymphocytes in the gutepithelium via E-cadherin [20]. Previously, we have revealeda crucial role of the adhesion molecule β7 integrin in theformation of GALT [19]. Studies performed with β7 integrindeficient mice or with antibodies targeting β7 integrinsindicate that β7 integrins are involved in the pathogenesis ofgut inflammation [21,22], protective immunity against mucosalpathogens [23,24], intestinal graft-versus-host disease [25],and also the pathogenesis of experimental autoimmuneencephalomyelitis [26].

We have recently shown that abrogated oral T celltolerance in a delayed type hypersensitivity (DTH) modelcan be a direct consequence of the impaired recirculation ofβ7 integrin deficient FoxP3+ Treg into the intestine [27].Here we concentrate on a detailed evaluation of the impactof β7 integrin on humoral immune responses, particularlywith regard to oral B cell tolerance.

2. Materials and methods

2.1. Mice

10 to 12 week-old β7 integrin deficient mice (C57BL/6-Itgbtm1Cgn/J fl, [19]), age-matched C57BL/6 J mice (WT),RAG-2tm/J mice [28], and β7 integrin Δ/Δ, RAG-2 Δ/Δ doublemutant mice were used in the experiments. The animalswere bred at the Helmholtz Centre for Infection Researchand at the RWTH Aachen under specific pathogen-freeconditions. All experiments were approved by the LocalInstitutional Animal Care and Research Advisory Committeeand authorized by the local government authority.

2.2. Histology and flow cytometry

For cryosections, small intestines were embedded in OCTfreezing medium on dry ice and 10 μm sections wereprepared. Air-dried sections were fixed and subsequentlystained with a mixture of fluorescent dye-coupled anti-bodies containing rat–anti-mouse–IgA–FITC (Caltag Labora-tories), rat–anti-mouse–CD3–CY5 (Clone 17A2), and DAPI inTBST with 2% rat serum. Pictures were processed withPhotoImpact software (version 10.0 SE, Ulead Systems).

To calculate the numbers of pDC by FACS analysis single‐cell suspensions of the intestinal IEL compartment werestained with the following antibodies: CD11c-PE-Cy7(eBioscience), pDCA-1-APC (eBioscience), and F4/80-Paci-ficBlue (Serotec) and analyzed on a FACS Canto-II (BDBiosciences). CD11c+PDCA-1+F8/80− cells in the lymphocytegate were considered as pDC.

2.3. Immunization protocols

CT: Mice were orally immunized by intragastric administra-tion of 10 μg/ml CT on days 0 and 18.

NP-CG: Mice were systemically immunized by intraperi-toneal (i.p.) injection of 100 μg alum-precipitated NP-CG.20 days later, mice were immunized by a second i.p.injection of 10 μg NP-CG.

OVA: (A) Immunization: Mice were systemically immu-nized by i.p. injection of 100 μg alum-precipitated OVA.16 days later mice were orally immunized by intragastricadministration of 5 mg OVA. (B) Tolerization: Mice receivedeither intragastric administration of 5 mg OVA on days 0, 1,and 3, or drinking water supplemented with 10 mg/ml OVAfor 6 days. On day 6, mice were systemically immunized byi.p. injection of 100 μg alum-precipitated OVA, followed bya second i.p. injection of 100 μg OVA on day 28.

At different time points CT-specific, NP-specific, or OVA-specific IgA and IgG titers from serum and intestinal washwere determined by ELISA, and CT-specific, NP-specific, or

89Humoral immune responses in the gut of β7 integrin –mice

OVA-specific IgA ASC and total IgA ASC in the LP and spleenby ELISPOT.

2.4. ELISA and ELISPOT analysis

For determination of specific IgA or IgG, microtest plateswere coated with either 1 μg/ml CT, or 10 μg/ml NP14-BSA,or 50 μg/ml OVA, and appropriate dilutions of samples fromserum or intestinal wash were added (1:50–1:6400). Afterwashing, HRP-labeled goat–anti-mouse IgA (M31007, Caltag)or rat-anti-mouse IgG (H+L, cat. # 415-035-166, Jackson)followed by o-phenylenediamine dihydrochloride was usedfor detection. The reaction was terminated by adding 1 NHCl, and OD was determined with a microplate reader(Sunrise, Tecan) set at 492 nm. IgG standard was monoclonalanti-OVA mouse IgG1, Sigma, A-6075.

CT-specific, NP-specific or OVA-specific IgA, IgG, and totalIgA ASC were identified by ELISPOT, following the protocolprovided by BD Biosciences. Briefly, dilutions of single-cellsuspensions prepared from spleen or lamina propria ofimmunized or untreated mice were cultured overnight onhydrophobic PVDFmembrane 96-well plates (Multiscreen HTS-IP, Millipore) coated either with CT (1 μg/ml), NP14-BSA(10 μg/ml), ovalbumin (50 μg/ml), or antimouse IgA (Clone11-44-2, eBioscience, 5 μg/ml PBS). After 16 h the cells wereremoved and the captured IgA was detected with biotinylatedgoat anti-mouse IgA (B2766, Sigma) or goat anti-mouse IgG (Fcspecific, B 9904, Sigma), followed by SA-HRP (cat. # 189733,1 μg/ml, Calbiochem) treatment. The plates were devel-oped with a chromogenic substrate solution containing AEC((3-Amino-9-Ethylcarbazole) Sigma). The spots werescanned automatically using an ELISPOT reader (CellularTechnology Ltd.) and ImmunoSpot image analyzer software(version 3.2).

2.5. Adoptive cell transfer

Single-cell suspensions were prepared from the spleens ofdonor C57BL/6J (WT) mice and 8×107 cells were transferredi.p. into β7 integrin Δ/Δ, RAG-2 Δ/Δ double mutant andRAG-2 −/− recipient mice.

2.6. Statistical analysis

Statistical analysis was performed with GraphPad Prismsoftware. All significant values were determined with thenon-paired two-tailed t-test.

3. Results and discussion

3.1. Normal Ig levels in β7 integrin deficient mice

β7 integrin deficient mice exhibit reduced numbers of IgA‐secreting PC in the LP of the small intestine when comparedwith wildtype (WT) mice as detected by histology andquantified by ELISPOT (Figs. 1A and B), which corroboratesour earlier histological observations [19]. In contrast, β7integrin deficient mice display significantly higher numbersof these cells in the spleen (Fig. 1C) when compared tocontrol mice. This suggests that β7 integrin deficiency does

not lead to an overall reduction in the number of IgA‐secreting PC.

To test whether the reduced number of IgA-secreting PCin the intestine of β7 integrin deficient mice translates intoreduced serum IgA levels, we compared the levels ofimmunoglobulins in WT and β7 integrin deficient mice.There was no significant difference detectable in the serumlevels of IgG1, IgG2a, IgG2b, IgG3, IgE, IgM, and IgA (data notshown) indicating that under non-inflammatory conditionsβ7 integrin is dispensable for the synthesis of regularamounts of immunoglobulins, including IgA.

3.2. Lack of IgA response after mucosal challenge ofβ7 integrin deficient mice

Next we examined whether β7 integrin deficient and WTmice exhibit differences in the induction of an antigen-specific IgA response after oral immunization using a T celldependent antigen. Cholera toxin (CT) is a potent T celldependent immunogen and intestinal adjuvant in mice and isknown to stimulate strong IgA responses [29]. We immunizedβ7 integrin mutants and control mice twice at an interval of18 days with an oral dose of 10 μg CT (Fig. 2). 35 days afterthe first application we determined the level of CT-specificIgA in the intestinal wash, CT-specific IgA and IgG in serum,and the number of CT-specific IgA+ cells in the intestinal LPand spleen. Whereas control mice secreted substantial titersof CT-specific IgA in blood (Fig. 2A) and intestine (Fig. 2B),this response was markedly reduced in the β7 integrindeficient mice. The lower level of CT-specific IgA in theintestine of β7 integrin deficient mice was accompanied by areduction in the number of CT-specific IgA+ cells in the LP ofthese mice (Fig. 2C). This finding is in accordance with thereduced specific IgA response and PC migration observed onoral immunization in MAdCAM-1 deficient mice [30] and thedefective specific IgA reaction to rotavirus noticed in β7integrin deficient mice [24]. It also supports the hypothesisthat the interaction of α4β7 integrin with MAdCAM-1 iscritical for the migration of primed plasma cells into the LP.Most IgA‐secreting resident PC in the LP express α4β7integrin [31]. Therefore, disturbed PC migration fromsecondary lymphoid organs to the LP effector sites mostprobably contributes to the impaired mucosal IgA responseor β7 integrin deficient mice upon immunization. Inaddition, impaired T cell help, caused by reduced T cellhoming in β7 integrin deficient mice [32], could account forthe observed impaired IgA responses to the different T celldependent antigens. In contrast, following oral immuniza-tion the two mouse strains showed comparable serum levelsof CT-specific IgG (Fig. 2D) and CT-specific IgA+ cells in thespleen (Fig. 2E).

3.3. Normal IgG response after systemic challengeof β7 integrin deficient mice

To evaluate whether the disturbed GALT in β7 integrindeficient mice affects the immune response to systemicantigen challenge, systemic (i.p.) primary and secondaryimmunizations with the T cell dependent antigen (NP-CG(4-hydroxy-3-nitrophenyl-chickenglobulin) were performedas indicated (Fig. 3). NP-specific serum IgG titers measured

90 A. Schippers et al.

before the secondary immunization (day 20) and at the endof the experiment (day 35) were high and no different inWT and β7 integrin deficient mice (Fig. 3A). In addition, thetwo mouse strains displayed comparable numbers of NP-specific IgG+ cells in the spleen (Fig. 3B). These resultsindicate that β7 integrin is dispensable for systemicallyinduced IgG responses. By contrast, NP-specific IgA re-sponses in β7 integrin deficient mice were markedlydecreased, as shown by decreased levels of NP-specificIgA in serum (Fig. 3C) and in intestinal wash (Fig. 3D), andby the lower number of NP-specific IgA‐secreting cells inthe LP (Fig. 3E).

3.4. Impaired IgA response aftermucosal challenge ofprimary i.p. immunized β7 integrin deficient mice

To test the secondary mucosal immune response, weperformed primary i.p. immunizations with ovalbumin(OVA), followed by a secondary immunization through gastricfeeding on day 18 (Fig. 4). At day 34, 16 days after thesecondary immunization, WTmice showed high serum titers ofOVA-specific IgA, while the response of β7 integrin deficientmice was significantly reduced (Fig. 4A). In contrast, bothmouse strains already displayed high titers of OVA-specificserum IgG after the first systemic immunization, which did not

Figure 1 Altered distribution of PC in β7 integrin deficient micedeficient (β7 Δ/Δ) mice stained with anti-IgA antibody (green), antiScale bars=100 μm. (B and C) Quantification of total IgA‐secreting Pthree separate experiments of 6 WT (black bars) and 5–8 β7 Δ/Δ (gr(C) with mean±SEM. P values were calculated using the non-paired

increase any further after a second oral immunization(Fig. 4B). Thus, β7 integrin appears to be necessary to mountan IgA response following oral immunization even afterprimary systemic immunization, while it is dispensable forthe systemic IgG response to OVA.

3.5. Lack of oral tolerance in β7 integrindeficient mice

Since the IgA immune response to oral antigen challenge isabsent in β7 integrin deficient mice, we were alsointerested in the impact of β7 integrin on the developmentof oral B cell tolerance. To induce oral tolerance, β7 integrindeficient mice and WT mice received three oral doses of5 mg OVA without any adjuvant (days 0, 1, and 3) (Fig. 5). Onday 6 mice were primed i.p. with OVA coupled to theadjuvant aluminum hydroxide followed by a secondary i.p.immunization with OVA on day 28. On day 35 aftertolerization β7 integrin deficient mice exhibited high anti-OVA IgG titers in serum, while control mice developed B celltolerance (Fig. 5A). This was accompanied by a highernumber of OVA-specific IgG+ cells in the spleen, as detectedby ELISPOT analysis at the end of the experiment (Fig. 5B).By contrast, when fed with PBS instead of OVA prior toimmunization, both mouse strains showed robust anti-OVA

. (A) Cryosections from small intestines of WT and β7 integrin-CD3 antibody (red), and DAPI for visualization of nuclei (blue).C by ELISPOT. The figure shows one representative example ofey bars) mice. Enumeration of IgA PC from the LP (B) and spleentwo-tailed t-test.

Figure 2 Lack of IgA response after mucosal challenge of β7 integrin deficient mice. WT and β7 integrin deficient (β7Δ/Δ) micewere immunized by two oral doses of CT, as indicated. 35 days after the first immunization, the levels of CT-specific IgA and IgGwere measured in equally diluted samples of intestinal washes and sera by ELISA, and CT-specific ASC was quantified by ELISPOT.The ELISA data show the mean OD. Results with means±SEM are the averages of 6 mice per group and are representative of threeexperiments. P values were calculated using the non-paired two-tailed t-test. (A) CT-specific IgA in serum. (B) CT-specific IgA inintestinal wash. (C) CT-specific IgA‐secreting cells in LP. (D) CT-specific IgG in serum. (E) CT-specific IgA‐secreting cells in spleen.

91Humoral immune responses in the gut of β7 integrin –mice

IgG titers (Fig. 5A) and displayed increased numbers of OVA-specific IgG+ cells in the spleen (Fig. 5C). This indicates thatdevelopment of oral B cell tolerance is dependent on β7integrin.

Recently, we have demonstrated, that the β7 integrin-MAdCAM-1 mediated homing of FoxP3+ Treg cells into the LPrepresents an essential step for the induction of intestinaltolerance in a T cell mediated DTH model [27]. Mice

deficient in β7 integrin or MAdCAM-1 were impaired intheir capacity to suppress DTH responses. Whereas adoptivetransfer of OTII cells expressing an OVA‐specific T cellreceptor prior to the induction of tolerance attenuated DTHresponses in β7 integrin deficient mice, this did not preserveoral tolerance induction in MAdCAM-1 deficient mice. Thisfinding indicates that α4β7 integrin-MAdCAM-1 mediated guthoming of Treg is an important step for the induction of oral

Figure 3 Normal IgG but impaired IgA response after systemic challenge of β7 integrin deficient mice. WT and β7 integrin deficient(β7Δ/Δ) mice were i.p. immunized by two consecutive doses of NP-CG-alum and NP-CG, as indicated. Levels of NP-specific IgA and IgGwere measured in equally diluted samples of intestinal washes and sera by ELISA, and quantification of NP-specific ASC was performedby ELISPOT at the start (d0), 20 days, and 35 days after the first immunization. The ELISA data shown are the mean OD. Results are theaverages with mean±SEM of 5–8 mice per group and are representative of three experiments. P values were calculated using the non-paired two-tailed t-test. (A) NP-specific IgG in serum. (B) NP-specific IgG secreting cells in spleen. (C) NP-specific IgA in serum. (D)NP-specific IgA in intestinal wash. (E) NP-specific IgA‐secreting cells in lamina propria (LP).

92 A. Schippers et al.

T cell tolerance, eventually leading to the suppression ofDTH responses. The fact that oral tolerance could berestored in β7 integrin deficient mice by cell transfer alsosupports the hypothesis that the observed impairment inthe induction of oral tolerance cannot be attributed to the

severely reduced PP of these mice. This result is in linewith observations showing that in the presence of MLN,which are not altered by β7 integrin deficiency, Peyerspatches are not required for the induction of oral tolerance[33].

Figure 4 Reduced secondary IgA response after mucosal challenge of initially systemic immunized β7 integrin deficient mice. WTand β7 integrin deficient (β7Δ/Δ) mice were immunized, first i.p. and then orally, by two consecutive doses of OVA-alum and OVA, asindicated. At the beginning, before the second immunization (d18), and at the end of the experiment (d34), the levels of OVA-specificIgA and IgG were measured in equally diluted serum samples by ELISA. The ELISA data shown are arbitrary units for IgA and μg/ml forOVA-specific IgG. Results with mean±SEM, are the averages of 4–5 mice per group and are representative of two independentexperiments. P values were calculated using the non-paired two-tailed t-test. (A) OVA-specific IgA in serum. (B) OVA-specific IgG inserum.

93Humoral immune responses in the gut of β7 integrin –mice

3.6. Oral B cell tolerance requires β7 Integrinexpression on cells of the innate immune system

Although it seems likely that the incapability of β7 integrindeficient mice to establish oral B cell tolerance, i.e. tosuppress OVA-specific IgG responses following an OVA feedalso arises from the disturbed migration of α4β7 integrindeficient T cells, this does not rule out other contributions ofβ7 integrin towards tolerance induction. The observationthat the impairment in oral T cell tolerance detected inMAdCAM-1 deficient mice is less pronounced than theimpairment in β7 integrin deficient mice supports this idea[27]. In addition to the α4β7 integrin–MAdCAM-1 interac-tions, which are required for gut homing of lymphocytes,αEβ7 integrin dependent interactions could also contributeto the development of oral tolerance.

To approach this question we performed another set oforal tolerance experiments. Mice lacking mature T and Blymphocytes provide an ideal system to study the impact ofdifferent sets of leukocytes on the development of oraltolerance by means of cell transfer experiments [34]. Weused this system to test whether defective oral B celltolerance, caused by β7 integrin deficiency, can be restoredby substitution with β7 integrin expressing lymphocytesalone. To this end we isolated single‐cell suspensions fromthe spleen of WT mice, which contain mainly T and B cells,and transferred these i.p. into RAG-2 deficient [28] or RAG-2/β7 integrin double deficient recipient mice, prior totolerance induction and immunization (Fig. 6). Transfer ofsplenic lypmhocytes was sufficient to elicit marked B cellresponses upon immunization as evidenced by high anti-

OVA‐specific IgG titers in both mouse strains. Yet, RAG-2deficient mice fed with OVA showed reduced anti-OVA IgGresponses compared to non-fed mice, whereas no attenua-tion of the response was detectable in RAG-2/β7 integrindouble deficient mice. This indicates that β7 integrinexpressing T and B cells alone are not sufficient to generateoral B cell tolerance and points to an additional impact of β7integrin on cells of the innate immune system. In line withthis idea are results from a recent publication, whereadoptively transferred CD4+ T cells from WT mice wereonly partially able to rescue oral tolerance of β7 integrindeficient mice in a DTH model [35].

Recently, different subsets of APC have been implicatedin regulating oral tolerance. Oral tolerance has beendemonstrated to rely on the antigen transport of αE integrinexpressing DC [36], migrating by a chemokine receptor (C–Cmotif) receptor 7-dependent process from the LP of theintestine to the MLN [9,37]. In the context of the special MLNenvironment, these migratory DC favor the differentiation ofgut‐homing α4β7 integrin expressing FoxP3+ Treg, forexample by their high capacity to produce retinoic acid[7]. In this context, it is interesting to note that β7 integrindeficient mice harbor decreased numbers of CD11c+ DCs inthe small intestine when compared to WT mice (manuscriptin preparation), suggesting that DC function in β7 integrindeficient mice might also be altered, e.g. by impaireduptake of intestinal antigens or reduced production ofretinoic acid.

Additional subsets of gut resident innate immune cellswere shown to be implicated in processes of oral tolerance.CX3CR1 expressing macrophages are thought to promote oral

Figure 5 Lack of oral B cell tolerance in β7 integrin deficient mice. WT and β7 integrin deficient (β7Δ/Δ) mice were eithertolerized by three consecutive oral doses of OVA in PBS or not tolerized but instead treated with PBS only at the intervals outlinedabove. Subsequently, mice were systemically immunized by two i.p. injections with OVA-alum and OVA. At the beginning (d0) and atthe end of the experiment (d35), the levels of OVA-specific IgG were measured in equally diluted samples of sera by ELISA, andquantification of OVA-specific ASC was performed by ELISPOT. The ELISA data shown are OVA-specific IgG (μg/ml). Results with mean±SEM, are the average of 6–10 mice per group and are representative of three experiments. P values were calculated using the non-paired two-tailed t-test. (A) OVA-specific IgG in serum. (B+C) OVA-specific IgG secreting cells in spleen.

94 A. Schippers et al.

tolerance by the production of IL10, which in turn leads tothe expansion of FoxP3+ Treg in the LP [27], and also NKTcells were found to play a role in the induction of oraltolerance by triggering the IL10 and TGFβ production ofTreg, and by clonally deleting antigen-specific T cells [38].Interestingly, RAG-2/β7 integrin double deficient miceexhibit reduced numbers of pDC in the intraepitheliallymphocyte (IEL) compartment of the small intestine, whencompared to RAG-2 deficient mice (Fig. 7). Tolerancepromoting interactions between pDC and Treg have alreadybeen shown to be responsible for antigen‐specific suppres-sion of CD4+ and CD8+ T cell responses [10]. Therefore,differences in the numbers or function of pDC in β7 integrindeficient mice could contribute to the observed impairment

in oral tolerance as well. A more detailed analysis of the roleof β7 integrin expression on the different innate immunecells mentioned above is still pending and warrants furtherinvestigation.

3.7. Conclusion

In summary we demonstrate that immunogenic as well astolerogenic humoral immune responses established in thegut are dependent on β7 integrin. Moreover, our studiesindicate that expression of β7 integrin on innate immunecells is critically involved in the development of oral B celltolerance. These findings extend our understanding of the

Figure 6 β7 integrin expression on cells of the innate immune system contributes to oral B cell tolerance. RAG-2 deficient (RAG-2Δ/Δ) and RAG-2/β7 integrin double deficient (RAG-2Δ/Δ β7Δ/Δ) mice were adoptively transferred with WT spleen cells and wereeither left untreated or tolerized by continuous administration of OVA with the drinking water as indicated. Subsequently mice weresystemically immunized by two i.p. injections with OVA-alum and OVA. At the end of the experiment (d35), the levels of OVA-specificIgG were measured in equally diluted samples of sera by ELISA. The ELISA data shown are the mean OD. Results with mean±SEM aregiven as the average of 6 mice per group and are representative of two experiments. P values were calculated using the non-pairedtwo-tailed t-test.

Figure 7 Reduced numbers of pDC in the IEL compartment ofRAG-2/β7 integrin double deficient mice. The percentage ofpDC in the lymphocyte gate from intraepithelial preparations ofsmall intestines in either RAG-2 deficient (RAG-2Δ/Δ) (blackbars) or RAG-2/β7 integrin double deficient (RAG-2Δ/Δ β7Δ/Δ)(grey bars) mice is shown. Results are depicted as the average of5 mice per group (mean±SEM) and are representative of fourexperiments. P values were calculated using the non-pairedtwo-tailed t-test.

95Humoral immune responses in the gut of β7 integrin –mice

process of oral tolerance induction and may eventually haveimplications for oral vaccine development or oral immunother-apy against allergy. Particularly immunotherapies blocking β7integrin receptors during chronic intestinal inflammation couldhave the potential to interfere with normal tolerogenicmechanisms in the gastrointestinal mucosa.

Conflict of interest statement

The authors declare that there are no conflicts of interest.

Acknowledgments

This work was supported by the Deutsche ForschungsgemeinschaftGrant WA 1127/2-1 to N.W. We thank Sieglinde Keilholtz-Gast andClaudia Uthoff-Hachenberg for expert technical help.

References

[1] G. Bouguen, J.B. Chevaux, L. Peyrin-Biroulet, Recent advancesin cytokines: therapeutic implications for inflammatory boweldiseases, World J. Gastroenterol. 17 (2011) 547–556.

96 A. Schippers et al.

[2] A.M. Faria, H.L. Weiner, Oral tolerance, Immunol. Rev. 206(2005) 232–259.

[3] S. Strobel, A.M. Mowat, Oral tolerance and allergic responsesto food proteins, Curr. Opin. Allergy Clin. Immunol. 6 (2006)207–213.

[4] Y. Chen, J. Inobe, R. Marks, P. Gonnella, V.K. Kuchroo, H.L.Weiner, Peripheral deletion of antigen-reactive T cells in oraltolerance, Nature 376 (1995) 177–180.

[5] I. Gutgemann, A.M. Fahrer, J.D. Altman, M.M. Davis, Y.H.Chien, Induction of rapid T cell activation and tolerance bysystemic presentation of an orally administered antigen,Immunity 8 (1998) 667–673.

[6] X. Zhang, L. Izikson, L. Liu, H.L. Weiner, Activation ofCD25(+)CD4(+) regulatory T cells by oral antigen administra-tion, J. Immunol. 167 (2001) 4245–4253.

[7] J.L. Coombes, K.R. Siddiqui, C.V. Arancibia-Carcamo, J. Hall,C.M. Sun, Y. Belkaid, F. Powrie, A functionally specializedpopulation of mucosal CD103+ DCs induces Foxp3+ regulatory Tcells via a TGF-beta and retinoic acid-dependent mechanism,J. Exp. Med. 204 (2007) 1757–1764.

[8] J.L. Viney, A.M. Mowat, J.M. O'Malley, E. Williamson, N.A. Fanger,Expanding dendritic cells in vivo enhances the induction of oraltolerance, J. Immunol. 160 (1998) 5815–5825.

[9] T. Worbs, U. Bode, S. Yan, M.W. Hoffmann, G. Hintzen, G.Bernhardt, R. Förster, O. Pabst, Oral tolerance originates inthe intestinal immune system and relies on antigen carriage bydendritic cells, J. Exp. Med. 203 (2006) 519–527.

[10] A. Goubier, B. Dubois, H. Gheit, G. Joubert, F. Villard-Truc,C. Asselin-Paturel, G. Trinchieri, D. Kaiserlian, Plasmacytoiddendritic cells mediate oral tolerance, Immunity 29 (2008)464–475.

[11] M. Swiecki, M. Colonna, Unraveling the functions of plasmacy-toid dendritic cells during viral infections, autoimmunity, andtolerance, Immunol. Rev. 234 (2010) 142–162.

[12] T.L. Denning, Y.C. Wang, S.R. Patel, I.R. Williams, B. Pulendran,Lamina propria macrophages and dendritic cells differentiallyinduce regulatory and interleukin 17-producing T cell responses,Nat. Immunol. 8 (2007) 1086–1094.

[13] A.L. Hart, S.C. Ng, E. Mann, H.O. Al-Hassi, D. Bernardo, S.C.Knight, Homing of immune cells: role in homeostasis andintestinal inflammation, Inflamm Bowel Dis 16 (2010)1969–1977.

[14] R.O. Hynes, Integrins: versatility, modulation, and signaling incell adhesion, Cell 69 (1992) 11–25.

[15] T. Schweighoffer, Y. Tanaka, M. Tidswell, D.J. Erle, K.J. Horgan,G.E. Luce, A.I. Lazarovits, D. Buck, S. Shaw, Selective expressionof integrin alpha 4 beta 7 on a subset of human CD4+ memory Tcells with Hallmarks of gut-trophism, J. Immunol. 151 (1993)717–729.

[16] K.L. Cepek, C.M. Parker, J.L. Madara, M.B. Brenner, Integrinalpha E beta 7 mediates adhesion of T lymphocytes toepithelial cells, J. Immunol. 150 (1993) 3459–3470.

[17] B. Johansson-Lindbom, M. Svensson, O. Pabst, C. Palmqvist,G. Marquez, R. Förster, W.W. Agace, Functional specializationof gut CD103+ dendritic cells in the regulation of tissue-selective T cell homing, J. Exp. Med. 202 (2005) 1063–1073.

[18] J. Lehmann, J. Huehn, M. de la Rosa, F. Maszyna, U. Kretschmer,V. Krenn, M. Brunner, A. Scheffold, A. Hamann, Expression of theintegrin alpha Ebeta 7 identifies unique subsets of CD25+ as wellas CD25− regulatory T cells, Proc. Natl. Acad. Sci. U. S. A. 99(2002) 13031–13036.

[19] N.Wagner, J. Löhler, E.J. Kunkel, K. Ley, E. Leung, G. Krissansen,K. Rajewsky, W. Müller, Critical role for beta7 integrins information of the gut-associated lymphoid tissue, Nature 382(1996) 366–370.

[20] M.P. Schön, A. Arya, E.A. Murphy, C.M. Adams, U.G. Strauch,W.W. Agace, J. Marsal, J.P. Donohue, H. Her, D.R. Beier, S.Olson, L. Lefrancois, M.B. Brenner, M.J. Grusby, C.M. Parker,

Mucosal T lymphocyte numbers are selectively reduced inintegrin alpha E (CD103)-deficient mice, J. Immunol. 162(1999) 6641–6649.

[21] B.C. Sydora, N. Wagner, J. Löhler, G. Yakoub, M. Kronenberg,W. Müller, R. Aranda, Beta7 Integrin expression is not requiredfor the localization of T cells to the intestine and colitispathogenesis, Clin. Exp. Immunol. 129 (2002) 35–42.

[22] D. Picarella, P. Hurlbut, J. Rottman, X. Shi, E. Butcher, D.J.Ringler, Monoclonal antibodies specific for beta 7 integrin andmucosal addressin cell adhesion molecule-1 (MAdCAM-1) reduceinflammation in the colon of scid mice reconstituted withCD45RBhigh CD4+ T cells, J. Immunol. 158 (1997) 2099–2106.

[23] D. Artis, N.E. Humphreys, C.S. Potten, N. Wagner, W. Müller,J.R. McDermott, R.K. Grencis, K.J. Else, Beta7 integrin-deficient mice: delayed leukocyte recruitment and attenuatedprotective immunity in the small intestine during enterichelminth infection, Eur. J. Immunol. 30 (2000) 1656–1664.

[24] N.A. Kuklin, L. Rott, N. Feng, M.E. Conner, N.Wagner,W.Müller,H.B. Greenberg, Protective intestinal anti-rotavirus B cellimmunity is dependent on alpha 4 beta 7 integrin expressionbut does not require IgA antibody production, J. Immunol. 166(2001) 1894–1902.

[25] S. Ueha, M. Murai, H. Yoneyama, M. Kitabatake, T. Imai, T.Shimaoka, S. Yonehara, S. Ishikawa, K. Matsushima, Interven-tion of MAdCAM-1 or fractalkine alleviates graft-versus-hostreaction associated intestinal injury while preserving graft-versus-tumor effects, J. Leukoc. Biol. 81 (2007) 176–185.

[26] J.R. Kanwar, J.E. Harrison, D. Wang, E. Leung, W. Müller, N.Wagner, G.W. Krissansen, Beta7 integrins contribute to demye-linating disease of the central nervous system, J. Neuroimmunol.103 (2000) 146–152.

[27] U. Hadis, B. Wahl, O. Schulz, M. Hardtke-Wolenski, A.Schippers, N. Wagner, W. Muller, T. Sparwasser, R. Förster,O. Pabst, Intestinal tolerance requires gut homing andexpansion of FoxP3+ regulatory T cells in the lamina propria,Immunity 34 (2011) 237–246.

[28] Y. Shinkai, G. Rathbun, K.P. Lam, E.M. Oltz, V. Stewart, M.Mendelsohn, J. Charron, M. Datta, F. Young, A.M. Stall, et al.,RAG-2-deficient mice lack mature lymphocytes owing toinability to initiate V(D)J rearrangement, Cell 68 (1992)855–867.

[29] N. Lycke, J. Holmgren, Strong adjuvant properties of choleratoxin on gut mucosal immune responses to orally presentedantigens, Immunology 59 (1986) 301–308.

[30] A. Schippers, C. Leuker, O. Pabst, A. Kochut, B. Prochnow,A.D. Gruber, E. Leung, G.W. Krissansen, N. Wagner, W. Müller,Mucosal addressin cell-adhesion molecule-1 controls plasma-cell migration and function in the small intestine of mice,Gastroenterology 137 (2009) 924–933.

[31] A.J. Macpherson, K.D. McCoy, F.E. Johansen, P. Brandtzaeg,The immune geography of IgA induction and function, MucosalImmunol. 1 (2008) 11–22.

[32] L. Lefrancois, C.M. Parker, S. Olson, W. Müller, N. Wagner,M.P. Schön, L. Puddington, The role of beta7 integrins in CD8 Tcell trafficking during an antiviral immune response, J. Exp.Med. 189 (1999) 1631–1638.

[33] T.W. Spahn, H.L. Weiner, P.D. Rennert, N. Lugering, A. Fontana,W. Domschke, T. Kucharzik, Mesenteric lymph nodes are criticalfor the induction of high-dose oral tolerance in the absence ofPeyer's patches, Eur. J. Immunol. 32 (2002) 1109–1113.

[34] K. Hirahara, T. Hisatsune, K. Nishijima, H. Kato, O. Shiho, S.Kaminogawa, CD4+ T cells anergized by high dose feedingestablish oral tolerance to antibody responses when trans-ferred in SCID and nude mice, J. Immunol. 154 (1995)6238–6245.

[35] B. Cassani, E.J. Villablanca, F.J. Quintana, P.E. Love, A. Lacy-Hulbert, W.S. Blaner, T. Sparwasser, S.B. Snapper, H.L.Weiner, J.R. Mora, Gut-tropic T cells that express integrin

97Humoral immune responses in the gut of β7 integrin –mice

alpha4beta7 and CCR9 are required for induction of oralimmune tolerance in mice, Gastroenterology 141 (2011)2109–2118.

[36] O. Schulz, E. Jaensson, E.K. Persson, X. Liu, T. Worbs, W.W.Agace, O. Pabst, Intestinal CD103+, but not CX3CR1+, antigensampling cells migrate in lymph and serve classical dendriticcell functions, J. Exp. Med. 206 (2009) 3101–3114.

[37] M.H. Jang, N. Sougawa, T. Tanaka, T. Hirata, T. Hiroi, K.Tohya, Z. Guo, E. Umemoto, Y. Ebisuno, B.G. Yang, J.Y. Seoh,

M. Lipp, H. Kiyono, M. Miyasaka, CCR7 is criticallyimportant for migration of dendritic cells in intestinal laminapropria to mesenteric lymph nodes, J. Immunol. 176 (2006)803–810.

[38] H.J. Kim, S.J. Hwang, B.K. Kim, K.C. Jung, D.H. Chung, NKTcells play critical roles in the induction of oral tolerance byinducing regulatory T cells producing IL-10 and transforminggrowth factor beta, and by clonally deleting antigen-specific Tcells, Immunology 118 (2006) 101–111.