of September 29, 2014.This information is current as

T CellsRestricted−Regulated by HLA-DRB1*04:02

Desmoglein 3 in Pemphigus Vulgaris Are Pathogenic IgG Antibodies against

HertlWohde, Rikard Holmdahl, Grete Sønderstrup and MichaelFeliciani, Kevin C. Visconti, Sebastian Willenborg, Jessica Rüdiger Eming, Tina Hennerici, Johan Bäcklund, Claudio

ol.1401081http://www.jimmunol.org/content/early/2014/09/24/jimmun

published online 24 September 2014J Immunol 

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The Journal of Immunology

Pathogenic IgG Antibodies against Desmoglein 3 in PemphigusVulgaris Are Regulated by HLA-DRB1*04:02–RestrictedT Cells

R€udiger Eming,* Tina Hennerici,*,1 Johan Backlund,†,1 Claudio Feliciani,‡

Kevin C. Visconti,x Sebastian Willenborg,* Jessica Wohde,* Rikard Holmdahl,†

Grete Sønderstrup,x and Michael Hertl*

Pemphigus vulgaris (PV) is considered as a model for an autoantibody-mediated organ-specific autoimmune disorder. IgG autoanti-

bodies directed against the desmosomal cadherin desmoglein 3 (Dsg3), the major autoantigen in PV, cause loss of epidermal ker-

atinocyte adhesion, resulting in blisters and erosions of the skin and mucous membranes. The association of human autoimmune

diseases with distinct HLA alleles is a well-known phenomenon, such as the association with HLA-DRB1*04:02 in PV. However,

direct evidence that HLA-DRB1*04:02–restricted autoreactive CD4+ T cells recognizing immunodominant epitopes of Dsg3

initiate the production of Dsg3-reactive IgG autoantibodies is still missing. In this study, we show in a humanized HLA-

DRB1*04:02–transgenic mouse model that HLA-DRB1*04:02–restricted T cell recognition of human Dsg3 epitopes leads to

the induction of pathogenic IgG Abs that induce loss of epidermal adhesion, a hallmark in the immune pathogenesis of PV.

Activation of Dsg3-reactive CD4+ T cells by distinct human Dsg3 peptides that bind to HLA-DRb1*04:02 is tightly regulated by

the HLA-DRB1*04:02 allele and leads, via CD40-CD40L–dependent T cell–B cell interaction, to the production of IgG Abs that

recognize both N- and COOH-terminal epitopes of the human Dsg3 ectodomain. These findings demonstrate key cellular and

humoral immune events in the autoimmune cascade of PV in a humanized HLA-transgenic mouse model. We show that CD4+

T cells recognizing immunodominant Dsg3 epitopes in the context of the PV-associated HLA-DRB1*04:02 induce the secretion of

Dsg3-specific IgG in vivo. Finally, these results identify Dsg3-reactive CD4+ T cells as potential therapeutic targets in the

future. The Journal of Immunology, 2014, 193: 000–000.

Pemphigus vulgaris (PV) is a potentially lethal autoimmunebullous disease of the skin caused by IgG autoantibodiesagainst desmoglein (Dsg)3 and Dsg1, components of the

desmosomal adhesion complex, leading to loss of adhesion of epi-dermal keratinocytes (1). Clinically, PV presents with chronic andprogressive mucosal lesions and, during the course of disease, with

extensive flaccid skin blisters. Pemphigus can be considered as aparadigm of an autoantibody-mediated organ-specific autoimmune

disease because the targeted autoantigens are known and its immune

pathogenesis is relatively well characterized (2, 3). Although rare, PV

is more common in distinct ethnic groups such as Jews, Iranians,

Irakis, and Indians where the disease-associated HLA class II alleles

HLA-DRB1*04:02 and HLA-DQB1*05:03 are more common (4, 5).We and others have provided evidence that these PV-associated

HLA class II alleles are involved in the activation of Dsg3-specific

autoaggressive CD4+ T cells, which are critical for the induction

and maintenance of autoreactive memory B cells as precursors of

autoantibody-producing plasma cells. Autoaggressive CD4+ Th

cell responses against the ectodomain of Dsg3 were identified in

PV patients by several independent investigators (6–8). Dsg3-

specific autoaggressive Th2 cells were preferentially detected in

PV patients with active disease, whereas healthy carriers of the

PV-associated HLA class II alleles HLA-DRB1*04:02 and HLA-

DQB1*05:03 showed autoreactive Th1 cell responses against

Dsg3 (8). On the basis of a peptide-binding algorithm for HLA-

DRB1*04:02, Wucherpfennig et al. (6) identified several Dsg3

peptides as potential T cell epitopes. We subsequently found that

distinct Dsg3 peptides, which all share a positively charged argi-

nine (R) or lysine (K) at position 4, induce a proliferative in vitro

response of peripheral T cells from PV patients. Moreover, CD4+

T cell recognition of these Dsg3 epitopes was restricted by HLA-

DRB1*04:02 and, in some cases, by HLA-DQB1*05:03, which

share similar peptide binding motifs and thus provides a molecular

basis for the dual HLA class II association observed in PV (9).Despite the strong evidence that IgG autoAb specific for Dsg3

or Dsg1 are pathogenic in PV, the immunological mechanisms

*Department of Dermatology and Allergology, Philipps University, D-35043 Mar-burg, Germany; †Department of Medical Biochemistry and Biophysics, KarolinskaInstitutet, S-171 77 Stockholm, Sweden; ‡Section of Dermatology, Department ofClinical and Experimental Medicine, University of Parma, 43100 Parma, Italy; andxDepartment of Microbiology and Immunology, School of Medicine, Stanford Uni-versity, Stanford, CA 94305

1T.H. and J.B. contributed equally.

Received for publication April 28, 2014. Accepted for publication August 27, 2014.

This work was supported by Grants EM 80/1-1 (to R.E.) and HE 1602/7-2 (to R.E.and M.H.) from the German Research Foundation (Deutsche Forschungsgemein-schaft); grants from the Knut and Alice Wallenberg Foundation (to R.H.), the Swed-ish Medical Research Council, and the Swedish Foundation for Strategic Research;and European Union Grants MASTERSWITCH (HEALTH-F2-2008-223404),BeTheCure (IMI115142) (to R.H. and J.B.), and HEALTH-F2-2008-200515 “Pem-phigus – from Autoimmunity to Disease” (to M.H.). M.H. received grants fromFresenius Medical Care and Medical & Biological Laboratories, Co., Ltd. G.S.received grants from the National Institutes of Health/National Institute of Ar-thritis and Musculoskeletal and Skin Diseases and the Juvenile Diabetes ResearchFoundation.

Address correspondence and reprint requests to Dr. R€udiger Eming, Departmentof Dermatology and Allergology, Philipps University Marburg, Baldingerstrasse,D-35043 Marburg, Germany. E-mail address: eming@med.uni-marburg.de

The online version of this article contains supplemental material.

Abbreviations used in this article: BMDC, bone marrow–derived dendritic cell; Dsg,desmosomal cadherin desmoglein; PV, pemphigus vulgaris; Tr1, type 1 regulatory T;TT, tetanus toxoid.

Copyright� 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1401081

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regulating IgG autoAb formation are largely unknown. In thisstudy, we show in an HLA-DRB1*04:02–transgenic mouse modelthat induction of pathogenic, Dsg3-reactive IgG Abs requires ac-tivation of Dsg3-reactive CD4+ T cells. Moreover, T cell activa-tion critically depends on the recognition of epitopes of the Dsg3ectodomain, which specifically bind to the PV-associated HLAclass II allele, DRB1*04:02 (6, 10). Thus T cell–dependent in-duction of pathogenic IgG Abs in pemphigus is tightly regulatedby polymorphisms of peptide-binding motifs of distinct HLA classII alleles, which are associated with PV.

Materials and MethodsHLA-DR4–transgenic mice

HLA-DRA1*01:01-DRB1*04:02/-DQA1*03:01, -DQB1*03:02 (DQ8)–transgenic DBA/1J mice were generated as described previously (11, 12).The mice also express the human CD4 coreceptor (13) and are I-Ab de-ficient (I-Ab2/2) (Supplemental Fig. 1A) (14). Transgenic C57BL/10 micecoexpressing DRB1*04:01 and human CD4, whereas lacking endogenousmurine MHC class II and a functional Ncf1 gene were also used (15–17).All animal experiments were reviewed approved by the local LaboratoryAnimal Ethics Committees at the Philipps University Marburg and theKarolinska Institutet Stockholm, respectively. The experiments were donein compliance with local policies and guidelines on the use of laboratoryanimals.

Dsg3 recombinants and peptides

Human and mouse Dsg3 recombinants linked to an E tag and a 63 histidinetag were produced and purified by affinity chromatography as describedpreviously (Table I) (8, 18–20). Fifteen- and 17-mer Dsg3 peptides weresynthesized by F-moc chemistry resulting in a .95% purity (Table III)(peptides & elephants, Potsdam, Germany) and were solubilized in DMSOacetic acid buffer (stock concentration, 2 mg/ml).

Immunization of HLA-DR4–transgenic mice

Eight to 12 wk old HLA-DRB1*04:02–transgenic mice were immunized byi.p. injection of recombinant human Dsg3 (20–40 mg) in alum on day 0,followed by immunization with 20–40 mg Dsg3 in IFA (Sigma-Aldrich,St. Louis, MO) on days 14 and 28 (Supplemental Fig. 1B). Blood sampleswere taken to analyze anti-Dsg3 IgG by ELISA as described previously(20). For the induction of Dsg3-reactive T cell responses, mice wereinjected s.c. into the foot paws with human Dsg3 protein and peptides (20–40mg), respectively, in adjuvant (IFA or TiterMax) (Sigma-Aldrich) on day0, and draining popliteal and inguinal lymph nodes were harvested on days7–10 (Supplemental Fig. 1C).

Generation of bone marrow–derived dendritic cells

Bone marrow–derived dendritic cells (BMDC) from HLA-DRB1*04:02–transgenic mice were generated as described previously (21). BMDC werematured with 0.1 mg/ml LPS (from Escherichia coli 026:B6; Sigma-Aldrich), pulsed with 10 mg/ml human Dsg3 protein and peptides, re-spectively, and incubated for 18 h at 37˚C, 5% CO2.

In vitro proliferative assays

CD4+ T cells were isolated from splenocytes of Dsg3-immunized HLA-DRB1*04:02–transgenic mice by MACS (Miltenyi Biotec, BergischGladbach, Germany), according to the manufacturer’s manual. Purity ofthe isolated CD4+ T cells was .90% as confirmed by FACS analysis.T cells were stained with CFSE (Molecular Probes/Life Technologies,Carlsbad, CA) following an established protocol (22). Specifically, 23 105

CD4+ T cells were cocultured with 104 BMDC in 96-well round-bottommicrotiter plates. Proliferation of CFSE-labeled CD4+ T cells was deter-mined by FACS analysis after 72 h of culture. In addition, IL-4+ and IFN-g+ T cells from popliteal and inguinal lymph nodes were restimulatedin vitro with Dsg3 protein and Dsg3 peptides, respectively, and weresubjected to ELISPOT analysis using an ELISPOT reader (A.EL.VIS,Hannover, Germany).

Ab treatment

HLA-DRB1*04:02–transgenic mice received rat anti-mouse CD4 mAb(GK1.5) or rat IgG2b isotype control (LTF-2) (both BioXCell, West Lebanon,NH) at 200 mg i.p. on days 21, 0, 1, 3, and 5; mice were immunizedwith 20–40 mg Dsg3 i.p. on day 0. Mice were also injected with hamster

anti-mouse CD40L mAb (MR-1) or poly hamster Ig isotype control (bothBioXCell) at 500 mg i.p. on days 22, 0, 2, 4, 7, and 14 and were im-munized with 20–40 mg Dsg3 i.p. on day 0.

Keratinocyte dissociation assay

The dispase based keratinocyte dissociation assay was performed as de-scribed previously (23, 24). Briefly, primary human epidermal keratino-cytes were grown to confluence in CnT-57 medium in 12-well plates(CELLnTEC Advanced Cell Systems, Bern, Switzerland), switched toCnT-02 medium (CELLnTEC Advanced Cell Systems) supplemented with1.2 mM CaCl2 24 h prior to the assay, and were incubated with mouse sera(diluted at 1:50) or the anti-Dsg3 mAb AK23 (1 mg/ml; MBL, Nagoya,Japan) overnight at 37˚C. For the final 2 h of the assay, recombinant ex-foliative toxin A (0.5 mg/ml; Toxin Technology, Sarasota, FL) was added.The adherent keratinocytes were then incubated with dispase I (1.5 U/ml;Roche Applied Sciences, Mannheim, Germany) at 37˚C for 20 min andsubjected to mechanical stress. Fragments were fixed in 1 ml of a 10%formalin solution and stained with crystal violet.

Ex vivo model using human skin biopsies

Four-millimeter punch biopsies were obtained from unaffected skin samplesof patients undergoing dermatosurgery in the Department of Dermatology andAllergology at the Philipps University Marburg, Germany. The patients gavewritten informed consent to donate excess skin samples resulting from sur-gical procedures for this research project. Punch biopsies were incubated in200 ml keratinocyte medium CnT-57 (CELLnTEC Advanced Cell Systems)supplemented with 1.2 mM CaCl2 in 96-well round-bottom plates. Serumsamples of Dsg3-immunized HLA-DRB1*04:02–transgenic mice (serumsamples of PBS-injected mice served as controls) were injected into thedermal site of the biospies at a dilution of 1:20–1:25. The skin biopsies wereincubated overnight at 5% CO2 and 37˚C, then rinsed in PBS several times,fixed in 10% formalin, and then subjected to H&E staining for histologicalanalysis. For detection of tissue-bound IgG Abs, cryosections of the skinbiopsies were subjected to immunofluorescence staining.

Immunofluorescence and histopathology

Human skin specimens and buccal or palatinal mouse mucosa were em-bedded in OCT TissueTek compound (Sakura, Tokyo, Japan), frozen, cutinto 3- to 4-mm sections and stained using a 1:250 dilution of a rabbit anti-mouse IgG FITC-labeled Ab for direct immunofluorescence microscopy(Zymed, San Francisco, CA). Indirect immunofluorescence with serumsamples of immunized mice (1:25 dilution) was performed on monkeyesophagus, according to the manufacturer’s protocol (The Binding Site,Birmingham, U.K.), and modified by using a rabbit anti-mouse IgG FITC-labeled Ab (Zymed). Formalin-fixed skin and mucosa sections of Dsg3-immunized mice were also stained with H&E.

Dsg3 ELISA

Mouse sera were diluted 1:20 and analyzed for anti-Dsg3 IgG by ELISAas described previously (20). In addition, two commercial human Dsg3ELISA kits were also used (MBL, Nagoya, Japan; Euroimmun, L€ubeck,Germany) and were modified by using an anti-mouse IgG HRP-conjugatedsecondary Ab.

ResultsImmunization of HLA-DRB1*04:02-transgenic mice withhuman Dsg3 leads to the induction of Dsg3-specific,pathogenic IgG Abs

We generated a humanized mouse model aimed at reproducingthe immunological key findings of PV under the immunogeneticrestriction by HLA-DRB1*04:02. DBA/J1 mice transgenic forHLA-DRB1*04:02 and HLA-DQB1*03:02 (which is in a linkagedisequilibrium with DRB1*04:02) and the human CD4 coreceptor,which were devoid of functional murine MHC class II (I-Ab2/2)were generated (Supplemental Fig. 1A). Mice were immunized withhuman recombinant Dsg3 (aa 1–566) (Table I, Supplemental Fig.1B) and mounted a robust IgG response against human Dsg3 (Fig.1A); these Abs belonged preferentially to the IgG1 and IgG2a

subclasses (data not shown). Sera from the Dsg3-immunized miceinduced loss of adhesion of monolayers of human keratinocytes(Fig. 1B) to a greater extent than sera from mice transgenic for theunrelated HLA-DRB1*04:01 allele (Supplemental Fig. 2) whereas

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sera from PBS-injected HLA-DRB1*04:02–transgenic control micedid not (Fig. 1E). Injection of sera from the Dsg3-immunized HLA-DRB1*04:02–transgenic mice into human skin biopsies led toantiepithelial cell surface IgG deposits (Fig. 1C) and intraepidermalloss of adhesion, which is a hallmark of PV (Fig. 1D). In contrast,injection of sera from PBS-injected mice into human skin neitherled to tissue-bound antiepithelial cell surface IgG (Fig. 1F) nor tointraepidermal loss of adhesion (Fig. 1G).The in vivo–induced anti-human Dsg3–reactive IgG Abs recog-

nized the same spectrum of epitopes as IgG autoantibodies from PVsera (20, 25). After the first immunization with human Dsg3, mouse

sera preferentially reacted with the COOH-terminal Dsg3(EC5)subdomain and, after additional immunizations, also with theN-terminal Dsg3(EC1) and Dsg3(EC2) domains, which containthe major pathogenic B cell epitopes (Fig. 1H, 1I, Table I). Sera fromthe human Dsg3-immunized mice showed only little IgG cross-reactivity with mouse Dsg3, which shows an overall homologywith human Dsg3 of 85.6% (Supplemental Fig. 3A). Despite theobserved IgG reactivity of the mouse sera with several N- andCOOH-terminal epitopes of the human Dsg3 ectodomain, there wasno evidence for tissue-bound antiepithelial cell surface IgG Absin oral mucosa of the Dsg3-immunized HLA-DRB1*04:02–

Table I. Dsg3 recombinants and control proteins

Recombinant Proteina Description of Recombinant Amino Acid Sequence References

Dsg3 Ectodomain of human Dsg3 (EC1–5) 1–566 (8, 9, 19, 26)Dsg3(EC1) Extracellular domain 1 (EC1) of human Dsg3 1–161 (20, 52, 53, 54)Dsg3(EC2) Extracellular domain 2 (EC2) of human Dsg3 87–227 (20, 52, 53, 54)Dsg3(EC3) Extracellular domain 3 (EC3) of human Dsg3 184–349 (20, 52, 53, 54)Dsg3(EC4) Extracellular domain 4 (EC4) of human Dsg3 313–451 (20, 52, 53, 54)Dsg3(EC5) Extracellular domain 5 (EC5) of human Dsg3 424–566 (20, 52, 53, 54)Dsg3 Ectodomain of mouse Dsg3 (EC1–5) 1–566 (55)Col VII Noncollagenous domain 1 (NC1) of human

collagen VII17–610 (56, 57)

aAll recombinants were produced as soluble proteins in a baculovirus expression system and were purified from insect cell culture supernatants byaffinity chromatography.

FIGURE 1. Induction of human Dsg3–specific,

pathogenic IgG Abs in HLA-DRB1*04:02–trans-

genic mice. (A) Immunization of DRB1*04:02–

transgenic mice with human Dsg3 leads to a

robust Ag-specific IgG response as shown by

ELISA; PBS-treated mice serve as controls

(n = 2). (B) In vitro, sera from Dsg3-immunized

mice induce loss of adhesion of human epi-

dermal keratinocytes as determined by dispase

dissociation assay. Injection of Dsg3-immu-

nized mouse sera into human skin specimens

leads to antiepithelial surface IgG deposits (C)

and intraepidermal loss of cell adhesion (D). In

contrast, sera from PBS-injected mice neither

induce keratinocyte monolayer dissociation (E),

antiepithelial surface IgG deposits in injected

human skin (F) nor loss of epidermal adhesion

in these skin sections (G) [(B–G) representative

results of three to five mice]. (H) On day 14

after the first Dsg3 immunization, mouse sera

show IgG reactivity against the COOH-terminal

Dsg3(EC5) subdomain and, upon the third (day

35) immunization (I), additional IgG reactivity

against the N-terminal Dsg3(EC1) and Dsg3(EC2)

subdomains and to a lesser extent against

Dsg3(EC3) and Dsg3(EC4) [(H and I) n = 4].

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transgenic mice, and accordingly, no evolving clinical pheno-type (Supplemental Fig. 3B). Noteworthy, the amino acid sequencesof the human and mouse Dsg3 ectodomain vary significantly in theareas of importance for CD4+ T cell activation (Table II), which mayimpede loss of T cell tolerance for mouse Dsg3, which precedespolyclonal activation of mouse Dsg3-specific pathogenic B cells.Comparison of the immundominant T cell epitopes of human Dsg3with their mouse Dsg3 analogs revealed discordant amino acids atpositions that are critical MHC class II anchor motifs. For example,the mouse analog to the human immunodominant T cell epitope ofDsg3, Dsg3(97–111), which is recognized by T cells from the ma-jority of PV patients does not share critical HLA-DRB1*04:02–binding motifs at positions 4 and 6 (Table II).

Induction of pathogenic anti-Dsg3 IgG requires interaction ofT cells and B cells

HLA-DRB1*04:02–transgenic mice were treated with the anti-CD4mAb GK 1.5 at the time of immunization with human Dsg3 leadingto a complete inhibition of anti-Dsg3 IgG production (Fig. 2A).Furthermore, HLA-DRB1*04:02–transgenic mice were treated withthe anti-CD40L mAb MR-1 before and right after immunizationwith human Dsg3 (Fig. 2B). Anti-Dsg3 IgG Ab production wascompletely inhibited, which demonstrates that T cell–B cell inter-action is critical in the induction phase of Dsg3-specific IgG pro-duction. These findings in the HLA-DRB1*04:02–transgenic mousemodel are in line with the clinical observation in three representativePV patients from a previously described cohort who were treatedwith the anti-CD20 mAb rituximab (26). Depletion of B cells byrituximab treatment (Fig. 2C) was associated with a rapid decreaseof peripheral Dsg3-specific Th2 cells (Fig. 2D), followed by a moredelayed decrease of anti-Dsg3 serum IgG (Fig. 2E).

T cell recognition of desmoglein 3 peptides is tightly restrictedby HLA-DRB1*04:02

Immunization of the HLA-DRB1*04:02-transgenic mice with hu-man Dsg3 induced a T cell response against a set of T cell epitopesof Dsg3, which all bind to HLA-DRB1*04:02 (Fig. 3, Table III).Splenic CD4+ T cells from mice injected with human Dsg3 showeda proliferative response against a set of HLA-DRB1*04:02–bindingDsg3 peptides presented by BMDC from HLA-DRB1*04:02–transgenic mice (Table III) as shown by CSFE staining (Fig. 3A).In contrast, ex vivo challenge of the splenic CD4+ T cells witha set of Dsg3 peptides that do not bind to HLA-DRB1*04:02(Table III) did not induce a significant proliferative response(Fig. 3B). Controls include cocultures of the splenic CD4+

T cells with BMDC alone (Fig. 3C) and BMDC with anti-CD3mAb (Fig. 3D), respectively.

In the reverse experiment, draining lymph node cells fromHLA-DR04:02–transgenic mice, which had been immunized witha set of five HLA-DRB1*04:02-binding Dsg3 peptides (Table III,Supplemental Fig. 1C), showed both IL-4+ and IFN-g+ T cellresponses upon in vitro restimulation with human Dsg3 or HLA-DRB1*04:02–binding Dsg3 peptides, respectively, but not or onlyto a much lesser extent with Dsg3 peptides that do not bind HLA-DRB1*04:02 (Fig. 3E, 3F). HLA-DRB1*04:02–transgenic miceimmunized with HLA-DRB1*04:02–nonbinding Dsg3 peptidesshowed merely an IFN-g+ T cell response to the same set ofpeptides but neither IL-4+ nor IFN-g+ T cell responses to Dsg3(Fig. 3E, 3F). In contrast, splenic CD4+ T cells from HLA-DRB1*04:01–transgenic mice that had been immunized with thesame set of HLA-DRB1*04:02–binding Dsg3 peptides (Table III)did neither show IL-4+ nor IFN-g+ T cell responses against humanDsg3 in vitro (Fig. 3G, 3H). Immunization and in vitro restim-ulation with HLA-DRB1*04:02–binding Dsg3 peptides inducedonly an IL-4+ T cell response in a single HLA-DRB1*04:02–transgenic mouse, whereas immunization followed by in vitrochallenge with HLA-DRB1*04:02–nonbinding Dsg3 peptidesled to an IL-4+ and IFN-g+ T cell response (Fig. 3G, 3H). Thebackground stimulation of IL-4+ and IFN-g+ lymph node T cellsfrom HLA-DRB1*04:02–transgenic mice that were injected withPBS and adjuvant only is shown in Supplemental Fig. 4A and 4B.

Immunization of HLA-DRB1*04:02–transgenic mice with T cellepitopes of human Dsg3 leads to the induction of anti-Dsg3 IgG

Immunization of the HLA-DRB1*04:02–transgenic mice with a poolof HLA-DRB1*04:02–binding Dsg3 peptides (Table III) inducedIgG Abs against human Dsg3 as shown by ELISA (Fig. 4A) andindirect immunofluorescence microscopy on monkey esophagusepithelium (Fig. 4B). In contrast, immunization of the HLA-DRB1*04:02–transgenic mice with Dsg3 peptides, which do notbind to HLA-DRB1*04:02 (Table III), did neither induce an IgGresponse against human Dsg3 by ELISA (Fig. 4A) nor did the serareact with monkey esophagus epithelium (Fig. 4C). Most strikingly,immunization of mice transgenic for HLA-DRB1*0401 (which isnot related to PV and expresses different peptide binding motifs)with the HLA-DRB1*04:02–binding Dsg3 peptides did not induceIgG against Dsg3 as determined by ELISA (Fig. 4D) and indirectimmunofluorescence on monkey esophagus (Fig. 4E). Neither didimmunization of the HLA-DRB1*0401–transgenic mice with Dsg3peptides that do not bind HLA-DRB1*04:02 (Table III) induce anti-Dsg3 IgG as determined by ELISA (Fig. 4D) and indirect immu-nofluorescence (Fig. 4F). These findings demonstrate that T cellrecognition of HLA-DRB1*04:02–restricted epitopes of Dsg3 iscritical for the induction of IgG Abs against Dsg3.

Table II. Homology of HLA-DRB1*04:02–binding peptides of the human Dsg3 ectodomain (aa 1–566) with the corresponding peptides of the mouseDsg3 ectodomain

Dsg3 peptidesa P1b P4b P6b

huDsg3(97–111) F G I F V V D K N T G D I N ImDsg3(97–111) F G I F V V D P N N G D I N IhuDsg3(190–204) L N S K I A F K I V S Q E P AmDsg3(190–204) M N S K I A F K I V S Q E P AhuDsg3(206–220) T P M F L L S R N T G E V R TmDsg3(206–220) M S M F L I S R N T G E V R ThuDsg3(251–265) C E C N I K V K D V N D N F PmDsg3(251–265) C E C S I K I K D V N D N F PhuDsg3(375–391) I N V R E G I A F R P A S K T F TmDsg3(375–391) I D V R E G I S F R P P S K T F T

aAccording to published gene sequences of human Dsg3 (3) and mouse Dsg3 (40).bCritical amino acid anchor motifs for peptide binding to HLA-DRB1*04:02 identified by Wucherpfennig et al. (6) and Tong et al. (29) are underlined. Heterologous amino

acids are bold.

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DiscussionIn this study, we show in an HLA-DRB1*04:02–transgenic mousemodel of the autoimmune bullous skin disorder PV that T cellrecognition of epitopes of Dsg3, the autoantigen of PV, in associ-ation with HLA-DRB1*04:02, leads, via B cell help, to the for-

mation of pathogenic IgG Abs that induce loss of epidermalkeratinocyte dissociation, a key finding in PV. On the basis ofthese observations, PV, although pathogenetically linked to IgGautoantibodies against the desmosomal cadherin, Dsg3 shouldbe considered as a T cell–dependent autoimmune disorder andmay largely profit from a therapeutic downregulation of autoag-gressive T cells.The hypothesis that autoreactive CD4+ T cells are critical initiators

and perpetuators of the autoimmune pathology of PV is based onepidemiological observations which show a strong association of PVwith two distinct HLA class II alleles, DRB1*04:02 and HLA-DQB1*05:03 (4, 5, 27, 28). HLA-DRB1*04:02 possesses a negativecharge at positions DRb70 (aspartate) and DRb71 (glutamate)contributing to the shape and the charge of the p4 pocket whichis critical for the binding of Dsg3 peptides for presentation toautoaggressive T cells in PV (6, 10). Both, DRB1*04:02 and HLA-DQB1*05:03, show a great homology in binding epitopes of theDsg3 ectodomain, which is supported by previous findings of ourgroup and others that both HLA class II alleles restrict T cell rec-ognition of identical Dsg3 epitopes (Table III) (9, 29).Previous studies from our group identified T cell responses to Dsg3

not only in PV patients but also in healthy carriers of the afore-mentioned PV-associated HLA class II alleles whose activation wasfound to be restricted by HLA-DRB1*04:02 and HLA-DQB1*05:03,respectively (8, 19, 30). In PV, Dsg3-specific autoaggressive T cellswere predominantly of the Th2 type, whereas Dsg-reactive T cells inthe healthy individuals were mainly Th1 cells (7–9). A similar di-chotomy of autoreactive T cells in patients and healthy donors wasalso found in the pathogenetically related but distinct autoimmunebullous skin disorders, pemphigus foliaceus and bullous pemphigoid,which are also mediated by pathogenic IgG autoantibodies (31, 32).Overall, PV can be considered as a Th2-driven autoimmune

disorder because most of the autoantibodies belong to the IgG4

and IgE subclasses (7, 33–37). Moreover, patients with PV havesignificantly lower frequencies of peripheral IL-10–secretingDsg3-reactive type 1 regulatory T (Tr1) cells than healthy car-riers of HLA-DRB1*04:02 that suppress proliferative responsesof T effector cells in vitro (38). Thus, activation of Dsg3-reactiveeffector T cells may be controlled by Dsg3-specific Tr1 cellsleading to peripheral tolerance in healthy individuals who areprotected by the higher frequency of Dsg3-reactive Tr1 cells thanpatients (38, 39).Immunization of humanized HLA-DRB1*04:02–transgenic mice

with T cell epitopes of Dsg3 is sufficient to induce a robustCD4+ T and B cell response against human Dsg3 leading to theproduction of pathogenic IgG Abs, which induced loss of ad-hesion of human epidermal keratinocytes ex vivo and in vitro(Fig. 4A, 4B). These IgG Abs only showed little cross-reactivitywith mouse Dsg3, which exhibits an overall homology to humanDsg3 of 85.6% (40). Specifically, there is a higher degree ofconservation (86–89% identity) for the N-terminal EC1 and EC2ectodomains of Dsg3, which contain the major pathogenicautoantibody epitopes in PV patients (20, 25, 40). In contrast,the homology between the human and mouse COOH-terminalDsg3(EC5) ectodomains is much lower (56% identity) (40). Still,the in vivo–induced anti-human Dsg3-specific IgG Abs did notsufficiently bind to the relevant mouse Dsg3 epitopes to inducea clinical phenotype in our HLA-transgenic animals. Moreover,the amino acid sequences of human and mouse Dsg3 vary sig-nificantly in the areas of importance for CD4+ T cell activation(Table II), which may impede loss of T cell tolerance for mouseDsg3, which precedes polyclonal activation of mouse Dsg3-specific pathogenic B cells. In this respect, the present mousemodel differs from the human autoimmune disease PV because

FIGURE 2. Induction of human Dsg3-specific IgG Abs requires inter-

action between CD4+ T cells and B cells. (A) Treatment of the HLA-

DRB1*04:02–transgenic mice with the anti-CD4 mAb GK1.5 at the time

of immunization with human Dsg3 completely abrogates the formation

of anti-Dsg3 IgG (n = 2). (B) In addition, treatment of the HLA-

DRB1*04:02–transgenic mice with the anti-CD40L Ab MR-1 at the time

of immunization and immediately afterward completely inhibits anti-Dsg3

IgG Ab induction (n = 2). (C) In three PV patients, treatment with the anti-

CD20 Ab rituximab does not only (C) rapidly deplete peripheral B cells but

also induces (D) a rapid decrease of peripheral IL-4–secreting Dsg3-re-

active Th2 cells, (E) followed by a significant reduction of anti-Dsg3 serum

IgG [(C–E) referring to Ref. 26].

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immunization with human Dsg3 protein elicits an immune re-sponse to a foreign Ag in these animals. Thus, we are limited inanalyzing loss of tolerance to endogenous mouse Dsg3 on boththe CD4+ T cell and the B cell level. The current HLA-transgenicmouse model is suitable for investigating the effector phase ofthe autoimmune response in PV with particular emphasis on the

activation and interaction of T and B cells, specific for humanDsg3, respectively. However, the primary scope of this investi-gation was to characterize cellular and humoral immune responsesto human Dsg3 under in vivo, with emphasis on Dsg3 peptiderecognition in association with HLA-B1*04:02, which is highlyprevalent in PV.

FIGURE 3. CD4+ T cells from human Dsg3-immunized HLA-DRB1*04:02–transgenic mice recognize a limited set of Dsg3 peptides. (A) Splenic CD4+

T cells from Dsg3-immunized HLA-DRB1*04:02–transgenic mice recognize a set of five Dsg3 peptides that bind to HLA-DRB1*04:02 (Table III) upon

coculture with HLA-DRB1*04:02+ BMDC as shown by CSFE staining. (B) In contrast, CD4+ splenic T cells from Dsg3-immunized HLA-DRB1*04:02–

transgenic mice show background proliferation in coculture with a set of Dsg3 peptides that do not bind to DRB1*04:02 (Table III). CD4+ splenic T cells in

coculture with BMDC alone (C) and in coculture with BMDC and the anti-CD3 Ab 145-2c11 (D) [(A–D) representative results of two experiments]. (E and

F) Draining lymph node cells from HLA-DR04:02–transgenic mice that were immunized with the five HLA-DRB1*04:02–binding Dsg3 peptides show IL-

4+ and IFN-g+ T cell responses upon in vitro stimulation with human Dsg3 or HLA-DRB1*04:02–binding Dsg3 peptides, respectively, but not with Dsg3

peptides that do not bind HLA-DRB1*04:02. Mice immunized with HLA-DRB1*04:02–nonbinding peptides did not show an IL-4+ or IFN-g+ T cell

response to Dsg3 [(E and F) n = 6–10]. (G and H) Lymph node cells from HLA-DRB1*04:01–transgenic mice immunized with HLA-DRB1*04:02–binding

and –nonbinding Dsg3-peptides, respectively, showed both IL-4+ and IFN-g+ T cell responses to the respective set of Dsg3 peptides, but there were neither

IL-4+ (except for one mouse) nor IFN-g+ T cell responses upon in vitro restimulation with Dsg3 [(G and H) n = 2–4].

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The fine specificity of the HLA-DRB1*04:02–restricted CD4+

T cell response to Dsg3 is documented by the observation thatimmunization of the HLA-DRB1*04:02–transgenic mice withDsg3 peptides that do not bind HLA-DRB1*04:02 neither inducesDsg3-reactive T cell responses nor Dsg3-specific IgG Abs (Figs. 3E,3F, 4A–C). Reversely, immunization of mice transgenic for theunrelated HLA-DRB1*04:01 allele with HLA-DRB1*04:02–binding T cell epitopes of human Dsg3 does not lead to the for-mation of Dsg3-specific IgG (Fig. 4D–F). HLA-DRB1*04:01,which is associated with rheumatoid arthritis, differs fromDRB1*04:02 only by the positive charge of the p4 pocket,which is critical for the binding of antigenic peptides (6, 41).Immunization of HLA-DRB1*04:01 control mice with Dsg3

protein induced Dsg3-specific IgG as measured by Dsg3 ELISA(Fig. 4D). In the functional keratinocyte dissociation assay, how-ever, the pathogenicity of anti-Dsg3 of the HLA-DRB1*04:01mice tended to be lower compared with serum samples ofthe Dsg3-immunized HLA-DRB1*04:02–transgenic mice (Supple-mental Fig. 2B). Of note, we did not see principal differencesin the epitope specificity of the Dsg3-reactive IgG Abs inHLA-DRB1*04:01– and DR04:02–transgenic mice, respectively(data not shown). These findings suggest that the human Dsg3protein also contains CD4+ T cell epitopes that bind to HLA-DR04:01 and, via T cell activation and T cell–B cell interaction,induce an IgG response against the human Dsg3 Ag.Wucherpfennig et al. (6) proposed several candidate T cell

peptides of Dsg3 on the basis of an algorithm for anchor motifsof the Dsg3 peptides and the charge of critical peptide bindingpockets of DRB1*04:02. Moreover, they showed a proliferativein vitro response of peripheral lymphocytes from PV patients tothree of the identified HLA-DRB1*04:02–associated peptides re-siding within the Dsg3 ectodomain (6). An independent studyconfirmed these findings and identified 10 HLA-DRB1*04:02–binding epitopes of Dsg3, which included the ones proposedpreviously by Wucherpfennig et al. based on molecular models(28). Using long-term CD4+ T cell clones, our group demonstratedthat the majority of HLA-DRB1*04:02–positive PV patientsshowed a proliferative T cell response to these HLA-DRB1*04:02–binding Dsg3 T cell peptides (Table III) (8, 9, 42). Specifically, allof the identified Dsg3 epitopes share common anchor residues atrelative positions 1, 4, and 6, which were previously identified to bepotential anchor motifs for DRB1*04:02 and carry a positive chargeat position 4, which is critical for binding to the negatively chargedP4 pocket of DRB1*04:02 (Table III) (6, 42).In this study, we show that induction of anti-human Dsg3 IgG

Abs in immunized HLA-DRB1*04:02–transgenic mice dependson the interaction of CD4+ T cells and B cells (Fig. 2). This in vivofinding closes an important gap that was not yet fully proven in the

human disorder PV. Circumstantial evidence for a pathogeneti-cally relevant interaction of autoreactive CD4+ T and B cells camefrom the clinical observation that in PV patients therapeutic B celldepletion with the monoclonal anti-CD20 mAb rituximab leads toa downregulation of Dsg3-specific T cells (Fig. 2C, 2D), which isassociated with a prompt clinical improvement before anti-Dsg3serum IgG autoantibodies are decreased (Fig. 2E) (26). Note-worthy, the frequencies of tetanus toxoid (TT)–specific Th cellsand serum IgG were not affected by rituximab treatment (26). Thisobservation strongly suggests that Dsg3-reactive T cells largelydepend on B cells as APCs whereas TT-specific T cells do not.This finding is in line with previous studies in rheumatoid arthritiswhich showed that serum IgG titers against pathogens such aspneumococcal capsular polysaccharides or TT were not signifi-cantly affected by rituximab (43). The dramatic inhibitory effectof rituximab on anti-Dsg3 IgG serum levels strongly suggests thattheir secretion largely depends on short-lived autoreactive plasmacells (44). Moreover, Dsg3-specific IgG-producing B cells weredetected in PV patients by ELISPOT assay upon ex vivo stimu-lation of their peripheral lymphocytes with Dsg3 (45). When thepatients’ peripheral lymphocytes were depleted of CD4+ T cells,anti-Dsg3 IgG–producing B cells were no longer detectable (45).The critical role of T cell–B cell interaction in the PV patho-

genesis is also supported by a PV mouse model established byAmagai and coworkers (46, 47). Transfer of splenocytes ofDsg32/2 mice immunized with murine Dsg3 into Dsg3+/+Rag22/2

recipient mice led to a clinical phenotype with mucosal erosionsreminiscent of PV. In contrast, transfer of splenocytes depleted ofCD4+ T cells to the Dsg3+/+Rag22/2 mice did neither induceautoantibody production nor a PV-like phenotype. Neither did thetransfer of B cell–depleted splenocytes into Dsg3+/+Rag22/2 micelead to the induction of anti-Dsg3 IgG and/or a clinical phenotype.Moreover, using the same animal model, Takahashi et al. (48)showed that a single Dsg3-reactive T cell clone was sufficient toprime naive B cells to produce Dsg3-specific pathogenic IgGautoantibodies. In an independent mouse model, immunization ofmice with human Dsg3 led to the induction of Dsg3-reactive Th2cells that were able to render unprimed B cells to secrete anti-Dsg3 IgG (49).In summary, the current study demonstrates that CD4+ T

cell recognition of Dsg3, the autoantigen of PV is tighly reg-ulated by HLA-DRB1*04:02, which is prevalent in PV. UsingDRB1*04:02–transgenic mice, we show that T cell–dependentB cell activation is critical for the induction of pathogenic IgGAbs, which directly induce epidermal loss of adhesion, a keyfinding in the immune pathogenesis of PV. Thus, specific tar-geting of Dsg3-reactive CD4+ T cells holds major promise asa therapeutic option in PV. A similar approach has proven

Table III. HLA-DRB1*04:02–binding and –nonbinding peptides of the human Dsg3 ectodomain (aa 1–566)

Dsg3 Peptidesa P1b P4b P6b

Dsg3(97–111)* F G I F V V D K N T G D I N IDsg3(190–204)* L N S K I A F K I V S Q E P ADsg3(206–220)* T P M F L L S R N T G E V R TDsg3(251–265)* C E C N I K V K D V N D N F PDsg3(375–391)* I N V R E G I A F R P A S K T F TDsg3(85–101)** Y R I S G V G I D Q P P F G I F VDsg3(145–161)** V K I L D I N D N P P V F S Q Q IDsg3(240–256)** A D K D G E G L S T Q C E C N I KDsg3(295–311)** E E Y T D N W L A V Y F F T S G NDsg3(400–416)** K L V D Y I L G T Y Q A I D E D T

aDsg3 15- and 17-mer peptides that bind to HLA-DRB1*04:02 (*) and Dsg3 17-mer peptides that do not bind to HLA-DRB1*04:02 (**) based on a peptide bindingalgorithm proposed by Wucherpfennig et al. (6) and confirmed by Tong et al. (29).

bCritical amino acid anchor motifs for peptide binding to HLA-DRB1*04:02 identified by Wucherpfennig et al. (6) and Tong et al. (29) are underlined.

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successful in the therapeutic downregulation of autoaggressiveT cell responses in patients with multiple sclerosis (50, 51).

AcknowledgmentsWe thank Eva Podstawa and Susanne Schwietzke for excellent technical

assistance and Drs. Ralf M€uller, Angela Nagel, and Christian Mobs for

scientific advice and discussions during the evolution of the project. We

also thank Dr. Roland Martin for thoughtful discussion and critical com-

ments on the manuscript.

DisclosuresR.E. has consultant arrangements with and has received payments for lec-

tures fromNovartis and received grants and payments for lectures and travel

expenses from Fresenius Medical Care; M.H. has board memberships with

Novartis and GlaxoSmithKline Stiefel, has consultant arrangements with

Roche, Biogen Idec, and Union Chimique Belge, received payments for

lectures from Janssen-Cilag and Biogen Idec, and received travel support

from Janssen-Cilag; and J.W. is now employed by Miltenyi Biotec. The

other authors have no financial conflicts of interest.

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FIGURE 4. Immunization of HLA-DRB1*04:02–transgenic mice with

HLA-DRB1*04:02–binding Dsg3 T cell epitopes induces IgG Abs against

human Dsg3. (A) Immunization of HLA-DRB1*04:02–transgenic mice

with a set of five HLA-DRB1*04:02–binding Dsg3 peptides (Table III)

induces IgG against human Dsg3. In contrast, immunization of the mice

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Controls include serum IgG reactivity against human Dsg3 of the HLA-

DRB1*04:01–transgenic mice immunized with human Dsg3 [(D–F) n =

2–4]. (Serum samples were diluted 1:20 for ELISA analysis and 1:25 for

immunofluorescence staining.)

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