Autoreactive T and B Cells from Bullous. Clustered in Distinct Regions of BP180 and BP230. This information is current as of November 1, 2018.

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1 This information is current as of November 1, References Subscription Permissions Alerts Autoreactive T and B Cells from Bullous Pemphigoid (BP) Patients Recognize Epitopes Clustered in Distinct Regions of BP180 and BP230 Sybille Thoma-Uszynski, Wolfgang Uter, Susanne Schwietzke, Gerold Schuler, Luca Borradori and Michael Hertl J Immunol 2006; 176: ; ; doi: /jimmunol This article cites 39 articles, 8 of which you can access for free at: Why The JI? Submit online. Rapid Reviews! 30 days* from submission to initial decision No Triage! Every submission reviewed by practicing scientists Fast Publication! 4 weeks from acceptance to publication *average Information about subscribing to The Journal of Immunology is online at: Submit copyright permission requests at: Receive free -alerts when new articles cite this article. Sign up at: Downloaded from by guest on November 1, 2018 The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD Copyright 2006 by The American Association of Immunologists All rights reserved. Print ISSN: Online ISSN:

2 The Journal of Immunology Autoreactive T and B Cells from Bullous Pemphigoid (BP) Patients Recognize Epitopes Clustered in Distinct Regions of BP180 and BP230 1 Sybille Thoma-Uszynski,* Wolfgang Uter, Susanne Schwietzke,* Gerold Schuler,* Luca Borradori, and Michael Hertl 2 * Bullous pemphigoid (BP) is a well-characterized model of autoantibody-mediated autoimmunity, which presumably depends on autoreactive Th cells that promote the activation of autoreactive B cells. The two major autoantigens of BP are BP180 and BP230, two components of dermoepidermal adhesion complexes. Both, autoreactive Th cell responses and autoantibody profiles were characterized in 35 patients with acute onset BP using BP180 and BP230 proteins. Our findings indicate the following: 1) autoreactive Th cells recognized epitopes within the NH 2 -terminal (77.1%), COOH-terminal (65.7%), and central portion (57.1%) of the BP180 ectodomain; 2) IgG autoantibodies were found to exhibit similar or identical reactivity against the NH 2 -terminal (82.8%), COOH-terminal (77.1%), and central portion (37.1%) of the BP180 ectodomain; 3) T and B cell reactivity with the NH 2 -terminal portion of the BP180 ectodomain was associated with extensive BP, whereas the central portion was more frequently recognized in limited BP; 4) only 7 of 16 (43.7%) and 6 of 16 (37.5%) BP patients showed a Th cellular response against the COOH- and NH 2 -terminal regions of BP230, respectively, whereas 5) IgG reactivity against the COOH- and NH 2 -termini of BP230 was detected in 5 of 16 (31.3%) and 6 of 16 (37.5%) patients, respectively. These results demonstrate that Th and B cell reactivities against BP180, are, in contrast to BP230 reactivity, almost constantly detectable in BP patients, and differential epitope recognition of BP180 seems to be associated with distinct clinical severity. These observations support the concept that BP180, but not BP230, is the primary autoantigen of BP critical for disease development. The Journal of Immunology, 2006, 176: Bullous pemphigoid (BP) 3 is the most frequent autoimmune subepidermal blistering disease of the skin. Clinical features of BP include generalized, tense blisters on normal and erythematous skin and, in some cases, blisters and erosions of the mucous membranes (1, 2). A hallmark of BP is the presence of circulating autoantibodies against BP180 and BP230, two components of junctional adhesion complexes called hemidesmosomes, critically involved in the maintenance of dermoepidermal adhesion (1, 3 10). In BP, immunologic injury mediated by autoantibody and/or autoreactive Th cells results in an impairment of the dermoepidermal junction integrity with subepidermal blister formation. Specifically, both in vitro and in vivo studies showed that the autoantibody-mediated tissue damage of BP largely depends on *Department of Dermatology and Department of Medical Informatics, Biometry and Epidemiology, Friedrich-Alexander-University Erlangen-Nürnberg, Erlangen, Germany; Department of Dermatology, Philipps-University of Marburg, Marburg, Germany; and Department of Dermatology, University Hospital, Geneva, Switzerland Received for publication August 4, Accepted for publication November 14, The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by grants from the Wilhelm Sander-Stiftung (No to M.H.), European Community (QLG1-CT to L.B. and M.H.), the Interdisciplinary Center for Clinical Research (Interdisziplinäres Zentrum für Klinische Forschung; C9 to M.H.), and the Swiss National Foundation of Scientific Research ( to L.B.). 2 Address correspondence and reprint requests to Dr. Michael Hertl, Department of Dermatology and Allergology, Philipps University of Marburg, Deutschhausstrasse 9, D Marburg, Germany. address: hertl@med.uni-marburg.de 3 Abbreviations used in this paper: BP, bullous pemphigoid; ECD, extracellular domain; Pg, pemphigoid gestationis; MMP, mucous membrane pemphigoid; SI, stimulation index; YI, Youden Index; IIF, indirect immunofluorescence. complement activation and subsequent chemotactic recruitment of inflammatory cells and secretion of proteases (11 13). Autoantibody pathogenicity may also be due to direct inhibition of the targeted adhesion molecules upon binding to sequential and/or conformational epitopes. BP180 is a transmembrane protein with a large collagenous extracellular domain (ECD) serving as cell adhesion molecule, whereas BP230 is a cytoplasmic protein critically involved in intermediate filament anchorage to the cell membrane (8). The noncollagenous NC16A region adjacent to the transmembrane part on the BP180 ectodomain represents an immunodominant region harboring major autoantibody epitopes recognized not only in BP, but also in related bullous autoimmune diseases such as pemphigoid gestationis (Pg) and mucous membrane pemphigoid (MMP) (14 17). This is in line with the recent ex vivo isolation of BP180- NC16A-reactive B cells from the peripheral blood of BP patients (18). However, autoantibodies also target epitopes located outside the NC16A domain in the COOH terminus and the central portion of the BP180 ECD (19 22). Th cells are presumably involved in early disease development and perpetuation of acquired autoantibody-mediated autoimmune bullous diseases, although the exact molecular mechanisms of specific T cell activation in BP remain elusive. Upon proper costimulation, Th cells become activated and secrete distinct cytokines, which stimulate B cells and thus foster plasma cell development and autoantibody production. Autoreactive Th cells that recognize regions of the BP180 ECD, including the immunodominant NC16A region, have been described in selected patients with BP and Pg (23 27). Clinically, BP has been associated with several MHC class II alleles, HLA-DQB1*0301, -*0305, -*0602, and -*0603 (28). HLA-DQB1*0301, in particular, has been linked to Copyright 2006 by The American Association of Immunologists, Inc /06/$02.00

3 2016 T AND B CELLS IN BP IgG autoantibody production in patients with MMP (29). In addition, a previous study from our laboratory demonstrated that the activation of autoaggressive BP180-reactive Th cells from a cohort of HLA-DQ 1*0301 BP patients was indeed restricted by this BP-associated HLA class II allele (23). Detailed knowledge of autoaggressive T cell and B cell responses in BP may facilitate the development of specific immunomodulatory therapeutic strategies. The purpose of this study was to investigate the BP180- and BP230-specific epitope recognition of autoreactive Th and B cells in patients, and to compare epitope recognition of the cellular and humoral autoimmune response in patients with active BP. Our findings demonstrate that both BP180 and BP230 contain distinct regions that are recognized to a similar extent by autoreactive Th and B cells. Noteworthy, T and B cell reactivity against BP180, but not against BP230, was detected in the majority of BP patients, suggesting that BP180 is the primary autoantigen of BP. Materials and Methods Patients and controls This analysis includes 35 patients with BP and 32 age- and sex-matched controls with unrelated skin conditions such as herpes zoster, erysipelas, atopic dermatitis, or eczema. Blood samples were obtained from patients with clinically active BP (n 35), i.e., patients with newly diagnosed BP who had not yet received immunosuppressive treatment. Patients and volunteers gave written consent to participate in this study, which was adherent to the Declaration of Helsinki Guidelines and has been approved by the local institutional review board. Diagnosis of BP was based on the following criteria: 1) typical clinical presentation with tense cutaneous blisters; 2) histopathological evidence of subepidermal blister formation; 3) linear IgG and/or C3 deposits at the dermoepidermal junction of perilesional skin by direct immunofluorescence; and 4) IgG reactivity with the epidermal side of human saline split skin or IgG reactivity with BP180 or BP230. Severity of BP was classified according to the extent and number of blisters as either limited disease (only few bullae at limited areas of the body involving up to 20% of total body surface) or extensive disease with total body surface involvement greater than 20% and wide-spread bullae. Expression and purification of BP180 and BP230 recombinant proteins The recombinant BP proteins were produced in a baculovirus expression system as GST and 6xHis-tagged fusion proteins as described below (Fig. 1); cdna expression constructs for human BP180 and BP230 were generated as reported previously (30, 31). In brief, cdnas for BP180 or BP230 were used as a template to amplify by PFU DNA polymerase with forward and reverse primers containing either an EcoRI or a NotI site the desired target sequences of BP180 and BP230 (Stratagene). The amplified cdna fragments were subsequently subcloned into the pacghlt-a baculovirus transfer vector (BD Pharmingen) by ligation of double-digested PCR fragments into the correspondingly cut vectors. Correctness of both constructs was verified by sequencing. pacghlt-a vectors were cotransfected with Baculo-DNA (SixPack; BD Clontech) into Sf21 insect cells, and recombinant baculoviruses were amplified using a previously described protocol (32). The recombinant proteins and the GST/6xHis control protein (generated by cotransfection of an empty pacghlt-a vector) were expressed in Sf21 cells as described previously (33). BP180 and BP230 recombinant proteins were purified as follows: three days after infection, baculovirus-infected insect cells were suspended in 1 ml of 6 M guanidine-hydrochloride, incubated for 24 h at 20 C, and then centrifuged (2000 g, 15 min). Solubilized proteins were purified by affinity-chromatography over Nickel-NTA agarose (Qiagen) using the batch method according to the manufacturer s protocol. The BP180 and BP230 recombinant proteins and the GST/6xHis control protein were purified under denaturing conditions by adding 6 M guanidine-hydrochloride to the lysis buffer and 8 M urea to washing and elution buffers. Finally, purified proteins were gradually dialyzed against PBS supplemented with 8 M to 3 M urea at 4 C for 48 h. Affinity-purified proteins were stored in aliquots at 80 C. Protein concentrations were determined by a commercial kit (DC Protein Assay; Bio-Rad) according to a modified protocol by Lowry. BP180ex (baculoprotein containing the entire ECD of BP180; aa residues ), BP180-N1 (baculoprotein of BP180; aa residues 490 FIGURE 1. Recombinant BP180 proteins. A, BP180 is a type II transmembrane protein containing an intracellular transmembrane and extracellular domain with 15 interrupted collagenous regions. B, The recombinant fusion proteins BP180ex, BP180-N1, BP180-N2, BP180-M, BP180-C1, and BP180-C2 are 6xHis- and GST-tagged at their NH 2 -terminal ends (residues ). The affinity-purified BP180 proteins were transferred onto nitrocellulose membrane and were reacted with an anti-gst Abs. 812), BP230-N2 (baculoprotein containing the NH 2 terminus of BP230; aa residues ), BP180-M (baculoprotein of BP180; aa residues ), BP180-C1 (baculoprotein of BP180; aa residues ), BP180-C2 (baculoprotein of BP180; aa residues ), BP230-C1 (baculoprotein containing the COOH-terminus (aa residues ) of BP230), BP230-C2 (baculoprotein containing the COOH-terminus (aa residues ) of BP230), and BP230-N were fractionated by 10% SDS-PAGE and visualized by Coomassie staining and Western blot with a monoclonal mouse anti-gst Ab (1:2000, clone GST3-4C; Zymed) to verify m.w. and immunoreactivity (Figs. 1 and 2). ELISA analysis using BP230 and BP180 recombinant proteins ELISA procedures were performed as described recently (10, 34). Optimal conditions for the ELISA were established by various chessboard titrations. The affinity-purified BP230 and BP180 proteins were immobilized on 96- well polystyrene plates (Maxisorb Immunoplate; Nunc) by coating each well with 0.5 g of BP230-N, BP230-C1, BP230-C2 and BP180ex, BP180-N1, BP180-N2 (baculoprotein of BP180; aa residues ), BP180-M, BP180-C1, and BP180-C2, respectively, or a molar equivalent amount of the control protein GST/6xHis in 100 l of 0.1 M bicarbonate buffer (ph 9.8), at 4 C for 5 h. ELISA plates were then washed five times with TTBS (TBS (ph 7.5) with 0.05% Tween 20; Merck) and blocked for 2 h with 100 l of 5% skimmed milk powder in TTBS at room temperature. BP and control sera were diluted 50-fold in blocking buffer and added to the wells in duplicate. After an incubation period of 12 h at 4 C and consecutive washes, each well was reacted with alkaline phosphatase-labeled goat anti-human IgG ( ) (1/6000; Kirkegaard & Perry Laboratories). After another series of washes, 100 l ofp-nitrophenylphosphate (Kirkegaard & Perry Laboratories) were added to the wells, and OD was measured at 405 nm when a defined patient serum (positive reference control)

4 The Journal of Immunology 2017 FIGURE 2. Recombinant BP230 proteins. A, BP230 is an intracellular hemidesmosomal component, which consists of a central rod flanked by globular end domains. B, The recombinant BP230 fusion proteins are 6xHis- and GST-tagged at their NH 2 -terminii. Two COOH-terminal constructs (BP230-C1, containing residues and BP230-C2, containing residues ) and one NH 2 -terminal construct (BP230-N, residues ) were used. The purified BP230 proteins were transferred onto nitrocellulose membrane and reacted with an anti-gst Ab. reached an OD of 1.0 as determined by a Wallac 1420 microplate reader (Wallac). All extinction data were standardized based on the positive and negative internal standards, which were set to 1 and 0 OD units, respectively. Background reactivity of IgG to the control protein GST/6xHis was subtracted from the mean OD values obtained with BP180-N and BP180-C proteins. To evaluate plate-to-plate variability, each plate included identical internal controls (two positive serum samples, one control serum). T cell proliferative assays with BP180 and BP230 PBMC from BP patients were isolated by Ficoll centrifugation. A total of 10 5 cells/ml in RPMI 1640 supplemented with 2 mm L-glutamine, 2 mm penicillin, 50 g/ml streptomycin (Invitrogen Life Technologies), and 10% human serum (PAA Laboratories) were plated in triplicate in round-bottom microtiter plates. BP180 and BP230 recombinant proteins were added at a final concentration of 10 g/ml. PHA (1%) was used as a positive control, and GST at 10 g/ml was used as mock control. After 6 days, each well was pulsed with 0.5 Ci [ 3 H]thymidine for 12 h, and incorporation was measured with a scintillation counter. BP180- or BP230-specific T cell proliferation was expressed as a stimulation index (SI), which is the ratio of [ 3 H]thymidine uptake in cultures with Ag and cultures without Ag. SI values 2.5 were considered positive. Statistical analysis The focus of this study is a comparison between results obtained with 1) two different assays yielding quantitative data and 2) different epitopes. ELISA results (OD values) had a skewed distribution, which was scarcely amenable to log transformation, and were thus tested with nonparametric statistical methods, e.g., Wilcoxon-Mann-Whitney or Kruskal-Wallis test for overall equality of mean scores. SI values followed a skewed distribution, even after log transformation, and were treated in an identical fashion (the log transformation was used to rescale SI values for better graphical representation; this transformation has no influence on the rank statistics used). The distribution of ELISA data is shown as box plot : the box shows first and third quartile, the median (as line) and the arithmetic mean (as dot), the whiskers represent values below the first quartile and above the third quartile within the 1.5-fold interquartile range, respectively, and outliers beyond the whiskers are shown as squares. The correlation between ELISA and SI results obtained with the same epitopes, and between related epitopes (BP180-C2 and BP180-C1 and BP180-N1 and BP180-N2, respectively) obtained with the same assay was quantified with the Spearman rank correlation coefficient. For data analysis, the statistical software package SAS, version 8.2 (SAS Institute), was used. Receiver operating characteristic curves were compiled by additionally using a freeware SAS macro package (35) to evaluate the diagnostic properties of different IgG Abs to BP180 Ags with respect to correctly classifying cases of BP. Because there are no natural cutpoints for these Ab levels, and because there are no previous studies that had provided evidence concerning an appropriate diagnostic threshold (except in case of BP 230 and BP180ex 10), an optimum cutpoint was determined data-adaptively by choosing the OD values with the largest Youden Index (YI sensitivity specificity 1). For those analyses requiring dichotomization of SI results as positive and negative, values 2.5 were considered positive. For dichotomization of ELISA results as positive and negative, cutpoints were defined based on the YIs obtained with the different BP180 and BP230 recombinants (see Table II). Results Clinical and immunoserological phenotype of the studied BP patients The present analysis involved 35 patients with acute BP. None of the patients received topical or systemic immunosuppressive treatment at the time of blood sampling. Among the 35 BP patients, 12 had limited and 23 extensive involvement. Mucosal involvement was observed in five BP patients, two of whom had limited disease. All patients sera were reactive with human saline split skin (97% reactive with the epidermal site of the blister) by indirect immunofluorescence (IIF) microscopy (Table I). Overall, 94.2 and 80% of the studied BP patients had anti-bp180 and anti-bp230 IgG autoantibody, respectively. Thus, a relatively homogeneous patient cohort was available for detailed analyses regarding BP180- and BP230-specific T cell proliferative responses and IgG reactivity (Table I). IgG autoreactivity with BP180 and BP230 Several recombinant forms of BP180 and BP230 known to harbor B cell epitopes were successfully produced in a baculovirus expression system and subsequently affinity purified. The recombinant proteins, the apparent m.w. of which corresponded to that predicted on the basis of their cdna, were immunoreactive with appropriate mabs directed against the GST-tag (Figs. 1 and 2) and with control BP sera (data not shown). Using the BP180 and BP230 recombinants for ELISA analysis, the diagnostic performance was best regarding IgG reactivity against the entire BP180 ECD (BP180ex; 90%) followed by IgG reactivity against NH 2 -, COOH-terminal, and midportion of the Table I. Clinical and immunological characteristics of BP patients and healthy control subjects a Patients Controls Sex (male/female) 21 of of 32 Age (min-median-max) Involvement: limited 12 of 35 None extensive 23 of 35 None IIF (SSS) 35 NT Epidermal 31 NT Dermal epidermal 3 NT Dermal 1 NT Anti-BP180 IgG only 7 of 35 None Anti-BP230 IgG only 2 of 35 None Anti-BP180 and anti-bp230 IgG 26 of 35 None a Min, Minimum; Max, maximum; SSS, saline split skin; 2 NT, not tested.

5 2018 T AND B CELLS IN BP Table II. Cutpoints (OD values with maximum corresponding YI with sensitivity and specificity, including exact 95% confidence intervals) Cutpoint (total IgG) Y Sensitivity Specificity BP180ex ( ) ( ) BP180-N BP180-N BP180-M BP180-C BP180-C BP230-C ( ) ( ) BP230-C ( ) ( ) BP230-N ( ) ( ) BP180 ECD (Fig. 3 and Table II). The majority, e.g., 29 of 35 (82.8%), of the BP patients sera preferentially recognized the NH 2 - terminal portion (BP180-N2), whereas only about one-third of the BP patients had detectable IgG against the midportion of the BP180 ECD (BP180-M) (Fig. 3 and Table III). The COOH-terminal part of the BP180 ECD covered by the recombinant proteins BP180-C1 and BP180-C2 was a target for IgG autoantibodies of almost 75% of the studied BP patients (Fig. 3 and Table III). IgG reactivity against BP230 was seen in 70% of the studied BP patients, with the NH 2 terminus being recognized by 72.4% of patients sera and the COOH terminus in 65.4% of the patients (Fig. 3 and Table IV). Due to the smaller number of BP230-reactive patients, their clinical phenotype could not be related to BP230 epitope recognition. IgG reactivity against the recombinant BP180 proteins was analyzed regarding clinical severity. Almost all patients with limited and extensive disease had detectable IgG against BP180, particularly against the recombinant proteins encompassing the NH 2 -terminal portion of the BP180 ECD (Table III). The NH 2 terminus including the immunodominant NC16A domain (BP180-N2) was recognized by 86.9% of the BP patients with extensive and by 75% of the patients with limited disease ( p 0.39, Fisher s exact test, 2-sided). In addition, most patients had detectable IgG directed against the COOHterminal recombinant proteins of BP180 ranging from 73.9% to 83.3% without significant differences between the clinical categories. The midportion of the BP180 ECD (BP 180-M) was more frequently recognized by specific IgG in limited BP (50%) than in extensive BP (30.4%) (Table III). There was, however, no direct relationship between a distinct autoantibody profile and the involvement of mucous membranes in the studied BP patients. Noteworthy, there is no sequential homology between the NH 2 terminus, midportion, and COOH terminus of the BP180 ectodomain (6). T cell recognition of BP180 of the BP patients This study was restricted to patients with acute onset BP before treatment with systemic glucocorticoids and adjuvants because the Table IV. T cell and IgG autoreactivity against BP230 T Cell Response IgG Response Correlation of T and B Cell Reactivity Positive % Positive % (%) BP230-C1 7/ / /16 (75.0) BP230-C2 6/ / /16 (75.0) BP230-N 6/ / /16 (37.5) in vitro detection of proliferative T cell responses to BP180 was strongly compromised by immunosuppressive treatment (data not shown). The majority (28 of 35; 80%) of the studied BP patients developed a significant proliferative T cell response to the ECD of BP180 (Table III). The NH 2 -terminal portion of the BP180 ECD (BP180-N1/N2) including the NC16A region was most frequently ( %) recognized, followed by the COOH-terminal (BP180-C1/C2; %) and the midportions of the ECD of BP180 (BP180-M; 57.1%) (Table III). With regard to the clinical phenotype, almost 74% of the BP patients with extensive disease showed T cell responses against the NH 2 -terminal regions of the ECD of BP180, 56.5% to the COOHterminal regions, and 47.8% BP patients had also autoaggressive T cells specific for the midportion of BP180 (Table III). Noteworthy, all of the five BP patients with an exclusive T cellular response against the NC16A domain showed an extensive phenotype with numerous blisters/erosions on the trunk. In contrast, BP patients with limited disease showed less frequent T cell response against the NH 2 -terminal region of the ECD (66.7%), but an increased T cell response against midportion (75%) and COOH terminus (83.3%) of BP180. However, all of these differences were nonsignificant ( p 0.15, Fisher s exact test). Comparison of BP180-specific T cell and B cell reactivity Based on the hypothesis that T cell and IgG reactivities are directly linked, we analyzed the correlation of T cell and B cell responses against distinct regions of BP180 and BP230 (Tables III and IV). The majority of the BP patients showed autoreactive T cell and IgG reactivity against the ECD of BP180 (BP180ex; 71.4%). This association was strongest when reactivity with the NC16A region (BP180-N2; 71.4%) was assessed, compared with that with the COOH terminus (BP180-C1/C2; ) and midportion of BP180 (BP180-M; 51.4%) (Table III) (Fig. 4). IgG reactivity against the recombinant proteins BP180-N1 (aa ) and BP180-N2 (aa ) was strongly correlated (0.98; p ; BP180-N1 vs BP180-N2; Fig. 5A), an observation suggesting that the major epitope(s) was located within the NC16A region (aa ), which represents exactly the overlapping region between BP180-N1 and BP180-N2. In addition, T cell reactivity against these overlapping BP180 recombinants encompassing the NH 2 -terminal region of the BP180 ECD revealed a high degree of correlation, providing support to the idea of the Table III. Clinical severity of BP and T cell and IgG reactivity against distinct regions of BP180 Extensive Disease Limited Disease Total Positive (%) T cell/igg reactivity Positive (%) T cell/igg reactivity Positive (%) T cell/igg reactivity BP180ex 19 of 23 (82.6)/18 of 23 (78.3) 9 of 12 (75.0)/11 of 12 (91.7) 28 of 35 (80.0)/29 of 35 (82.8) BP180-N1 17 of 23 (73.4)/17 of 23 (73.9) 8 of 12 (66.7)/6 of 12 (50.0) 25 of 35 (71.4)/23 of 35 (65.7) BP180-N2 17 of 23 (73.4)/20 of 23 (86.9) 8 of 12 (66.7)/9 of 12 (75.0) 25 of 35 (71.4)/29 of 35 (82.8) BP180-M 11 of 23 (47.8)/7 of 23 (30.4) 9 of 12 (75.0)/6 of 12 (50.0) 20 of 35 (57.1)/13 of 35 (37.1) BP180-C2 13 of 23 (56.5)/17 of 23 (73.9) 10 of 12 (83.3)/9 of 12 (75.0) 23 of 35 (65.7)/26 of 35 (74.3) BP180-C1 13 of 23 (56.5)/17 of 23 (73.9) 9 of 12 (75.0)/10 of 12 (83.3) 22 of 35 (62.8)/27 of 35 (77.1)

6 The Journal of Immunology 2019 FIGURE 3. IgG reactivity with distinct regions of the BP180 ectodomain. The sera of 35 BP patients and 32 controls were reacted with BP180-derived recombinant proteins by ELISA, and IgG binding was visualized by a substrate reaction. Data are displayed as box plots: the box shows first and third quartiles, the median (as line), and the arithmetic mean (as dot), the whiskers represent values below the first and above the third quartile within the 1.5-fold interquartile range, respectively, and outliers beyond the whiskers are shown as squares. The dotted lines indicate the cutoff values as evaluated by the maximization of YI. existence of T cell epitopes within the NC16A region of BP180 (Fig. 5C). On the same line, IgG and T cell reactivity against the two overlapping COOH-terminal BP180 recombinants, BP180-C2 and BP180-C1, was strongly correlated (0.71; p ), suggesting that T and B cell epitopes were located in the same regions (Fig. 5B). Thus, the major relevant T cell and B cell epitopes of BP180 are presumably harbored within residues aa (BP180-N2) and residues aa (BP180-C2), respectively. Recognition of BP230 by autoreactive T and B cells Sixteen BP patients were further analyzed for BP230-specific autoreactive T cell responses. Seven of 16 (43.8%) and 6 of 16 (37.5%) patients had IgG autoantibody reactive with the COOH terminus and the NH 2 terminus of BP230, respectively, with 8 of 16 (50%) serum samples showing anti-bp230 IgG reactivity (Table IV). Overall, 9 of 16 (56.3%) BP230-IgG-reactive patients showed a T cellular response to COOH terminus and/or NH 2 terminus of BP230. Correlation of BP230-specific T cell proliferation and IgG reactivity was weak to negligible for any of the investigated BP230 recombinant proteins (Fig. 6). When the concordance of BP180 and BP230-specific IgG reactivity was analyzed, 43.8% of the BP patients (7 of 16) had both BP180- and BP230-specific IgG, whereas 43.8% of the sera were exclusively anti-bp180 IgG reactive (Table V). Seven of the 16 (43.8%) BP patients showed both T and IgG reactivity against the NH 2 and/or COOH terminus of BP230 (Table IV). Noteworthy, the majority (14 of 16) of the studied patients showed proliferative T cell responses to BP180 including the aforementioned BP230- reactive patients (Table IV). Discussion In this study, we present a comprehensive analysis of T and B cell recognition of BP180 and BP230, the autoantigens of the potentially devastating autoimmune bullous disorder, BP. Our results show that the vast majority of the 35 studied BP patients analyzed had both autoaggressive T cells and IgG autoantibody reactive with defined extracellular regions of BP180 and, to a lesser extent, with distinct NH 2 - and COOH-terminal domains of BP230. This finding is of particular interest in light of the current discussion as to whether the transmembranous adhesion molecule, BP180, or the intracellular hemidesmosomal component, BP230, is the major autoantigen of BP. In extension to previous investigations, this study provides evidence that autoaggressive T cells recognize three major regions of the ECD of BP180, i.e., NH 2 terminus, midportion, and COOH terminus. Previous reports only identified serum autoantibody (10, 21, 33, 34) and autoaggressive T cells (23) recognizing epitopes within the NH 2 -terminal region of the BP180 ECD in the majority

7 2020 T AND B CELLS IN BP FIGURE 4. T and B cell reactivity against distinct regions of the BP180 ectodomain. T cells and sera derived from 35 patients with acute onset BP were cocultured with the BP180 recombinants or reacted by ELISA, respectively. BP180-specific IgG reactivity (OD values at 405 nm) and T cell proliferation (BP180-specific SI) are displayed as dot plots. SI was defined as the ratio of [ 3 H]thymidine uptake in T cell cultures with/ without Ag and was considered to be significant at a value 2.5. of BP patients. Specifically, these studies focused on T cell recognition of the NC16A subdomain, which resides within the NH 2 - terminal region of BP180 (24, 25, 27, 36). In fact, a considerable number of BP and Pg patients studied displayed NC16A-specific peripheral T cell responses, which were mainly of the Th2 type or of a mixed Th1/Th2 type (24, 25, 27, 36). Specifically, Lin et al. (24) identified autoreactive T cells directed against the NC16A region of the BP180 ECD in 12 patients with active BP and two patients with Pg. Because the NC16A region also represents the major binding site for autoantibodies in BP, we concluded that this region contained immunodominant epitopes for both autoaggressive T and B cells in BP. This contention is supported by the observation that IgG autoantibodies against the NC16A are pathogenic in a mouse model of BP (11, 12). Leyendeckers et al. (18) recently demonstrated that BP180-NC16A-reactive B cells are present in the peripheral blood of the majority of BP patients. T cell recognition of BP180-NC16A seems to be rather heterogeneous because BP180-NC16A-specific T cell clones from BP patients preferentially expressed TCRBV13, whereas T cell clones derived from a Pg patient expressed TCRBV3 (26). Recently, IFN- secretion by NC16A-specific autoreactive T cells was detected in 2 of 10 patients with MMP and in 2 of 17 patients with BP, but not in healthy controls (27). These results are in apparent contrast to a previous study from our group showing that BP180-reactive T cells from BP patients preferentially secreted Th2-like cytokines, whereas BP180-responsive T cell clones from healthy individuals secreted Th1-like cytokines, such as IFN- (23). In contrast to the aforementioned studies, the present study demonstrates that both autoreactive Th cells and IgG autoantibodies not only recognize epitopes located within the NC16A region, but also within the COOH-terminal and central portion of the ECD of BP180. Although the NC16A region turned out to be a major T cell target in our analyses, the COOH terminus of BP180 was recognized both by T cells and autoantibodies in up to 65 and 75% of the studied BP patients, respectively. Pathogenetically, this BP180 region is of particular interest, because IgG reactivity with the COOH terminus has been associated with a distinct clinical phenotype of BP presenting with a preferential mucosal involvement (21, 34, 37). T cells responsive to the COOH terminus had not yet been identified. Unexpectedly, the midportion of the ECD of BP180 was recognized by T cells in 57.1% of the BP patients, whereas autoantibody reactivity against this region was only observed in

8 The Journal of Immunology 2021 FIGURE 5. IgG autoantibodies and autoreactive T cells recognize the same epitopes of the NH 2 and COOH terminus of the BP180 ectodomain. The proliferative T cell responses and IgG reactivity of 35 BP patients against NH 2 - and COOH-terminal regions of BP180 were compared and are displayed as dot blot. Correlation of IgG against overlapping proteins of the NH 2 terminus (A) and COOH terminus (B) was relatively high (BP180-N1 vs BP180-N2, p ; and BP180-C1 vs BP180-C2, p ). Similarly, T cell reactivity against overlapping NH 2 -(C) and COOH-terminal (D) BP180 proteins was relatively high (BP180-N1 vs BP180-N2, p ; and BP180-C1 vs BP180-C2, p ). 37% of the patients. This antigenic region of the BP180 ECD had been identified as the exclusive target of a subgroup of patients with BP (20). This observation implies that, in BP, relevant B cell epitopes are present in three distinct regions, i.e., NH 2 terminus, COOH terminus, and the central portion of the ECD of BP180. When BP180-specific T cell responses and IgG reactivity in individual BP patients were compared, there was a correlation of at least 50% for the various BP180 recombinant proteins. The NH 2 -terminal portion of the BP180 ECD was targeted by T and B cells to the greatest extent, whereas an overlapping T and B cell response to midportion and COOH terminus of BP180 was only FIGURE 6. T and B cell reactivity against BP230. BP230-specific IgG reactivity (OD values at 405 nm) and proliferative T cell responses (as determined as a SI) were investigated in 16 patients with acute onset BP. SI was defined as the ratio of [ 3 H]thymidine uptake in T cell cultures with/without Ag and was considered to be significant at a value 2.5. Data are displayed as dot plots for the different BP230 recombinant proteins. Correlation of BP230-specific IgG and T cell proliferation was moderate.

9 2022 T AND B CELLS IN BP Table V. Concordance between T cell a and IgG d reactivity against BP180 and BP230 pos. BP230 a)/b) Total seen in 50% of the patients (Table III). This finding is in line with a previous study by Lin et al. (25) who detected both autoantibody and T cell reactivity directed against the NC16A-domain of BP180 in the majority of the patients investigated. A similar degree of overlapping T and B cell recognition was also seen in most of the BP patients for the COOH-terminal region of BP180. The midportion of the BP180 ECD was more frequently recognized by autoreactive T cells than by IgG autoantibodies. Moreover, T and B cell recognition of the midportion of BP180 was seen more frequently in patients with limited than in patients with extensive disease (41.7 vs 13%) (Table III). Thus, differential BP180 epitope recognition may be associated with limited vs extensive BP. As shown in Fig. 7, there is a body of evidence demonstrating that both T and B cell epitopes are located in defined and restricted regions of the ectodomain of BP180. Specifically, a recent study by di Zenzo et al. (21) has shown by phage display library screening that IgG autoantibodies recognized epitopes clustered in the three aforementioned regions of the BP180 ectodomain. Ongoing studies will have to define whether T and B cell epitopes of BP180 are indeed identical, as partly shown for the NC16A domain, or just clustered in distinct regions. A major finding of the present study was the identification of autoaggressive T cells that targeted distinct regions of BP230, the second major autoantigen of BP. The current understanding of the neg. BP180 a,b pos. 9/7 6/7 15/14 neg. 0/1 1/1 1/2 a [ 3 H]Thymidine uptake (3-HT) was calculated as a SI (3-HT in cultures with/ without Ag). A SI 2.5 was considered to be significant. pos, positive; neg, negative. b pos.: either BP230-C1-, BP230-C2-, BP230-N-, BP180-ex, BP180-N1, BP180- N2, BP180-M, BP180-C1, or BP180-C2 reactive 3-HT response as determined by ELISA. pos.: either BP230-C1-, BP230-C2-, BP230-N-, BP180-ex, BP180-N1, BP180-N2, BP180-M, BP180-C1, or BP180-C2 reactive IgG above cutpoint. pathogenesis of BP supports the concept that IgG against BP230 are not the primary pathogenic autoantibody but may enhance the local inflammatory response that precedes the autoantibody-induced loss of dermoepidermal adhesion (2, 7). Autoreactive T cells and/or IgG autoantibodies against the NH 2 - and COOH-terminal regions of BP230 were detectable in 40% of the tested BP patients. In 50% of the studied patients, both autoaggressive T and B cells recognized identical regions, i.e., the NH 2 - and COOHterminal rod domains of BP230. The majority of the patients with BP230-specific T cellular responses had also BP230-reactive IgG in their sera. Noteworthy, 90% of the BP patients, including those with BP230-reactive T cells, had also T cells specific for BP180. A single BP patient was neither responsive to BP180 on the T cellular level nor showed a proliferative T cell response to BP230. Despite extensive clinical data, no relationship could be established between T and B cell recognition of BP230 and a distinct clinical phenotype. Recent studies from our group provided statistical evidence that the titers of BP230-specific IgG were related to a more limited phenotype of BP, whereas the extent of IgG reactivity against the NH 2 terminus of BP180 was associated with extensive BP (10, 34). Noteworthy, recent studies in pemphigus vulgaris, an unrelated autoimmune bullous skin disorder, showed that autoaggressive T cells specific for desmoglein 3 preferentially targeted regions located in the NH 2 -terminal domains EC1 and EC2 (38) that were also preferentially recognized by IgG autoantibodies (39). In a HLA class II-transgenic mouse model of myasthenia gravis, autoreactive T cells responded to three cytoplasmic peptide sequences of the human acetylcholine receptor, which were also targeted by the pathogenic autoantibodies (40). Together, these findings strongly suggest that both Th and B cells recognize similar or identical regions of the autoantigens of several human autoimmune disorders. In summary, the present findings demonstrate that BP180 and, to a lesser extent, BP230 are major targets for both autoreactive T cells and IgG autoantibodies in active BP. With regard to BP180 recognition, autoreactive T cells and IgG autoantibodies target similar or identical antigenic regions within the NH 2 and COOH termini and the midportion of the ECD of BP180, with the NC16A FIGURE 7. Synopsis of T and B cell epitopes located within the BP180 ectodomain. Three major antigenic regions of the ectodomain of BP180 are targeted by autoreactive T cells and IgG autoantibodies, i.e., the immunodominant NC16a region (aa ) adjacent to the transmembrane region, the midportion (aa ), and the COOH terminus (aa ) of BP180. The findings of the present and previous studies are listed with their references in the literature for T cell reactivity only (normal), B cell reactivity (italic), and both T and B cell reactivity (bold).

10 The Journal of Immunology 2023 domain being the immunodominant region. Noteworthy, preferential T and B cell recognition of the NH 2 terminus and midportion of BP180 seemed to be associated with extensive and limited BP, respectively. In addition, T and B cell recognition of BP230 was focused on the NH 2 - and COOH-terminal rod domains. Finally, our observations support the concept that BP180 is the primary autoantigen of BP. Acknowledgments We are indebted to Dr. J. R. Stanley (University of Pennsylvania, Philadelphia, PA) and Dr. J. Uitto (Jefferson Medical College, Philadelphia, PA), who generously provided cdnas for human BP230. Disclosures The authors have no financial conflict of interest. References 1. Jordon, R. E., E. H. Beutner, E. Witebsky, G. Blumental, W. L. Hale, and W. F. Lever Basement zone antibodies in bullous pemphigoid. J. Am. Med. Assoc. 200: Hertl, M Humoral and cellular autoimmunity in autoimmune bullous skin disorders. Int. Arch. Allergy Immunol. 122: Stanley, J. R., P. Hawley-Nelson, S. H. Yuspa, E. M. Shevach, and S. I. Katz Characterization of bullous pemphigoid antigen: a unique basement membrane protein of stratified squamous epithelia. Cell 24: Labib, R. S., G. J. Anhalt, H. P. Patel, D. F. Mutasim, and L. A. Diaz Molecular heterogeneity of the bullous pemphigoid antigens as detected by immunoblotting. J. Immunol. 136: Stanley, J. R., T. Tanaka, S. Mueller, V. Klaus-Kovtun, and D. Roop Isolation of complementary DNA for bullous pemphigoid antigen by use of patients autoantibodies. J. Clin. Invest. 82: Giudice, G. J., D. J. Emery, and L. A. Diaz Cloning and primary structural analysis of the bullous pemphigoid autoantigen BP180. J. Invest. Dermatol. 99: Guo, L., L. Degenstein, J. Dowling, Q. C. Yu, R. Wollmann, B. Perman, and E. Fuchs Gene targeting of BPAG1: abnormalities in mechanical strength and cell migration in stratified epithelia and neurologic degeneration. Cell 81: Borradori, L., and A. Sonnenberg Structure and function of hemidesmosomes: more than simple adhesion complexes. J. Invest. Dermatol. 112: Ruhrberg, C., and F. M. Watt The plakin family: versatile organizers of cytoskeletal architecture. Curr. Opin. Genet. Dev. 7: Thoma-Uszynski, S., W. Uter, S. Schwietzke, S. C. Hofmann, T. Hunziker, P. Bernard, R. Treudler, C. C. Zouboulis, G. Schuler, L. Borradori, and M. Hertl BP230- and BP180-specific auto-antibodies in bullous pemphigoid. J. Invest. Dermatol. 122: Liu, Z., J. M. Shipley, T. H. Vu, X. Zhou, L. A. Diaz, Z. Werb, and R. M. Senior Gelatinase B-deficient mice are resistant to experimental bullous pemphigoid. J. Exp. Med. 188: Liu, Z., S. D. Shapiro, X. Zhou, S. S. Twining, R. M. Senior, G. J. Giudice, J. A. Fairley, and L. A. Diaz A critical role for neutrophil elastase in experimental bullous pemphigoid. J. Clin. Invest. 105: Chen, R., G. Ning, M. L. Zhao, M. G. Fleming, L. A. Diaz, Z. Werb, and Z. Liu Mast cells play a key role in neutrophil recruitment in experimental bullous pemphigoid. J. Clin. Invest. 108: Zillikens, D., P. A. Rose, S. D. Balding, Z. Liu, M. Olague-Marchan, L. A. Diaz, and G. J. Giudice Tight clustering of extracellular BP180 epitopes recognized by bullous pemphigoid autoantibodies. J. Invest. Dermatol. 109: Sakuma-Oyama, Y., A. M. Powell, N. Oyama, S. Albert, B. S. Bhogal, and M. M. Black Evaluation of a BP180-NC16a enzyme-linked immunosorbent assay in the initial diagnosis of bullous pemphigoid. Br. J. Dermatol. 151: Giudice, G. J., D. J. Emery, B. D. Zelickson, G. J. Anhalt, Z. Liu, and L. A. Diaz Bullous pemphigoid and herpes gestationis autoantibodies recognize a common non-collagenous site on the BP180 ectodomain. J. Immunol. 151: Zillikens, D., F. Caux, J. M. Mascaro, U. Wesselmann, E. Schmidt, C. Prost, J. P. Callen, E. B. Brocker, L. A. Diaz, and G. J. Giudice Autoantibodies in lichen planus pemphigoides react with a novel epitope within the C-terminal NC16A domain of BP180. J. Invest. Dermatol. 113: Leyendeckers, H., K. Tasanen, L. Bruckner-Tuderman, D. Zillikens, C. Sitaru, J. Schmitz, and N. Hunzelmann Memory B cells specific for the NC16A domain of the 180 kda bullous pemphigoid autoantigen can be detected in peripheral blood of bullous pemphigoid patients and induced in vitro to synthesize autoantibodies. J. Invest. Dermatol. 120: Nakatani, C., T. Muramatsu, and T. Shirai Immunoreactivity of bullous pemphigoid (BP) autoantibodies against the NC16A and C-terminal domains of the 180 kda BP antigen (BP180): immunoblot analysis and enzyme-linked immunosorbent assay using BP180 recombinant proteins. Br. J. Dermatol. 139: Maczek, C., S. Thoma-Uszynski, G. Schuler, and M. Hertl Simultaneous onset of pemphigoid and factor VIII antibody hemophilia. Hautarzt. 53: di Zenzo, G., F. Grosso, M. Terracina, F. Mariotti, O. De Pita, K. Owaribe, A. Mastrogiacomo, F. Sera, L. Borradori, and G. Zambruno Characterization of the anti-bp180 autoantibody reactivity profile and epitope mapping in bullous pemphigoid patients. J. Invest. Dermatol. 122: Mariotti, F., F. Grosso, M. Terracina, M. Ruffelli, P. Cordiali-Fei, F. Sera, G. Zambruno, A. Mastrogiacomo, and G. Di Zenzo Development of a novel ELISA system for detection of anti-bp180 IgG and characterization of autoantibody profile in bullous pemphigoid patients. Br. J. Dermatol. 151: Büdinger, L., L. Borradori, C. Yee, R. Eming, S. Ferencik, H. Grosse-Wilde, H. F. Merk, K. Yancey, and M. Hertl Identification and characterization of autoreactive T cell responses to bullous pemphigoid antigen 2 in patients and healthy controls. J. Clin. Invest. 102: Lin, M. S., M. A. Gharia, S. J. Swartz, L. A. Diaz, and G. J. Giudice Identification and characterization of epitopes recognized by T lymphocytes and autoantibodies from patients with herpes gestationis. J. Immunol. 162: Lin, M. S., C. L. Fu, G. J. Giudice, M. Olague-Marchan, A. M. Lazaro, P. Stastny, and L. A. Diaz Epitopes targeted by bullous pemphigoid T lymphocytes and autoantibodies map to the same sites on the bullous pemphigoid 180 ectodomain. J. Invest. Dermatol. 115: Hacker-Foegen, M. K., D. Zillikens, G. J. Giudice, and M. S. Lin T cell receptor gene usage of BP180-specific T lymphocytes from patients with bullous pemphigoid and pemphigoid gestationis. Clin. Immunol. 113: Black, A. P., S. L. Seneviratne, L. Jones, A. S. King, S. Winsey, G. Arsecularatne, F. Wojnarowska, and G. S. Ogg Rapid effector function of circulating NC16A-specific T cells in individuals with mucous membrane pemphigoid. Br. J. Dermatol. 151: Delgado, J. C., D. Turbay, E. J. Yunis, J. J. Yunis, E. D. Morton, K. Bhol, R. Norman, C. A. Alper, R. A. Good, and R. Ahmed A common major histocompatibility complex class II allele HLA-DQB1*0301 is present in clinical variants of pemphigoid. Proc. Natl. Acad. Sci. USA 93: Setterfield, J., J. Theron, R. W. Vaughan, K. I. Welsh, E. Mallon, F. Wojnarowska, S. J. Challacombe, and M. M. Black Mucous membrane pemphigoid: HLA-DQB1*0301 is associated with all clinical sites of involvement and may be linked to antibasement membrane IgG production. Br. J. Dermatol. 145: Borradori, L., P. J. Koch, C. M. Niessen, S. Erkeland, M. R. van Leusden, and A. Sonnenberg The localization of bullous pemphigoid antigen 180 (BP180) in hemidesmosomes is mediated by its cytoplasmic domain and seems to be regulated by the 4 integrin subunit. J. Cell Biol. 136: Skaria, M., F. Jaunin, T. Hunziker, S. Riou, H. Schumann, L. Bruckner-Tuderman, M. Hertl, P. Bernard, J. H. Saurat, B. Favre, and L. Borradori IgG autoantibodies from bullous pemphigoid patients recognize multiple antigenic reactive sites located predominantly within the B and C subdomains of the COOH-terminus of BP230. J. Invest. Dermatol. 114: Amagai, M., T. Hashimoto, N. Shimizu, and T. Nishikawa Absorption of pathogenic autoantibodies by the extracellular domain of pemphigus vulgaris antigen (Dsg3) produced by baculovirus. J. Clin. Invest. 94: Haase, C., L. Büdinger, L. Borradori, C. Yee, H. F. Merk, K. Yancey, and M. Hertl Detection of IgG autoantibodies in the sera of patients with bullous and gestational pemphigoid: ELISA studies utilizing a baculovirus- encoded form of bullous pemphigoid antigen 2. J. Invest. Dermatol. 110: Hofmann, S., S. Thoma-Uszynski, T. Hunziker, P. Bernard, C. Koebnick, A. Stauber, G. Schuler, L. Borradori, and M. Hertl Severity and phenotype of bullous pemphigoid relate to autoantibody profile against the NH2- and COOH-terminal regions of the BP180 ectodomain. J. Invest. Dermatol. 119: Witte, S Macro for generation of ROC-curves. Proceedings of the 6th Conference of SAS Application in Research and Development, Dortmund, Germany. 36. Lin, M. S., C. L. Fu, M. Olague-Marchan, M. K. Hacker, D. Zillikens, G. J. Giudice, and J. A. Fairley Autoimmune responses in patients with linear IgA bullous dermatosis: both autoantibodies and T lymphocytes recognize the NC16A domain of the BP180 molecule. Clin. Immunol. 102: Balding, S. D., C. Prost, L. A. Diaz, P. Bernard, C. Bedane, D. Aberdam, and G. J. Giudice Cicatricial pemphigoid autoantibodies react with multiple sites on the BP180 extracellular domain. J. Invest. Dermatol. 106: Veldman, C. M., K. L. Gebhard, W. Uter, R. Wassmuth, J. Grotzinger, E. Schultz, and M. Hertl T cell recognition of desmoglein 3 peptides in patients with pemphigus vulgaris and healthy individuals. J. Immunol. 172: Sekiguchi, M., Y. Futei, Y. Fujii, T. Iwasaki, T. Nishikawa, and M. Amagai Dominant autoimmune epitopes recognized by pemphigus antibodies map to the N-terminal adhesive region of desmogleins. J. Immunol. 167: Yang, H., E. Goluszko, C. David, D. K. Okita, B. Conti-Fine, T. S. Chan, M. A. Poussin, and P. Christadoss Mapping myasthenia gravis-associated T cell epitopes on human acetylcholine receptors in HLA transgenic mice. J. Clin. Invest. 109: