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Identification of Immunodominant Th2-Cell Epitopes in Chinese Patients with Bullous Pemphigoid

Open ArchivePublished:March 26, 2018DOI:https://doi.org/10.1016/j.jid.2018.03.1515
      Bullous pemphigoid (BP) is a subepidermal autoimmune blistering disease caused by autoantibodies targeting the juxtamembranous extracellular noncollagenous 16A (NC16A) domain of human collagen XVII (also known as BP180). Because T-helper (Th) cells are essential for antibody responses to antigens, we adopted an assay to map the immunodominant Th2-cell epitopes in NC16A. We synthesized 22 overlapping peptides spanning the entire sequence of BP180-NC16A and investigated the reactivity of Th2 cells from patients with BP to these peptides using the Enzyme-Linked ImmunoSpot (ELISPOT) assay. We screened two epitope peptides, P2 (492–506 aa: VRKLKARVDELERIR) and P5 (501–515 aa: ELERIRRSILPYGDS), and confirmed that these epitopes play a dominant role in stimulating CD4+ T-cell proliferation and Th2 IL-4 cytokine production, and activating B cells to secrete autoantibodies. These immunodominant epitopes are HLA-DR-restricted and were observed in subjects with different HLA alleles. This work contributes to elucidation of the epitope-mediated immunologic pathogenesis of BP, and the identified Th2-cell epitopes are candidates for epitope-specific therapeutic strategy.

      Abbreviations:

      autoAb (autoantibody), BP (bullous pemphigoid), ELISA (enzyme-linked immunosorbent assay), ELISPOT (Enzyme-Linked ImmunoSpot), NC16A (juxtamembranous extracellular noncollagenous 16A), PBMC (peripheral blood mononuclear cell), sCD23 (soluble CD23), SFC (spot-forming cell), Th (T helper)

      Introduction

      Bullous pemphigoid (BP) is an autoimmune blistering skin disorder, which commonly develops in the elderly. The disorder clinically presents with blister formation and pruritic erythema on the entire body (
      • Bernard P.
      • Antonicelli F.
      Bullous pemphigoid: a review of its diagnosis, associations and treatment.
      ,
      • Schmidt E.
      • Zillikens D.
      Pemphigoid diseases.
      ). Immunologically, patients with BP have IgG autoantibodies (autoAbs) that target two hemidesmosomal components: transmembrane collagen XVII (COL17, also known as BP180 or BPAG2) and plakin family protein BP230 (BPAG1), both located at the dermal-epidermal junction of basal keratinocytes. BP180 is considered to be the major autoantigen (
      • Bagci I.S.
      • Horvath O.N.
      • Ruzicka T.
      • Sardy M.
      Bullous pemphigoid.
      ).
      BP180 is a type II-oriented transmembrane protein with 15 interrupted collagenous and 16 noncollagenous extracellular domains. The immunologic recognition of the BP180 extracellular noncollagenous 16A (BP180-NC16A) domain and its pathogenic relevance are supported by evidence that passive transfer into neonatal mice of IgG against the BP180-NC16A domain induced key immunopathologic features of human BP, including subepidermal blistering with involvement of the complement cascade, mast cells, and neutrophils (
      • Liu Z.
      • Sui W.
      • Zhao M.
      • Li Z.
      • Li N.
      • Thresher R.
      • et al.
      Subepidermal blistering induced by human autoantibodies to BP180 requires innate immune players in a humanized bullous pemphigoid mouse model.
      ). In addition, epitope mapping using the enzyme-linked immunosorbent assay (ELISA) targeting several fragments of BP180 elucidated that sera from most patients with BP recognized the NC16A domain. Inactivation of BP180-NC16A-reactive IgG with a recombinant NC16A decoy peptide blocks the pathogenic activity of IgG from the BP sera in vivo (
      • Lin M.S.
      • Fu C.L.
      • Giudice G.J.
      • Olague-Marchan M.
      • Lazaro A.M.
      • Stastny P.
      • et al.
      Epitopes targeted by bullous pemphigoid T lymphocytes and autoantibodies map to the same sites on the bullous pemphigoid 180 ectodomain.
      ). The disease activity of BP tends to correlate with the level of circulating IgG autoAbs targeting NC16A (
      • Di Zenzo G.
      • Della Torre R.
      • Zambruno G.
      • Borradori L.
      Bullous pemphigoid: from the clinic to the bench.
      ,
      • Feng S.
      • Wu Q.
      • Jin P.
      • Lin L.
      • Zhou W.
      • Sang H.
      • et al.
      Serum levels of autoantibodies to BP180 correlate with disease activity in patients with bullous pemphigoid.
      ,
      • Tsuji-Abe Y.
      • Akiyama M.
      • Yamanaka Y.
      • Kikuchi T.
      • Sato-Matsumura K.C.
      • Shimizu H.
      Correlation of clinical severity and ELISA indices for the NC16A domain of BP180 measured using BP180 ELISA kit in bullous pemphigoid.
      ). These findings support the conclusion that BP autoAbs against the BP180-NC16A antigen directly initiate the autoimmune response in BP pathogenesis.
      T-helper (Th) cells can be distinguished based on their cytokine profiles, which endow different functional properties. Th2 cells, characterized by the secretion of IL-4, IL-5, and IL-13, play pivotal roles in eliciting antibody response. They are essential to stimulate proliferation of B cells, to induce B-cell antibody class switching, and to increase neutralizing antibody production (
      • Raphael I.
      • Nalawade S.
      • Eagar T.N.
      • Forsthuber T.G.
      T cell subsets and their signature cytokines in autoimmune and inflammatory diseases.
      ). One early event in the development of antibody production is the activation of antigen-specific CD4+ T cells. CD4+ T-cell support is initiated by antigen-presenting cells, which expose peptide fragments derived from foreign proteins on major histocompatibility complex class II that bind T-cell receptors. The presence of autoreactive CD4+ T cells has been reported in BP, and particular major histocompatibility complex II alleles occur more frequently in patients with BP, indicating that CD4+ T cells may be contributing to the pathogenesis of BP (
      • Lin M.S.
      • Fu C.L.
      • Giudice G.J.
      • Olague-Marchan M.
      • Lazaro A.M.
      • Stastny P.
      • et al.
      Epitopes targeted by bullous pemphigoid T lymphocytes and autoantibodies map to the same sites on the bullous pemphigoid 180 ectodomain.
      ,
      • Pickford W.J.
      • Gudi V.
      • Haggart A.M.
      • Lewis B.J.
      • Herriot R.
      • Barker R.N.
      • et al.
      T cell participation in autoreactivity to NC16a epitopes in bullous pemphigoid.
      ,
      • Thoma-Uszynski S.
      • Uter W.
      • Schwietzke S.
      • Schuler G.
      • Borradori L.
      • Hertl M.
      Autoreactive T and B cells from bullous pemphigoid (BP) patients recognize epitopes clustered in distinct regions of BP180 and BP230.
      ). The Hiroshi Shimizu group revealed that BP180-NC16A-reactive CD4+ T cells play a pivotal role in pathogenesis in a BP model via the CD40-CD40L interaction. However, the specific epitopes to BP180-NC16A in Th2 cells remain unclear (
      • Ujiie H.
      • Shibaki A.
      • Nishie W.
      • Shinkuma S.
      • Moriuchi R.
      • Qiao H.
      • et al.
      Noncollagenous 16A domain of type XVII collagen-reactive CD4+ T cells play a pivotal role in the development of active disease in experimental bullous pemphigoid model.
      ).
      In this study, we aimed to map the immunodominant Th2-cell epitopes from BP180-NC16A antigens in patients with BP from the Chinese Han population. First, we synthesized a series of 15-mer peptides spanning the entire sequence of the BP180-NC16A region, which overlapped by 12 amino acids. We screened the candidate peptides using IL-4 ELISPOT assays. To explore the immunogenic activities of these epitopes, we tested the proliferation rate of CD4+ T cells and examined B-cell activation and autoAbs secretion by ELISA after co-culture of the candidate peptides and peripheral blood mononuclear cells (PBMCs) isolated from patients with BP. Our study identified two Th2-cell epitopes from the BP180-NC16A, which contribute to the immunological pathogenesis of BP.

      Results

      Demographics

      Details of the enrolled patients with BP (n = 27) are listed in Table 1. Patients with BP were 48.15 % male, with the mean age of 70.50 ± 11.50 (standard deviation) years, and 51.85 % female, with the mean age of 60.14 ± 14.03 (standard deviation) years.
      Table 1Clinical status, HLA class II alleles, and anti-BP180 NC16A titers from 27 patients with BP and 9 healthy donors (HD)
      SamplesHLA class II allelesAnti-BP180-NC16A antibody titersPositive peptides in IL-4 ELISPOT
      NumberSexAge (y)HLA-DQB1HLA-DRB1
      BP1M570602,02010301,1501202, 9
      BP2M780303,03030901,09012532, 3, 5, 6
      BP3M840604,03011302,1501501, 2, 3, 5
      BP4F660301,03030701,12011891, 2, 3, 5, 10
      BP5F660502,06021454,150117710, 14, 15, 20, 21
      BP6F370301,03020406,1312691, 2, 3, 5
      BP7F360301,04010401,0405251, 7
      BP8F750502,03030901,15011842, 6, 9
      BP9M850303,03030901,09012003, 5, 6
      BP10M800501,06011001,150216517, 19
      BP11M730502,03011101,1602852, 3, 12, 16
      BP12F690301,03020406,110110210, 17, 19
      BP13F660501,06010803,10011562, 6, 7
      BP14F580501,03011001,11041723, 4, 9
      BP15M710602,03011101,1501904, 9
      BP16F470501,06010101,16021883, 4, 9
      BP17F800401,04020405,0802259, 19, 20
      BP18M450301,04010405,12022342, 7, 8, 14, 16, 18
      BP19F740301,03011101,1106345, 11, 15, 19
      BP20M740501,03011001,120110214, 16
      BP21F520501,03011001,12011201, 2, 6
      BP22M690301,05010101,1101525, 7, 9, 17, 19
      BP23F480602,02010301,1501312, 11
      BP24M690601,03011101,15011612, 7
      BP25M640501,04010405,10011452, 16
      BP26F680501,05021001,1602462, 9, 10
      BP27M660202,03020406,07012161, 2, 5, 6, 7, 9
      HD1M300602,02010301,1501
      HD2F270502,03030901,1602
      HD4F320202,03020406,0701
      HD5M340303,03030901,0901
      HD6F300602,02020701,1501
      HD7F270301,03011101,1202
      HD9M260201,03010301,1202
      Abbreviations: BP, bullous pemphigoid; ELISPOT, Enzyme-Linked ImmunoSpot; NC16A, juxtamembranous extracellular noncollagenous 16A.

      Identification of Th2-cell epitopes

      We used an IL-4 ELISPOT assay to determine Th2-cell epitopes in the BP180-NC16A. A positive response was ≥25 spot-forming cells (SFC) per 106 cells, which was more than three times the blank control. CD8+ T cells were depleted from PBMCs before in vitro peptide stimulation to eliminate interference.
      Th2 cells from all of the 27 patients with BP responded to two or more peptides. Th2 cells from most patients responded to P2 and P5 stimulation (Figure 1a), with the response rates for each peptide at 67% and 33%, respectively. Responses of Th2 cells from 27 patients stimulated by 22 peptides are summarized in Figure 1b, with P2 and P5 exhibiting the most significant and strongest positive effects on Th2 cells from patients with BP compared with irrelevant peptides (NC). To correct for individual patient variation, SFC values were normalized to the total number of spots. As shown in Figure 1c, P2 accounted for 9.83% of all the spots in the screen (19,787 SFC per 106 cells), while P5 accounted for 7.79% of all the spots in the screen, strong positivity compared with other peptides. Thus, we identified P2 (492–506 aa: VRKLKARVDELERIR) and P5 (501–515 aa: ELERIRRSILPYGDS) as candidate Th2-cell epitopes for BP (Figure 1d).
      Figure 1
      Figure 1Summary of the T-cell responses to the NC16A overlapped peptides in 27 patients with BP. Candidate Th2-cell epitopes were identified using the ELISPOT assay through the signaling of spot-forming cells (SFC). (a, b) Depleted-CD8+ T-peripheral blood mononuclear cell IL-4 secretion in response to a series of 22 overlapping peptides that span the entire sequence of BP180-NC16A (n = 27) (NC, irrelevant peptide). P2 and P5 were identified as candidate epitopes. (a) Patients with BP responding to 22 overlapping peptides. Results show that Th2 cells from most patients responded to P2 and P5 stimulation. (b) SFC/106 cells responding to the 22 overlapping peptides. P2 and P5 had the strongest positive effects on Th2 cells. (c) SFC values for each peptide were normalized to the total number of spots. All 22 peptides were sorted and ordered by reaction intensity. The third row of the table is the P value of adjacent peptides. (d) Schematic diagrams depicting the structure and regions of the candidate Th2-cell epitopes in BP180-NC16A. BP, bullous pemphigoid; ELISPOT, Enzyme-Linked ImmunoSpot; NC16A, juxtamembranous extracellular noncollagenous 16A; Th, T helper.
      Furthermore, we used ELISA to investigate the precise IgG reaction to the 22 overlapped peptides. By ELISA, we found the BP patient’s serum reactive with P6, P7, P11, and P12 peptides, which covered 504–521 aa and 519–536 aa domains in NC16A (Supplementary Figure S2).

      HLA class II restriction of identified epitopes

      To further determine which HLA molecules were required for the T-cell response, we measured the functional ability of antibodies specific for HLA-DR, DP, or DQ in inhibiting the IL-4 response (Figure 2). Five patients with BP who responded with P2 and P5 were used for HLA restriction analysis and following functional assay. The responses of samples from all five patients with BP to P2 and P5 were inhibited by the anti-DR antibody but not by antibody against neither DP nor DQ. These data show that the T-cell response to P2 and P5 was restricted to class II HLA-DR. We predicted the binding sites of peptides to HLA class II molecules using IEDB.org. The two epitope peptides were predicted to bind to a dozen DR molecules (Supplementary Tables S2–S4 online).
      Figure 2
      Figure 2HLA class II restriction of CD4+ T-cell responses to single peptides, P2 and P5. CD8+ T-depleted peripheral blood mononuclear cells were incubated with antibodies against HLA-DR, DP, and DQ (anti-IgG as a control group), followed by P2 or P5 stimulation, respectively (n = 5). (a) P2 stimulation inhibited by the anti-HLA-DR antibody; (b) P5 stimulation inhibited by the anti-HLA-DR antibody. SFC, spot-forming cell.

      P2 and P5 promote CD4+ T-cell proliferation in vitro

      To further determine the immunologic effects of the candidate epitope peptides, we measured proliferation of CD4+ T cells after culturing PBMCs from patients with BP and gender and HLA-DR-type matched healthy donors with P2, P5, or an irrelevant peptide (NC) in vitro. Proliferation of CD4+ T cells from patients with BP stimulated with P2 and P5 significantly increased after 7 days compared with healthy donors (P < 0.05) (n = 5) (Figure 3), indicating that these peptides have specific effects on BP CD4+ T cells. Furthermore, we compared proliferation of CD4+ T cells from patients with BP stimulated by P2, P5, NC, or other NC16A peptides P11, P16, and P22. CD4+ T cells from patients with BP only responded to P2 and P5, whereas the NC (Figure 3c) and other NC16A peptides (Supplementary Figure S1 online) showed no notable effect on CD4+ T-cell proliferation. These results indicate that P2 and P5 promote proliferation of CD4+ T cells in BP.
      Figure 3
      Figure 3P2 and P5 activated CD4+ T-cell proliferation in vitro. (a) Flow cytometry to detect proliferation of CD4+ T cells from patients with BP after co-culture with candidate peptides, irrelevant peptide (NC), and anti-CD3 mAb as negative and positive controls, respectively. (b) Statistical analysis showed proliferation of CD4+ T cells from patients with BP after stimulation with candidate peptides significantly increased compared with healthy donors (HD) (n = 5). (c) Statistical analysis showed that compared with NC, P2 and P5 significantly stimulated proliferation of CD4+ T cells from patients with BP (n = 5). BP, bullous pemphigoid; CFSE, carboxy fluorescein succinimidyl ester; NC, juxtamembranous extracellular noncollagenous.

      P2 and P5 significantly promote secretion of BP180-NC16A IgG autoAbs from B cells in vitro

      To determine whether the T-cell epitope could induce secretion of specific IgG antibodies, we stimulated PBMCs from patients with BP or gender and HLA-DR-type matched normal controls with the two candidate epitope peptides, P2 and P5, and then detected levels of anti-BPAG2-NC16A IgG in supernatants. Titers of anti-BPAG2-NC16A IgG in patients with BP after stimulation by P2 and P5 were higher than those in healthy donors, while there were no differences in anti-BPAG2-NC16A antibody titers between patients with BP and healthy donors after stimulation with negative peptides (NC) (Figure 4a and b ) and other NC16A peptides (Supplementary Figure S1). These results indicate that the T-cell epitopes can not only promote B-cell activation but also promote production of BP-specific antibodies.
      Figure 4
      Figure 4P2- and P5-induced B-cell activation in vitro. ELISA assays were performed to concentrate the anti-BP180-NC16A IgG antibody and sCD23 in supernatants after stimulation of peripheral blood mononuclear cells from patients with BP or from healthy donors (HD) with candidate epitope peptides, irrelevant peptide (NC), and blank as a control (n = 5). P2 (a) and P5 (b) significantly promoted anti-BP180-NC16A IgG synthesis compared with NC. P2 (c) and P5 (d) significantly increased the supernatant sCD23 levels compared with NC. BP, bullous pemphigoid; NC16A, juxtamembranous extracellular noncollagenous 16A; sCD23, soluble CD23.

      P2 and P5 elevate sCD23 levels in PBMC supernatant in vitro

      PBMCs were initially stimulated for 7 days with P2 and P5; then soluble CD23 (sCD23) concentration, a hallmark of B-cell activation, was examined by ELISA. Controls included PBMCs from healthy donors or PBMCs stimulated with irrelevant peptides. The sCD23 levels significantly increased after stimulation with P2 and P5 in PBMCs from patients with BP compared with healthy donors (n = 5) (Figure 4c and d). The results indicate that P2 and P5 can promote B-cell activation from patients with BP in vitro.

      Discussion

      The data presented in this study identified immunodominant Th2-cell epitopes in the BP180-NC16A domain of patients with BP. We identified overlapping peptides that contain immunodominant, DR-restricted epitopes and demonstrated that the epitope peptides have immunologic activity to stimulate CD4+ T-cell proliferation, B-cell activation, and anti-BP180-NC16A IgG antibody secretion. T and B cells from healthy donors did not respond to the epitope peptides. Likewise, T and B cells from patients with BP did not respond to irrelevant peptide or other peptides in the NC16A domain. This study provides evidence that immunologically activated Th2-cell epitopes in the BP180-NC16A domain may be involved in the autoimmune response in BP.
      AutoAbs against two hemidesmosomal antigens, BP180 and BP230, have been identified as causative of BP. The NC16A domain of BP180 is considered to contain the main pathogenic epitopes of BP (
      • Liu Z.
      • Sui W.
      • Zhao M.
      • Li Z.
      • Li N.
      • Thresher R.
      • et al.
      Subepidermal blistering induced by human autoantibodies to BP180 requires innate immune players in a humanized bullous pemphigoid mouse model.
      ). Previous studies using immunoblotting identified that NC16A-responsive BP T lymphocytes were CD4+ cells (
      • Lin M.S.
      • Fu C.L.
      • Giudice G.J.
      • Olague-Marchan M.
      • Lazaro A.M.
      • Stastny P.
      • et al.
      Epitopes targeted by bullous pemphigoid T lymphocytes and autoantibodies map to the same sites on the bullous pemphigoid 180 ectodomain.
      ) and reported that the NC16A region of BP180 contained the major epitopes recognized by BP autoAbs, with NC16A2-2.5 (507–532 aa) as the dominant area (
      • Wang G.
      • Ujiie H.
      • Shibaki A.
      • Nishie W.
      • Tateishi Y.
      • Kikuchi K.
      • et al.
      Blockade of autoantibody-initiated tissue damage by using recombinant fab antibody fragments against pathogenic autoantigen.
      ). Passive transfer of affinity-purified Abs against fragmented NC16A peptides into neonatal COL XVII -humanized mice followed by evaluating skin fragility led to identification of the Asp522-Gln545 domain as the precise pathogenic epitopes within NC16A (
      • Ujiie H.
      • Shibaki A.
      • Nishie W.
      • Shinkuma S.
      • Moriuchi R.
      • Qiao H.
      • et al.
      Noncollagenous 16A domain of type XVII collagen-reactive CD4+ T cells play a pivotal role in the development of active disease in experimental bullous pemphigoid model.
      ). These studies aimed to identify precise B-cell epitopes in the NC16A domain. We also measured the IgG reaction to the 22 overlapped peptides by ELISA and identified candidate B-cell epitopes. Supplementary Figure S2 online shows that P6–P7 (504–521 aa) and P11–P12 (519–536 aa) are candidate B-cell epitopes in the NC16A domain, which overlap with the NC16A-2-2.5 region.
      In this study, we mainly focused on Th2-cell epitopes. Based on ELISPOT results, CD4+ T cells from most patients with BP recognized P2 (492–506 aa) and P5 (501–515 aa) peptides, covering the amino acid sequence 492–515 aa. Thus, Th2-cell epitopes overlap with the NC16A B-cell epitope, indicating that epitope-responding Th2 cells may support production of antibodies by expressing T-cell receptors and cytokines. Pickford identified autoreactive T cells responding to five epitopes using IL-4 ELISA assays in acute-onset, chronic active, and remittent patients with BP, but did not carry out peptide mapping experiments (
      • Pickford W.J.
      • Gudi V.
      • Haggart A.M.
      • Lewis B.J.
      • Herriot R.
      • Barker R.N.
      • et al.
      T cell participation in autoreactivity to NC16a epitopes in bullous pemphigoid.
      ). Our findings with two peptides may be due to the higher sensitivity of the ELISPOT assay and inclusion of only acute-onset patients.
      We evaluated the HLA II restriction of the responsive BP donors to further understand the molecular basis of the immunodominant epitope. Previous reports showed that DQB1*0301 occurs more frequently in Caucasian patients with BP and that HLA-DRB1*04, DRB1*1101, and DQB1*0302 occur more frequently in Japanese patients with BP compared with healthy controls, indicating that HLA differences influence susceptibility to BP (
      • Zakka L.R.
      • Reche P.
      • Ahmed A.R.
      Role of MHC Class II genes in the pathogenesis of pemphigoid.
      ). The association between HLA class II genes and BP in the Chinese population has not been clearly shown, with only two protective alleles previously reported (
      • Gao X.-H.
      • Winsey S.
      • Li G.
      • Barnardo M.
      • Zhu X.-J.
      • Chen H.-D.
      • et al.
      HLA-DR and DQ polymorphisms in bullous pemphigoid from northern China.
      ). In our study, CD4+ T-cell response was restricted to HLA-DR in patients with BP. The donors that responded to peptides P2 and P5 showed a wide range of haplotypes of HLA-DRB1, including homozygosity and numerous combinations of DR haplotypes. These epitopes were promiscuous among several HLA class II haplotypes. Moreover, using IEDB.org, we predicted the peptide binding sites in HLA class II molecules. The epitope peptides were predicted to bind to a dozen DR molecules, indicating a strong, promiscuous epitope association with HLA-DR (Supplementary Tables S2–S4). It is possible that blocking of these single epitopes will affect a diverse population, allowing patients to benefit from deimmunization of the BP180-NC16A.
      Th2 cells are critical for promoting B cells to secrete IgE and IgG autoAbs in certain autoimmune diseases. Antigen-specific Th2 cells could activate autologous B cells and produce auto-antigen-specific autoAbs in patients with pemphigus vulgaris and systemic sclerosis (
      • Kuwana M.
      • Medsger T.A.J.
      • Wright T.M.
      Analysis of soluble and cell surface factors regulating anti-DNA topoisomerase I autoantibody production demonstrates synergy between Th1 and Th2 autoreactive T cells.
      ,
      • Veldman C.
      • Stauber A.
      • Wassmuth R.
      • Uter W.
      • Schuler G.
      • Hertl M.
      Dichotomy of autoreactive Th1 and Th2 cell responses to desmoglein 3 in patients with pemphigus vulgaris (PV) and healthy carriers of PV-associated HLA class II alleles.
      ). Here, we found that P2 and P5 in BP180-NC16A could activate Th2 cells and further promote BP180-NC16A IgG autoAb production in PBMCs from patients with BP. These results suggest that antigen-specific Th2 cells against these epitopes may exist in BP and help to activate autologous B cells, which produce anti-BP180-NC16A autoAbs. Further in vitro co-culture of autoreactive Th2 cells and B cells will help clarify the role of these epitope-specific Th2 cells.
      Historically, clinical and experimental studies investigating the pathogenesis of BP mainly focused on IgG class autoAbs; however, IgE class autoAbs were detectable in 90% of patients with BP (
      • Ujiie H.
      IgE autoantibodies in bullous pemphigoid: supporting role, or leading player?.
      ,
      • van Beek N.
      • Schulze F.S.
      • Zillikens D.
      • Schmidt E.
      IgE-mediated mechanisms in bullous pemphigoid and other autoimmune bullous diseases.
      ,
      • van Beek N.
      • Luttmann N.
      • Huebner F.
      • Recke A.
      • Karl I.
      • Schulze F.S.
      • et al.
      Correlation of serum levels of IgE autoantibodies against BP180 with bullous pemphigoid disease activity.
      ). Several studies have revealed the pathogenic roles of IgE anti-BP180 autoAbs in BP and possible pathomechanisms (
      • Messingham K.N.
      • Srikantha R.
      • DeGueme A.M.
      • Fairley J.A.
      FcR-independent effects of IgE and IgG autoantibodies in bullous pemphigoid.
      ,
      • Ujiie H.
      IgE autoantibodies in bullous pemphigoid: supporting role, or leading player?.
      ). Furthermore, patients with BP can be effectively treated by blocking IgE binding to FcεRI (
      • Fairley J.A.
      • Baum C.L.
      • Brandt D.S.
      • Messingham K.A.N.
      Pathogenicity of IgE in autoimmunity: successful treatment of bullous pemphigoid with omalizumab.
      ). Generally, IgE synthesis in antigen-activated B cells was positively controlled by CD23, a low-affinity Fc receptor for IgE (
      • Liu C.
      • Richard K.
      • Wiggins M.
      • Zhu X.
      • Conrad D.H.
      • Song W.
      CD23 can negatively regulate B-cell receptor signaling.
      ). CD23 not only influences IgE production, but is also a specific marker expressed by activated B cells. Studies have suggested that serum levels of sCD23 correlate with serum IgE levels and disease severity in patients with BP (
      • Cooper A.M.
      • Hobson P.S.
      • Jutton M.R.
      • Kao M.W.
      • Drung B.
      • Schmidt B.
      • et al.
      Soluble CD23 controls IgE synthesis and homeostasis in human B cells.
      ). In our co-culture system, elevated sCD23 was detected in PBMC supernatants in the presence of P2 and P5, suggesting that Th2-cell epitopes P2 and P5 promote B-cell activation and may further affect IgE autoAbs synthesis.
      Our study has some limitations. Because BP is rare, the sample size is small. Almost all the patients with BP were elderly, and a large amount of peripheral blood is difficult to take from these patients, making enrolling patients difficult. The immunodominant epitopes should be further confirmed in a larger group of patients with BP. Our previous work generated Fabs against the human BP180-NC16A domain, which competitively inhibited the binding of human BP autoAbs to BP180 (
      • Wang G.
      • Ujiie H.
      • Shibaki A.
      • Nishie W.
      • Tateishi Y.
      • Kikuchi K.
      • et al.
      Blockade of autoantibody-initiated tissue damage by using recombinant fab antibody fragments against pathogenic autoantigen.
      ). This study shows the therapeutic potential of using epitope-specific antibodies for patients with BP. Further studies should explore developing P2- and P5-specific therapeutic strategies for patients with BP.
      Overall, we identified two HLA-DR-restricted 15-mer BP180-NC16A-derived CD4+ T-cell epitopes, which were peptides 501–515 aa: ELERIRRSILPYGDS and 492–506 aa: VRKLKARVDELERIR. These epitopes were immunogenic, inducing CD4+ T-cell proliferation and activating B cells to be autoAbs-secreting cells. This work contributes to the full understanding of the immunologic pathogenesis of BP. The identified Th2-cell epitopes are candidates for the development of epitope-specific therapeutic strategies for patients with BP.

      Materials and Methods

      Clinical samples

      All the patients with BP provided written, informed consent in the Department of Dermatology, Xijing Hospital, including inpatients and outpatients from November 2013 to November 2015. A total of 27 patients were enrolled in this study, and their baseline characteristics were summarized in Table 1. All subjects were newly diagnosed with BP based on the typical clinical and histologic presentation, direct or indirect immunofluorescence examination, and the presence of circulating autoAbs (BP180-NC16A). None of the enrolled patients had received any immunosuppressive treatment. Normal control blood samples were collected from sex- and HLA-matched healthy donors working at our department (Table 1). This study was performed in accordance with the Declaration of Helsinki principles and approved by the local ethics committee of Xijing Hospital, Fourth Military Medical University, Xi’an, China.

      Peptide synthesis

      A series of 15-mer peptides, which overlap by 12 amino acids and span the entire sequence of BP180-NC16A, were synthesized by APeptides, China (Supplementary Table S1 online). Peptides were purified by high-performance liquid chromatography (>95% purity) and validated by mass spectrometry. Synthesized peptides were dissolved in DMSO at 10 mg/ml and stored at –80°C.

      DNA extraction and genotyping

      Genomic DNA was extracted from blood samples using the Qiagen FlexiGene DNA Kit (Qiagen, Hilden, Germany). HLA class II (DQB1 and DRB1) typing was performed by Sanger sequence-based typing of each second exon in BGI LifeTech, Beijing.

      Lymphocyte preparation

      PBMCs were isolated from 20 ml of blood using human lymphocyte separation medium (Dakewe, Beijing, China) according to the manufacturer’s recommendations. The isolated PBMCs were washed and then CD8+ T-cell depletion was performed by flow cytometry using monoclonal phycoerythrin anti-CD8 (Biolegend, San Diego, CA). PBMCs depleted of CD8+ T cells were freshly cultured in PRMI-1640 containing 20% fetal bovine serum for the ELISPOT assay. Additional 40 ml of blood was collected for functional assays. PBMCs were isolated and freshly cultured in PRMI-1640 containing 20% fetal bovine serum as mentioned above.

      IL-4 ELISPOT assay

      PBMCs depleted of CD8+ T cells were utilized in the IL-4 ELISPOT assay according to the manufacturer’s instructions (ELISpotPRO for human IL-4, Mebtech, Stockholm, Sweden) to quantify peptide-specific T cells after in vitro expansion. Briefly, PBMCs at a density of 2 × 105 cells/well were cultured in the presence of peptides at a final concentration of 4 μg/well in an ELISPOT plate. The CD3 antibody at a dilution of 1:1,000 was used as a positive control. Irrelevant peptides (NC) were used as a negative control. After incubation for 48 hours at 37°C, cells were discarded and IL-4 secretions were captured using a biotin-streptavidin system followed by BCIP/NBT-plus. The assay was duplicated. Spots were counted using an ELISPOT Image Analyzer and Software (Cell Technology. Jessup, MD). SFCs are defined as follows : SFC = experimental group – negative control group. Positive response was defined as at least 25 SFC/106 input cells, exceeding three times the background response after subtracting the number of spots in the background controls from those in the stimulated samples.

      ELISA assay for detection of the serum IgG reaction to the 22 overlapped NC16A peptides

      The serum from patients with BP was plated in 96-well plates coated with the 22 peptides at a 10-fold dilution. Serum from healthy donors and noncoated wells were used as the control. After incubation overnight at 4°C, the solution was discarded and plates were washed four times with phosphate buffered saline tween-20. Subsequently, 100 μl Goat Anti-Human IgG secondary antibody (diluted 1:10,000) was added to each well and incubated for 1 hour at room temperature. After washing four times with phosphate buffered saline tween-20, 100 μl of tetramethyl benzidine One-Step Substrate Reagent was added to each well and incubated for 30 minutes at room temperature in the dark. Finally, 50 μl of Stop Solution was added to each well and the absorbance measured immediately at 450 nm with an ELISA reader. All samples were tested in duplicate. A cutoff value of response was defined as follows: (the average of the healthy donor’s absorbance) × 2.1. Positive response was defined as greater than or equal to the cutoff value.

      HLA restriction assays

      Five patients with BP who responded with P2 and P5 were used for HLA restriction analysis and following functional assay.
      To block HLA-II-restricted responses, 10 μg/ml anti-HLA-DP (Leinco Technologies, St. Louis, MO), anti-HLA-DQ (Abcam, Cambridge, UK), or anti-HLA-DR (Abcam) was added for 60 minutes before adding peptides in CD8+ T-cell-depleted PBMCs followed by the IL-4 ELISPOT assay. After incubation for 48 hours, spots were counted using ELISPOT Image Analyzer and Software (Cell Technology). All samples were tested in triplicate.

      T-cell proliferation assay

      PBMCs were isolated and labeled with carboxy fluorescein succinimidyl ester (Invitrogen, Carlsbad, CA) at 37°C for 15 minutes. The reaction was terminated by the addition of fetal bovine serum and then cells were counted and seeded into a 96-well U-bottom plate at a density of 1 × 105 cells/well. The U-bottom plate was precoated with anti-CD3/CD28 antibodies followed by 4 μg candidate peptides and rhIL-2. Cells were harvested after 7 days and stained with the CD4–antigen-presenting cell antibody (Biolegend) for 30 minutes at 4°C. Proliferation of CD4+ T cells was analyzed by flow cytometry of antigen-presenting cell and carboxy fluorescein succinimidyl ester. All samples were tested in triplicate.

      ELISA assay for detection of cytokines and antibody production

      ELISA assays to detect sCD23 were performed to quantify B-cell activation after co-culture of PBMCs with specific peptides. PBMCs were plated in 96-well plates precoated with anti-CD3/CD28 antibodies at a density of 2 × 106 cells/well, followed by the addition of specific peptides. After incubation for 7 days, supernatants were collected, and the concentrations of sCD23 and specific anti-BP180-NC16A antibody were measured using a sCD23 ELISA kit (Shanghai Westang Biology Technology., China) and a BP180-NC16A ELISA kit (MBL International, Japan) according to the manufacturer’s recommendations. The color reaction was developed with tetramethyl benzidine and analyzed at 450 nm. All samples were tested in triplicate.

      Statistical analyses

      The age of enrolled patients with BP was presented as mean ± standard deviation. Differences between reaction intensities of two peptides were compared through the χ2 test. The nonparametric Student’s t-test was used for comparison between the two subject groups. For experiments with more than two groups, the differences between groups were compared by a one-way analysis of variance followed by Dunnett’s test, in which all groups were tested against a control group as a reference. P-value < 0.05 was considered statistically significant. All statistical analyses were performed using GraphPad Prism 7.0 (GraphPad Software, San Diego, CA).

      Conflict of Interset

      The authors state no conflict of interest.

      Acknowledgments

      We are grateful to Professor Boquan Jin for helpful intellectual discussions, and Dr. Ying Ma for excellent technical assistance. Yao Ke assisted in manuscript preparation. We thank all blood donors for their voluntary support of our study. We appreciate language editing assistance provided by DerMEDit (www.DerMEDit.com). This work was financially supported by a grant from the National Natural Science Foundation of China (81220108016).

      Supplementary Material

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