TH1/TH17 cell recognition of desmoglein 3 and bullous pemphigoid antigen 180 in patients with lichen planus
2018; Elsevier BV; Volume: 142; Issue: 2 Linguagem: Inglês
10.1016/j.jaci.2018.02.044
ISSN1097-6825
AutoresThomas Schmidt, Farzan Solimani, Robert Pollmann, Ronja Stein, Ansgar Schmidt, Inna Stulberg, Katja Kühn, Rüdiger Eming, Verena Eubel, P Kind, Nicole B. Arweiler, Cassian Sitaru, Michael Hertl,
Tópico(s)Nail Diseases and Treatments
ResumoLichen planus (LP) is a chronic relapsing inflammatory disorder of the skin and mucous membranes affecting 1% to 2% of the general population.E1Bouquot J.E. Gorlin R.J. Leukoplakia, lichen planus, and other oral keratoses in 23,616 white Americans over the age of 35 years.Oral Surg Oral Med Oral Pathol. 1986; 61: 373-381Abstract Full Text PDF PubMed Scopus (231) Google Scholar, E2Mustafa M.B. Porter S.R. Smoller B.R. Sitaru C. Oral mucosal manifestations of autoimmune skin diseases.Autoimmun Rev. 2015; 14: 930-951Crossref PubMed Scopus (63) Google Scholar, E3Scully C. Beyli M. Ferreiro M.C. Ficarra G. Gill Y. Griffiths M. et al.Update on oral lichen planus: etiopathogenesis and management.Crit Rev Oral Biol Med. 1998; 9: 86-122Crossref PubMed Scopus (405) Google Scholar, E4Sugerman P.B. Savage N.W. Walsh L.J. Zhao Z.Z. Zhou X.J. Khan A. et al.The pathogenesis of oral lichen planus.Crit Rev Oral Biol Med. 2002; 13: 350-365Crossref PubMed Scopus (521) Google Scholar Clinically, it is characterized by reticular purple papules and plaques of the skin and white papules and erosions of the mucous membranes. Overall, LP poses a major therapeutic challenge based on the chronic refractory course and has a major effect on patients' quality of life. In LP lesions the T-cell infiltrate presents with a band-like dermal distribution underneath the dermal-epidermal basement membrane zone (BMZ), where apoptosis of basal epidermal keratinocytes is commonly seen. Thus T cells are believed to be major inducers of epithelial damage, but their target antigens have not yet been identified.E4Sugerman P.B. Savage N.W. Walsh L.J. Zhao Z.Z. Zhou X.J. Khan A. et al.The pathogenesis of oral lichen planus.Crit Rev Oral Biol Med. 2002; 13: 350-365Crossref PubMed Scopus (521) Google Scholar Two clinically and pathogenetically distinct autoimmune bullous skin disorders, pemphigus vulgaris (PV) and bullous pemphigoid (BP), share clinical similarities with LP. Mucosal lesions of patient with PV exhibit a similar phenotype as erosive mucosal LP (Fig 1, A). In patients with LP with mucosal involvement, serum IgG autoantibodies against desmoglein 3 (Dsg3; major autoantigen of PV) and BP180 (major autoantigen of BP) were detected occasionally.E5Muramatsu K. Nishie W. Natsuga K. Fujita Y. Iwata H. Yamada T. et al.Two cases of erosive oral lichen planus with autoantibodies to desmoglein 3.J Dermatol. 2016; 43: 1350-1353Crossref PubMed Scopus (12) Google Scholar, E6Herrero-González J.E. Parera Amer E. Segura S. Mas Bosch V. Pujol R.M. Martínez Escala M.E. Epithelial antigenic specificities of circulating autoantibodies in mucosal lichen planus.Int J Dermatol. 2016; 55: 634-639Crossref PubMed Scopus (12) Google Scholar Moreover, a rare clinical variant of LP, lichen planus pemphigoides (LPP), shows major features of BP, including tense skin blisters with subepidermal loss of adhesion (Fig 1, A), deposits of IgG and/or C3 at the BMZ, and IgG autoantibodies against BP180.E7Zaraa I. Mahfoudh A. Sellami M.K. Chelly I. El Euch D. Zitouna M. et al.Lichen planus pemphigoides: four new cases and a review of the literature.Int J Dermatol. 2013; 52: 406-412Crossref PubMed Scopus (63) Google Scholar, E8Skaria M. Salomon D. Jaunin F. Friedli A. Saurat J.H. Borradori L. IgG autoantibodies from a lichen planus pemphigoides patient recognize the NC16A domain of the bullous pemphigoid antigen 180.Dermatology. 1999; 199: 253-255Crossref PubMed Scopus (22) Google Scholar On the basis of these findings, we sought to study peripheral blood T-cell responses against Dsg3 and BP180, respectively, in a cohort of patients with mucocutaneous LP (Fig 1, B-I). By using the ELISpot assay, patients with LP showed an increased peripheral blood TH1-dominated cell response against Dsg3 (Fig 1, B-D, and see Table E1 in this article's Online Repository at www.jacionline.org) and a mixed TH1/TH17 cell response against BP180 (Fig 1, G-I, and see Table E2 in this article's Online Repository at www.jacionline.org) in contrast to patients with PV or BP (Fig 1, E, F, J, and K). Furthermore, LP lesions were associated with a TH1 (CD3+T-bet+)– and TH17 (IL-17A+)–dominated T-cell infiltrate, whereas skin lesions of patients with BP or PV were characterized by CD4+GATA-3+ TH2 cells (Fig 2, D and E). In patients with LP, the dermal inflammatory cell infiltrate was composed mainly of CD8+ and CD4+ T cells, whereas skin lesions from patients with BP or PV showed a predominance of CD4+ T cells (Fig 2, A and C). Accordingly, we observed increased plasma levels of the TH1-specific and IFN-γ–associated chemokine CCL5 (see Fig E1, A, in this article's Online Repository at www.jacionline.org) and increased expression of CCR4 as a central T-cell skin-homing factor and potential receptor for CCL5 on peripheral blood CD4+ and CD8+ T cells in patients with LP (see Fig E1, B). The relative decrease in numbers of peripheral blood CD8+, but not CD4+, T cells might be a consequence of intense transmigration of CD8+ T cells into the skin (see Fig E1, C). Of note, in skin lesions of patients with LP, IL-17A+ cells accumulated along the BMZ (Fig 2, A). This TH17 cell distribution pattern differed significantly from skin lesions of patients with BP (Fig 2, B). In contrast, there was no difference in numbers and distribution patterns of forkhead box protein 3 (FoxP3)+ regulatory T cells in skin lesions of patients with LP and those with BP (see Fig E2 in this article's Online Repository at www.jacionline.org). To prove the hypothesis of whether the TH1/TH2 cell dichotomy in patients with LP and those with BP is of clinical relevance, we studied the T-cell profile of 3 patients with LPP (Fig 2, F). Here we observed a mixed peripheral blood TH1/TH2 cell response against BP180-NH2 (Fig 2, I), which was accompanied by an inflammatory skin infiltrate composed of TH1 cells (CD3+T-bet+) along the BMZ (as in patients with LP) and a perivascular TH2 (CD4+GATA-3+)–dominated infiltrate around the dermal blood vessels (as in patients with BP), thus combining features of LP and BP (Fig 2, G and H). Here, for the first time, we show in a large patient cohort that LP is associated with TH1/TH17 cell recognition of 2 well-known cutaneous autoantigens, Dsg3 and BP180. Thus far, there have been only sparse data on the immune recognition of these autoantigens in patients with LP, including detection of serum IgG against Dsg3 and BP180 in patients with chronic mucosal LPE5Muramatsu K. Nishie W. Natsuga K. Fujita Y. Iwata H. Yamada T. et al.Two cases of erosive oral lichen planus with autoantibodies to desmoglein 3.J Dermatol. 2016; 43: 1350-1353Crossref PubMed Scopus (12) Google Scholar, E6Herrero-González J.E. Parera Amer E. Segura S. Mas Bosch V. Pujol R.M. Martínez Escala M.E. Epithelial antigenic specificities of circulating autoantibodies in mucosal lichen planus.Int J Dermatol. 2016; 55: 634-639Crossref PubMed Scopus (12) Google Scholar and peripheral blood T-cell recognition of BP180 in a few patients with vulvar LP.1Baldo M. Bailey A. Bhogal B. Groves R.W. Ogg G. Wojnarowska F. T cells reactive with the NC16A domain of BP180 are present in vulval lichen sclerosus and lichen planus.J Eur Acad Dermatol Venereol. 2010; 24: 186-190Crossref PubMed Scopus (44) Google Scholar The observed dichotomy of autoreactive peripheral blood TH1 responses in patients with LP versus TH2 responses in patients with PV or BP extends previous findings from our laboratory and others identifying Dsg3-specific TH2 cells as central players in the pathogenesis of PV.E10Lin M.S. Swartz S.J. Lopez A. Ding X. Fernandez-Vina M.A. Stastny P. et al.Development and characterization of desmoglein-3 specific T cells from patients with pemphigus vulgaris.J Clin Invest. 1997; 99: 31-40Crossref PubMed Scopus (149) Google Scholar, E9Rizzo C. Fotino M. Zhang Y. Chow S. Spizuoco A. Sinha A.A. Direct characterization of human T cells in pemphigus vulgaris reveals elevated autoantigen-specific Th2 activity in association with active disease.Clin Exp Dermatol. 2005; 30: 535-540Crossref PubMed Scopus (54) Google Scholar, 2Hertl M. Eming R. Veldman C. T cell control in autoimmune bullous skin disorders.J Clin Invest. 2006; 116: 1159-1166Crossref PubMed Scopus (145) Google Scholar, 3Veldman C. Stauber A. Wassmuth R. Uter W. Schuler G. Hertl M. Dichotomy of autoreactive Th1 and Th2 cell responses to Dsg3 in patients with pemphigus vulgaris (PV) and healthy carriers of PV-associated HLA class II alleles.J Immunol. 2003; 170: 635-642Crossref PubMed Scopus (101) Google Scholar In contrast, peripheral TH1 cell responses against Dsg3 were seen preferentially in patients with LP with mucosal involvement (Fig 1, B-D, and see Table E1). In patients with BP, autoreactive BP180-specific peripheral TH2 cells recognize epitopes of the NH2-terminus and, to a lesser extent, the COOH-terminus of the BP180 ectodomain, which are both targeted by IgG autoantibodies.E11Lin 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.J Invest Dermatol. 2000; 115: 955-961Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, E12Thoma-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.J Immunol. 2006; 176: 2015-2023Crossref PubMed Scopus (91) Google Scholar, E13Shen Z. Gao X. Ma L. Zhou Z. Shen X. Liu W. Expression of Foxp3 and interleukin-17 in lichen planus lesions with emphasis on difference in oral and cutaneous variants.Arch Dermatol Res. 2014; 306: 441-446Crossref PubMed Scopus (39) Google Scholar, 4Büdinger L. Borradori L. Yee C. Eming R. Ferencik S. Grosse-Wilde H. et al.Identification and characterization of autoreactive T cell responses to bullous pemphigoid antigen 2 in patients and healthy controls.J Clin Invest. 1998; 102: 2082-2089Crossref PubMed Scopus (192) Google Scholar Hence T-cell recognition of the NH2-terminus of ectodomain BP180 in patients with LP (Fig 1, G-I, and see Table E2) strongly suggests its pathogenic relevance in patients with LP. Various studies have revealed a TH1 phenotype in lesions of patients with LP, and T cells isolated from lesions of patients with LP secreted predominantly TH1 cytokines (like IFN-γ and TNF-α) in vitro.E14Simark-Mattsson C. Bergenholtz G. Jontell M. Eklund C. Seymour G.J. Sugerman P.B. et al.Distribution of interleukin-2, -4, -10, tumour necrosis factor-alpha and transforming growth factor-beta mRNAs in oral lichen planus.Arch Oral Biol. 1999; 44: 499-507Crossref PubMed Scopus (117) Google Scholar, E4Sugerman P.B. Savage N.W. Walsh L.J. Zhao Z.Z. Zhou X.J. Khan A. et al.The pathogenesis of oral lichen planus.Crit Rev Oral Biol Med. 2002; 13: 350-365Crossref PubMed Scopus (521) Google Scholar Iijima et alE15Iijima W. Ohtani H. Nakayama T. Sugawara Y. Sato E. Nagura H. et al.Infiltrating CD8+ T cells in oral lichen planus predominantly express CCR5 and CXCR3 and carry respective chemokine ligands RANTES/CCL5 and IP-10/CXCL10 in their cytolytic granules: a potential self-recruiting mechanism.Am J Pathol. 2003; 163: 261-268Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar described infiltrating CD8+ T cells in the skin lesions of patients with LP, which expressed CCR4, CCR5, CCL5, and CXCL10 in their cytolytic granules, suggesting a self-recruiting mechanism.E15Iijima W. Ohtani H. Nakayama T. Sugawara Y. Sato E. Nagura H. et al.Infiltrating CD8+ T cells in oral lichen planus predominantly express CCR5 and CXCR3 and carry respective chemokine ligands RANTES/CCL5 and IP-10/CXCL10 in their cytolytic granules: a potential self-recruiting mechanism.Am J Pathol. 2003; 163: 261-268Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar Levels of the TH1 transcription factor T-bet have been reported to be increased in peripheral blood T cells of patients with LP, and IFN-γ was described as the central proinflammatory cytokine in patients with LP.E16Khan A. Farah C.S. Savage N.W. Walsh L.J. Harbrow D.J. Sugerman P.B. Th1 cytokines in oral lichen planus.J Oral Pathol Med. 2003; 32: 77-83Crossref PubMed Scopus (179) Google Scholar, E4Sugerman P.B. Savage N.W. Walsh L.J. Zhao Z.Z. Zhou X.J. Khan A. et al.The pathogenesis of oral lichen planus.Crit Rev Oral Biol Med. 2002; 13: 350-365Crossref PubMed Scopus (521) Google Scholar, 5Piccinni M. Lombardelli L. Logiodice F. Tesi D. Kullolli O. Biagiotti R. et al.Potential pathogenetic role of Th17, Th0, and Th2 cells in erosive and reticular oral lichen planus.Oral Dis. 2014; 20: 212-218Crossref PubMed Scopus (57) Google Scholar, 6Xie S. Ding L. Xiong Z. Zhu S. Implications of Th1 and Th17 cells in pathogenesis of oral lichen planus.J Huazhong Univ Sci Technol Med Sci. 2012; 32: 451-457Crossref PubMed Scopus (41) Google Scholar, 7Terlou A. Santegoets Lindy A.M. van der Meijden W.I. Heijmans-Antonissen C. Swagemakers S.M. Vander Spek P.J. et al.An autoimmune phenotype in vulvar lichen sclerosus and lichen planus: a Th1 response and high levels of microRNA-155.J Invest Dermatol. 2012; 132: 658-666Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar This concept is supported by our demonstration of a TH1-dominated cutaneous T-cell infiltrate in lesions of patients with LP and a more TH2-dominated skin infiltrate in patients with BP or PV (Fig 2, D and E). Both PV and BP are associated with distinct HLA class II alleles, which are critical for the presentation of autoantigenic peptides. HLA-DRB1*04:02 and HLA-DQB1*05:03, which are prevalent in patients with PV,E17Sinha A.A. Brautbar C. Szafer F. Friedmann A. Tzfoni E. Todd J.A. et al.A newly characterized HLA DQ beta allele associated with pemphigus vulgaris.Science. 1988; 239: 1026-1029Crossref PubMed Scopus (171) Google Scholar, E18Ahmed A.R. Yunis E.J. Khatri K. Wagner R. Notani G. Awdeh Z. et al.Major histocompatibility complex haplotype studies in Ashkenazi Jewish patients with pemphigus vulgaris.Proc Natl Acad Sci U S A. 1990; 87: 7658-7662Crossref PubMed Scopus (189) Google Scholar and HLA-DQB1*03:01, which is prevalent in patients with BP,E19Delgado J.C. Turbay D. Yunis E.J. Yunis J.J. Morton E.D. Bhol K. et al.A common major histocompatibility complex class II allele HLA-DQB1* 0301 is present in clinical variants of pemphigoid.Proc Natl Acad Sci U S A. 1996; 93: 8569-8571Crossref PubMed Scopus (174) Google Scholar, E20Esmaili N. Mortazavi H. Chams-Davatchi C. Daneshpazhooh M. Damavandi M.R. Aryanian Z. et al.Association between HLA-DQB1*03:01 and bullous pemphigoid in Iranian patients.Iran J Immunol. 2013; 10: 1-9PubMed Google Scholar have been shown to restrict the activation of autoreactive T cells in patients with these disorders.E21Veldman C.M. Gebhard K.L. Uter W. Wassmuth R. Grötzinger J. Schultz E. et al.T cell recognition of desmoglein 3 peptides in patients with pemphigus vulgaris and healthy individuals.J Immunol. 2004; 172: 3883-3892Crossref PubMed Scopus (99) Google Scholar, E22Eming R. Hennerici T. Bäcklund J. Feliciani C. Visconti K.C. Willenborg S. et al.Pathogenic IgG antibodies against desmoglein 3 in pemphigus vulgaris are regulated by HLA-DRB1*04:02-restricted T cells.J Immunol. 2014; 193: 4391-4399Crossref PubMed Scopus (46) Google Scholar, 4Büdinger L. Borradori L. Yee C. Eming R. Ferencik S. Grosse-Wilde H. et al.Identification and characterization of autoreactive T cell responses to bullous pemphigoid antigen 2 in patients and healthy controls.J Clin Invest. 1998; 102: 2082-2089Crossref PubMed Scopus (192) Google Scholar A recent phenome-wide association study also linked LP to certain MHC regions, particularly HLA-DQB1*05:01, but an association with BP- or PV-associated HLA class II alleles has not been described.E23Liu J. Ye Z. Mayer J.G. Hoch B.A. Green C. Rolak L. et al.Phenome-wide association study maps new diseases to the human major histocompatibility complex region.J Med Genet. 2016; 53: 681-689Crossref PubMed Scopus (19) Google Scholar Instead, LP was found to be associated with HLA-DRB1*01:01.E24Carcassi C. Cottoni F. Floris L. La Nasa G. Mulargia M. Baldini G. et al.The HLA-DRB1*0101 allele is responsible for HLA susceptibility to lichen ruber planus.Eur J Immunogenet. 1994; 21: 425-429Crossref PubMed Scopus (12) Google Scholar, E25Luis-Montoya P. Yamamoto-Furusho J.K. Vega-Memije E. Rodríguez-Carreón A. Ruiz-Morales J.A. Vargas-Alarcón G. et al.HLA-DRB1*0101 is associated with the genetic susceptibility to develop lichen planus in the Mexican Mestizo population.Arch Dermatol Res. 2007; 299: 405-407Crossref PubMed Scopus (11) Google Scholar Of note, a clinically distinct LP variant, lichen planopilaris, which affects the hair shaft, was associated with HLA-DRB1*11:01 and HLA-DQB1*03:01.E26Pavlovsky L. Israeli M. Sagy E. Berg A.L. David M. Shemer A. et al.Lichen planopilaris is associated with HLA DRB1*11 and DQB1*03 alleles.Acta Derm Venereol. 2015; 95: 177-180Crossref PubMed Scopus (13) Google Scholar Identification of IL-17A+ BP180-specific T cells (Fig 1, H) closes a gap between the first description of TH17 cells in skin lesions of patients with LP and their potential functional relevance in LP pathogenesis. A few studies have demonstrated IL-17A+ T cells in the inflammatory infiltrate of skin lesions of patients with LPE13Shen Z. Gao X. Ma L. Zhou Z. Shen X. Liu W. Expression of Foxp3 and interleukin-17 in lichen planus lesions with emphasis on difference in oral and cutaneous variants.Arch Dermatol Res. 2014; 306: 441-446Crossref PubMed Scopus (39) Google Scholar, E27Wang H. Han Q. Luo Z. Xu C. Liu J. Dan H. et al.Oral lichen planus may enhance the expression of Th17-associated cytokines in local lesions of chronic periodontitis.Clin Oral Investig. 2014; 18: 1647-1654Crossref PubMed Scopus (20) Google Scholar and increased IL-17A levels in the sera of patients with LP.E28Shaker O. Hassan A.S. Possible role of interleukin-17 in the pathogenesis of lichen planus.Br J Dermatol. 2012; 166: 1367-1368Crossref PubMed Scopus (24) Google Scholar, 6Xie S. Ding L. Xiong Z. Zhu S. Implications of Th1 and Th17 cells in pathogenesis of oral lichen planus.J Huazhong Univ Sci Technol Med Sci. 2012; 32: 451-457Crossref PubMed Scopus (41) Google Scholar Here IL-17A+ T cells were found preferentially adjacent to the epidermal basal layer of skin lesions of patients with LP, where apoptosis induction of basal keratinocytes occurs (Fig 2, A and B). IL-17A is produced by mainly TH17 cells and γδ+ T cells,E29Jin W. Dong C. IL-17 cytokines in immunity and inflammation.Emerg Microbes Infect. 2013; 2: e60Crossref PubMed Scopus (402) Google Scholar which were also described in skin lesions of patients with LP.E30Gadenne A.S. Strucke R. Dunn D. Wagner M. Bleicher P. Bigby M. T-cell lines derived from lesional skin of lichen planus patients contain a distinctive population of T-cell receptor gamma delta-bearing cells.J Invest Dermatol. 1994; 103: 347-351Abstract Full Text PDF PubMed Scopus (42) Google Scholar, 5Piccinni M. Lombardelli L. Logiodice F. Tesi D. Kullolli O. Biagiotti R. et al.Potential pathogenetic role of Th17, Th0, and Th2 cells in erosive and reticular oral lichen planus.Oral Dis. 2014; 20: 212-218Crossref PubMed Scopus (57) Google Scholar, 6Xie S. Ding L. Xiong Z. Zhu S. Implications of Th1 and Th17 cells in pathogenesis of oral lichen planus.J Huazhong Univ Sci Technol Med Sci. 2012; 32: 451-457Crossref PubMed Scopus (41) Google Scholar Moreover, IL-17+ and Foxp3+ cells have been identified also in skin lesions of patients with BP, but their numbers correlated with neither disease activity nor severity.E31Arakawa M. Dainichi T. Ishii N. Hamada T. Karashima T. Nakama T. et al.Lesional Th17 cells and regulatory T cells in bullous pemphigoid.Exp Dermatol. 2011; 20: 1022-1024Crossref PubMed Scopus (80) Google Scholar Of note, recent studies using a Dsg3–T-cell receptor–transgenic mouse model showed that CD4+ Dsg3-specific TH1 cells induced a lichenoid dermatitis with lymphocytic interface dermatitis reminiscent of LP, whereas transfer of polarized TH17 cells induced severe psoriasis-like skin inflammation in the Dsg3-expressing tissues,E32Nishimoto S. Kotani H. Tsuruta S. Shimizu N. Ito M. Shichita T. et al.Th17 cells carrying TCR recognizing epidermal autoantigen induce psoriasis-like skin inflammation.J Immunol. 2013; 191: 3065-3072Crossref PubMed Scopus (34) Google Scholar, 8Takahashi H. Kouno M. Nagao K. Wada N. Hata T. Nishimoto S. et al.Desmoglein 3-specific CD4+ T cells induce pemphigus vulgaris and interface dermatitis in mice.J Clin Invest. 2013; 121: 3677-3688Crossref Scopus (66) Google Scholar supporting our concept that the cytokine profile of T-cell responses against cutaneous autoantigens has an effect on the phenotypic outcome of the resulting autoimmune disorder. In summary, we identified the autoantigens of PV and BP, Dsg3 and BP180, respectively, as target antigens of the inflammatory skin disorder LP. We show that LP is characterized by a peripheral blood TH1/TH17-dominated cell response, whereas PV and BP are associated primarily with TH2 responses against these autoantigens. This TH1/TH2 dichotomy was also found in skin lesions of patients with LP versus those of patients with BP and PV. The present findings strongly suggest that the cytokine profile of the T-cell autoimmune response to Dsg3 and BP180 is critical for the evolving clinical phenotype. The protocol of the study was approved by the Ethics Committee of the Medical Faculty of Philipps University Marburg, Germany (Az: 83/13). We thank Dr Giovanna Zambruno, Rome, Italy, and Dr Pascal Joly, Rouen, France, for providing PBMC samples from a total of 11 patients with PV patients; Dr Christian Möbs and Dr Ronald Wolf, Marburg, Germany, for critical reading of the manuscript and helpful discussions; and Andrea Gerber, Elke Hermann, and Viktoria Wischmann for excellent technical assistance. Fifty-three patients with LP, 21 patients with BP, 16 patients with PV, 3 patients with LPP, and 18 healthy control (HC) subjects were studied. Diagnosis of LP was based on the clinical phenotype and histopathologic findings. Diagnosis of BP was established based on the presence of pruritus associated with either urticarial plaques, tense bullae, pruritic papules or eczematous lesions, and IgG and/or C3 deposits at the BMZ by means of direct immunofluorescence (DIF) and anti-BP180 and/or anti-/BP230 IgG reactivity by means of ELISA. Diagnosis of LPP was made clinically based on the presence of lichenoid papules that transformed into tense bullae and immunologically through detection of IgG and/or C3 deposits along the BMZ by using DIF and anti-BP180 serum IgG by using ELISA. Accordingly, PV was diagnosed based on fragile blisters and/or erosions of the mucous membranes and skin and epidermal cell-surface deposits of IgG and/or C3 by using DIF and anti-Dsg3 serum IgG by using ELISA. None of the studied patients with LP, BP, PV, or LPP were receiving systemic immunosuppressive treatment. Patients with LP did not show serum IgG against BP180, BP230, Dsg1, Dsg3, collagen VII, laminin-332, or laminin-γ1. Patients with LP were subdivided by clinical phenotype: cutaneous LP was defined as LP with skin lesions only, whereas mucosal LP was defined as LP with mucosal lesions with or without skin involvement; total LP includes all studied patients with LP. For immunohistologic analyses, skin biopsy specimens from 12 patients with LP, 12 patients with BP, 7 patients with PV, and 3 patients with LPP were analyzed. Citrate-phosphate-dextrose-adenine–treated peripheral blood samples from patients with LP and HCs were incubated for 30 minutes at 4°C for antibody staining: mouse-anti human CD3 phycoerythrin (PE)–Cy5 (clone UCHT1), mouse anti-human CD3 allophycocyanin (APC; clone UCHT1), mouse anti-human CD4 fluorescein isothiocyanate (FITC; clone RPA-T4), mouse anti-human CD8 FITC (clone SK1), mouse anti-human CD25 APC (clone M-A251), mouse anti-human CD127 PE (clone HIL-7R-M21), mouse anti-human T-cell receptor γδ (clone B1), mouse IgG1 FITC (clone MOPC-21), mouse IgG1 PE (clone G18-145), mouse IgG1 PE-Cy5 (clone MOPC-21), mouse IgG1 APC (clone G18-145; all from Becton Dickinson, Franklin Lakes, NJ), mouse anti-human CCR4 (clone 205410), and mouse IgG2 APC (clone 133303; both from R&D Systems, Minneapolis, Minn). Red blood cells were lysed with ACK lysis buffer (0.15 mol/L NH4Cl, 1 mmol/L KHCO3, and 0.1 mmol/L EDTA). Afterward, cells were washed twice (with PBS, 1% BSA, and 0.1% NaN3), resuspended, and analyzed by using fluorescence-activated cell sorting (FACSCalibur, Becton Dickinson). FCS files were analyzed with FlowJo 7.6 single-cell analysis software (FlowJo, Ashland, Ore). IgG autoantibodies were analyzed in plasma samples from patients and HCs by means of ELISA, according to the manufacturers' instructions. The following ELISA assays were used: human Dsg1, Dsg3, BP180, BP230 (Euroimmun, Lübeck, Germany), and collagen VII (MBL, Nagoya, Japan). For chemokine detection, the following ELISA kits were used: human CCL5, human CXCL10, human IL-8, and human IL-17 (all from BioLegend, San Diego, Calif). Staining of paraffin-embedded skin sections was performed with the automated IHC stainer BOND-MAX (Leica, Wetzlar, Germany) and Autostainer Plus automated immunostaining device (Dako, Hamburg, Germany) by using the following primary antibodies: mouse anti-human CD3, CD4, and CD8 (all from Novocastra, Leica, Wetzlar, Germany); rabbit anti-human IL-17A and rabbit anti-human FoxP3 (both from Novus, Littleton, Colo); and rabbit anti-human T-bet and rabbit anti-human GATA-3 (both from Cell Signaling Technology, Danvers, Mass). Secondary antibodies used were as follows: Bond Polymer Refine Detection Kit (Leica; CD3, CD4, and CD8) biotinylated anti-rabbit IgG, and biotinylated anti-mouse IgG (Vector Laboratories, Burlingame, Calif; IL-17A, FoxP3, T-bet, and GATA-3). Antibodies from Vector Laboratories were subsequently detected by using peroxidase- or alkaline phosphatase–labeled ABC systems (Dako, Glostrup, Denmark). Visualization was carried out with 3,3′-diaminobenzide or Liquid Permanent Red staining (both from Dako) as chromogenes. The T-cell infiltrate of skin lesions was quantified based on microscopic images (Axiostar; Zeiss, Jena, Germany) in combination with CellˆD software (Soft Imaging System, Berlin, Germany) and free ImageJ software (imagej.nih.gov/ij/; see Fig E3). At ×100 magnification, CD3+, CD4+, CD8+, FoxP3+, and IL-17A+ T cells were counted (×200 magnification for CD3+T-bet+ and CD4+GATA-3+ T cells). After generating a grid (ImageJ software; area per point, 50,000 square pixels), all stained T cells were counted in 2 squares (4 squares for CD3+T-bet+ and CD4+GATA-3+ T cells) adjacent to the BMZ (ImageJ software, cell counter; Fig E3, A), and their proportion of all infiltrating cells was determined afterward. Moreover, with regard to IL-17A+ and FoxP3+ T cells, the T-cell infiltrate adjacent to the dermal-epidermal BMZ was further analyzed on images taken at ×200 magnification and after grid formation (ImageJ software; area per point, 50,000 square pixels) counting 4 squares in the BMZ and dermal region (ImageJ software, cell counter; Fig E3, B). BP180-NH2 (NH2-terminus of the BP180 ectodomain; amino acids 490 to 812, including the immunodominant NC16A domain), BP180-COOH (COOH-terminus of the BP180 ectodomain, amino acids 1352-1465), Dsg1 (entire ectodomain, amino acids 1-496), and Dsg3 (entire ectodomain, amino acids 1-566) were produced in a baculovirus expression system, as previously described.E33Müller R. Svoboda V. Wenzel E. Gebert S. Hunzelmann N. Müller H. et al.IgG reactivity against non-conformational NH-terminal epitopes of the desmoglein 3 ectodomain relates to clinical activity and phenotype of pemphigus vulgaris.Exp Dermatol. 2006; 15: 606-614Crossref PubMed Scopus (46) Google Scholar ELISpot assays were performed, as previously described.E34Möbs C. Schmidt T. Research techniques made simple: monitoring of T-cell subsets using the ELISPOT assay.J Invest Dermatol. 2016; 136: e55-e59Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, E35Möbs C. Ipsen H. Mayer L. Slotosch C. Petersen A. Würtzen P.A. et al.Birch pollen immunotherapy results in long-term loss of Bet v 1-specific TH2 responses, transient TR1 activation, and synthesis of IgE-blocking antibodies.J Allergy Clin Immunol. 2012; 130: 1108-1116.e6Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar IFN-γ–, IL-5–, and IL-17A– positive spots were detected according to the manufacturers' instructions (Human IFNγ-ELISpot, Human IL-5-ELISpot; Becton Dickinson; Human IL-17A ELISpot Ready-Set-Go, eBioscience, San Diego, Calif). PBMCs were seeded at 1 × 105 cells per well on ELISpot plates, and developed plates were finally analyzed with the ELISpot plate reader A.EL.VIS (A.EL.VIS, Hannover, Germany). For data analysis, spots of nonstimulated controls (mean) were subtracted from spots (mean) of cultures with antigen (all in duplicate). One spot was added to all values to exclude values of 0 to form ratios of distinct cytokine-producing T-cell subsets (Fig 1, F and K). All patients with LP (n = 33), patients with BP (n = 10), and patients with PV (n = 6) who showed a distinct T-cell response of greater than the cutoff (BP180, 20 IFN-γ+ spots; Dsg3, 8 IFN-γ+ spots) were analyzed. Statistical analysis was performed with GraphPad Prism 6.02 software (GraphPad Software, La Jolla, Calif). Data are displayed as bar graphs showing means with SDs or as dot blots and box plots showing medians. Differences were considered significant at a P value of less than .05, as determined by using the Mann-Whitney U or Student t test. Gaussian distribution was tested by using the Shapiro-Wilk test.Fig E2Analysis of regulatory T cells in skin lesions of patients with LP and those with BP. A, Representative image of FoxP3+ cells in the upper dermis and dermal-epidermal BMZ (interrupted line) in patients with LP and those with BP, showing a diffuse distribution of Foxp3+ regulatory T cells. B, Number of FoxP3+ cells along the BMZ and upper dermis (patients with LP, n = 8; patients with BP, n = 10). No statistically significant difference in distribution of regulatory T cells was observed between the BMZ and upper dermis, as determined by using the Mann-Whitney U test.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E3Analysis of T-lymphocyte subsets in skin lesions of patients with LP, patients with BP, and patients with PV. A, Enumeration of T cells was based on use of a microscope (Axiostar, Zeiss) in combination with CellˆD (Soft Imaging System) and ImageJ software. For counting of specifically stained T cells, images of skin lesions at ×100 or ×200 magnification were taken. After generating a grid (ImageJ software; area per point, 50,000 square pixels), all infiltrating and specifically stained cells were counted in 2 defined squares (S1/S2; 4 squares for ×200 magnification) adjacent to the dermal-epidermal BMZ (ImageJ software, cell counter), and the proportion of all infiltrating cells to specifically stained T cells was determined. B, For localization analysis of IL-17A+ and FoxP3+ T cells, images were taken at ×200 magnification and after grid formation (ImageJ software; area per point, 50,000 square pixels). All infiltrating and specifically stained cells were counted in 4 squares adjacent to the BMZ (B1-B4) and 4 squares in the adjacent upper dermal region (D1-D4; ImageJ software, cell counter). The proportion of specifically stained T cells of all infiltrating cells was determined, as were the numbers of IL-17A+ T cell along the BMZ and upper dermis.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table E1T-cell recognition of Dsg1 and Dsg3 in patients with LPELISpotLP skin∗P values of ELISpot analysis of patients with LP with skin involvement (LP skin), mucosal involvement (LP mucosa), and the entire LP cohort (LP total) compared with HCs. Significant P values are shown in boldface.LP mucosa∗P values of ELISpot analysis of patients with LP with skin involvement (LP skin), mucosal involvement (LP mucosa), and the entire LP cohort (LP total) compared with HCs. Significant P values are shown in boldface.LP total∗P values of ELISpot analysis of patients with LP with skin involvement (LP skin), mucosal involvement (LP mucosa), and the entire LP cohort (LP total) compared with HCs. Significant P values are shown in boldface.Dsg1 IFN-γP = .22P = .825P = .44Dsg3 IFN-γP = .0724P = .0255P = .0183Dsg1 IL-17P = .395P = .35P = .2939Dsg3 IL-17P = .32P = .98P = .646∗ P values of ELISpot analysis of patients with LP with skin involvement (LP skin), mucosal involvement (LP mucosa), and the entire LP cohort (LP total) compared with HCs. Significant P values are shown in boldface. Open table in a new tab Table E2T-cell recognition of BP180 (NH2- and COOH-terminus) in patients with LPELISpotLP skin∗P values of ELISpot analysis of patients with LP with skin involvement (LP skin), mucosal involvement (LP mucosa), and the entire LP cohort (LP total) compared with HC subjects. Significant P values are shown in boldface.LP mucosa∗P values of ELISpot analysis of patients with LP with skin involvement (LP skin), mucosal involvement (LP mucosa), and the entire LP cohort (LP total) compared with HC subjects. Significant P values are shown in boldface.LP total∗P values of ELISpot analysis of patients with LP with skin involvement (LP skin), mucosal involvement (LP mucosa), and the entire LP cohort (LP total) compared with HC subjects. Significant P values are shown in boldface.BP180-NH2 IFN-γP = .23P = .0008P = .0033BP180-COOH IFN-γP = .407P = .21P = .21BP180-NH2 IL-17P = .0033P = .0470P = .0108BP180-COOH IL-17P = .3731P = .3275P = .9303∗ P values of ELISpot analysis of patients with LP with skin involvement (LP skin), mucosal involvement (LP mucosa), and the entire LP cohort (LP total) compared with HC subjects. Significant P values are shown in boldface. Open table in a new tab
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