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Pivotal Role of Lesional and Perilesional T/B Lymphocytes in Pemphigus Pathogenesis

Open ArchivePublished:June 21, 2017DOI:https://doi.org/10.1016/j.jid.2017.05.032
      Pemphigus is a skin and mucosal membrane-targeting autoimmune bullous disease. Previous studies have shown that circulating anti-desmoglein1/3 antibodies are pathogenic and mediate blister formation. However, the role of infiltrating immune cells in lesional skin has not been fully investigated. In this study we showed that there existed a large number of B and T lymphocytes and plasma cells in the skin lesions by immunohistochemistry and immunofluorescence staining. In addition, a significantly increased number of Dsg1- and Dsg3-specific B cells could be identified by flow cytometric analysis or enzyme-linked immunospot technique (i.e., ELISPOT) assay. Furthermore, anti-Dsg1 and Dsg3 antibodies could be detected from the supernatant of in vitro cultures with isolated lymphocytes from lesional skin. We found that most T lymphocytes infiltrating pemphigus vulgaris lesions were CD4+ T helper cells expressing IL-21 and IL-17a but not typical T follicular helper cells expressing CXCR5. Additionally, our microarray assay showed that the level of chemokine CCL19 was significantly elevated, suggesting active T-/B-lymphocyte trafficking and aggregation in the pemphigus vulgaris lesions. Collectively, our results suggest a critical role of locally infiltrating lymphocytes in pemphigus pathogenesis.

      Abbreviations:

      BP (bullous pemphigoid), ELISPOT (enzyme-linked immunospot technique), PV (pemphigus vulgaris), Tfh (follicular helper T cell), Th (T helper), TLO (tertiary lymphoid organ)

      Introduction

      Pemphigus is a severe organ-specific autoimmune bullous disease, characterized by intraepidermal blister formation involving mucosal membranes and skin (
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      Pemphigus: autoimmunity to epidermal cell adhesion molecules.
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      Pemphigus—paradigm of autoantibody-mediated autoimmunity.
      ,
      • Stanley J.R.
      Autoantibodies against adhesion molecules and structures in blistering skin diseases.
      ). Pathogenic autoantibodies against Dsg1 or/and Dsg3 in desmosomes lead to loss of cell adhesion of keratinocytes in the human skin epidermis, designated as acantholysis. Although current evidence supports a key role of circulating anti-Dsg autoantibodies in the pemphigus pathogenesis (
      • Hertl M.
      Humoral and cellular autoimmunity in autoimmune bullous skin disorders.
      ), the exact mechanism of how autoantibodies are secreted and induce acantholysis requires further investigation.
      Pemphigus is a T/B cell- and antibody-mediated autoimmune disease (
      • Hertl M.
      • Veldman C.
      Pemphigus—paradigm of autoantibody-mediated autoimmunity.
      ), which has also been suggested to be a T helper (Th) type 2-dependent disorder (
      • Rizzo 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.
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      • et al.
      T cell recognition of desmoglein 3 peptides in patients with pemphigus vulgaris and healthy individuals.
      ). Other T-cell subsets, including CD4+CD25+ regulatory T cells and Th17 cells, have also been implicated in the pathogenesis of pemphigus (
      • Asothai R.
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      • Sharma V.K.
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      Distinctive Treg associated CCR4-CCL22 expression profile with altered frequency of Th17/Treg cell in the immunopathogenesis of Pemphigus Vulgaris.
      ,
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      • Yuan H.J.
      • Zheng J.
      • et al.
      The imbalance of Th17 and regulatory T cells in pemphigus patients.
      ). T follicular helper cells (Tfh) are a newly discovered subset of CD4+ Th cells whose function appears to help B-cell activation and antibody production during humoral immune responses (
      • Pan M.
      • Zhu H.
      • Xu R.
      Immune cellular regulation on autoantibody production in pemphigus.
      ). Recently, circulating Tfh cells (defined as CD4+CXCR5+ T cells) have been reported to be significantly increased in pemphigus, along with elevated IL-21 in plasma (
      • Hennerici T.
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      • Schmidt T.
      • Seipelt M.
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      • Mobs C.
      • et al.
      Increased frequency of T follicular helper cells and elevated interleukin-27 plasma levels in patients with pemphigus.
      ). Despite the large body of studies showing the important role of circulating T and B cells in the production of pathogenic antibodies in pemphigus, few studies have been attempted to characterize the phenotype and function of lesional or perilesional T and B cells in pemphigus, which may function as the tertiary lymphoid organ (TLO) that plays a key role in induction, maintenance, and development of autoimmune diseases in target organs (
      • Chang A.
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      In situ B cell-mediated immune responses and tubulointerstitial inflammation in human lupus nephritis.
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      Revisiting the role of B cells in skin immune surveillance.
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      Tertiary lymphoid organs in infection and autoimmunity.
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      Inducible bronchus-associated lymphoid tissue (iBALT) in patients with pulmonary complications of rheumatoid arthritis.
      ,
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      • et al.
      Cellular basis of ectopic germinal center formation and autoantibody production in the target organ of patients with Sjogren's syndrome.
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      • et al.
      Lymphoid chemokine B cell-attracting chemokine-1 (CXCL13) is expressed in germinal center of ectopic lymphoid follicles within the synovium of chronic arthritis patients.
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      • et al.
      Lymphoid neogenesis in rheumatoid synovitis.
      ).
      A previous study showed that CD4+ T-cell lines isolated from the perilesional skin of pemphigus vulgaris (PV) patients, when co-cultured with CD56+CD3 major histocompatibility complex class II+ natural killer cells, secreted a large amount of IL-6, IL-8, and IFN-γ in response to antigen stimulation (
      • Stern J.N.
      • Keskin D.B.
      • Barteneva N.
      • Zuniga J.
      • Yunis E.J.
      • Ahmed A.R.
      Possible role of natural killer cells in pemphigus vulgaris - preliminary observations.
      ). These results strongly suggest that the locally infiltrating CD4+ T cells from PV patients could respond in a pattern similar to that observed in the peripheral blood. In this study, we found that a significant number of Dsg-specific B cells could be detected in pemphigus lesional skin. Our in vitro co-culture studies showed that these B cells could secrete large amounts of anti-Dsg1 and Dsg3 antibodies. We further showed that IgG+ plasma cells were in close contact with T cells by immunohistochemistry staining. Additionally, T cells that infiltrated the PV lesions were IL-21–producing helper T cells that also expressed IL-17a. Our microarray study also showed that the expression of CCL19 was significantly elevated in the lesional skin of PV, which may be involved in the trafficking and aggregation of antigen-specific B and T cells.

      Results

      PV lesions contain desmoglein-specific B cells

      To examine lymphocytic infiltration and determine whether B cells accumulate in the PV lesions, we examined cells from the skin with PV lesions and skin from healthy donors by flow cytometry assay. There were a significant number of lymphocytes being detected in the PV lesions (17.95 ± 3.85% vs. 7.83 ± 1.29%; P = 0.0234) (Figure 1b). The frequency of CD19+ B cells was found to be much higher in PV lesions than in healthy skin (4.27 ± 1.13% vs. 0.61 ± 0.31%, P = 0.0002, Mann-Whitney test) (Figure 1c). A fraction of CD19+ B cells from the PV lesions could specifically bind to Dsg1 and Dsg3 but not the irrelevant protein (see Supplementary Figure S1 online). In contrast, no B cell from bullous pemphigoid (BP) lesions bound to Dsg1/3 (Figure 1d). Further, the short-term enzyme-linked immunospot technique (ELISPOT) assay showed that the frequencies of Dsg1- and Dsg3-specific IgG secreting lymphocytes isolated from lesional skin of PV patients were 0.22 ± 0.031% and 0.54 ± 0.095%, respectively, whereas almost no Dsg1/3-specific IgG-secreting cell was detected from BP lesions (Figure 1e). These results indicate that lymphocytes and Dsg-specific B cells significantly infiltrated the lesional skin of PV patients and that these B cells are capable of secreting anti-Dsg antibodies.
      Figure 1
      Figure 1Sampling tissues and B cells in skin. (a) A total of 34 PV patients, three BP patients, and 32 health donors had skin samples. For these participants, part of the skin tissue was fixed by formalin and embedded in paraffin for immunohistochemistry or immunofluorescence staining; other parts of tissue were used for cell isolation and then for flow cytometry analysis, in vitro culture, or microarray assay. Scale bar = 1 cm. (b) Representative dot plots were gated to identify lymphocytes in PV lesions and skin of healthy donors. Summarized percentages of lymphocytes are shown. (c) Representative flow cytometric plots of lymphocytes were gated to show CD19+ B-cell subsets. Summarized percentages of B cells are shown. (d) Representative dot plots of skin CD19+ B cells were positive for Dsg1 and Dsg3 binding in PV and BP groups. Summarized data indicating statistical differences of Dsg1- and Dsg3-specific B cells of PV and BP lesion are shown. (e) Representative plates show Dsg1- and Dsg3-specific IgG secreting cells of skin lymphocytes from PV and BP lesional skin by enzyme-linked immunospot technique assay. Summarized Dsg1- and Dsg3-specifc IgG-secreting cells are shown. BP, bullous pemphigoid; FSC, forward scatter; IHC, immunohistochemistry; PV, pemphigus vulgaris; SSC, side scatter.

      B lymphocytes from PV lesions produce anti-Dsg1 and Dsg3 antibodies after in vitro culture

      To further examine whether infiltrating B cells from lesional skin are able to produce anti-Dsg antibodies, lymphocytes from skin tissues of PV patients or healthy donors were cultured in vitro for 6 days. The supernatants were assayed for anti-Dsg1 and Dsg3 antibodies (Figure 2a). To reduce the effect of plate-to-plate variability, the index value was used for statistical analysis. The descriptive statistics for the PV and control groups are shown in Supplementary Table S1 online. Because the samples were supernatants from in vitro culture, to ensure the validation of supernatant anti-Dsg specific ELISA, receiver operating characteristic analysis was used to illustrate the diagnostic performance of the ELISA (
      • Messingham K.A.
      • Noe M.H.
      • Chapman M.A.
      • Giudice G.J.
      • Fairley J.A.
      A novel ELISA reveals high frequencies of BP180-specific IgE production in bullous pemphigoid.
      ). The areas under the curve to measure the performance of the anti-Dsg1 and Dsg3 tests were determined to be 0.9381 (standard error = 0.0411, 95% confidence interval = 0.8576–1.019, P < 0.0001) and 0.975 (standard error = 0.02331, 95% confidence interval = 0.9293–1.021, P < 0.0001), respectively (Figure 2b). Paired sensitivity and specificity values were derived from the receiver operating characteristic analysis for calculating cut-off values to discriminate positive and negative results. After maximization of the Youden’s index (J = sensitivity + specificity-1), the cut-off points were chosen to be 0.64 and 0.92 index units for anti-Dsg1 and Dsg3, respectively, corresponding to a Youden’s index of 0.76 and 0.85, respectively (see Supplementary Table S2 online). The selected cut-off of anti-Dsg1 resulted in a sensitivity of 90% and a specificity of 85.71%, and anti-Dsg3 resulted in a sensitivity of 90% with a specificity of 95%. Analysis of supernatant showed that 17 of 20 PV patients (85.0%) and 19 of 20 PV patients (95.0%) had detectable levels of anti-Dsg1 and Dsg3 antibodies using our selected cut-off. In addition, lymphocytes both from bullous pemphigoid lesion and healthy skin were used as controls for in vitro culture and for total IgG and anti-Dsg antibody detection by ELISA. By all appearances, lymphocytes from PV lesion produced much more total IgG and anti-Dsg1 and Dsg3 antibodies than those from healthy skin. However, total IgG of the supernatant from in vitro culture of lesional skin from bullous pemphigoid was at about the same level as PV, but the titer results of anti-Dsg1 and anti-Dsg3 antibodies were much lower than those from in vitro culture of PV lesional lymphocytes (see Supplementary Figure S2 online), which provided evidence suggesting that anti-Dsg antibodies in PV patients was antigen specific. To further confirm antibody specificity, Western blotting analysis was performed. We used supernatant from three PV patients with high ELISA titer results for this study, and our results showed that the antibodies of supernatant from two out of three patients could specifically bind to a 130-kDa protein that was also recognized by the mouse anti-Dsg3 mAb 5G11 (Figure 2c); none of them recognized Dsg1.
      Figure 2
      Figure 2In vitro culture of lymphocytes from skin and production of anti-Dsg1 and Dsg3 antibodies. (a) Schema shows skin lymphocyte isolation and in vitro culture, which are described in detail in the Materials and Methods. Scale bar = 1 cm. (b) Diagnostic properties of the anti-Dsg1 and -Dsg3 ELISA for supernatants from an in vitro culture were depicted as receiver operating characteristic curves, showing sensitivity and specificity of the ELISA. (c) Western blotting analysis of human keratinocyte extracts. Lane 1, human keratinocyte extracts was stained by Coomassie blue reagent. Lane 2, mouse anti-human Dsg3 mAb 5G11. Lane 3, supernatant from in vitro culture of PV lesional lymphocytes. Lane 4, supernatant from in vitro culture of BP lesional lymphocytes. (d) The titers of anti-Dsg1 and -Dsg3 antibodies produced by PV lesional lymphocytes are positively correlated with PDAI. AUC, area under the curve; BP, bullous pemphigoid; PDAI, Pemphigus Disease Area Index; PV, pemphigus vulgaris.
      Our data also showed that both anti-Dsg1 and -Dsg3 antibodies from in vitro culture had positive correlation with the Pemphigus Disease Area Index (P = 0.0206, R = 0.683 and P = 0.0159, R = 0.676, respectively) (Figure 2d). These results suggest that B cells from PV lesions are able to produce anti-Dsg1 and -Dsg3 antibodies, which may contribute to the disease pathogenicity.

      B and T cells infiltrate PV lesions, along with IgG-secreting CD138+ plasma cells

      We further sought to test three PV patients to discover whether there are other immune cells involved in PV lesions to provide an environment for antibody production, assisting B-cell differentiation and maturation, by immunohistochemistry and immunofluorescence. We found accumulations of CD3+ and CD4+ T cells, CD19+ B cells, and CD138+ plasma cells in all of the three PV lesions (Figure 3a). It appears that a TLO-like structure was formed in pemphigus lesional skin. Further analysis showed that CD3+ T cells were in close contact with CD20+ B cells by immunofluorescence staining (Figure 3b). In addition, CD138+ plasma cells with IgG in the cytoplasm were present in the dermis of PV lesional area (Figure 3c). These data suggest that in addition to B cells, there are other immune cells infiltrating the lesional skin of PV patients, leading to the formation of a TLO-like structure.
      Figure 3
      Figure 3T and B lymphocytes interact closely in PV lesions, with CD138+ IgG+ plasma cells infiltrating PV lesions. (a) Immunohistochemistry staining shows CD3-, CD4-, CD19-, and CD138-expressed T, B, and plasma cells infiltrating PV lesions. Scale bar = 100 μm. (b) Co-staining of CD20 and CD3 in PV lesions. CD3+ T cells could be detected in close contact with CD20+ B cells in PV lesions. Red = CD3, green = CD20, and blue = DAPI. Scale bar = 10 μm. (c) Immunofluorescent staining of IgG in cytoplasm of CD138+ plasma cells in PV lesions. Red = CD138, green = IgG, and blue = DAPI. Scale bar = 10 μm. PV, pemphigus vulgaris.

      T cells in PV skin are mainly CD4+ Th cells producing IL-21, but not classical Tfh cells

      Because Tfh cells reside close to B cells in lymph nodes, promoting B cell activities and supporting the generation of antibodies in the germinal centers, we analyzed the proportion of Tfh cells expressing CXCR5. CXCR5+ T cells were gated from CD3+CD4+ T cells, as shown in Figure 4a. PV lesions contained a significantly higher proportion of CD3+ T cells than healthy skin (71.18 ± 3.95% vs. 57.18 ± 3.56%, P = 0.0127), although no difference in cell percentage was found between CD4+ T cells and Tfh cells (59.04 ± 3.57% vs. 62.06 ± 3.85%, P = 0.5783 for CD4+ T cells and 2.12 ± 0.32% vs. 2.47 ± 0.48%, P = 0.5861, unpaired t test, for Tfh cells) (Figure 4b). Because cytokine IL-21 was shown to be strongly associated with Tfh cells, we also examined the secretion of IL-21 by CD4+ T cells through flow cytometry. Our data showed a significantly higher proportion of CD4+IL-21+ T cells in PV lesions than in healthy skin (3.98 ± 0.87% vs. 1.21 ± 0.06%, P = 0.0195) (Figure 4c). In addition, 47.87 ± 7.803% of IL-21–secreting CD4+ T cells could also produce IL-17a. In contrast, IL-21–negative CD4+ T cells secreted minimal levels of IL-17a (3.327 ± 0.277%, P = 0.0294) (Figure 4d). Collectively, these results suggest that CD4+ T cells that may promote B cells for autoantibody production in pemphigus lesional skin are not typical Tfh cells but IL-21–secreting Th cells that also have the ability to produce IL-17a.
      Figure 4
      Figure 4T-cells that infiltrate PV lesions are IL-21–secreting T helper cells but not conventional Tfh cells. (a) Representative flow plots of CD3+ T cells were gated from lymphocytes and subsequently gated to show percentages of helper T-cell subsets (CD4+) and follicular helper T cells (CD4+CXCR5+) in PV lesions. (b) Summarized data show percentages of CD3+ T cells, CD3+CD4+ helper T cells, and CD3+CD4+CXCR5+ follicular helper T cells in PV and healthy skin. (c) Representative dot plots were gated from CD3+CD4+ lymphocytes of PV lesions and healthy skin to show IL-21 production. Summarized percentages of CD4+IL-21+ T cells in PV lesions and healthy skin are shown. (d) Representative dot plots were gated to show IL-17+ T-cell subsets from CD4+IL-21+ and CD4+IL-21 cells. Summarized data indicating the statistics between these two groups are shown. FSC, forward scatter; PV, pemphigus vulgaris; SSC, side scatter.

      Increased CCL19 may lead to lymphoid tissue neogenesis in PV lesions

      We next used the microarray gene expression analysis to screen putative chemokines that may be involved in lymphocyte trafficking and lymphoid tissue formation and identified five predictable target genes that were up-regulated in lymphocytes derived from PV lesions (Figure 5a, 5b). Because CCL19 has been reported to participate in the process of lymphoid tissue formation (
      • van de Pavert S.A.
      • Mebius R.E.
      New insights into the development of lymphoid tissues.
      ), we validated this gene by real-time PCR analysis to confirm microarray data. We found that CCL19 was remarkably up-regulated at the transcriptional level and increased by more than 4.0-fold in PV compared with normal control samples, as determined by real-time PCR assays (P = 0.0378) (Figure 5c).
      Figure 5
      Figure 5Expression of CCL19 is up-regulated in PV lesions. (a, b) Total RNAs of lymphocytes derived from skin of three PV patients and three healthy control subjects were used for microarray analysis of 57 genes of chemokines and receptors in total. Transcripts of 31 genes were found to be up-regulated in lymphocytes of PV lesions, and six genes were up-regulated remarkably. (c) Real-time PCR analysis of mRNA expression levels of CCL19 in lymphocytes of PV lesion and healthy skin. PV, pemphigus vulgaris.

      Discussion

      In this study, we found that B and T cells, as well as IgG+ plasma cells, are accumulated in the lesions of pemphigus to form a TLO-like structure. Moreover, we found that Dsg-specific B cells infiltrated the lesional skin. Ex vivo experiments showed that these B cells are capable of producing autoantibodies. Based on these findings, we hypothesize that locally infiltrating B cells may interact with T cells in lesional skin, leading to Dsg-specific autoantibody production, which may play a pivotal role in pemphigus pathogenesis.
      We first examined the immune cells infiltrating PV lesions and found that the frequency of CD19+ B cells is remarkably elevated in PV lesions. The frequency of infiltrating CD19+ B cells is positively correlated with Pemphigus Disease Area Index (see Supplementary Figure S3 online). In addition, we discovered a sizable percentage of Dsg1- and Dsg3-specific B cells in lesional skin by flow cytometry assay. We also performed immunostaining for Dsg3-specific B cells. Unfortunately, we could not co-stain Dsg3 and B cells because of technical limitations, but we detected CD4+T cells and Dsg3+ cells infiltrating PV lesions (see Supplementary Figure S4 online). This might be due to the scarcity of Dsg3+ cells, which is consistent with our flow cytometry data indicating that Dsg3+ cells were less than 10% of total B cells. Furthermore, ex vivo experiments showed that B cells from PV lesions are capable of secreting anti-Dsg1 and -Dsg3 antibodies. This is further confirmed by both short-term ELISPOT and an in vitro culture. Although the source of these antigen-specific B cells has not been defined, it is possible that locally activated antigen-presenting cells such as dermal dendritic cells in pemphigus drive B-cell activation and differentiation (
      • Stern J.N.
      • Keskin D.B.
      • Barteneva N.
      • Zuniga J.
      • Yunis E.J.
      • Ahmed A.R.
      Possible role of natural killer cells in pemphigus vulgaris - preliminary observations.
      ). Our data further showed that in addition to B cells, T cells also infiltrated PV lesions, with T cells residing intimately with B cells (Figure 3). The infiltrating T cells were mainly CD4+ Th cells that are capable of producing IL-21 and IL-17a. However, these CD4+ T cells are not classical Tfh cells expressing Bcl-6 or CXCR5 (Figure 4a, Supplementary Figure S5 online). At ectopic sites, lymphoid chemokines (CCL19, CCL21, CXCL12, and CXCL13) have been found to be essential chemokines in lymphoid neogenesis (
      • Jones G.W.
      • Jones S.A.
      Ectopic lymphoid follicles: inducible centres for generating antigen-specific immune responses within tissues.
      ). They induce upstream of lymphoid tissue organization, leading to T/B-cell recruitment and segregation and promote leukocyte accumulation (
      • Aloisi F.
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      Lymphoid neogenesis in chronic inflammatory diseases.
      ). We found that CCL19 is significantly elevated in PV lesions, suggesting a role of CCL19 expression in aggregation of T/B lymphocytes. In addition to CCL19, CCL21, and CXCL13 were also up-regulated in the lesional skin of PV patients (see Supplementary Figure S6 online). CCL19 and CCL21 have been reported to regulate the T-cell homing and maturation of dendritic cells, and CXCL13 can recruit B cells into the follicles (
      • Aloisi F.
      • Pujol-Borrell R.
      Lymphoid neogenesis in chronic inflammatory diseases.
      ). It is likely that CCL19, along with CXCL13 and CCL21, attracts lymphocyte aggregation, leading to TLO formation in pemphigus lesional skin, which may provide an anatomical microenvironment for autoantibody production.
      Although nonclassical Tfh cells were found in PV lesions in our study, so-called milky spots in the omentum indicated that it was not absolutely necessary for the development of germinal center-like structure and T/B responses (
      • Rangel-Moreno J.
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      Omental milky spots develop in the absence of lymphoid tissue-inducer cells and support B and T cell responses to peritoneal antigens.
      ). One of the reasons for the lack of typical Tfh cells that express CXCR5 may be that T cells simply do not need this chemokine receptor to get contact with B cells (
      • Hutloff A.
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      • Baelde H.J.
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      • Jacobi A.
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      ,
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      • Carragher D.M.
      • Kusser K.
      • Hartson L.
      • Moquin A.
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      Omental milky spots develop in the absence of lymphoid tissue-inducer cells and support B and T cell responses to peritoneal antigens.
      ). With the irregular infiltrates of T and B cells in PV lesions, T cells may not necessarily migrate from a T-cell area into a B-cell follicle as in the lymph node. Our data also showed that IL-21–producing Th cells are significantly increased in PV lesions. IL-21 is a cytokine secreted mainly by Tfh and Th17 cells (
      • Bettelli E.
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      • Oukka M.
      • Kuchroo V.K.
      Induction and effector functions of T(H)17 cells.
      ,
      • Deenick E.K.
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      ). Indeed, most IL-21–producing Th cells also secrete IL-17a. IL-17 has been shown to play an important role in initiating and controlling ectopic lymphoid neogenesis in inducible bronchus-associated lymphoid tissue (
      • Grogan J.L.
      • Ouyang W.
      A role for Th17 cells in the regulation of tertiary lymphoid follicles.
      ), multiple sclerosis (
      • Peters A.
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      ), and rheumatoid arthritis (
      • Canete J.D.
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      • van Duivenvoorde L.
      • Ramirez J.
      • et al.
      Ectopic lymphoid neogenesis is strongly associated with activation of the IL-23 pathway in rheumatoid synovitis.
      ). Th17 cells have also been shown to develop a “Tfh-like” phenotype and contribute to TLO formation and function (
      • Peters A.
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      • Franz B.
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      ) in the central nervous system. Similarly, Th17 cells in Peyer’s patches display the features of Tfh-like cells and support the antigen-specific IgA response at the germinal center (
      • Hirota K.
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      ). Thus, it is possible that these IL-21/IL-17–producing Th cells provide helper functions for B-cell activation and differentiation, leading to pathogenic autoantibody production in PV lesions.
      We further showed that the antibody levels secreted from in vitro cultured cells from PV lesions have a positive correlation with Pemphigus Disease Area Index (Figure 2), which indicates a positive correlation with disease severity. Using anti-human HLA-DR antibody to block the T-B cell interaction during in vitro culture, we found that antibody production was significantly reduced (see Supplementary Figure S7 online). Additionally, we found that the B cells from lesional skin express IgG on their surface (see Supplementary Figure S8 online). Most of these IgG+ B cells are also CD27+ memory B cells (see Supplementary Figure S9 online). It is plausible to speculate that these memory B cells in PV lesional skin may play a critical role in disease relapse. CD138+ plasma cells have been reported to infiltrate in other skin diseases such as squamous cell carcinoma (
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      ) and scleroderma skin (
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      ). Our study showed that plasma cells with IgG in their cytoplasm aggregated in lesional skin, which may lead to the local antigen-specific antibody production in PV. In addition, we collected lymphocytes from both lesional and peripheral blood of three PV patients for in vitro culture simultaneously, and after 6 days, the titer of the anti-Dsg3 antibodies of supernatant from in vitro culture of lesional lymphocytes was much higher than that from the peripheral blood (see Supplementary Figure S10 online).
      • Geherin S.A.
      • Fintushel S.R.
      • Lee M.H.
      • Wilson R.P.
      • Patel R.T.
      • Alt C.
      • et al.
      The skin, a novel niche for recirculating B cells.
      also found that skin B cells had increased MHCⅡ, CD1, and CD80/86 expression, which may be well-suited for T-cell activation at the site of lesion, and the accumulation of B cells and antibody-secreting cells could increase local antibody titers, further augmenting autoimmunity.
      In summary, we discovered that the frequency of B cells is significantly increased in the lesional skin of PV patients. We found that Dsg-specific B lymphocytes infiltrated lesional skin and are capable of producing autoantibodies against Dsg1 and Dsg3. In addition, the presence of IL-21/IL-17–producing Th cells, antigen-specific B cells, and plasma cells in PV lesions and up-regulation of CCL19 expression suggest the potential formation of a TLO-like structure. Although our study shows the existence of antigen-specific B cells and plasma cells in the lesional skin of PV patients, it remains unclear how locally produced autoantibodies contribute to pemphigus pathogenesis. Nevertheless, our study identifies locally infiltrating antigen-specific B cells, which to our knowledge has not been previously reported. These findings further support the role of local immune effectors in acantholysis of pemphigus and suggest that TLO-targeted topical therapies may be valuable in treating this potentially life-threatening autoimmune disease.

      Materials and Methods

      Human subjects

      Patients with PV were diagnosed based on the clinical manifestations and histology criteria, and pictures of patients before and after treatments are shown in Supplementary Figure S11 online. Skin biopsy samples were collected from lesions of 34 PV patients (Table 1), three BP patients, and 32 healthy donors (Figure 1a). Because of the limited sample size, we used different samples for different experiments. All patients had the mucocutaneous type disease and had not been treated by systemic therapy before the study. The study was approved by Shanghai Jiao Tong University School of Medicine Research Ethics Committee. Written informed consent was obtained from all subjects before the study.
      Table 1Summary of all patients involved in the study
      No.Age, yearsSexCutaneous LesionOral LesionDIFIIFIndex of ELISARelapsePDAI
      TrunkExtremityScalp and faceDsg1Dsg3
      141F++++++17817337
      238M++++++626840
      370M++++++12818221
      467M+++NDND105127+55
      548M+++++13516622
      658F+++++77238+27
      742M+++++19016+46
      869F+++52230+36
      928F+++21680+23
      1044F++++++174182+22
      1129F+++++471427
      1247F++++2894+23
      1365F++++++165110unknown35
      1445F++++87104+28
      1534F++++++175316+30
      1659F++++++151221unknown32
      1740F+++++51161+30
      1872F++++++266163unknown33
      1948F+++++101174+50
      2041M+++++12918946
      2126M+++++109105+46
      2248M++++++21319548
      2343M+++++2419550
      2460M++++++94200+23
      2545F++++9216040
      2644M++++++2023952
      2749F+++++1518660
      2855M++++++200193+55
      2949M++++++80175+52
      3053F++++36198+54
      3163F++++++150122+24
      3257F+++++196164+30
      3351F++++76166+16
      3434M++++104155+32
      Abbreviations: +, positive; –, negative; DIF, directive immunofluorescence; F, female; IIF, indirective immunofluorescence on monkey esophagus; M, male; ND, not done; PDAI, Pemphigus Disease Area Index.

      Skin cell preparation

      Human skin samples of 1 × 1 cm from PV and BP patients and healthy donors were collected and incubated in a buffer containing collagenase IV, hyaluronidase, and DNase-I (Sigma Aldrich, St. Louis, MO) for digesting in 37 °C by 2 hours and after passing through a 70-μm cell strainer (BD Bioscience, San Jose, CA), single-cell suspensions were obtained. Cells were isolated by density gradient using Lymphoprep solution (Axis-shield, Oslo, Norway) and resuspended in RPMI 1640 (Invitrogen, Camarillo, CA) medium supplemented with 5% fetal bovine serum (Sigma Aldrich, St. Louis, MO).

      In vitro culture

      Cells from PV and BP patients’ and healthy donors’ skin were adjusted to 1.0 × 106/ml and cultured in 200 μl of RPMI 1640 complete medium supplemented with 10% fetal bovine serum in a 96-well plate. Supernatants were harvested on the sixth day for anti-Dsg1 and -Dsg3 IgG measurement and Western blotting analysis.

      ELISA

      Anti-Dsg1 and -Dsg3 IgG levels were measured by ELISA performed according to standard procedures (MBL, Nagoya, Japan).

      Dsg-specific B-cell detection

      Recombinant Dsg 1-His-Flag (rDsg1-His-Flag) and Dsg 3-His-Flag (rDsg3-His-Flag) were produced by Qinghong Biotech (Shanghai, China). Cells from PV and BP patients’ lesions (2.0 × 105) were incubated for 1 hour at room temperature with rDsg1-His-Flag and rDsg3-His-Flag. After washing, His-Tag Rabbit mAb (PE Conjugate) (Cell Signaling Technology, Danvers, MA; clone: D3I1O) and DYKDDDDK-Tag Rabbit mAb (Alexa Fluor 647 conjugate) (Cell Signaling Technology, Danvers, MA; clone: D6W5B) were used to identify Dsg-binding cells. B cells were identified with anti-human CD19 antibody (PerCP conjugate) (BD Biosciences, San Jose, CA; clone: HIB19).

      ELISPOT assay

      MultiScreenHTS IP Filter 96-well plate with polyvinylidene fluoride membrane (Merck Millipore, Darmstadt, Germany) were coated with 30μg/ml rDsg1-His-Flag and rDsg3-His-Flag at 4 °C overnight. The plates were blocked by 1% bovine serum albumin in Tris-buffered saline with 1 mM CaCl2 for 1 hour at 37 °C after washing three times with phosphate buffered saline-Ca. To detect anti-Dsg3 IgG–secreting cells, isolated cells from PV and BP lesions were incubated on the rDsg1- and rDsg3-coated 96-well plates at 37 °C for 20 hours. The plates were washed with phosphate buffered saline-Ca and phosphate buffered saline-Ca containing 0.05% Tween 20, and other steps were followed according to the procedures of the IgG B cell ELISPOT kit (U-CyTech Biosciences, Utrecht, The Netherlands). The numbers of the spots were quantified by ImmunoSpot 5.1.36 software (Cellular Technology, Shaker Heights, OH).

      Western blotting

      Detailed information can be found in the Supplementary Materials online.

      Flow cytometric analysis and surface and intracellular staining

      Detailed information can be found in the Supplementary Materials.

      Immunohistochemistry staining and immunofluorescence microscopy

      Detailed information can be found in the Supplementary Materials.

      Microarray analysis

      Detailed information can be found in the Supplementary Materials.

      Reverse transcription and real-time PCR assay

      Detailed information can be found in the Supplementary Materials.

      Statistical analysis

      Data were analyzed using GraphPad Prism version 5.0 (GraphPad Software, La Jolla, CA). Data of the two groups are shown as means ± standard error of the mean. Statistical differences in quantitative parameters among two groups were determined using unpaired t test with Welch’s correction, unless otherwise stated in the results. Pearson product moment correlation coefficient was used to measure the linear relationship between two variables. Differences were considered statistically significant at P < 0.05.

      Conflict of Interest

      The authors state no conflict of interest.

      Acknowledgments

      We would like to acknowledge John R. Stanley, Department of Dermatology, University of Pennsylvania for supporting our study and kindly providing keratinocyte extracts, mouse anti-human Dsg1 mAb P124 and mouse anti-human Dsg3 mAb 5G11. We also thank Xuming Mao, Department of Dermatology, University of Pennsylvania for editing the manuscript. This work was supported by grants from the National Natural Science Foundation of China (81472875, 81402598, and 81673064).

      Supplementary Material

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      Linked Article

      • Desmoglein 3-Reactive B Cells “Hiding” in Pemphigus Lesions
        Journal of Investigative DermatologyVol. 137Issue 11
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          Pemphigus vulgaris is an autoimmune blistering disease caused by anti-desmoglein 3 IgG autoantibodies. It is accepted that interactions between autoreactive B and T cells are key to humoral autoimmunity targeting desmoglein 3. This orchestrated process usually occurs in secondary lymphoid organs, including the spleen and lymph nodes. Thus, it seems likely that autoreactive B cells reside and produce autoantibodies in these tissues. Yuan et al. analyzed lymphocytes in the lesional skin of patients with pemphigus vulgaris using several experimental techniques and concluded that desmoglein 3-reactive B cells were present.
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