Characterization of cellular and humoral immune responses in pemphigus patients and an HLA-transgenic mouse model

The rare, but potentially fatal autoimmune disorder pemphigus is considered as a prototypical antibody-mediated organ-specific disease, in which immunoglobulin (Ig) G autoantibodies (auto-ab) mainly target the desmosomal cadherins Desmoglein 3 (Dsg3) and Dsg1 within the epidermis. This process, referred to as acantholysis, manifests clinically with the formation of flaccid blisters and erosions of the skin and mucous membranes. Still to date, therapeutic intervention is predominantly limited to unspecific immunosuppression causing severe side effects and comorbidities. Although it has long been recognized that auto-ab crucially contribute to the pathology of pemphigus, the exact immunological events leading to the loss of self-tolerance have not yet been fully identified. Aim of the first part of this doctoral thesis was to investigate antigen-presenting cells (APC) and APC-derived cytokines, as well as their relation to T helper (Th) cell subsets and the auto-ab response in the pathogenesis of pemphigus. Therefore, blood samples were obtained from 34 pemphigus patients, of which twelve clinically well characterized patients with no or minimal intake of immunosuppressive agents were selected for comprehensive analysis. This study group consisted of nine pemphigus vulgaris (PV), as well as three pemphigus foliaceus (PF) patients who were categorized according to either active or remittent state of disease. A cohort of twelve patients suffering from the autoimmune muscle weakness myasthenia gravis (MG) served as a control for a further unrelated auto-ab–mediated organ-specific disorder. Concerning the APC compartment, a major finding was reduced circulating frequencies of both myeloid and plasmacytoid dendritic cells (mDC and pDC, respectively) in active pemphigus patients. In concordance with this observation, augmented surface expression of the C-C chemokine receptor 2 (CCR2) of mDC suggest an increased migration activity to inflamed peripheral tissues, such as the skin. Similarly, active pemphigus patients displayed an upregulation of CCR2 on blood CD14+ monocytes, as well as elevated numbers of circulating CD16+ monocytes with expansion of the nonclassical subset. However, functional analysis on the cellular level could not confirm an enhanced immunogenicity status of APC in active pemphigus patients, yet we found in these patients increased plasma levels of the proinflammatory tumor necrosis factor (TNF)-α. Interestingly, plasma concentrations of interleukin (IL)-27 known to exert primarily regulatory functions were also elevated in active pemphigus patients, pointing to a rather inflammatory role of IL-27 in disease pathogenesis. Strikingly, in pemphigus patients, a significant correlation was not only found between serum IgG auto-ab titers and plasma levels of both IL-6 and TNF-α, but also between auto-ab titers and plasma concentrations of IL-27. Furthermore, similarly augmented plasma levels of IL-21 playing a crucial role in B cell activation possibly indicate the mechanism of an IL-27–mediated activation of IL-21–producing Tfh cells. Finally, as a further major finding, we observed an increase of both Th17 cells and IL-10–secreting T cells in active pemphigus patients. Aim of the second part of this doctoral thesis was to investigate Dsg3-specific cellular and humoral immune responses in a novel human leukocyte antigen (HLA)-DRB1*04:02–transgenic (tg) mouse model of PV under the genetic restriction by HLA-DRB1*04:02. Rationale for the generation of this model is the high prevalence of distinct HLA class II alleles in PV patients, such as HLA-DRB1*04:02, –DRB1*14:01, and –DQB1*05:03. The occurrence of PV being associated with the presence of specific HLA class II alleles thereby reflects the high relevance of autoreactive CD4+ T cells in disease pathogenesis. In summary, this experimental study convincingly confirmed that the PV mouse model reproduces the principle mechanisms of HLA-dependent and human Dsg3-specific induction of CD4+ T and B cell responses observed in PV patients. Accordingly, the generation of Dsg3-reactive IgG antibody responses highly depended on prior activation of Dsg3-reactive CD4+ T cells. In turn, APC-mediated activation of T cells critically relied on the recognition of epitopes of the Dsg3 ectodomain, which displayed strong binding affinity to the PV-linked HLA class II allele HLA-DRB1*04:02. Furthermore, limited cross-reactivity of human Dsg3-specific IgG antibodies with the mouse analogue protein explained lack of the clinical phenotype of PV upon immunization of HLA-DRB1*04:02–tg mice with human Dsg3 protein. Yet, this model accurately reflects that polymorphisms of peptide-binding motifs of specific PV-related HLA-class II alleles tightly regulate CD4+ T cell-mediated induction of Dsg3-reactive IgG antibodies. Therefore, this novel mouse model represents a suitable tool for further investigations of the pathoimmunological mechanisms of PV in vivo considering the strong HLA class II association of this disease

Characterization of cellular and humoral immune responses in pemphigus patients and an HLA-transgenic mouse model

The rare, but potentially fatal autoimmune disorder pemphigus is considered as a prototypical antibody-mediated organ-specific disease, in which immunoglobulin (Ig) G autoantibodies (auto-ab) mainly target the desmosomal cadherins Desmoglein 3 (Dsg3) and Dsg1 within the epidermis. This process, referred to as acantholysis, manifests clinically with the formation of flaccid blisters and erosions of the skin and mucous membranes. Still to date, therapeutic intervention is predominantly limited to unspecific immunosuppression causing severe side effects and comorbidities. Although it has long been recognized that auto-ab crucially contribute to the pathology of pemphigus, the exact immunological events leading to the loss of self-tolerance have not yet been fully identified. Aim of the first part of this doctoral thesis was to investigate antigen-presenting cells (APC) and APC-derived cytokines, as well as their relation to T helper (Th) cell subsets and the auto-ab response in the pathogenesis of pemphigus. Therefore, blood samples were obtained from 34 pemphigus patients, of which twelve clinically well characterized patients with no or minimal intake of immunosuppressive agents were selected for comprehensive analysis. This study group consisted of nine pemphigus vulgaris (PV), as well as three pemphigus foliaceus (PF) patients who were categorized according to either active or remittent state of disease. A cohort of twelve patients suffering from the autoimmune muscle weakness myasthenia gravis (MG) served as a control for a further unrelated auto-ab–mediated organ-specific disorder. Concerning the APC compartment, a major finding was reduced circulating frequencies of both myeloid and plasmacytoid dendritic cells (mDC and pDC, respectively) in active pemphigus patients. In concordance with this observation, augmented surface expression of the C-C chemokine receptor 2 (CCR2) of mDC suggest an increased migration activity to inflamed peripheral tissues, such as the skin. Similarly, active pemphigus patients displayed an upregulation of CCR2 on blood CD14+ monocytes, as well as elevated numbers of circulating CD16+ monocytes with expansion of the nonclassical subset. However, functional analysis on the cellular level could not confirm an enhanced immunogenicity status of APC in active pemphigus patients, yet we found in these patients increased plasma levels of the proinflammatory tumor necrosis factor (TNF)-α. Interestingly, plasma concentrations of interleukin (IL)-27 known to exert primarily regulatory functions were also elevated in active pemphigus patients, pointing to a rather inflammatory role of IL-27 in disease pathogenesis. Strikingly, in pemphigus patients, a significant correlation was not only found between serum IgG auto-ab titers and plasma levels of both IL-6 and TNF-α, but also between auto-ab titers and plasma concentrations of IL-27. Furthermore, similarly augmented plasma levels of IL-21 playing a crucial role in B cell activation possibly indicate the mechanism of an IL-27–mediated activation of IL-21–producing Tfh cells. Finally, as a further major finding, we observed an increase of both Th17 cells and IL-10–secreting T cells in active pemphigus patients. Aim of the second part of this doctoral thesis was to investigate Dsg3-specific cellular and humoral immune responses in a novel human leukocyte antigen (HLA)-DRB1*04:02–transgenic (tg) mouse model of PV under the genetic restriction by HLA-DRB1*04:02. Rationale for the generation of this model is the high prevalence of distinct HLA class II alleles in PV patients, such as HLA-DRB1*04:02, –DRB1*14:01, and –DQB1*05:03. The occurrence of PV being associated with the presence of specific HLA class II alleles thereby reflects the high relevance of autoreactive CD4+ T cells in disease pathogenesis. In summary, this experimental study convincingly confirmed that the PV mouse model reproduces the principle mechanisms of HLA-dependent and human Dsg3-specific induction of CD4+ T and B cell responses observed in PV patients. Accordingly, the generation of Dsg3-reactive IgG antibody responses highly depended on prior activation of Dsg3-reactive CD4+ T cells. In turn, APC-mediated activation of T cells critically relied on the recognition of epitopes of the Dsg3 ectodomain, which displayed strong binding affinity to the PV-linked HLA class II allele HLA-DRB1*04:02. Furthermore, limited cross-reactivity of human Dsg3-specific IgG antibodies with the mouse analogue protein explained lack of the clinical phenotype of PV upon immunization of HLA-DRB1*04:02–tg mice with human Dsg3 protein. Yet, this model accurately reflects that polymorphisms of peptide-binding motifs of specific PV-related HLA-class II alleles tightly regulate CD4+ T cell-mediated induction of Dsg3-reactive IgG antibodies. Therefore, this novel mouse model represents a suitable tool for further investigations of the pathoimmunological mechanisms of PV in vivo considering the strong HLA class II association of this disease

Transgenic mouse technology in skin biology: generation of complete or tissue-specific knockout mice

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Increased Frequency of T Follicular Helper Cells and Elevated Interleukin-27 Plasma Levels in Patients with Pemphigus.

Pemphigus is an autoimmune disease in which IgG auto-antibodies (auto-ab) against the desmosomal cadherins desmoglein (Dsg) 3 and Dsg1 cause loss of epidermal keratinocyte adhesion. Aim of this study was to investigate cytokines derived from antigen-presenting cells (APC) and their relation to CD4+ T cell subpopulations and to the auto-ab response in pemphigus. In this regard, patients with pemphigus were compared to patients with myasthenia gravis (MG), an unrelated auto-ab-mediated autoimmune disease, and healthy controls. In pemphigus and MG, the plasma concentrations of the APC-derived immunomodulatory cytokine IL-27 were highly increased. Strikingly, IL-27 strongly correlated with Dsg-specific IgG auto-ab titers. T helper (Th) 17 cells were augmented in both pemphigus and MG patients while T follicular helper (Tfh) cells, which are essential in providing B cell help, were increased only in pemphigus along with increasing plasma concentrations of IL-21, a cytokine produced by Th17 and Tfh cells. Moreover, we could detect Dsg3-specific autoreactive T cells producing IL-21 upon ex vivo stimulation with Dsg3. These findings suggest that IL-27 and IL-21-producing T cells, are involved in the pathogenesis of pemphigus. The further characterization of IL-21-producing T cells and of the role of IL-27 will lead to a more defined understanding of the auto-ab response in pemphigus

Elevated Tfh cells in pemphigus patients.

<p><b>(A)</b> cTfh cells (defined as CD4⁺CXCR5⁺ T cells) were analyzed by flow cytometry in patients with pemphigus (Pem; n = 10), MG (n = 12) and HC (n = 12). (<b>A</b>) The frequency of cTfh cells was significantly increased in Pem patients while altered PD-1 (<b>B</b>) or ICOS (<b>C</b>) expression on cTfh cells could not be observed. Pem patients are categorized as remittent (open symbols) and active (filled symbols). Differences between the groups were analyzed using Mann-Whitney-U-Test with <i>p</i> values indicated as *<i>p</i><0.05.</p

Elevated IL-21 plasma levels and increased IL-17-producing T cells in pemphigus.

<p>Patients with pemphigus (Pem; n = 10), MG (n = 12) and HC (n = 12) were analysed for IL-21 and IL-17 (<b>A</b>) Active Pem patients as well as MG patients displayed significantly increased plasma concentrations of IL-21. (<b>B</b>) An increasing frequency of IL-17-producing T cells could be obersevd in Pem patients while (<b>C</b>) IL-17/IL-21 double positive T cells and (<b>D</b>) IL-21 single positive T cells showed no statistical difference compared to HC. (<b>E</b>) Pem patients showed increased numbers of autoreactive IL-21-producing T cells by ELISpot assay. PBMC were used for flow cytometry analysis of the intracellular produced cytokines upon stimulation with PMA and ionomycin. Pem patients are categorized as remittent (open symbols) and active (filled symbols). Differences between the groups were analyzed using Mann-Whitney-U-Test with <i>p</i> values indicated as *<i>p</i><0.05, **<i>p</i>0.01 and ***<i>p</i>0.001.</p

Correlation of plasma cytokine levels in patients with active pemphigus and MG.

<p>Correlation of plasma cytokine levels in patients with active pemphigus and MG.</p

Increased plasma levels of IL-27 in active pemphigus and MG patients.

<p>Plasma samples of pemphigus (Pem) patients (n = 12) categorized as active and remittent (rem), MG patients (n = 12) and HC (n = 12) were tested for different cytokines by ELISA. <b>(A)</b> Both active Pem and MG patients had significantly increased plasma concentrations of IL-27. <b>(B)</b> The plasma levels of IL-6 were not increased in active Pem while <b>(C)</b> TNF-α was statistically increased in active pemphigus. Differences between the groups were analyzed using Mann-Whitney-U-Test with <i>p</i> values indicated as *<i>p</i><0.05.</p