Rüdiger Eming

T cell control in autoimmune bullous skin disorders

Autoimmune bullous disorders are a group of severe skin diseases characterized clinically by blisters and erosions of skin and/or mucous membranes. A hallmark of these disorders is the presence of IgG and occasionally IgA autoantibodies that target distinct adhesion structures of the epidermis, dermoepidermal basement membrane, and anchoring fibrils of the dermis. This Review focuses on the potential role of autoreactive T cells in the pathogenesis of these disorders. Pemphigus vulgaris (PV) and bullous pemphigoid (BP) are the best-characterized bullous disorders with regard to pathogenesis and T cell involvement. Activation of autoreactive T cells in PV and BP is restricted by distinct HLA class II alleles that are prevalent in individuals with these disorders. Autoreactive T cells are not only present in patients but can also be detected in healthy individuals. Recently, a subset of autoreactive T cells with remarkable regulatory function was identified in healthy individuals and to a much lesser extent in patients with PV, suggesting that the occurrence of autoimmune bullous disorders may be linked to a dysfunction of Tregs.

Rituximab Exerts a Dual Effect in Pemphigus Vulgaris

Pemphigus vulgaris (PV) is a severe autoimmune blistering disease affecting the skin and mucous membranes. Autoreactive CD4(+) T helper (Th) lymphocytes are crucial for the autoantibody response against the desmosomal adhesion molecules, desmoglein (dsg)-3 and dsg1. Eleven patients with extensive PV were treated with the anti-CD20 antibody, rituximab (375 mg per m(2) body surface area once weekly for 4 weeks). Frequencies of autoreactive CD4(+) Th cells in the peripheral blood of the PV patients were determined 0, 1, 3, 6, and 12 months after rituximab treatment. Additionally, the clinical response was evaluated and serum autoantibody titers were quantified by ELISA. Rituximab induced peripheral B-cell depletion for 6-12 months, leading to a dramatic decline of serum autoantibodies and significant clinical improvement in all PV patients. The frequencies of dsg3-specific CD4(+) Th1 and Th2 cells decreased significantly for 6 and 12 months, respectively, while the overall count of CD3(+)CD4(+) T lymphocytes and the frequency of tetanus toxoid-reactive CD4(+) Th cells remained unaffected. Our findings indicate that the response to rituximab in PV involves two mechanisms: (1) the depletion of autoreactive B cells and (2) the herein demonstrated, presumably specific downregulation of dsg3-specific CD4(+) Th cells.

Safety and clinical outcomes of rituximab therapy in patients with different autoimmune diseases: experience from a national registry (GRAID)

IntroductionEvidence from a number of open-label, uncontrolled studies has suggested that rituximab may benefit patients with autoimmune diseases who are refractory to standard-of-care. The objective of this study was to evaluate the safety and clinical outcomes of rituximab in several standard-of-care-refractory autoimmune diseases (within rheumatology, nephrology, dermatology and neurology) other than rheumatoid arthritis or non-Hodgkins lymphoma in a real-life clinical setting.MethodsPatients who received rituximab having shown an inadequate response to standard-of-care had their safety and clinical outcomes data retrospectively analysed as part of the German Registry of Autoimmune Diseases. The main outcome measures were safety and clinical response, as judged at the discretion of the investigators.ResultsA total of 370 patients (299 patient-years) with various autoimmune diseases (23.0% with systemic lupus erythematosus, 15.7% antineutrophil cytoplasmic antibody-associated granulomatous vasculitides, 15.1% multiple sclerosis and 10.0% pemphigus) from 42 centres received a mean dose of 2,440 mg of rituximab over a median (range) of 194 (180 to 1,407) days. The overall rate of serious infections was 5.3 per 100 patient-years during rituximab therapy. Opportunistic infections were infrequent across the whole study population, and mostly occurred in patients with systemic lupus erythematosus. There were 11 deaths (3.0% of patients) after rituximab treatment (mean 11.6 months after first infusion, range 0.8 to 31.3 months), with most of the deaths caused by infections. Overall (n = 293), 13.3% of patients showed no response, 45.1% showed a partial response and 41.6% showed a complete response. Responses were also reflected by reduced use of glucocorticoids and various immunosuppressives during rituximab therapy and follow-up compared with before rituximab. Rituximab generally had a positive effect on patient well-being (physicians visual analogue scale; mean improvement from baseline of 12.1 mm).ConclusionsData from this registry indicate that rituximab is a commonly employed, well-tolerated therapy with potential beneficial effects in standard of care-refractory autoimmune diseases, and support the results from other open-label, uncontrolled studies.

Recommendations for the use of rituximab (anti-CD20 antibody) in the treatment of autoimmune bullous skin diseases.

Autoimmune bullous skin disorders are induced by autoantibodies against distinct adhesion complexes of the epidermal and dermal‐epidermal junction. Since most of these disorders are characterized by a severe, potentially lethal course,they require long‐term immunosuppressive treatment to reduce the de novo synthesis of pathogenic autoantibodies by B lymphocytes. Rituximab, a chimeric monoclonal antibody against CD20 on B lymphocytes, has shown promise in several case reports or cohort studies in the treatment of paraneo‐plastic pemphigus,refractory cases of pemphigus vulgaris and foliaceus and in other autoimmune bullous disorders.Treatment with rituximab leads to depletion of pathogenic B‐cells which may last up to 12 months resulting in a reduction of plasma cells secreting pathogenic autoantibodies.Rituximab is usually administered in an adjuvant setting at a dose of 375 mg/m2 i.v.in weekly intervals for four consecutive weeks in addition to the standard immunosuppressive treatment.The present consensus statement of German‐speaking derma‐tologists,rheumatologists and oncologists summarizes and evaluates the current evidence for the use and mode of application of rituximab in autoimmune bullous skin disorders.

Long-Term Remissions of Severe Pemphigus After Rituximab Therapy Are Associated with Prolonged Failure of Desmoglein B Cell Response

Changes in the B cell repertoire mediate long-lasting therapeutic effects of rituximab in pemphigus patients. A Blistering Attack on Autoimmunity The devastating effects of autoimmune diseases like type 1 diabetes and multiple sclerosis are front-page news. However, rare autoimmune diseases may be even more distressing in their relative anonymity. One such condition is pemphigus, which is a severe blistering condition caused by autoantibodies to adhesion proteins in the skin and mucus. The B cell–depleting antibody rituximab has been shown to treat pemphigus short term in early clinical trials. Now, Colliou et al. find that rituximab therapy can help pemphigus patients even after 6 years, in part by reshaping the B cell repertoire during reconstitution. The authors followed pemphigus patients from their early trial out at least 6 years after rituximab therapy. They found that nearly two-thirds of patients achieved a long-term complete response—either completely off of therapy or when treated with low levels of supplementary prednisone. They then compared patients with complete response to those with incomplete response to look at differences in reconstitution of the B cell compartment. Patients with complete response had a greater proportion of naïve and transitional B cells than those with incomplete response, which suggests a barrier to B cell maturation. Indeed, many of these transitional B cells secreted the regulatory cytokine interleukin-10. Moreover, complete responders had a specific loss of anti–desmoglein-specific B cells, which are pathogenic in pemphigus patients, but not B cells that respond to infectious agents. Together, these data suggest that B cell repertoire is reshaped after rituximab therapy, allowing for long-term effects in addition to the short-term loss of pathogenic B cells. If these observations are confirmed in large studies, these data could form the basis for a new frontline therapy for recalcitrant pemphigus. Pemphigus is a severe blistering condition of the skin and mucosa caused by autoantibodies directed against desmogleins, which are a type of keratinocyte adhesion protein. B cell depletion by rituximab has short-term efficacy against pemphigus. We aimed to assess the long-term course of pemphigus patients after B cell depletion and to understand the immunological mechanisms that mediate long-lasting remissions. We evaluated the clinical course of 22 pemphigus patients treated with rituximab after a 79-month median follow-up and compared the anti-desmoglein B cell response and B and T lymphocyte subpopulations and repertoire between patients who achieved complete remission (CR) and those who had incomplete remission (IR). Thirteen patients (59%) experienced CR during the study, including 10 patients off treatment and 3 patients with prednisone doses <10 mg/day; 9 patients had IR. A marked increase was observed in the ratio of CD19+CD27− naïve B cells to CD19+CD27+ memory B cells. Indeed, patients in CR had a fourfold higher number of transitional B cells and interleukin-10–secreting regulatory B cells than those in IR. Furthermore, CR was associated with modification of the initial B cell repertoire and the disappearance of desmoglein-specific circulating immunoglobulin G–positive (IgG+) B lymphocytes, whereas a skewed B cell repertoire was observed in patients in IR. Thus, a blockage of B cell maturation, a prolonged repopulation with naïve B cells, and a delayed reappearance of memory B cells, which resulted in the disappearance of circulating desmoglein-specific IgG+ B lymphocytes, contribute to the long-lasting effectiveness of rituximab for treating pemphigus.

Pemphigus autoantibodies generated through somatic mutations target the desmoglein-3 cis-interface

Pemphigus vulgaris (PV) is an autoimmune blistering disease of skin and mucous membranes caused by autoantibodies to the desmoglein (DSG) family proteins DSG3 and DSG1, leading to loss of keratinocyte cell adhesion. To learn more about pathogenic PV autoantibodies, we isolated 15 IgG antibodies specific for DSG3 from 2 PV patients. Three antibodies disrupted keratinocyte monolayers in vitro, and 2 were pathogenic in a passive transfer model in neonatal mice. The epitopes recognized by the pathogenic antibodies were mapped to the DSG3 extracellular 1 (EC1) and EC2 subdomains, regions involved in cis-adhesive interactions. Using a site-specific serological assay, we found that the cis-adhesive interface on EC1 recognized by the pathogenic antibody PVA224 is the primary target of the autoantibodies present in the serum of PV patients. The autoantibodies isolated used different heavy- and light-chain variable region genes and carried high levels of somatic mutations in complementary-determining regions, consistent with antigenic selection. Remarkably, binding to DSG3 was lost when somatic mutations were reverted to the germline sequence. These findings identify the cis-adhesive interface of DSG3 as the immunodominant region targeted by pathogenic antibodies in PV and indicate that autoreactivity relies on somatic mutations generated in the response to an antigen unrelated to DSG3.

Desmocollin 3-mediated binding is crucial for keratinocyte cohesion and is impaired in pemphigus.

Desmocollin (Dsc) 1–3 and desmoglein (Dsg) 1–4, transmembrane proteins of the cadherin family, form the adhesive core of desmosomes. Here we provide evidence that Dsc3 homo- and heterophilic trans-interaction is crucial for epidermal integrity. Single molecule atomic force microscopy (AFM) revealed homophilic trans-interaction of Dsc3. Dsc3 displayed heterophilic interaction with Dsg1 but not with Dsg3. A monoclonal antibody targeted against the extracellular domain reduced homophilic and heterophilic binding as measured by AFM, caused intraepidermal blistering in a model of human skin, and a loss of intercellular adhesion in cultured keratinocytes. Because autoantibodies against Dsg1 are associated with skin blistering in pemphigus, we characterized the role of Dsc3 binding for pemphigus pathogenesis. In contrast to AFM experiments, laser tweezer trapping revealed that pemphigus autoantibodies reduced binding of Dsc3-coated beads to the keratinocyte cell surface. These data indicate that loss of heterophilic Dsc3/Dsg1 binding may contribute to pemphigus skin blistering.

B-Cell-Directed Therapy for Inflammatory Skin Diseases

The basic understanding of inflammatory dermatoses and autoimmune-mediated skin disorders has greatly advanced and broadened our understanding of underlying immune mechanisms that shape the complex network of chronic inflammation and autoimmunity. The new treatment options for psoriasis exemplify how new insights into (auto)immune responses, especially the role and function of various immune cells and proinflammatory cytokines, may lead to new therapeutic strategies. The concept of targeting B cells in autoimmune-mediated disorders is closely related to the discovery of autoantibodies and their cellular origin. However, the appreciation of B cells in autoimmunity has significantly changed and is not limited to their role as progenitors of autoantibody secreting plasma cells. Recent investigations of various inflammatory skin diseases, that is, autoimmune blistering disorders, collagen vascular diseases, and atopic dermatitis, actually support the concept that B cells might be as important as T cells in the etiopathogenesis of these disorders. The striking clinical improvement seen in patients with rheumatoid arthritis following B-cell depletion with the anti-CD20 mAb rituximab has tremendously catalyzed the interest in B-cell-targeted therapies in different autoimmune diseases. Future translational and clinical investigations are mandatory to precisely define the role and the contribution of impaired B-cell function in (auto)immune-mediated skin diseases.

IgG Autoantibodies Against Desmocollin 3 in Pemphigus Sera Induce Loss of Keratinocyte Adhesion

Pemphigus is considered an autoimmune bullous skin disorder associated with IgG against the desmosomal components, desmoglein 3 (Dsg3) and desmoglein 1 (Dsg1). This concept is supported by the in vitro and in vivo pathogenicity of anti-Dsg3/Dsg1 IgG and the mucosal blistering phenotype of mice with a genetic deficiency of Dsg3. Mice deficient for another desmosomal adhesion molecule, desmocollin 3 (Dsc3), show a similar pemphigus phenotype, and we investigated the pathogenicity of Dsc3-reactive IgG autoantibodies that were identified previously in a subset of patients with atypical pemphigus. We here demonstrate that IgG against Dsc3 causes loss of adhesion of epidermal keratinocytes. Specifically, IgG against Dsc3 was purified from Dsc3-reactive pemphigus sera by affinity column chromatography using recombinant human Dsc3. Affinity purified IgG was functionally active and did not only react with recombinant Dsc3 but also with epidermis and cultured human keratinocytes. Moreover, Dsc3-reactive IgG induced loss of adhesion of epidermal keratinocytes in a dispase-based keratinocyte dissociation assay that was reversed on pre-adsorption with human Dsc3 but not Dsg3. These findings demonstrate that IgG autoantibodies against an additional component of the desmosomes, Dsc3, induce loss of keratinocyte adhesion and thus may contribute to blister formation in pemphigus.

Prolonged Clinical Remission of Patients with Severe Pemphigus upon Rapid Removal of Desmoglein-Reactive Autoantibodies by Immunoadsorption

Background: In pemphigus, loss of epidermal adhesion is induced by binding of circulating autoantibodies to the desmosomal cadherins desmoglein 3 (Dsg3) in pemphigus vulgaris (PV) and desmoglein 1 (Dsg1) in pemphigus foliaceus (PF), respectively. Therapeutic removal of Dsg-reactive autoantibodies by immunoadsorption (IA) has been demonstrated to exert clinical remission of the disease. Objectives: The aim of this intervention study was to evaluate the efficacy and safety of the peptide-based Globaffin® adsorber system in the treatment of severe pemphigus cases. Patients and Methods: We applied IA in 4 PV and 2 PF patients with severe chronic disease resistant to conventional immunosuppressive therapy. IA was performed on 4 consecutive days, representing 1 treatment cycle, followed by a 4-week treatment-free interval. Serum samples for determining serum IgG and anti-Dsg1/Dsg3 IgG autoantibodies were drawn daily before and after IA, respectively. During follow-up, patients were examined carefully, and laboratory parameters were controlled monthly for up to 1 year. Results: IA led to excellent clinical responses. Skin and mucosal lesions cleared almost completely within weeks. One IA cycle reduced anti-Dsg1 and anti-Dsg3 autoantibodies by an average of 50–70% as determined by ELISA. Conclusions: Using the Globaffin adsorber system, IA represents an effective and safe treatment opportunity in severe and therapy-resistant pemphigus.