Characterization of pathogenic auto-antibodies directed against desmoglein 3 and desmocollin 3 in sera of pemphigus patients.

Pemphigus vulgaris (PV) represents the most frequent clinical type of the pemphigus group of autoimmune bullous skin disorders. There is substantial evidence that blister formation in pemphigus patients is mediated by auto-antibodies (auto-Abs) targeted against certain desmosomal cadherins, namely desmoglein1 (Dsg1) and desmoglein3 (Dsg3). Several pathogenic epitopes of Dsg3 are located at the amino-(NH2)-terminal end of the Dsg3 ectodomain, namely the extracellular domain 1 (EC1). On the other hand a great number of pemphigus patients exhibit auto-Abs directed against the more carboxy-(COOH)-terminal epitopes of Dsg3, e.g. within the EC4- and EC5-domain and these domains may play an essential role in maintaining desmosomal adhesion. Some pemphigus patients exhibit additional or solely auto-Abs against other desmosomal cadherins, especially desmocollin 3 (Dsc3). However, the pathogenic relevance of Dsc3-reactive immunoglobulin G (IgG) has not been directly shown. This study aimed to first establish a method to specifically isolate Dsg3-reactive IgG from PV sera and to further investigate their pathogenic capacity using a keratinocyte based in vitro assay. This method was further applied to sera of four Japanese patients suffering from atypical pemphigus all of them exhibiting a positive IgG reactivity against Dsc3. Sepharose based affinity chromatochraphy columns coated with recombinant baculovirus produced proteins of the extracellular domains of Dsg3 and Dsc3, respectively, were used to specifically isolate auto-Abs from pemphigus sera. Affinity purified IgG fractions were subsequently tested for antigen specificity using enzyme-linked immunosorbent assay (ELISA) and immunoblotting (IB). Reactivity with native Dsg3- and Dsc3-protein, respectively, was proven by immunofluorescence (IF) on cultured human keratinocytes, monkey esophagus and frozen sections of normal human skin. Finally, a keratinocyte based so-called dissociation assay served to investigate the in vitro pathogenicity of the affinity purified IgG fractions. Eight Dsg3-reactive PV patients showed Dsg3-domain-specific auto-antibodies by ELISA. Four of these patients were selected for further investigation based on their antibody profile, i.e. their epitope specificity. Two patients (#1 and #8) exhibited IgG directed against Dsg3EC1 and Dsg3EC4. Patient #2 exclusively expressed auto-Abs directed against Dsg3EC1 whereas patient #6 showed IgG reactivity against Dsg3EC4, only. Serum IgG was then affinity purified using the respective recombinant Dsg3-subdomains. Antigen specificity of the eluted IgG-fractions was subsequently verified by IB and ELISA. Isolated IgG fractions showed a characteristic intercellular staining pattern by IF using cultured human keratinocytes indicating positive reactivity with native Dsg3-protein. Finally Dsg3-, Dsg3EC1- and Dsg3EC4-specific IgG caused keratinocyte dissociation which was comparable to the positive control, a monoclonal antibody (AK23) directed against the NH2-terminus of Dsg3. These techniques were then applied to the sera of four atypical, i.e. two pemphigus vegetans and two pemphigus herpetiformis patients, in order to isolate Dsc3-specific IgG. All but one of these patients, who showed additional Dsg1 reactivity, exhibited IgG reactivity exclusively against Dsc3 but no other desmosomal cadherin. From all sera IgG fractions were successfully isolated and antigen specificity to Dsc3 was verified. Dsc3-reacitve IgG showed a characteristic intercellular staining pattern by IF on cultured human keratinocytes, monkey esophagus and human skin. Finally all isolated IgG fractions were able to induce loss of keratinocyte adhesion in vitro.Taken together this data strongly suggests a significant acantholytic effect of IgG directed against COOH-terminal epitopes of Dsg3 in addition to the well known pathogenic epitopes at the NH2-terminus of this auto-antigen. Moreover Dsc3-reactive IgG isolated from patients with atypical pemphigus variants proved to be pathogenic in vitro. For the first time these results directly show the acantholytic effect of Dsc3-reactive IgG and provides evidence for the pathogenic relevance of Dsc3-IgG in pemphigus patients lacking reactivity against other desmosomal cadherins. Further investigations are needed to elucidate the mechanisms by which auto-Abs directed against COOH-terminal epitopes of Dsg3 induce acantholysis. The pathogenic relevance of other epitopes of Dsg3 needs to be addressed. Finally screening of pemphigus patients’ sera for Dsc3-reactive IgG should provide further knowledge about their correlation with atypical pemphigus variants

Context Dependent Role of Type 2 Innate Lymphoid Cells in Allergic Skin Inflammation.

The discovery of innate lymphoid cells (ILC) has profoundly influenced the understanding of innate and adaptive immune crosstalk in health and disease. ILC and T cells share developmental and functional characteristics such as the lineage-specifying transcription factors and effector cytokines, but importantly ILC do not display rearranged antigen-specific receptors. Similar to T cells ILC are subdivided into 3 different helper-like subtypes, namely ILC1-3, and a killer-like subtype comprising natural killer (NK) cells. Increasing evidence supports the physiological relevance of ILC, e.g., in wound healing and defense against parasites, as well as their pathogenic role in allergy, inflammatory bowel diseases or psoriasis. Group 2 ILC have been attributed to the pathogenesis of allergic diseases like asthma and atopic dermatitis. Other inflammatory skin diseases such as allergic contact dermatitis are profoundly shaped by inflammatory NK cells. This article reviews the role of ILC in allergic skin diseases with a major focus on ILC2. While group 2 ILC are suggested to contribute to the pathogenesis of type 2 dominated inflammation as seen in atopic dermatitis, we have shown that lack of ILC2 in type 1 dominated contact hypersensitivity results in enhanced inflammation, suggesting a regulatory role of ILC2 in this context. We provide a concept of how ILC2 may influence context dependent the mutual counterbalance between type I and type II immune responses in allergic skin diseases

Hemidesmosomal Reactivity and Treatment Recommendations in Immune Checkpoint Inhibitor-Induced Bullous Pemphigoid—A Retrospective, Monocentric Study

Immune checkpoint inhibitors (ICI) induce T-cell-mediated antitumour responses. While ICI were initially successfully applied in metastasized melanoma, they are now approved for several tumour entities. Numerous autoimmune disorders have been reported to occur as adverse events of the treatment, among them bullous pemphigoid (BP), with less than 1% of the patients experiencing ICI-induced BP. This number is higher than the estimated prevalence of autoimmune bullous diseases in the general population of Germany, which lies around 0.05%. We here describe our cohort of eight patients, who developed a bullous pemphigoid under or shortly after ICI treatment. Half of them had a severe subtype (as shown by BPDAI >57) and showed a median onset of ICI-BP after 10 months of ICI initiation. Six patients had a palmar and/or plantar involvement, while oral involvement occurred in one case. All patients had linear epidermal IgG depositions in split skin in the indirect immunofluorescence. In four out of five biopsies available for direct immunofluorescence, linear IgG and C3 depositions were detected at the basement membrane, while one patient showed linear IgM staining. Moderate to high levels of FLBP180 autoantibodies were found in seven of eight cases. The disease can still be active after ICI discontinuation, while rituximab might be required for remission. Finally, four tumour samples were stained histochemically for collagen XVII (BP180), but no enhanced expression was found

Additive Intralesional Interleukin-2 Improves Progression-Free Survival in a Distinct Subgroup of Melanoma Patients with Prior Progression under Immunotherapy

A considerable amount of melanoma patients show primary resistance to PD-1 and CTLA-4 inhibitors. We have previously reported a beneficial role of intralesional Interleukin-2 (IL-2) in 9 melanoma patients developing new locoregional metastases under immunotherapy. We have now expanded this retrospective cohort to 27 patients. Patients were evaluated for their tumor characteristics, treatment response and progression-free and overall survival (PFS/OS). In 16 patients, tumor biopsies before and under IL-2 treatment were evaluated for immune markers. The median follow-up time was 16 (1–59) months from start of IL-2 treatment. Treatment response of locoregional metastases was seen in 74% of all patients and response of distant organ metastases in 37% of stage IV patients, respectively. A prolonged PFS and OS was significantly associated with absence of active distant metastases (p = 0.008), response of locoregional metastases (p = 0.002), increase of absolute eosinophil count (AEC) (p < 0.001) and an influx of CD8+ tumor infiltrating lymphocytes (TILs) (p = 0.003). Additional intralesional treatment with IL-2 in patients with locoregional progression under immunotherapy is a well-tolerated, easily feasible therapeutic option especially in patients lacking active distant metastases. A careful patient selection can lead to an improved PFS and OS

Characterization of pathogenic auto-antibodies directed against desmoglein 3 and desmocollin 3 in sera of pemphigus patients.

Pemphigus vulgaris (PV) represents the most frequent clinical type of the pemphigus group of autoimmune bullous skin disorders. There is substantial evidence that blister formation in pemphigus patients is mediated by auto-antibodies (auto-Abs) targeted against certain desmosomal cadherins, namely desmoglein1 (Dsg1) and desmoglein3 (Dsg3). Several pathogenic epitopes of Dsg3 are located at the amino-(NH2)-terminal end of the Dsg3 ectodomain, namely the extracellular domain 1 (EC1). On the other hand a great number of pemphigus patients exhibit auto-Abs directed against the more carboxy-(COOH)-terminal epitopes of Dsg3, e.g. within the EC4- and EC5-domain and these domains may play an essential role in maintaining desmosomal adhesion. Some pemphigus patients exhibit additional or solely auto-Abs against other desmosomal cadherins, especially desmocollin 3 (Dsc3). However, the pathogenic relevance of Dsc3-reactive immunoglobulin G (IgG) has not been directly shown. This study aimed to first establish a method to specifically isolate Dsg3-reactive IgG from PV sera and to further investigate their pathogenic capacity using a keratinocyte based in vitro assay. This method was further applied to sera of four Japanese patients suffering from atypical pemphigus all of them exhibiting a positive IgG reactivity against Dsc3. Sepharose based affinity chromatochraphy columns coated with recombinant baculovirus produced proteins of the extracellular domains of Dsg3 and Dsc3, respectively, were used to specifically isolate auto-Abs from pemphigus sera. Affinity purified IgG fractions were subsequently tested for antigen specificity using enzyme-linked immunosorbent assay (ELISA) and immunoblotting (IB). Reactivity with native Dsg3- and Dsc3-protein, respectively, was proven by immunofluorescence (IF) on cultured human keratinocytes, monkey esophagus and frozen sections of normal human skin. Finally, a keratinocyte based so-called dissociation assay served to investigate the in vitro pathogenicity of the affinity purified IgG fractions. Eight Dsg3-reactive PV patients showed Dsg3-domain-specific auto-antibodies by ELISA. Four of these patients were selected for further investigation based on their antibody profile, i.e. their epitope specificity. Two patients (#1 and #8) exhibited IgG directed against Dsg3EC1 and Dsg3EC4. Patient #2 exclusively expressed auto-Abs directed against Dsg3EC1 whereas patient #6 showed IgG reactivity against Dsg3EC4, only. Serum IgG was then affinity purified using the respective recombinant Dsg3-subdomains. Antigen specificity of the eluted IgG-fractions was subsequently verified by IB and ELISA. Isolated IgG fractions showed a characteristic intercellular staining pattern by IF using cultured human keratinocytes indicating positive reactivity with native Dsg3-protein. Finally Dsg3-, Dsg3EC1- and Dsg3EC4-specific IgG caused keratinocyte dissociation which was comparable to the positive control, a monoclonal antibody (AK23) directed against the NH2-terminus of Dsg3. These techniques were then applied to the sera of four atypical, i.e. two pemphigus vegetans and two pemphigus herpetiformis patients, in order to isolate Dsc3-specific IgG. All but one of these patients, who showed additional Dsg1 reactivity, exhibited IgG reactivity exclusively against Dsc3 but no other desmosomal cadherin. From all sera IgG fractions were successfully isolated and antigen specificity to Dsc3 was verified. Dsc3-reacitve IgG showed a characteristic intercellular staining pattern by IF on cultured human keratinocytes, monkey esophagus and human skin. Finally all isolated IgG fractions were able to induce loss of keratinocyte adhesion in vitro.Taken together this data strongly suggests a significant acantholytic effect of IgG directed against COOH-terminal epitopes of Dsg3 in addition to the well known pathogenic epitopes at the NH2-terminus of this auto-antigen. Moreover Dsc3-reactive IgG isolated from patients with atypical pemphigus variants proved to be pathogenic in vitro. For the first time these results directly show the acantholytic effect of Dsc3-reactive IgG and provides evidence for the pathogenic relevance of Dsc3-IgG in pemphigus patients lacking reactivity against other desmosomal cadherins. Further investigations are needed to elucidate the mechanisms by which auto-Abs directed against COOH-terminal epitopes of Dsg3 induce acantholysis. The pathogenic relevance of other epitopes of Dsg3 needs to be addressed. Finally screening of pemphigus patients’ sera for Dsc3-reactive IgG should provide further knowledge about their correlation with atypical pemphigus variants

Additive Intralesional Interleukin-2 Improves Progression-Free Survival in a Distinct Subgroup of Melanoma Patients with Prior Progression under Immunotherapy

A considerable amount of melanoma patients show primary resistance to PD-1 and CTLA-4 inhibitors. We have previously reported a beneficial role of intralesional Interleukin-2 (IL-2) in 9 melanoma patients developing new locoregional metastases under immunotherapy. We have now expanded this retrospective cohort to 27 patients. Patients were evaluated for their tumor characteristics, treatment response and progression-free and overall survival (PFS/OS). In 16 patients, tumor biopsies before and under IL-2 treatment were evaluated for immune markers. The median follow-up time was 16 (1–59) months from start of IL-2 treatment. Treatment response of locoregional metastases was seen in 74% of all patients and response of distant organ metastases in 37% of stage IV patients, respectively. A prolonged PFS and OS was significantly associated with absence of active distant metastases (p = 0.008), response of locoregional metastases (p = 0.002), increase of absolute eosinophil count (AEC) (p + tumor infiltrating lymphocytes (TILs) (p = 0.003). Additional intralesional treatment with IL-2 in patients with locoregional progression under immunotherapy is a well-tolerated, easily feasible therapeutic option especially in patients lacking active distant metastases. A careful patient selection can lead to an improved PFS and OS

Variable Outcome of Immunotherapy in Advanced Multiple Cutaneous Squamous Cell Carcinomas in Two Patients with Recessive Dystrophic Epidermolysis Bullosa

Cutaneous squamous cell carcinoma (cSCC) is a major complication of recessive dystrophic epidermolysis bullosa (RDEB) that has high morbidity and mortality rates and unmet therapeutic needs. The aim of this study was to evaluate the molecular pattern of cSCC and the clinical course of immunotherapy in 2 RDEB patients with multiple advanced cSCC. Clinical course and disease staging were evaluated retrospectively. The tumour tissues were subjected to immunohistochemical staining. DNA from the blood and cSCC samples was subjected to massive parallel sequencing, and somatic mutations were determined. Patient 1 survived for over 2 years as disease control was achieved with cemiplimab and intralesional interleukin-2. The target advanced cSCC demonstrated a high rate of somatic mutations and strong expression of the immune markers, indoleamine 2,3-dioxygenase, programmed cell death protein ligand 1, and lymphocyte-activation gene 3. The patient ultimately succumbed to complications of oesophageal carcinoma. Patient 2 had an undifferentiated cSCC on the foot, which displayed a low mutational burden and did not express immune markers. The tumour progressed quickly even with cemiplimab therapy. These 2 cases underscore the challenges of cSCC treatment for RDEB. Multiple tumours with different molecular and immune profiles occur concomitantly or sequentially, and surgical excision is not always possible because of the anatomical and tissue constraints imposed by the disease itself. In conclusion, programmed cell death protein 1 inhibitors are approved and effective in treating metastatic and locally advanced cSCC. Our experience and the literature suggest that cemiplimab is an option in patients with RDEB if surgery is not. Somatic mutations and the immune microenvironment should be characterized to predict therapeutic response, particularly in aggressive undifferentiated tumours

Incidence and severity of immune-related hepatitis after dual checkpoint therapy is linked to younger age independent of herpes virus immunity

Background and Aims: Dual immune checkpoint blockade (ICB) therapy can result in immune-related-adverse events (irAE) such as ICB-hepatitis. An expansion of effector-memory (TEM) CD4 T cells associated with antiviral immunity against herpesviridae was implicated in ICB-hepatitis. Notably, these memory subsets are frequently associated with age. Here, we sought to understand baseline patient, immune and viral biomarkers associated with the development of ICB-hepatitis to identify currently lacking baseline predictors and test if an expansion of TEM or positive serology against herpesviridae can predict ICB-hepatitis. Methods: A discovery (n = 39) and validation cohort (n = 67) of patients with advanced melanoma undergoing anti-PD-1&anti-CTLA4 combination therapy (total n = 106) were analyzed for baseline clinical characteristics, occurrence of irAE and oncological outcomes alongside serological status for CMV, EBV and HSV. Immune populations were profiled by high-parametric flow cytometry (n = 29). Results: ICB-hepatitis occurred in 59% of patients within 100 days; 35.9% developed severe (CTCAE 3–4) hepatitis. Incidence of ICB-hepatitis was higher in the younger ( = 55y: 27.8%) age group (p = 0.0003), occured earlier in younger patients (p < 0.0001). The association of younger age with ICB-Hepatitis was also observed in the validation cohort (p = 0.0486). Incidence of ICB-hepatitis was also associated with additional non-hepatic irAE (p = 0.018), but neither positive IgG serostatus for CMV, EBV or HSV nor TEM subsets despite an association of T cell subsets with age. Conclusion: Younger age more accurately predicts ICB-hepatitis after anti-PD-1&anti-CTLA4 checkpoint therapy at baseline compared to herpes virus serology or TEM subsets. Younger patients should be carefully monitored for the development of ICB-hepatitis