Type 2 T-Cell Responses against Distinct Epitopes of the Desmoglein 3 Ectodomain in Pemphigus Vulgaris.

Pemphigus vulgaris (PV) is an autoimmune blistering disorder of the skin and/or mucous membranes caused by IgG autoantibodies that predominantly target two transmembrane desmosomal cadherins: desmoglein (DSG)1 and DSG3. DSG-specific T cells play a central role in PV pathogenesis because they provide help to autoreactive B cells for autoantibody production. In this study, we characterized DSG3-specific peripheral T cells in a cohort of 52 patients with PV and 41 healthy controls with regard to cytokine profile and epitope specificity. By ELISpot analysis, type 2 T cells reactive with the DSG3 ectodomain were significantly increased in patients with PV compared with those in healthy controls. By dextramer analysis, CD4+ T cells specific for an epitope within the extracellular domain of DSG3, DSG3(206-220), were found at significantly higher frequencies in patients with PV than in HLA-matched healthy controls. T-cell recognition of two distinct DSG3 epitopes, that is, DSG3(206-220) and DSG3(378-392), correlated significantly, suggesting a synergistic effect in B-cell help. Immunization of HLA-DRB1∗04:02-transgenic mice with PV with the same set of DSG3 peptides induced pathogenic DSG3-specific IgG antibodies, which induced loss of keratinocyte adhesion in vitro. Thus, DSG3 peptide-specific T cells are of particular interest as surrogate markers of disease activity and potential therapeutic targets in PV.

Correction: Zakrzewicz et al. Stabilization of Keratinocyte Monolayer Integrity in the Presence of Anti-Desmoglein-3 Antibodies through FcRn Blockade with Efgartigimod: Novel Treatment Paradigm for Pemphigus? Cells 2022, 11, 942.

The authors wish to make the following changes to their paper [...].

Stabilization of Keratinocyte Monolayer Integrity in the Presence of Anti-Desmoglein-3 Antibodies through FcRn Blockade with Efgartigimod: Novel Treatment Paradigm for Pemphigus?

Pemphigus vulgaris is an autoimmune blistering disease of the epidermis, caused by autoantibodies against desmosomal proteins, mainly desmogleins 1 and 3, which induce an impairment of desmosomal adhesion and blister formation. Recent findings have shown that inhibition of immunoglobulin G binding on the neonatal Fc receptor, FcRn, results in reduced autoantibody recycling and shortens their half-life, providing a valid treatment option for PV. We have here analyzed the role of FcRn in human keratinocytes treated with antibodies isolated from pemphigus vulgaris patient or with recombinant anti-desmoglein-3 antibodies that induce pathogenic changes in desmosomes, such as loss of monolayer integrity, aberrant desmoglein-3 localization and degradation of desmoglein-3. We show that blocking IgG binding on FcRn by efgartigimod, a recombinant Fc fragment undergoing clinical studies for pemphigus, stabilizes the keratinocyte monolayer, whereas the loss of desmoglein-3 is not prevented by efgartigimod. Our data show that FcRn may play a direct role in the pathogenesis of pemphigus at the level of the autoantibody target cells, the epidermal keratinocytes. Our data suggest that in keratinocytes, FcRn may have functions different from its known function in IgG recycling. Therefore, stabilization of keratinocyte adhesion by FcRn blocking entities may provide a novel treatment paradigm for pemphigus.

Human Desmocollin 3‒Specific IgG Antibodies Are Pathogenic in a Humanized HLA Class II Transgenic Mouse Model of Pemphigus.

Pemphigus is a potentially lethal autoimmune bullous skin disorder, which is associated with IgG autoantibodies against desmoglein (DSG) 3 and DSG1. Notably, a subset of patients with pemphigus presents with a similar clinical phenotype in the absence of anti-DSG IgG, suggesting the presence of serum IgG reactive with desmosomal components other than DSG1 or DSG3. We and others have previously shown that such patients have serum IgG autoantibodies against desmocollin 3 (DSC3), a component of desmosomes, which induce loss of keratinocyte adhesion ex vivo. Moreover, DSC3 hypomorphic mice show a severe blistering phenotype of the mucous membrane, which is highly characteristic of pemphigus. These findings prompted us to study the induction and regulation of anti-human DSC3 IgG in humanized mice transgenic for HLA-DRB1∗04:02, which is a highly prevalent haplotype in pemphigus. We show that IgG from sera of immunized mice induces acantholysis in a dispase-based keratinocyte dissociation assay through the activation of p38 MAPKs and EGFR. Passive IgG transfer from mice immunized with recombinant human DSC3 into neonates did not induce intraepidermal loss of adhesion presumably owing to the lack of homology between human and mouse DSC3. Ex vivo stimulation of splenocytes from DSC3-immunized mice with human DSC3 leads to a significant proliferative IFN-γ and IL-4 T-cell response, which is restricted by HLA-DR/HLA-DQ. These findings suggest that the induction of pathogenic anti-DSC3 IgG is associated with DSC3-specific T cells that recognize DSC3 in association with HLA-DRB1∗04:02.

Immortalized Human hTert/KER-CT Keratinocytes a Model System for Research on Desmosomal Adhesion and Pathogenesis of Pemphigus Vulgaris.

Pemphigus Vulgaris is an autoimmune disease that results in blister formation in the epidermis and in mucosal tissues due to antibodies recognizing desmosomal cadherins, mainly desmoglein-3 and -1. Studies on the molecular mechanisms of Pemphigus have mainly been carried out using the spontaneously immortalized human keratinocyte cell line HaCaT or in primary keratinocytes. However, both cell systems have suboptimal features, with HaCaT cells exhibiting a large number of chromosomal aberrations and mutated p53 tumor suppressor, whereas primary keratinocytes are short-lived, heterogeneous and not susceptible to genetic modifications due to their restricted life-span. We have here tested the suitability of the commercially available human keratinocyte cell line hTert/KER-CT as a model system for research on epidermal cell adhesion and Pemphigus pathomechanisms. We here show that hTert cells exhibit a calcium dependent expression of desmosomal cadherins and are well suitable for typical assays used for studies on Pemphigus, such as sequential detergent extraction and Dispase-based dissociation assay. Treatment with Pemphigus auto-antibodies results in loss of monolayer integrity and altered localization of desmoglein-3, as well as loss of colocalization with flotillin-2. Our findings demonstrate that hTert cells are well suitable for studies on epidermal cell adhesion and Pemphigus pathomechanisms.

Altered Expression of Ganglioside Metabolizing Enzymes Results in GM3 Ganglioside Accumulation in Cerebellar Cells of a Mouse Model of Juvenile Neuronal Ceroid Lipofuscinosis.

Juvenile neuronal ceroid lipofuscinosis (JNCL) is caused by mutations in the CLN3 gene. Most JNCL patients exhibit a 1.02 kb genomic deletion removing exons 7 and 8 of this gene, which results in a truncated CLN3 protein carrying an aberrant C-terminus. A genetically accurate mouse model (Cln3Δex7/8 mice) for this deletion has been generated. Using cerebellar precursor cell lines generated from wildtype and Cln3Δex7/8 mice, we have here analyzed the consequences of the CLN3 deletion on levels of cellular gangliosides, particularly GM3, GM2, GM1a and GD1a. The levels of GM1a and GD1a were found to be significantly reduced by both biochemical and cytochemical methods. However, quantitative high-performance liquid chromatography analysis revealed a highly significant increase in GM3, suggesting a metabolic blockade in the conversion of GM3 to more complex gangliosides. Quantitative real-time PCR analysis revealed a significant reduction in the transcripts of the interconverting enzymes, especially of ß-1,4-N-acetyl-galactosaminyl transferase 1 (GM2 synthase), which is the enzyme converting GM3 to GM2. Thus, our data suggest that the complex a-series gangliosides are reduced in Cln3Δex7/8 mouse cerebellar precursor cells due to impaired transcription of the genes responsible for their synthesis.

Aß42 oligomers impair the bioenergetic activity in hippocampal synaptosomes derived from APP-KO mice.

Employing hippocampal synaptosomes from amyloid precursor protein (APP)-deleted mice we analyzed the immediate effects of amyloid beta peptide 42 (Aß42) peptide in its oligomeric or fibrillar assembly or of soluble amyloid precursor protein alpha (sAPPα) protein on their bioenergetic activity. Upon administration of oligomeric Aß42 peptide for 30 min we observed a robust decrease both in mitochondrial activity and in mitochondrial membrane potential (MMP). In contrast the respective fibrillary or scrambled peptides showed no effect, indicating that inhibition strictly depends on the oligomerization status of the peptide. Hippocampal synaptosomes from old APP-KO mice revealed a further reduction of their already impaired bioenergetic activity upon incubation with 10 µm Aß42 peptide. In addition we evaluated the influence of the sAPPα protein on mitochondrial activity of hippocampal synaptosomes derived from young or old APP-KO animals. In neither case 20 nm nor 200 nm sAPPα protein had an effect on mitochondrial metabolic activity. Our findings demonstrate that hippocampal synaptosomes derived from APP-KO mice are a most suitable model system to evaluate the impact of Aß42 peptide on its bioenergetic activity and to further elucidate the molecular mechanisms underlying the impairments by oligomeric Aß42 on mitochondrial function. Our data demonstrate that extracellular Aß42 peptide is taken up into synaptosomes where it immediately attenuates mitochondrial activity.

APP Deletion Accounts for Age-Dependent Changes in the Bioenergetic Metabolism and in Hyperphosphorylated CaMKII at Stimulated Hippocampal Presynaptic Active Zones.

Synaptic release sites are characterized by exocytosis-competent synaptic vesicles tightly anchored to the presynaptic active zone (PAZ) whose proteome orchestrates the fast signaling events involved in synaptic vesicle cycle and plasticity. Allocation of the amyloid precursor protein (APP) to the PAZ proteome implicated a functional impact of APP in neuronal communication. In this study, we combined state-of-the-art proteomics, electrophysiology and bioinformatics to address protein abundance and functional changes at the native hippocampal PAZ in young and old APP-KO mice. We evaluated if APP deletion has an impact on the metabolic activity of presynaptic mitochondria. Furthermore, we quantified differences in the phosphorylation status after long-term-potentiation (LTP) induction at the purified native PAZ. We observed an increase in the phosphorylation of the signaling enzyme calmodulin-dependent kinase II (CaMKII) only in old APP-KO mice. During aging APP deletion is accompanied by a severe decrease in metabolic activity and hyperphosphorylation of CaMKII. This attributes an essential functional role to APP at hippocampal PAZ and putative molecular mechanisms underlying the age-dependent impairments in learning and memory in APP-KO mice.

Regional Specializations of the PAZ Proteomes Derived from Mouse Hippocampus, Olfactory Bulb and Cerebellum.

Neurotransmitter release as well as structural and functional dynamics at the presynaptic active zone (PAZ) comprising synaptic vesicles attached to the presynaptic plasma membrane are mediated and controlled by its proteinaceous components. Here we describe a novel experimental design to immunopurify the native PAZ-complex from individual mouse brain regions such as olfactory bulb, hippocampus, and cerebellum with high purity that is essential for comparing their proteome composition. Interestingly, quantitative immunodetection demonstrates significant differences in the abundance of prominent calcium-dependent PAZ constituents. Furthermore, we characterized the proteomes of the immunoisolated PAZ derived from the three brain regions by mass spectrometry. The proteomes of the release sites from the respective regions exhibited remarkable differences in the abundance of a large variety of PAZ constituents involved in various functional aspects of the release sites such as calcium homeostasis, synaptic plasticity and neurogenesis. On the one hand, our data support an identical core architecture of the PAZ for all brain regions and, on the other hand, demonstrate that the proteinaceous composition of their presynaptic active zones vary, suggesting that changes in abundance of individual proteins strengthen the ability of the release sites to adapt to specific functional requirements.