IgM to IgG Class Switching Is a Necessary Step for Pemphigus Phenotype Induction in Desmoglein 3-Specific B Cell Receptor Knock-in Mouse.

Pemphigus vulgaris is an autoimmune blistering disease caused by IgG targeting desmoglein 3 (Dsg3), an adhesion molecule of keratinocytes. Anti-Dsg3 IgG production is prevented in healthy individuals, but it is unclear how Dsg3-specific B cells are regulated. To clarify the immunological condition regulating Dsg3-specific B cells, a pathogenic anti-Dsg3 Ig (AK23) knock-in mouse was generated. AK23 knock-in B cells developed normally without undergoing deletion or acquiring an anergic phenotype in vivo. The knock-in B cells showed Ca2+ influx upon IgM cross-linking and differentiated into AK23-IgG+ B cells after LPS and IL-4 stimulation in vitro that induced a pemphigus phenotype after adoptive transfer into Rag2 -/- mice. However, the knock-in mouse itself produced AK23-IgM but little IgG without blisters in vivo. Dsg3 immunization and skin inflammation caused AK23-IgG production and a pemphigus phenotype in vivo. Furthermore, Fcgr2b deficiency or haploinsufficiency spontaneously induced AK23-IgG production and a pemphigus phenotype with poor survival rates in AK23 knock-in mice. To assess Fcgr2b involvement in Ig class-switch efficiency, postswitch transcripts of B cells were quantified and significantly higher in Fcgr2b -/- and Fcgr2b +/- mice than wild-type mice in a gene dose-dependent manner. Finally, RNA sequencing revealed reduced expression of FCGR2B and FcγRIIB-related genes in patient B cells. These results indicated that Dsg3-specific B cells do not spontaneously perform pathogenic class switching in vivo, and pemphigus phenotype induction was prevented under normal conditions. Attenuated FcγRIIB signaling is also one of the drivers for pathogenic class switching and is consistent with immunological features identified from clinical samples. This study unveiled a characteristic immune state silencing autoreactive B cells in mice.

Peripheral tolerance by Treg via constraining OX40 signal in autoreactive T cells against desmoglein 3, a target antigen in pemphigus.

Antigen-specific peripheral tolerance is crucial to prevent the development of organ-specific autoimmunity. However, its function decoupled from thymic tolerance remains unclear. We used desmoglein 3 (Dsg3), a pemphigus antigen expressed in keratinocytes, to analyze peripheral tolerance under physiological antigen-expression conditions. Dsg3-deficient thymi were transplanted into athymic mice to create a unique condition in which Dsg3 was expressed only in peripheral tissue but not in the thymus. When bone marrow transfer was conducted from high-avidity Dsg3-specific T cell receptor-transgenic mice to thymus-transplanted mice, Dsg3-specific CD4+ T cells developed in the transplanted thymus but subsequently disappeared in the periphery. Additionally, when Dsg3-specific T cells developed in Dsg3-/- mice were adoptively transferred into Dsg3-sufficient recipients, the T cells disappeared in an antigen-specific manner without inducing autoimmune dermatitis. However, Dsg3-specific T cells overcame this disappearance and thus induced autoimmune dermatitis in Treg-ablated recipients but not in Foxp3-mutant recipients with dysfunctional Tregs. The molecules involved in disappearance were sought by screening the transcriptomes of wild-type and Foxp3-mutant Tregs. OX40 of Tregs was suggested to be responsible. Consistently, when OX40 expression of Tregs was constrained, Dsg3-specific T cells did not disappear. Furthermore, Tregs obtained OX40L from dendritic cells in an OX40-dependent manner in vitro and then suppressed OX40L expression in dendritic cells and Birc5 expression in Dsg3-specific T cells in vivo. Lastly, CRISPR/Cas9-mediated knockout of OX40 signaling in Dsg3-specific T cells restored their disappearance in Treg-ablated recipients. Thus, Treg-mediated peripheral deletion of autoreactive T cells operates as an OX40-dependent regulatory mechanism to avoid undesired autoimmunity besides thymic tolerance.

[Laparoscopic Abdominoperineal Resection of the Rectum in a Case with Pagetoid Spread of Anal Canal Carcinoma].

The patient, a man in his 60s, first noticed an elevated lesion around the anus 3 years ago. The lesion failed to subside with the topical drug preparations prescribed at a local clinic, and the patient was referred to the Department of Dermatology of our hospital for further workup and treatment. The findings of biopsy from the lesion suggested skin infiltration of anal canal carcinoma, and the patient was referred to the Department of Surgery. Examination here revealed only induration of the anal canal, with no abnormality of the rectal mucosa even when the endoscope was reversed to visualize the rectum. Examination by various imaging modalities failed to reveal any metastases to the lymph nodes or distant organs, and the primary lesion remained unidentified. Laparoscopic abdominoperineal excision of the rectum was performed, beginning with anal manipulation. First, a 15-mm margin was set on the skin from the tumor edge, and the skin stump was divided into 4 equal portions. After confirming by rapid intraoperative frozen-section examination that the margin was negative along the full circumference, anal manipulation was performed, leaving a distance in the vertical direction immediately below the tumor. Upon completion of the anal manipulation, intraperitoneal manipulation was performed in a routine manner. The anal skin was relaxed subcutaneously, as done during mastectomy, and the subsequent suture closure could be done smoothly. The tumor was classified as pT1bN0M0, pStage Ⅰ. The experience with this case indicates that biopsy should be proactively employed for the diagnosis in such cases, and that proactive skin biopsy is useful when dealing with intractable anal skin lesions.

Autoimmunity and immunological tolerance in autoimmune bullous diseases.

Autoimmune diseases are devastating conditions in which the immune system is directed against the host, leading to life-threatening destruction of organs. Although autoantigens are ill-defined in most autoimmune diseases, this is not the case in the skin. Autoimmune bullous diseases have been extensively studied with detailed characterization of autoantigens, the epitopes that are targeted, and the mechanisms of action that mediate autoimmune tissue destruction. Pemphigus is an autoimmune bullous disease caused by circulating IgG that targets two desmosomal proteins, desmoglein 1 and 3, which are crucial for cell-cell adhesion of keratinocytes. Binding of auto-antibodies to desmogleins impairs keratinocyte adhesion, leading to severe blistering disease. Mouse models that recapitulate the human disease have been instrumental in elucidating the detailed pathophysiology. Taking advantage of the fact that desmogleins are specifically targeted in pemphigus, studying humoral and cellular autoimmunity against these autoantigens provides us with an opportunity to understand not only the effector mechanisms of B and T cells in mediating pathology but also how autoreactive lymphocytes are regulated during development in the thymus and post-development in the periphery. This review introduces pemphigus and its subtypes as prototypic autoimmune diseases from which recent basic and translational developments should provide insight into how autoimmunity develops.

Intravenous Ig Regulates Anti-Desmoglein 3 IgG Production in B220- Antibody-Producing Cells in Mice with Pemphigus Vulgaris.

Intravenous Ig (IVIG) is a treatment option for intractable cases of pemphigus vulgaris (PV), an autoimmune blistering disease caused by autoantibodies against desmoglein 3 (DSG3). To investigate the efficacy of IVIG on autoantibody secretion, we produced PV model mice by adoptive transfer of immunized Dsg3-/- splenocytes to Rag2-/- mice. We found that circulating anti-DSG3 IgG ELISA titer decreased in PV model mice after 5 days of treatment with IVIG compared with PBS-treated mice, whereas the F(ab')2 fragment did not suppress the anti-DSG3 IgG titer. enzyme-linked immunospot assay revealed that IVIG treatment reduced the frequency of anti-DSG3 antibody-secreting cells in the spleen but not in lymph nodes and bone marrow. Moreover, this reduction was observed only in the splenic B220- fraction but not in the B220+ fraction. Furthermore, IVIG decreased the serum levels of anti-DSG3 IgG, even after a significant reduction of its titer, owing to antibody-mediated CD20+ B cell depletion. In addition, IVIG suppressed anti-DSG3 IgG production in B220-CD138+ plasma cells derived from PV model mice ex vivo. These results indicate that IVIG reduced autoantibody production in B220- cells containing plasma cells in PV model mice, and this function may indicate one of the mechanisms of action of IVIG on PV.

A Case of Kerion Celsi Caused by Trichophyton tonsurans, a Plate Culture of Which Showed Yellow-Green Fluorescence Under UVA Light.

We herein report a case of kerion celsi of the scalp and tinea corporis due to Trichophyton tonsurans. A 17-year-old Japanese male high school student who practiced judo had alopecic patches with severe inflammation on the scalp. We performed a fungal culture and identified the causative fungus as T. tonsurans. A plate culture of T. tonsurans showed lemon-yellow colonies with yellow-green fluorescence under UVA light, which are typical findings for Microsporum canis. However, genetic analysis of the ribosomal RNA gene of the isolate facilitated differential diagnosis of T. tonsurans.In contrast to dermatophytosis due to other dermatophytes, the clinical features of infection caused by T. tonsurans, an anthropophilic dermatophyte, are initially not very apparent and, thus, are frequently overlooked. We herein present a case of a severe type of kerion celsi caused by T. tonsurans with a fluorescence pattern mimicking M. canis colonies under UVA light. We suspect that yellow pigment metabolites, such as riboflavin, which are fluorescent under UV when secreted into the culture medium, are the virulence factors for not only M. canis, but also T. tonsurans, as shown in the present case.

Reduced methylation of PFKFB3 in cancer cells shunts glucose towards the pentose phosphate pathway.

Haem oxygenase (HO)-1/carbon monoxide (CO) protects cancer cells from oxidative stress, but the gas-responsive signalling mechanisms remain unknown. Here we show using metabolomics that CO-sensitive methylation of PFKFB3, an enzyme producing fructose 2,6-bisphosphate (F-2,6-BP), serves as a switch to activate phosphofructokinase-1, a rate-limiting glycolytic enzyme. In human leukaemia U937 cells, PFKFB3 is asymmetrically di-methylated at R131 and R134 through modification by protein arginine methyltransferase 1. HO-1 induction or CO results in reduced methylation of PFKFB3 in varied cancer cells to suppress F-2,6-BP, shifting glucose utilization from glycolysis toward the pentose phosphate pathway. Loss of PFKFB3 methylation depends on the inhibitory effects of CO on haem-containing cystathionine ß-synthase (CBS). CBS modulates remethylation metabolism, and increases NADPH to supply reduced glutathione, protecting cells from oxidative stress and anti-cancer reagents. Once the methylation of PFKFB3 is reduced, the protein undergoes polyubiquitination and is degraded in the proteasome. These results suggest that the CO/CBS-dependent regulation of PFKFB3 methylation determines directional glucose utilization to ensure resistance against oxidative stress for cancer cell survival.