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

Abstract

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.

Immunostimulatory effects of Toll‐like receptor ligands as adjuvants in establishing a novel mouse model for pemphigus vulgaris

“…This approach includes bystander cells and limits the precision of information regarding the effects of adjuvants. 5,61,[71][72][73]…”

“…Secondly, in our analysis, we gated on total B cells and did not specifically examine antigen‐specific cells. This approach includes bystander cells and limits the precision of information regarding the effects of adjuvants 5,61,71–73 …”

In Vitro, Ex Vivo, and In Vivo Models for the Study of Pemphigus

“…This technique demonstrated that, for example, hydrocortisone present in a skin culture was able to inhibit acantholysis induced by Pemphigus sera [16]. This approach was further ameliorated and standardized, leading to the establishment of a protocol allowing acantholysis to be induced in human skin organ cultures (HSOC) injected with a bi-specific anti-DSG 1 and three single-chain antibody variable fragments (scFv) [17], PVIgG or the pathogenic IgG AK23 [18] (Figure 1A). Using this method, blister formation can be easily evaluated by immunofluorescence or haematoxylin and eosin staining.…”