Pemphigus vulgaris (PV) is a chronic, life-altering autoimmune disease due to the production of anti-desmoglein antibodies causing the loss of cell–cell adhesion in keratinocytes (acantholysis) and blister formation in both skin and mucous membranes. The dispase-based keratinocyte dissociation assay (DDA) is the method of choice to examine the pathogenic effect of antibodies and additional co-stimuli on cell adhesion in vitro. Despite its widespread use, there is a high variability of experimental conditions, leading to inconsistent results. In this paper, we identify and discuss pitfalls in the application of DDA, including generation of a monolayer with optimized density, appropriate culturing conditions to obtain said monolayer, application of mechanical stress in a standardized manner, and performing consistent data processing. Importantly, we describe a detailed protocol for a successful and reliable DDA and the respective ideal conditions for three different types of human keratinocytes: (1) primary keratinocytes, (2) the HaCaT spontaneously immortalized keratinocyte cell line, and (3) the recently characterized HaSKpw spontaneously immortalized keratinocyte cell line. Our study provides detailed protocols which guarantee intra- and inter-experimental comparability of DDA.

The NLRP1 (nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-1) inflammasome is the most important inflammasome in human keratinocytes. It plays a crucial role in regulating innate immunity in the skin. This study aimed to evaluate NLRP1 inflammasome activation and the corresponding levels of detection in different keratinocyte cell lines to identify a suitable in vitro model for analyzing inflammasome activation in keratinocytes. We compared NLRP1 inflammasome activation, expression, and cell death among primary keratinocytes and immortalized keratinocyte cell lines HaCaT, HaSKpw, and SVTERT upon stimulation with ultraviolet B (UVB) irradiation or talabostat. The effects of both NLRP1 inducers on cell death and the modification of NLRP1 molecules were examined using fluorescence-activated cell sorting analysis, Western blotting, and an enzyme-linked immunosorbent assay. The key inflammasome components had varied expression levels among the keratinocyte cell models, with the highest expression observed in primary keratinocytes. Moreover, our data showed that both UVB and talabostat triggered cell death, and NLRP1 inflammasome activation was readily detected in primary keratinocytes but not in the analyzed immortalized keratinocyte cell lines. Therefore, we do not recommend the use of the immortalized keratinocyte cell lines HaCaT, HaSKpw, and SVTERT for analyzing inflammasome activation in keratinocytes; we strongly recommend the use of primary keratinocytes for these studies.

Background/Objectives: Melanoma is a highly malignant skin tumor with poor response to conventional chemotherapeutic regimens. Melanoma cells induce cytotoxic T cell-mediated immune responses, and immunotherapy has significantly improved survival rates for patients with advanced disease. Methods: Here, we investigate NK cell-mediated melanoma cell killing and its regulation by PD-L1/PD-1 blockade and IFNβ. Four melanoma cell lines were used in this study. To evaluate NK cell cytotoxicity, cells were exposed to NK cells with or without IFNβ. The calcein release assay was used to measure cell death, while specific inhibitors and siRNA silencing were applied to determine the contribution of individual effector pathways. Results: NK cells were able to kill melanoma cells with sensitivity to killing varying between different cell lines. Cytotoxic effects were mainly mediated through activation of the TRAIL signaling cascade. In cell lines with low sensitivity to NK cell killing, expression of PD-L1 was noted and killing by NK cells could be significantly increased by inhibition of the PD-L1/PD-1 checkpoint. Killing of melanoma cells could be further increased by incubation of NK cells with IFNβ. Conclusions: Our results point to a role of NK cells in the killing of melanoma cells and a potential clinical benefit of a combination therapy of IFNβ and anti-PD-1 antibody.

CFLIP is required for epidermal integrity and skin inflammation silencing via protection from TNF-induced keratinocyte apoptosis. Here, we genera | POSTER

Pemphigus vulgaris is a chronic, life-altering bullous autoimmune disease with production of antibodies against the desmosomal proteins Desmoglei | POSTER

A20 is a known key regulator of NF-κB activity and inflammatory responses. A20 plays a role also in the control of cell death and cell death rece |