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Atopic dermatitis results in diminished barrier function and altered production of antimicrobial peptides. Dendritic epidermal T cells (DETCs) play an important role in the wound repair and inflammation process. Our previous work identified an IL-4–dependent loss of DETCs in Stat6VT mice and in the MC903-induced skin inflammation mouse model. However, the mechanisms through which IL-4 mediates the loss of DETCs are unclear. In this study, we show that IL-4Rα germline knockout mice (Il4ra^–/–) have increased DETCs, faster wound healing, and increased epidermal differentiation complex gene and fibronectin expression. The absence of IL-4Rα minimized the MC903-induced loss of DETCs, and reciprocal bone marrow chimera experiments in Il4ra^–/– and wild-type mice demonstrated structural nonhematopoietic IL-4–responsive cell-mediated DETC homeostasis. Skin keratinocyte-derived IL-15 decreased dramatically in the MC903 model, while injection of IL-15 rescued DETC loss by promoting DETC proliferation and limiting apoptosis. Conditional deletion of IL-4Rα from keratinocytes using Il4ra^fl/fl K14-Cre mice showed an increase of DETCs, increased IL-15 production, and diminished skin inflammation following wounding. These results suggest that IL-4–dependent effects on DETCs in allergic skin inflammation are mediated by the IL-4Rα receptor of keratinocytes.

The development of agonists capable of activating the human complement system by binding to the C1 complex presents a novel approach for targeted cell killing. Bispecific nanobodies and Abs can successfully use C1 for this purpose; however, efficacy varies significantly between epitopes, Ab type, and bispecific design. To address this variability, we investigated monomeric agonists of C1 in the form of bispecific nanobodies, which lack Fc domains that lead to oligomerization in Abs. These therefore offer an ideal opportunity to explore the geometric parameters crucial for C1 activation. In this study, we explored the impact of linker length as a metric for Ag and epitope location. DNA nanotechnology and protein engineering allowed us to design linkers with controlled lengths and flexibilities, revealing a critical range of end-to-end distances for optimal complement activation. We discovered that differences in complement activation were not caused by differential C1 activation or subsequent cleavage of C4, but instead impacted C4b deposition and downstream membrane lysis. Considering the importance of Ab class and subclass, this study provides insights into the structural requirements of C1 binding and activation, highlighting linker and hinge engineering as a potential strategy to enhance potency over specific cellular targets. Additionally, using DNA nanotechnology to modify geometric parameters demonstrated the potential for synthetic biology in complement activation. Overall, this research offers valuable insights into the design and optimization of agonists for targeted cell killing through complement activation.