D2.225 - Characterizing skin-resident antigen presenting cell responses to plant-based Fel d 1 Bioparticles

Particulate-based allergen immunotherapies (AIT) are a promising novel strategy for desensitization. Plant-based enveloped bioparticles (eBPs) carrying the cat dander allergen Fel d 1 demonstrated to be strongly hypoallergenic and to induce maturation and IL-10 secretion in human monocyte-derived dendritic cells (moDCs). Skin prick tests in 20 cat-allergic individuals with Fel d 1 eBPs confirmed their hypo-allergenicity in vivo. In this pre-clinical study, we used moDC and CD4+ T cell co-cultures to study regulatory T cell (Treg) induction and ex vivo skin vaccination to assess the capacity of Fel d 1 eBPs to activate skin-derived antigen presenting cells (APCs) and to elucidate their underlying mechanism of action.

Spherical 150-200 nm Fel d 1 eBPs were synthesized in Nicotiana benthamiana. MoDCs were exposed to Fel d 1 eBPs, natural Fel d 1 (nFel d 1), recombinant Fel d 1 (rFel d 1) after which moDC maturation and cytokine production was measured, by flow cytometry and ELISA, respectively. Additionally, these moDCs were co -cultured with allogeneic naïve CD4+ T cells, after which IL-10 production (ELISA) and Treg (Foxp3+CD25+CD127low) induction (flow cytometry) was determined. Skin-explants were intradermally injected with Fel d 1 eBPs, nFel d 1, or alum-nFel d 1 and crawl out cells of these injected biopsies were analyzed using a 22-color spectral flow cytometry panel optimized for the characterization of skin-derived APCs. In addition, 0.3-cm skin grafts were obtained using a dermatome, after which the epidermal and dermal sheets were separated and enzymatically digested to isolate dermal APCs. These cells were stimulated with Fel d 1 eBPs and natural Fel d 1 (nFel d 1) for 6 or 16 hours to determine which intracellular pathways are activated.   

This study confirmed previous findings that Fel d 1 eBPs induced moDC maturation (HLA-DR, CD80, CD83, CD86) and cytokine production (IL-10, IL-6). Co-cultures of moDCs and naïve CD4+ T cells demonstrated that Fel d 1 eBPs induced Tregs and production of IL-10 by T cells. To assess whether these effects are also observed when the eBPs are applied intradermally, a 22-color spectral flow cytometry panel was successfully optimized to phenotype skin-derived crawl-out cells, to enable high-dimensional characterization of APCs. The panel allows discrimination of major skin-resident APC subsets, including epidermal and dermal populations, while maintaining viability and signal resolution. The ex vivo skin explant and intradermal injection workflow was established and reproducibly yielded sufficient cell numbers for downstream phenotypic analysis. In parallel, protocols for isolation of dermal APC subsets suitable for transcriptomic profiling were finalized . Samples generated using this workflow have been prepared and are currently being processed for RNA sequencing.

In this study, we demonstrated induction of Tregs by Fel d 1 eBPs and established an ex vivo human skin vaccination platform combined with a high-dimensional flow cytometry panel for the analysis of skin-derived APCs. The optimized workflow enables phenotypic characterization and downstream transcriptomic analyses. Ongoing RNA sequencing will further elucidate the molecular responses of skin-resident immune cells to Fel d 1 eBPs.