Targeted delivery of a vaccine protein to Langerhans cells in the human skin via the C-type lectin receptor Langerin

Human skin is a preferred vaccination site as it harbors multiple dendritic cell (DC) subsets, which display distinct C-type lectin receptors (CLR) that recognize pathogens. Antigens can be delivered to CLR by antibodies or ligands to boost antigen-specific immune responses. This concept has been established in mouse models but detailed insights into the functional consequences of antigen delivery to human skin DC in situ are sparse. In this study, we cloned and produced an anti-human Langerin antibody conjugated to the EBV nuclear antigen 1 (EBNA1). We confirmed specific binding of anti-Langerin-EBNA1 to Langerhans cells (LC). This novel LC-based vaccine was then compared to an existing anti-DEC-205-EBNA1 fusion protein by loading LC in epidermal cell suspensions before coculturing them with autologous T cells. After restimulation with EBNA1-peptides, we detected elevated levels of IFN-γ- and TNF-α-positive CD4+ T cells with both vaccines. When we injected the fusion proteins intradermally into human skin explants, emigrated skin DC targeted via DEC-205-induced cytokine production by T cells, whereas the Langerin-based vaccine failed to do so. In summary, we demonstrate that antibody-targeting approaches via the skin are promising vaccination strategies, however, further optimizations of vaccines are required to induce potent immune responses

Laser‐facilitated epicutaneous immunotherapy with hypoallergenic beta‐glucan neoglycoconjugates suppresses lung inflammation and avoids local side effects in a mouse model of allergic asthma

Background Allergen-specific immunotherapy via the skin targets a tissue rich in antigen-presenting cells, but can be associated with local and systemic side effects. Allergen-polysaccharide neoglycogonjugates increase immunization efficacy by targeting and activating dendritic cells via C-type lectin receptors and reduce side effects. Objective We investigated the immunogenicity, allergenicity, and therapeutic efficacy of laminarin-ovalbumin neoglycoconjugates (LamOVA). Methods The biological activity of LamOVA was characterized in vitro using bone marrow-derived dendritic cells. Immunogenicity and therapeutic efficacy were analyzed in BALB/c mice. Epicutaneous immunotherapy (EPIT) was performed using fractional infrared laser ablation to generate micropores in the skin, and the effects of LamOVA on blocking IgG, IgE, cellular composition of BAL, lung, and spleen, lung function, and T-cell polarization were assessed. Results Conjugation of laminarin to ovalbumin reduced its IgE binding capacity fivefold and increased its immunogenicity threefold in terms of IgG generation. EPIT with LamOVA induced significantly higher IgG levels than OVA, matching the levels induced by s.c. injection of OVA/alum (SCIT). EPIT was equally effective as SCIT in terms of blocking IgG induction and suppression of lung inflammation and airway hyperresponsiveness, but SCIT was associated with higher levels of therapy-induced IgE and TH2 cytokines. EPIT with LamOVA induced significantly lower local skin reactions during therapy compared to unconjugated OVA. Conclusion Conjugation of ovalbumin to laminarin increased its immunogenicity while at the same time reducing local side effects. LamOVA EPIT via laser-generated micropores is safe and equally effective compared to SCIT with alum, without the need for adjuvant

Mast cells and γδ T cells are largely dispensable for adaptive immune responses after laser-mediated epicutaneous immunization

Background The skin resembles an attractive target for vaccination due to its accessibility and abundance of resident immune cells. Cells like γδ T cells and mast cells (MCs) are part of the first line of defence against exogenous threats. Despite being important mediators for eliciting TH2 immune responses after epithelial stress, γδ T cell and MC functions still remain to be completely understood. Here, we aimed to characterize their roles in shaping adaptive immune responses after laser-mediated epicutaneous immunization (EPI). Methods γδ T cell knock out, MC-depleted, and wildtype control mice were immunized with mannan-conjugated grass pollen allergen Phl p 5 (P5-MN) by laser-mediated EPI. After 2–3 immunizations, cytokine expression, T helper polarization, and antigen-specific IgG1/IgE levels were analysed. Furthermore, the local cytokine/chemokine milieu after laser microporation was determined. Results The majority of inflammatory chemokines and cytokines induced by laser treatment were not affected by the presence of γδ T cells or MCs. However, RANTES was elevated in γδ T cell knock out mice and GROα, TSLP, and IL-33 were significantly decreased after MC depletion. The absence of γδ T cells or depletion of MCs had no substantial effect on adaptive immune responses after laser-mediated EPI, except for slightly reduced IgG1 and effector T cell levels in MC-depleted mice. Conclusions γδ T cells did not play a pivotal role in shaping the humoral and cellular adaptive immune response after laser-mediated EPI. MC depletion decreased the numbers of effector T cells, indicating a potential role of MCs in the activation and maturation of T cells after EPI

Guidelines for DC preparation and flow cytometry analysis of mouse nonlymphoid tissues

This article is part of the Dendritic Cell Guidelines article series, which provides a collection of state-of-the-art protocols for the preparation, phenotype analysis by flow cytometry, generation, fluorescence microscopy and functional characterization of mouse and human dendritic cells (DC) from lymphoid organs and various nonlymphoid tissues. DC are sentinels of the immune system present in almost every mammalian organ. Since they represent a rare cell population, DC need to be extracted from organs with protocols that are specifically developed for each tissue. This article provides detailed protocols for the preparation of single-cell suspensions from various mouse nonlymphoid tissues, including skin, intestine, lung, kidney, mammary glands, oral mucosa and transplantable tumors. Furthermore, our guidelines include comprehensive protocols for multiplex flow cytometry analysis of DC subsets and feature top tricks for their proper discrimination from other myeloid cells. With this collection, we provide guidelines for in-depth analysis of DC subsets that will advance our understanding of their respective roles in healthy and diseased tissues. While all protocols were written by experienced scientists who routinely use them in their work, this article was also peer-reviewed by leading experts and approved by all coauthors, making it an essential resource for basic and clinical DC immunologists