Specific protein antigen delivery to human Langerhans cells in intact skin

Immune modulating therapies and vaccines are in high demand, not least to the recent global spread of SARS-CoV2. To achieve efficient activation of the immune system, professional antigen presenting cells have proven to be key coordinators of such responses. Especially targeted approaches, actively directing antigens to specialized dendritic cells, promise to be more effective and accompanied by reduced payload due to less off-target effects. Although antibody and glycan-based targeting of receptors on dendritic cells have been employed, these are often expensive and time-consuming to manufacture or lack sufficient specificity. Thus, we applied a small-molecule ligand that specifically binds Langerin, a hallmark receptor on Langerhans cells, conjugated to a model protein antigen. Via microneedle injection, this construct was intradermally administered into intact human skin explants, selectively loading Langerhans cells in the epidermis. The ligand-mediated cellular uptake outpaces protein degradation resulting in intact antigen delivery. Due to the pivotal role of Langerhans cells in induction of immune responses, this approach of antigen-targeting of tissue-resident immune cells offers a novel way to deliver highly effective vaccines with minimally invasive administration

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

Macrophages and Fibroblasts Differentially Contribute to Tattoo Stability

International audienceBackground: Little information is available about the complexity and function of skin cells contributing to the high stability of tattoos. It has been shown that dermal macrophages play an important role in the storage and maintenance of pigment particles. By contrast, the impact of dermal fibro-blasts, forming the connective tissue of the skin, on the stability of the tattoo is not known. Method: In this study, we compared the cell number and the particle load in dermal macrophages versus dermal fibroblasts, isolated from tail skin of tattooed mice. Results: Microscopic analysis revealed that both cell populations contained the tattoo particles, although in largely different amounts. A small number of mac-rophages with high side scatter intensity contained a large quantity of pigment particles, whereas a high number of der-mal fibroblasts harbored only a few pigment particles. Using the CD64 dtr mouse model that allows for selective, diphtheria toxin-mediated depletion of macrophages, we have previously shown that macrophages hold the tattoo in place by capture-release and recapture cycles. In the tattooed skin of macrophage-depleted mice, the content of pigment particles in fibroblasts did not change; however, the total number of fibroblasts carrying particles increased. Conclusion: The present study demonstrates that dermal macrophages and fibroblasts contribute in different ways to the tattoo stability and further improves our knowledge on tattoo persistence

Immune Reactions against Gene Gun Vaccines Are Differentially Modulated by Distinct Dendritic Cell Subsets in the Skin

<div><p>The skin accommodates multiple dendritic cell (DC) subsets with remarkable functional diversity. Immune reactions are initiated and modulated by the triggering of DC by pathogen-associated or endogenous danger signals. In contrast to these processes, the influence of intrinsic features of protein antigens on the strength and type of immune responses is much less understood. Therefore, we investigated the involvement of distinct DC subsets in immune reactions against two structurally different model antigens, <i>E</i>. <i>coli</i> beta-galactosidase (betaGal) and chicken ovalbumin (OVA) under otherwise identical conditions. After epicutaneous administration of the respective DNA vaccines with a gene gun, wild type mice induced robust immune responses against both antigens. However, ablation of langerin⁺ DC almost abolished IgG1 and cytotoxic T lymphocytes against betaGal but enhanced T cell and antibody responses against OVA. We identified epidermal Langerhans cells (LC) as the subset responsible for the suppression of anti-OVA reactions and found regulatory T cells critically involved in this process. In contrast, reactions against betaGal were not affected by the selective elimination of LC, indicating that this antigen required a different langerin⁺ DC subset. The opposing findings obtained with OVA and betaGal vaccines were not due to immune-modulating activities of either the plasmid DNA or the antigen gene products, nor did the differential cellular localization, size or dose of the two proteins account for the opposite effects. Thus, skin-borne protein antigens may be differentially handled by distinct DC subsets, and, in this way, intrinsic features of the antigen can participate in immune modulation.</p></div

βGal- and OVA gene gun vaccines react autonomously and do not influence each other.

<p>LangDTR or B6 WT mice were injected once with 1μg DT and GG-immunized 1wk later with either pCI-βGal or pCI-OVA, or an equimolar mix of pCI-βGal/pCI-OVA plasmids co-precipitated onto the same gold particles. Mice were boosted 1 wk after the first immunization and analyzed 1wk later. Specific lysis of (A) DAPIYTNV-pulsed or (B) SIINFEKL-pulsed syngeneic target cells injected into GG-immunized langDTR and WT mice. Splenocytes were in vitro restimulated with (C) DAPIYTNV, (D), SIINFEKL, (E) βGal protein or (F) OVA protein and analyzed for IFNγ secretion by ELISPOT technique. Data represent means ± SD of groups of 5 mice and are representative of two experiments.</p

Epidermal LC are dispensable for βGal-specific immunity but down-regulate OVA-specific T cell functions.

<p>LangDTR or B6 WT mice were injected once with 1μg DT. Epidermal sheets were stained for langerin expression 3 wks later. (B) Repopulation of langerin⁺ DC in the dermis analyzed by FACS. LdDC from back skin were identified as CD11c⁺ EpCam^neg langerin⁺ cells. Groups of langDTR and B6 WT mice, treated once with 1μg DT were GG-immunized with either, pCI-OVA or pCI-βGal starting 1wk after DT treatment and boosted after 1wk. Splenocytes were in vitro restimulated with (C) CTL peptides DAPIYTNV for βGal- (left panel) or SIINFEKL for OVA-immunized mice (right panel) or with (E) βGal protein (left panel) or OVA protein (right panel) and analyzed for IFNγ secretion by ELISPOT technique. (D) Specific lysis of DAPIYTNV-pulsed (left panel) or SIINFEKL-pulsed (right panel) syngeneic target cells injected into GG-immunized langDTR and WT mice. (F) IgG1 antibody titers were analyzed by ELISA. Data represent means ± SD of groups of 4–5 mice and representative at least of two experiments.</p

Antigen-specific immune modulation is not due to differential cellular localization of the vaccine gene product.

<p>(A) Western blots of cell lysates (L) and supernatants (SN) of BHK cells transfected with the respective gene vaccines pCI-OVA or pCI-cherryOVA. LC-depleted langDTR and DT-treated B6 WT mice were GG-immunized with pCI-cherryOVA twice at a 1 wk interval, starting 1wk after a single DT injection. One week after the boost splenocytes were restimulated in vitro with (B) SIINFEKL or (<i>D</i>, <i>E</i>) OVA protein and analyzed for (B, D) IFNγ and (E) IL4 secretion by ELISPOT technique. (C) Specific lysis of SIINFEKL-pulsed syngeneic target cells injected into langDTR and WT mice immunized with pCI-cherryOVA. (F) Serum IgG1 titer from pCI-cherryOVA-immunized mice was analyzed by ELISA. Data represent means ± SD of groups of 5 mice. Data shown are representative of two independent experiments.</p

Tumor-targeted therapy with BRAF-inhibitor recruits activated dendritic cells to promote tumor immunity in melanoma

Background Tumor-targeted therapy causes impressive tumor regression, but the emergence of resistance limits long-term survival benefits in patients. Little information is available on the role of the myeloid cell network, especially dendritic cells (DC) during tumor-targeted therapy.Methods Here, we investigated therapy-mediated immunological alterations in the tumor microenvironment (TME) and tumor-draining lymph nodes (LN) in the D4M.3A preclinical melanoma mouse model (harboring the V-Raf murine sarcoma viral oncogene homolog B (BRAF)V600E mutation) by using high-dimensional multicolor flow cytometry in combination with multiplex immunohistochemistry. This was complemented with RNA sequencing and cytokine quantification to characterize the immune status of the tumors. The importance of T cells during tumor-targeted therapy was investigated by depleting CD4+ or CD8+ T cells in tumor-bearing mice. Tumor antigen-specific T-cell responses were characterized by performing in vivo T-cell proliferation assays and the contribution of conventional type 1 DC (cDC1) to T-cell immunity during tumor-targeted therapy was assessed using Batf3−/− mice lacking cDC1.Results Our findings reveal that BRAF-inhibitor therapy increased tumor immunogenicity, reflected by an upregulation of genes associated with immune activation. The T cell-inflamed TME contained higher numbers of activated cDC1 and cDC2 but also inflammatory CCR2-expressing monocytes. At the same time, tumor-targeted therapy enhanced the frequency of migratory, activated DC subsets in tumor-draining LN. Even more, we identified a cDC2 population expressing the Fc gamma receptor I (FcγRI)/CD64 in tumors and LN that displayed high levels of CD40 and CCR7 indicating involvement in T cell-mediated tumor immunity. The importance of cDC2 is underlined by just a partial loss of therapy response in a cDC1-deficient mouse model. Both CD4+ and CD8+ T cells were essential for therapy response as their respective depletion impaired therapy success. On resistance development, the tumors reverted to an immunologically inert state with a loss of DC and inflammatory monocytes together with the accumulation of regulatory T cells. Moreover, tumor antigen-specific CD8+ T cells were compromised in proliferation and interferon-γ-production.Conclusion Our results give novel insights into the remodeling of the myeloid landscape by tumor-targeted therapy. We demonstrate that the transient immunogenic tumor milieu contains more activated DC. This knowledge has important implications for the development of future combinatorial therapies