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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

Additional file 1: Supplement Figure 1. Detection of adipocytes in unfixed granulation tissue. Intra-vital confocal microscopy single channel images of unfixed granulation tissue compared with subcutaneous fat tissue (merge displayed in Fig. 1b) stained with BODIPY-493/503 to highlight lipid droplets, wheat germ agglutinin (CF®555 WGA) to highlight ECM substrates and cell morphology (N-acetyl-D-glucosamine and sialic acid present in hyaluronan and other heterogeneous polysaccharides), and Hoechst 33342 staining for nuclei. Supplement Figure 2. PPARG overexpression in day 3 differentiated cells. Representative immunoblotting of PPARG- or puromycin overexpressing human ASC subjected to in vitro differentiation for 3 days in the presence of macrophage-CM. Supplement Figure 3. Numbers of NCT-regulated cytokines. Comparison of cytokines with greater than two-fold change in response to NCT-treatment in various CM. Shown are the total number of regulated cytokines per CM (Set Size), and the number of regulated cytokines found exclusively in one CM or shared between different CM types (Intersection Size). Intersections between different CM types are indicated with connected black dots, single dots indicate a single CM. As shown, 22, 7, 6 and 2 cytokines were found exclusively regulated by M(IFNG/LPS), Mo, M0 and M(IL4/IL13) respectively. Four cytokines were similarly regulated in M(IFNG/LPS) and Mo, 3 in M(IFNG/LPS) and M0, 2 in M0 and M(IL4/IL13), 1 in M(IFNG/LPS) and M(IL4/IL13), and 2 in M(IFNG/LPS), M0 and M(IL4/IL13). Supplement Figure 4. Efficacy of IL1RA to inhibit anti-adipogenic effect of IL1B. IL1B receptor mediated signaling was inhibited by the addition of 100ng/ml recombinant IL1RA. Effect on adipocyte differentiation was evaluated by flow cytometry assessing numbers of lipid-laden cells. Values depict the percentage of lipid-laden adipocytes as a percentage of untreated control cells (0ng IL1B) that emit green fluorescence after staining with BODIPY-493/503, n=3. Statistical significance was determined by using non parametric unpaired Student’s t-test. Data are shown as mean ± SEM. Asterisks indicate p-values < 0.05 (*). Supplement Table 1. Table with log2 cytokine protein expression levels used to generate the heatmap in Fig. 2c. (1) and (2) depict values obtained from two independent measurements. Supplement Table 2. Regulation of cytokines by NCT co-treatment in various CM. Contains log2 fold change (M) and log2 average (A) values representing the extent of regulation, and average expression, of cytokines following NCT co-treatment of CM (e.g. a comparison of NCT co-treated Mo cells against untreated Mo cells). Supplement Table 3. List of Primers used for quantitative RT-PCR.

Vascular endothelial cells (ECs) and several cancer cells express B7h, which is the ligand of the ICOS T cell costimulatory molecule. We have previously shown that B7h triggering via a soluble form of ICOS (ICOS-Fc) inhibits the adhesion of polymorphonuclear and tumor cell lines to HUVECs; thus, we suggested that ICOS-Fc may act as an anti-inflammatory and antitumor agent. Because cancer cell migration and angiogenesis are crucial for metastasis dissemination, the aim of this work was to evaluate the effect of ICOS-Fc on the migration of cancer cells and ECs. ICOS-Fc specifically inhibited the migration of HUVECs, human dermal lymphatic ECs, and the HT29, HCT116, PC-3, HepG2, JR8, and M14 tumor cell lines expressing high levels of B7h, whereas it was ineffective in the RPMI7932, PCF-2, LM, and BHT-101 cell lines expressing low levels of B7h. Furthermore, ICOS-Fc downmodulated hepatocyte growth factor facilitated the epithelial-to-mesenchymal transition in HepG2 cells. Moreover, ICOS-Fc downmodulated the phosphorylation of focal adhesion kinase and the expression of β-Pix in both HUVECs and tumor cell lines. Finally, treatment with ICOS-Fc inhibited the development of lung metastases upon injection of NOD-SCID-IL2Rγnull mice with CF-PAC1 cells, as well as C57BL/6 mice with B16-F10 cells. Therefore, the B7h−ICOS interaction may modulate the spread of cancer metastases, which suggests the novel use of ICOS-Fc as an immunomodulatory drug. However, in the B16-F10–metastasized lungs, ICOS-Fc also increased IL-17A/RORc and decreased IL-10/Foxp3 expression, which indicates that it also exerts positive effects on the antitumor immune response.

13 p.-7 fig.-1 tab., Inducible T-cell costimulator (ICOS) upon binding to its ligand (ICOSL) mediates adaptive immunity and antitumor response. Thus, antitumor therapies targeting the ICOS/ICOSL pathway hold great promise for cancer treatment. In this regard, ICOSL triggering by a soluble recombinant form of ICOS (ICOS-Fc) hampered adhesiveness and migration of dendritic, endothelial, and tumor cells in vitro. Furthermore, in vivo treatment with ICOS-Fc previously showed the capability to inhibit lung metastatization of ICOSL+ B16-F10 melanoma cells when injected intravenously in mice, but it failed to block the growth of established subcutaneous B16-F10 murine tumors. Thus, we asked whether passive targeting of solid tumors with ICOS-Fc-loaded biocompatible and biodegradable nanoparticles (NPs) could instead prove effectiveness in reducing tumor growth. Here, ICOS-Fc was loaded in two types of polymer nanoparticles, i.e. cross-linked β-cyclodextrin nanosponges (CDNS) and poly(lactic-co-glycolic acid) (PLGA) NPs and in vitro characterized. In vivo experiments showed that treatment of C57BL6/J mice with ICOS-Fc loaded into the two nanoformulations inhibits the growth of established subcutaneous B16-F10 tumors. This anticancer activity appears to involve both anti-angiogenic and immunoregulatory effects, as shown by decreased tumor vascularization and downmodulation of IL-10 and Foxp3, two markers of regulatory T cells (Tregs). Overall, the substantial in vivo anticancer activity of ICOS-Fc-loaded CDNS and PLGA NPs against different components of the tumor microenvironment makes these nanoformulations attractive candidates for future combination cancer therapy., This work was supported by the Associazione Italiana Ricerca sul Cancro (IG 20714, AIRC, Milano), Fondazione Amici di Jean (Torino), and Fondazione Cariplo (2017–0535).

A defective switching off of the immune response is involved in several autoimmune diseases. This switching off involves Fas-mediated apoptosis, perforin-mediated fratricide of activated lymphocytes, and the suppressive activity of regulatory T (Treg) cells. These mechanisms are altered in autoimmune lymphoproliferative syndrome that often displays autoimmune thrombocytopenia. The aim of this research was to evaluate these mechanisms in adult patients with primary immune thrombocytopenia (ITP), compared with healthy controls. The results show that a substantial subgroup of the ITP patients displayed a defective Fas function; most of them displayed decreased Fas expression in T cells activated in vitro. Moreover, ITP patients displayed an increased frequency of rare missense variations of the PRF1 gene and decreased levels of Treg. Immunological analysis showed that levels of Interleukin (IL)10 and IL17 were decreased and marginal zone B cells were increased. Moreover, myeloid and plasmacytoid dendritic cells were decreased in ITP patients. In conclusion, in adult ITP patients, several mechanisms involved in shutting off the immune response are defective and several immunological parameters are dysregulated; these alterations may play a role in the clinical heterogeneity of the disease.