PD-L1- and calcitriol-dependent liposomal antigen-specific regulation of systemic inflammatory autoimmune disease.

Autoimmune diseases resulting from MHC class II-restricted autoantigen-specific T cell immunity include the systemic inflammatory autoimmune conditions rheumatoid arthritis and vasculitis. While currently treated with broad-acting immunosuppressive drugs, a preferable strategy is to regulate antigen-specific effector T cells (Teffs) to restore tolerance by exploiting DC antigen presentation. We targeted draining lymph node (dLN) phagocytic DCs using liposomes encapsulating 1α,25-dihydroxyvitamin D3 (calcitriol) and antigenic peptide to elucidate mechanisms of tolerance used by DCs and responding T cells under resting and immunized conditions. PD-L1 expression was upregulated in dLNs of immunized relative to naive mice. Subcutaneous administration of liposomes encapsulating OVA323-339 and calcitriol targeted dLN PD-L1hi DCs of immunized mice and reduced their MHC class II expression. OVA323-339/calcitriol liposomes suppressed expansion, differentiation, and function of Teffs and induced Foxp3+ and IL-10+ peripheral Tregs in an antigen-specific manner, which was dependent on PD-L1. Peptide/calcitriol liposomes modulated CD40 expression by human DCs and promoted Treg induction in vitro. Liposomes encapsulating calcitriol and disease-associated peptides suppressed the severity of rheumatoid arthritis and Goodpasture's vasculitis models with suppression of antigen-specific memory T cell differentiation and function. Accordingly, peptide/calcitriol liposomes leverage DC PD-L1 for antigen-specific T cell regulation and induce antigen-specific tolerance in inflammatory autoimmune diseases.

Combined Exposure of Activated Intestinal Epithelial Cells to Nondigestible Oligosaccharides and CpG-ODN Suppresses Th2-Associated CCL22 Release While Enhancing Galectin-9, TGFß, and Th1 Polarization.

BACKGROUND: Short-chain galacto- and long-chain fructo-oligosaccharides (scGOS/lcFOS) and CpG-ODN affect intestinal epithelial cells (IEC). Epithelial IL1α may contribute to allergic sensitization via autocrine mediator release affecting dendritic cells (DC). We studied whether IL1α contributes to Th2-associated mediator release by activated IEC and IEC/DC cocultures and possible modulation by scGOS/lcFOS±CpG-ODN. METHODS: Solid phase or transwell cultured IEC were preincubated with IL1α and/or IFNγ/TNFα for 6 h. The transwell IEC were also apically exposed to scGOS/lcFOS±CpG-ODN for 6 h, washed, and re-exposed, while cocultured with immature moDC (ccDC) for 48 h. These ccDC were subsequently added to allogeneic naïve T cells (MLR). IEC- and/or DC-derived mediators and T cell cytokines were measured. RESULTS: IL1α tended to enhance IL25 and enhanced IL33 and CCL20 release by IEC, while IL1α or TNFα or IFNγ enhanced CCL22. These were all further increased upon combined exposure of IFNγ/TNFα±IL1α coinciding with increased IL33 secretion in the solid phase culture. In the transwell, IL25 and IL33 remained under detection, while CCL20 and CCL22 were induced by IL1α or IFNγ/TNFα, respectively, and a synergistic increase was observed upon combined exposure of IFNγ/TNFα and IL1α. Furthermore, IFNγ was found to enhance galectin-9 secretion, which was more pronounced in IFNγ/TNFα±IL1α-exposed IEC and coincided with TGFß increase. Epithelial CpG-ODN exposure further increased CCL20, while reducing CCL22 release by IFNγ/TNFα/IL1α-activated IEC; however, scGOS/lcFOS suppressed both. Combined scGOS/lcFOS and CpG-ODN reduced CCL22, while CCL20 and regulatory galectin-9 and TGFß remained high in the supernatant of IFNγ/TNFα/IL1α-activated IEC and the following IEC/DC coculture. ccDC of scGOS/lcFOS- and CpG-ODN-exposed IFNγ/TNFα/IL1α-activated IEC increased IFNγ, IL10, TGFß, and galectin-9 secretion in the MLR compared to ccDC exposed to control-activated IEC. CONCLUSION: IL1α enhanced CCL20 and Th2-associated CCL22 release by IFNγ/TNFα-activated IEC. Combined scGOS/lcFOS and CpG-ODN exposure suppressed CCL22, while maintaining high CCL20, TGFß, and galectin-9 concentrations. In addition, ccDC derived from this IEC/DC coculture enhanced Th1 and regulatory mediator secretion mimicking known in vivo effects.

The Human Glycoprotein Salivary Agglutinin Inhibits the Interaction of DC-SIGN and Langerin with Oral Micro-Organisms.

Salivary agglutinin (SAG), also known as gp340 or SALSA, is a glycoprotein encoded by the Deleted in Malignant Brain Tumours 1 gene and is abundantly present in human saliva. SAG aggregates bacteria and viruses, thereby promoting their clearance from the oral cavity. The mucosa lining the oral cavity contains dendritic cells (DC) and Langerhans cells (LC), which express the C-type lectin receptors (CLR) DC-SIGN and Langerin, respectively. Both DC-SIGN and Langerin recognise mannose and fucose carbohydrate structures on pathogens and self-glycoproteins to regulate immunity and homeostasis. The purpose of this study was to investigate whether SAG interacts with these CLR and whether this interferes with the binding to oral pathogens. We show that whole parotid saliva and SAG, when coated to microplates, strongly interact with DC-SIGN and Langerin, probably via mannose and fucose structures. Also, primary human DC and LC bind parotid saliva and SAG via DC-SIGN and Langerin, respectively. Furthermore, SAG binding to DC-SIGN or Langerin prevented binding to the micro-organisms Candida albicans and Escherichia coli which express mannose and fucose-containing glycan structures. Thus, binding of saliva glycoprotein SAG to DC-SIGN and Langerin may inhibit pathogen-DC/LC interactions, and could prove to be a new immunomodulatory mechanism of SAG.

Glycan modification of antigen alters its intracellular routing in dendritic cells, promoting priming of T cells.

Antigen uptake by dendritic cells and intracellular routing of antigens to specific compartments is regulated by C-type lectin receptors that recognize glycan structures. We show that the modification of Ovalbumin (OVA) with the glycan-structure Lewis(X) (Le(X)) re-directs OVA to the C-type lectin receptor MGL1. Le(X)-modification of OVA favored Th1 skewing of CD4(+) T cells and enhanced cross-priming of CD8(+) T cells. While cross-presentation of native OVA requires high antigen dose and TLR stimuli, Le(X) modification reduces the required amount 100-fold and obviates its dependence on TLR signaling. The OVA-Le(X)-induced enhancement of T cell cross-priming is MGL1-dependent as shown by reduced CD8(+) effector T cell frequencies in MGL1-deficient mice. Moreover, MGL1-mediated cross-presentation of OVA-Le(X) neither required TAP-transporters nor Cathepsin-S and was still observed after prolonged intracellular storage of antigen in Rab11(+)LAMP1(+) compartments. We conclude that controlled neo-glycosylation of antigens can crucially influence intracellular routing of antigens, the nature and strength of immune responses and should be considered for optimizing current vaccination strategies.

IL-10/IFNγ co-expressing CD4(+) T cells induced by IL-10 DC display a regulatory gene profile and downmodulate T cell responses.

Induced regulatory T cells (iTreg) are imperative for tolerance induction and spreading of infectious tolerance. Ex vivo generated tolerogenic dendritic cells (tDCs) have strong therapeutic potential to induce antigen-specific iTreg. We previously demonstrated that IL-10 tDC-primed T cells are very suppressive and produce IL-10. Here, we show that the majority of IL-10(+) T cells co-express IFNγ, giving rise to the question whether these cells are proinflammatory or regulatory. Whole genome gene expression analysis revealed a strong regulatory gene profile and a suppressed Th1 gene profile for IL-10/IFNγ co-expressing CD4(+) T cells. Protein analysis confirmed an extensive regulatory phenotype for IL-10(+)/IFNγ(+) T cells, with specific enhanced expression of GARP and PD-1. In line with these data, isolated IL-10(+)/IFNγ(+) T cells displayed potent suppressive capacity. Thus, IL-10/IFNγ co-expressing CD4(+) T cells induced by IL-10 tDC show dominance of immunomodulation over Th1-mediated immunoactivation and can contribute to induction or spreading of immunological tolerance.

MPLA incorporation into DC-targeting glycoliposomes favours anti-tumour T cell responses.

Dendritic cells (DC) are attractive targets for cancer immunotherapy as they initiate strong and long-lived tumour-specific T cell responses. DC can be effectively targeted in vivo with tumour antigens by using nanocarriers such as liposomes. Cross-presentation of tumour antigens is enhanced with strong adjuvants such as TLR ligands. However, often these adjuvants have off-target effects, and would benefit from a DC-specific targeting strategy, similar to the tumour antigen. The goal of this study was to develop a strategy for specifically targeting DC with tumour antigen and adjuvant by using glycoliposomes. We have generated liposomes containing the glycan Lewis(Le)(X) which is highly specific for the C-type lectin receptor DC-SIGN expressed by DC. Le(X)-modified liposomes were taken up by human monocyte-derived DC in a DC-SIGN-specific manner. As adjuvants we incorporated the TLR ligands Pam3CySK4, Poly I:C, MPLA and R848 into liposomes and compared their adjuvant capacity on DC. Incorporation of the TLR4 ligand MPLA into glycoliposomes induced DC maturation and production of pro-inflammatory cytokines, in a DC-SIGN-specific manner, and DC activation was comparable to administration of soluble MPLA. Incorporation of MPLA into glycoliposomes significantly enhanced antigen cross-presentation of the melanoma tumour antigen gp100280-288 peptide to CD8(+) T cells compared to non-glycosylated MPLA liposomes. Importantly, antigen cross-presentation of the gp100280-288 peptide was significantly higher using MPLA glycoliposomes compared to the co-administration of soluble MPLA with glycoliposomes. Taken together, our data demonstrates that specific targeting of a gp100 tumour antigen and the adjuvant MPLA to DC-SIGN-expressing DC enhances the uptake of peptide-containing liposomes, the activation of DC, and induces tumour antigen-specific CD8(+) T cell responses. These data demonstrate that adjuvant-containing glycoliposome-based vaccines targeting DC-SIGN(+) DC represent a powerful new approach for CD8(+) T cell activation.

Controlled release of a model vaccine by nanoporous ceramic microneedle arrays.

Current vaccination technology can advance from the use of novel ceramic nanoporous microneedle arrays (npMNA), where the material serves as a storage reservoir for vaccines. Moreover, npMNA will enhance vaccine efficacy by more precisely reaching skin dendritic cells, the kickstarters of T and B cell immunity. In the present study we assessed the efficacy of vaccination using npMNAs by in vivo application of OVA257-264 peptides mixed with agonistic anti-CD40 antibodies as adjuvant. The induction of OVA-specific CD8(+) T cells via npMNA was comparable with the frequency induced via intradermal injection using needle-syringe. However, only when expanding the vaccination area by using two npMNAs the frequencies of induced IFN-γ-specific effector CD8(+) T cells were comparable with those induced via needle-syringe injection. Analysis of vaccine release from npMNA in a human ex vivo skin explant model revealed that OVA257-264 peptides were indeed delivered intradermal, and release also increased by prolonging the npMNA application time on the human skin. Together, our studies demonstrate the potential of npMNA for vaccine delivery in human skin and in vivo induction of CD8(+) effector T cell responses.

In situ Delivery of Tumor Antigen- and Adjuvant-Loaded Liposomes Boosts Antigen-Specific T-Cell Responses by Human Dermal Dendritic Cells.

Dendritic cells (DCs) have an important role in tumor control via the induction of tumor-specific T-cell responses and are therefore an ideal target for immunotherapy. The human skin is an attractive site for tumor vaccination as it contains various DC subsets. The simultaneous delivery of tumor antigen with an adjuvant is beneficial for cross-presentation and the induction of tumor-specific T-cell responses. We therefore developed liposomes that contain the melanoma-associated antigen glycoprotein 100280-288 peptide and Toll-like receptor 4 (TLR4) ligand monophosphoryl lipid A (MPLA) as adjuvant. These liposomes are efficiently taken up by monocyte-derived DCs, and antigen presentation to CD8(+) T cells was significantly higher with MPLA-modified liposomes as compared with non-modified liposomes or the co-administration of soluble MPLA. We used a human skin explant model to evaluate the efficiency of intradermal delivery of liposomes. Liposomes were efficiently taken up by CD1a(+) and especially CD14(+) dermal DCs. Induction of CD8(+) T-cell responses by emigrated dermal DCs was significantly higher when MPLA was incorporated into the liposomes as compared with non-modified liposomes or co-administration of soluble MPLA. Thus, the modification of antigen-carrying liposomes with TLR ligand MPLA significantly enhances tumor-specific T-cell responses by dermal DCs and is an attractive vaccination strategy in human skin.

Inhibition of TNF receptor signaling by anti-TNFα biologicals primes naïve CD4(+) T cells towards IL-10(+) T cells with a regulatory phenotype and function.

TNFα is a potent pro-inflammatory cytokine playing a pivotal role in several autoimmune diseases. Little is known about the mechanism of TNFα blocking agents on naïve T cell differentiation. Here, we report that neutralizing TNFα during priming of naïve CD4(+) T cells by dendritic cells favors development of IL-10(+) T helper cells. TNFα counteracts IL-10(+) T cell priming mainly via TNFRI receptor signaling. While initial T cell activation was not affected, neutralization of TNFα negatively affected sustained T cell differentiation in later stages of T cell priming. Whole genome gene expression analysis revealed an extended regulatory gene profile for anti-TNFα-treated T cells. Indeed, neutralizing TNFα during naïve T cell priming enhanced the suppressive function of anti-TNFα-treated T cells. Taken together, inhibition of TNFα-TNFR interaction shifts the balance of Th cell differentiation towards IL-10 expressing suppressive T cells, which may be one of the beneficial mechanisms in TNFα blocking therapies.

IL-10-generated tolerogenic dendritic cells are optimal for functional regulatory T cell induction--a comparative study of human clinical-applicable DC.

Tolerogenic dendritic cells (tDC) are a promising tool for specific cellular therapy to induce immunological tolerance in transplantation and autoimmunity. To date, most described tDC methods have not been converted into clinically applicable protocols and systematic comparison of required functional characteristics, i.e. migration and functional regulatory T cell (Treg) induction, is lacking. We compare clinical-grade tDC generated with vitamin D(3), IL-10, dexamethasone, TGFß or rapamycin. For good migratory capacity and a stable phenotype, additional maturation of tDC was required. Maturation with a cocktail of TNFα, IL-1ß and PGE(2) induced optimal migration. Importantly, all tDC showed a stable phenotype under pro-inflammatory conditions. Especially IL-10 DC showed most powerful tolerogenic characteristics with high IL-10 production and low T cell activation. Moreover, in a functional suppression assay only IL-10 DC induced Treg that strongly suppressed T cell reactivity. Thus, clinical-grade IL-10 DC show functional characteristics that make them best suited for tolerance-inducing therapies.

MGL-mediated internalization and antigen presentation by dendritic cells: a role for tyrosine-5.

Professional antigen-presenting cells are essential for the initiation of adaptive immune responses; however, they also play a vital role in the maintenance of tolerance towards self-antigens. C-type lectins can function as antigen receptors by capturing carbohydrate ligands for processing and presentation. Here, we focused on the dendritic cell (DC)-expressed macrophage galactose-type lectin (MGL), a C-type lectin with a unique specificity for terminal GalNAc residues, such as the tumor-associated Tn antigen. Soluble model antigens are efficiently internalized by MGL and subsequently presented to responder CD4+ T cells. The tyrosine-5 residue in the YENF motif, present in the MGL cytoplasmic domain, was essential for the MGL-mediated endocytosis in CHO cells. In conclusion, MGL contributes to the antigen processing and presentation capacities of DC and may provide a suitable target for the initiation of anti-tumor immune responses.