Nadine van Montfoort

Antigen storage compartments in mature dendritic cells facilitate prolonged cytotoxic T lymphocyte cross-priming capacity

Dendritic cells (DCs) are crucial for priming of naive CD8+ T lymphocytes to exogenous antigens, so-called “cross-priming.” We report that exogenous protein antigen can be conserved for several days in mature DCs, coinciding with strong cytotoxic T lymphocyte cross-priming potency in vivo. After MHC class I peptide elution, protein antigen-derived peptide presentation is efficiently restored, indicating the presence of an intracellular antigen depot. We characterized this depot as a lysosome-like organelle, distinct from MHC class II compartments and recently described early endosomal compartments that allow acute antigen presentation in MHC class I. The storage compartments we report here facilitate continuous supply of MHC class I ligands. This mechanism ensures sustained cross-presentation by DCs, despite the short-lived expression of MHC class I–peptide complexes at the cell surface.

Immune Complex-Loaded Dendritic Cells Are Superior to Soluble Immune Complexes as Antitumor Vaccine

Dendritic cells (DCs) play an important role in the induction of T cell responses. FcγRs, expressed on DCs, facilitate the uptake of complexed Ag, resulting in efficient MHC class I and MHC class II Ag presentation and DC maturation. In the present study, we show that prophylactic immunization with DCs loaded with Ag-IgG immune complexes (ICs) leads to efficient induction of tumor protection in mice. Therapeutic vaccinations strongly delay tumor growth or even prevent tumors from growing out. By depleting CD4+ and CD8+ cell populations before tumor challenge, we identify CD8+ cells as the main effector cells involved in tumor eradication. Importantly, we show that DCs that are preloaded in vitro with ICs are at least 1000-fold more potent than ICs injected directly into mice or DCs loaded with the same amount of noncomplexed protein. The contribution of individual FcγRs to Ag presentation, T cell response induction, and induction of tumor protection was assessed. We show that FcγRI and FcγRIII are capable of enhancing MHC class I-restricted Ag presentation to CD8+ T cells in vitro and that these activating FcγRs on DCs are required for efficient priming of Ag-specific CD8+ cells in vivo and induction of tumor protection. These findings show that targeting ICs via the activating FcγRs to DCs in vitro is superior to direct IC vaccination to induce protective tumor immunity in vivo.

Antigen processing by nardilysin and thimet oligopeptidase generates cytotoxic T cell epitopes

Cytotoxic T lymphocytes (CTLs) recognize peptides presented by HLA class I molecules on the cell surface. The C terminus of these CTL epitopes is considered to be produced by the proteasome. Here we demonstrate that the cytosolic endopeptidases nardilysin and thimet oligopeptidase (TOP) complemented proteasome activity. Nardilysin and TOP were required, either together or alone, for the generation of a tumor-specific CTL epitope from PRAME, an immunodominant CTL epitope from Epstein-Barr virus protein EBNA3C, and a clinically important epitope from the melanoma protein MART-1. TOP functioned as C-terminal trimming peptidase in antigen processing, and nardilysin contributed to both the C-terminal and N-terminal generation of CTL epitopes. By broadening the antigenic peptide repertoire, nardilysin and TOP strengthen the immune defense against intracellular pathogens and cancer.

Lipid profile and carotid intima-media thickness in a prospective cohort of very preterm subjects at age 19 years: effects of early growth and current body composition

Cardiovascular disease (CVD) risk is associated with prenatal and infancy growth. However, the relative importance of these time periods for the CVD risk is uncertain. To elucidate this, we tested in a very preterm cohort the effects of birth weight for gestational age and weight gain between birth and 3 mo post-term (early postnatal weight gain) and between 3 mo and 1 y post-term (late infancy weight gain) on the lipid profile and carotid intima-media thickness (CIMT) at age 19 y. A less favorable lipid profile was strongly associated with higher current body mass index (BMI), greater waist circumference, and greater absolute fat mass. CIMT was positively associated with current height, and with low-density lipoprotein (LDL) cholesterol and apolipoprotein B (ApoB) levels, and LDL/high-density lipoprotein (HDL) cholesterol and ApoB/apolipoprotein AI (ApoAI) ratios. Lipid profile and CIMT were unrelated to gestational age, birth weight standard deviation score (SDS) and early postnatal weight gain. CIMT was positively associated with late infancy weight gain, but the relationship disappeared after correction for current height. Our findings in 19 y olds born very preterm argue for an effect of current body composition, rather than of early growth, on the CVD risk. Attempts to reduce the CVD risk in this specific population should focus on weight reduction in young adulthood rather than on optimizing the early growth pattern.

Fcγ Receptor IIb Strongly Regulates Fcγ Receptor-Facilitated T Cell Activation by Dendritic Cells

FcγR ligation by Ag–Ab immune complexes (IC) not only mediates effective Ag uptake, but also strongly initiates dendritic cell (DC) maturation, a requirement for effective T cell activation. Besides the activating FcγRI, FcγRIII, and FcγRIV, the inhibitory FcγRIIb is expressed on DCs. It is unclear how the ratio between signals from the activating FcγR and the inhibitory FcγRIIb determines the outcome of FcγR ligation on DCs. By microarray analysis, we compared the transcriptomes of steady state and IC-activated bone marrow-derived wild-type (WT) DCs expressing all FcγR or DCs expressing only activating FcγR (FcγRIIb knockout [KO]) or only the inhibitory FcγRIIb (FcR γ-chain KO). In WT DCs, we observed a gene expression profile associated with effective T cell activation, which was absent in FcR γ-chain KO, but strikingly more pronounced in FcγRIIb KO bone marrow-derived DCs. These microarray results, confirmed at the protein level for many cytokines and other immunological relevant genes, demonstrate that the transcriptome of IC-activated DCs is dependent on the presence of the activating FcγR and that the modulation of the expression of the majority of the genes was strongly regulated by FcγRIIb. Our data suggest that FcγRIIb-deficient DCs have an improved capacity to activate naive T lymphocytes. This was confirmed by their enhanced FcγR-dependent Ag presentation and in vivo induction of CD8+ T cell expansion compared with WT DCs. Our findings underscore the potency of FcγR ligation on DCs for the effective induction of T cell immunity by ICs and the strong regulatory role of FcγRIIb.

A Novel Role of Complement Factor C1q in Augmenting the Presentation of Antigen Captured in Immune Complexes to CD8+ T Lymphocytes

Ag-IgG immune complexes (IC) are efficiently taken up, and Ag-derived peptides are subsequently processed and presented by APC. In vitro experiments indicate that IgG Fc Receptors (FcγR) facilitate the efficient uptake of IC by dendritic cells. Previous experiments showed that the cross-presentation of Ag-derived peptides after s.c. administration of IC is FcγR-dependent. To study the role of different FcγR and complement in MHC class I Ag presentation after i.v. administration, we used mice deficient for FcγRs and complement components. These mice were injected with CFSE-labeled OVA-specific CD8+ T cells followed by administration of IC composed of OVA and rabbit anti-OVA IgG i.v. to measure MHC class I presentation of OVA-derived peptides. The Ag presentation was partly reduced in FcRγ-chain-deficient mice, but not affected in FcγRI/II/III-deficient mice, complement factor C3-deficient mice, or FcγRI/II/III × C3-deficient mice. Importantly, CD8+ T cell proliferation was significantly reduced in mice deficient for C1q. This proliferation could be restored when IC were incubated with purified human C1q before injection. Likewise, purified C1q could strongly enhance the uptake and presentation of IC by dendritic cells in vitro. Heat inactivation abrogated the C1q-mediated uptake of IC. In addition, in vivo uptake of OVA-IC in the spleen was significantly reduced in C1q-deficient mice compared with wild-type mice. Together, these results indicate a novel function of C1q, which is present in high levels in the bloodstream, by directly enhancing the uptake and MHC class I presentation of Ag captured in IC by APC to CD8+ T cells.

Circulating specific antibodies enhance systemic cross‐priming by delivery of complexed antigen to dendritic cells in vivo

Increasing evidence suggests that antibodies can have stimulatory effects on T‐cell immunity. However, the contribution of circulating antigen‐specific antibodies on MHC class I cross‐priming in vivo has not been conclusively established. Here, we defined the role of circulating antibodies in cross‐presentation of antigen to CD8+ T cells. Mice with hapten‐specific circulating antibodies, but naϊve for the T‐cell antigen, were infused with haptenated antigen and CD8+ T‐cell induction was measured. Mice with circulating hapten‐specific antibodies showed significantly enhanced cross‐presentation of the injected antigen compared with mice that lacked these antibodies. The enhanced cross‐presentation in mice with circulating antigen‐specific antibodies was associated with improved antigen capture by APCs. Importantly, CD11c+ APCs were responsible for the enhanced and sustained cross‐presentation, although CD11c− APCs had initially captured a significant amount of the injected antigen. Thus, in vivo formation of antigen‐antibody immune complexes improves MHC class I cross‐presentation, and CD8+ T‐cell activation, demonstrating that humoral immunity can aid the initiation of systemic cellular immunity. These findings have important implications for the understanding of the action of therapeutic antibodies against tumor‐associated antigens intensively used in the clinic nowadays.

Kupffer Cells Interact With Hepatitis B Surface Antigen In Vivo and In Vitro, Leading to Proinflammatory Cytokine Production and Natural Killer Cell Function

BACKGROUND Based on their localization, Kupffer cells (KCs) likely interact with hepatitis B virus (HBV). However, the role of KCs in inducing immunity toward HBV is poorly understood. Therefore, the interaction of hepatitis B surface antigen (HBsAg) and KCs, and possible functional consequences, were assessed. METHODS KCs in liver tissue from patients with chronic HBV were analyzed for presence of HBsAg and their phenotype, and compared with KCs in control liver tissue. Liver graft perfusate-derived KCs and in vitro-generated monocyte-derived macrophages were investigated for functional interaction with patient-derived HBsAg. RESULTS Intrahepatic KCs were HBsAg positive and more activated than those from control livers. KCs internalized HBsAg in vitro, which did not change their phenotype, but strongly induced proinflammatory cytokine production. Additionally, monocyte-derived macrophages also interacted with HBsAg, leading to activation and cytokine production. Furthermore, HBsAg-exposed macrophages and KC activated natural killer (NK) cells, resulting in increased CD69 expression and interferon-γ production. CONCLUSIONS KCs directly interact with HBsAg in vivo and in vitro. HBsAg-induced cytokine production by KCs and monocyte-derived macrophages and subsequent NK cell activation may be an early event in viral containment and may support induction of HBV-specific immunity upon HBV infection, but may also contribute to liver pathology.

DC-ATLAS: a systems biology resource to dissect receptor specific signal transduction in dendritic cells

BackgroundThe advent of Systems Biology has been accompanied by the blooming of pathway databases. Currently pathways are defined generically with respect to the organ or cell type where a reaction takes place. The cell type specificity of the reactions is the foundation of immunological research, and capturing this specificity is of paramount importance when using pathway-based analyses to decipher complex immunological datasets. Here, we present DC-ATLAS, a novel and versatile resource for the interpretation of high-throughput data generated perturbing the signaling network of dendritic cells (DCs).ResultsPathways are annotated using a novel data model, the Biological Connection Markup Language (BCML), a SBGN-compliant data format developed to store the large amount of information collected. The application of DC-ATLAS to pathway-based analysis of the transcriptional program of DCs stimulated with agonists of the toll-like receptor family allows an integrated description of the flow of information from the cellular sensors to the functional outcome, capturing the temporal series of activation events by grouping sets of reactions that occur at different time points in well-defined functional modules.ConclusionsThe initiative significantly improves our understanding of DC biology and regulatory networks. Developing a systems biology approach for immune system holds the promise of translating knowledge on the immune system into more successful immunotherapy strategies.

BDCA3+CLEC9A+ human dendritic cell function and development

Dendritic cells (DC) are the most potent antigen presenting cells (APC). They comprise a family of different subsets and play an essential role in the induction and regulation of immune responses. Recently, gene expression profiling identified BDCA3(+)CLEC9A(+) DC as a separate human DC subset. This subset was identified in blood, where they represent the smallest population of human DC, as well as in lymphoid and peripheral tissues. This review summarizes the phenotypic, functional and developmental characteristics of BDCA3(+)CLEC9A(+) DC in relation to their mouse equivalents CD8α(+) DC and CD103(+) DC and other human DC subsets. Apart from being potent antigen presenting cells, their specialized functional capacities compared to other human DC subsets, indicate that these BDCA3(+)CLEC9A(+) DC are of major importance in the induction of anti-viral and anti-tumor immunity. Further characterization of their functional properties, developmental pathways and underlying molecular mechanisms may identify target molecules to fully exploit the immune modulatory function of BDCA3(+)CLEC9A(+) DC and potential use of these cells in immunotherapy.