Sjef Verbeek

Universal Vaccine Based on Ectodomain of Matrix Protein 2 of Influenza A: Fc Receptors and Alveolar Macrophages Mediate Protection

The ectodomain of matrix protein 2 (M2e) of influenza A virus is an attractive target for a universal influenza A vaccine: the M2e sequence is highly conserved across influenza virus subtypes, and induced humoral anti-M2e immunity protects against a lethal influenza virus challenge in animal models. Clinical phase I studies with M2e vaccine candidates have been completed. However, the in vivo mechanism of immune protection induced by M2e-carrier vaccination is unclear. Using passive immunization experiments in wild-type, FcRγ−/−, FcγRI−/−, FcγRIII−/−, and (FcγRI, FcγRIII)−/− mice, we report in this study that Fc receptors are essential for anti-M2e IgG-mediated immune protection. M2e-specific IgG1 isotype Abs are shown to require functional FcγRIII for in vivo immune protection but other anti-M2e IgG isotypes can rescue FcγRIII−/− mice from a lethal challenge. Using a conditional cell depletion protocol, we also demonstrate that alveolar macrophages (AM) play a crucial role in humoral M2e-specific immune protection. Additionally, we show that adoptive transfer of wild-type AM into (FcγRI, FcγRIII)−/− mice restores protection by passively transferred anti-M2e IgG. We conclude that AM and Fc receptor-dependent elimination of influenza A virus-infected cells are essential for protection by anti-M2e IgG.

A Critical Role for Syk Protein Tyrosine Kinase in Fc Receptor-Mediated Antigen Presentation and Induction of Dendritic Cell Maturation

Dendritic cells (DCs) are the only APCs capable of initiating adaptive immune responses. The initiation of immune responses requires that DCs 1) internalize and present Ags; and 2) undergo a differentiation process, called “maturation”, which transforms DCs into efficient APCs. DC maturation may be initiated by the engagement of different surface receptors, including certain cytokine receptors (such as TNFR), Toll-like receptors, CD40, and FcRs. The early activation events that link receptor engagement and DC maturation are not well characterized. We found that FcR engagement by immune complexes induced the phosphorylation of Syk, a protein tyrosine kinase acting immediately downstream of FcRs. Syk was dispensable for DC differentiation in vitro and in vivo, but was strictly required for immune complexes internalization and subsequent Ag presentation to T lymphocytes. Importantly, Syk was also required for the induction of DC maturation and IL-12 production after FcR engagement, but not after engagement of other surface receptors, such as TNFR or Toll-like receptors. Therefore, protein tyrosine phosphorylation by Syk represents a novel pathway for the induction of DC maturation.

FcγRIIB Regulates Nasal and Oral Tolerance: A Role for Dendritic Cells

Mucosal tolerance prevents the body from eliciting productive immune responses against harmless Ags that enter the body via the mucosae, and is mediated by the induction of regulatory T cells that differentiate in the mucosa-draining lymph nodes (LN) under defined conditions of Ag presentation. In this study, we show that mice deficient in FcγRIIB failed to develop mucosal tolerance to OVA, and demonstrate in vitro and in vivo a critical role for this receptor in modulating the Ag-presenting capacity of dendritic cells (DC). In vitro it was shown that absence of FcγRIIB under tolerogenic conditions led to increased IgG-induced release of inflammatory cytokines such as MCP-1, TNF-α, and IL-6 by bone marrow-derived DC, and increased their expression of costimulatory molecules, resulting in an altered immunogenic T cell response associated with increased IL-2 and IFN-γ secretion. In vivo we could show enhanced LN-DC activation and increased numbers of Ag-specific IFN-γ-producing T cells when FcγRIIB−/− mice were treated with OVA via the nasal mucosa, inferring that DC modulation by FcγRIIB directed the phenotype of the T cell response. Adoptive transfer of CD4+ T cells from the spleen of FcγRIIB−/− mice to naive acceptor mice demonstrated that OVA-responding T cells failed to differentiate into regulatory T cells, explaining the lack of tolerance in these mice. Our findings demonstrate that signaling via FcγRIIB on DC, initiated by local IgG in the mucosa-draining LN, down-regulates DC activation induced by nasally applied Ag, resulting in those defined conditions of Ag presentation that lead to Tr induction and tolerance.

The Inhibitory Receptor FcγRII Reduces Joint Inflammation and Destruction in Experimental Immune Complex-Mediated Arthritides Not Only by Inhibition of FcγRI/III but Also by Efficient Clearance and Endocytosis of Immune Complexes

Studies of FcgammaRII-/- mice identified the inhibitory function of this receptor in joint inflammation and cartilage destruction induced with immune complexes (ICs). To extend our insight in the role of FcgammaRII in arthritis, we explored the role of FcgammaRII in the absence of activating receptors I and III using FcgammaRI/III-/- as well as FcgammaRI/II/III-/- mice. When antigen-induced arthritis (AIA) was elicited, which is a mixture of T cell and IC-driven inflammation, arthritis was almost absent at day 7 in FcgammaRI/III-/- mice. Remarkably, in FcgammaRI/II/III-/- mice, this model induced a tremendously increased arthritis as compared to wild-type controls. This implies that FcgammaRII regulates joint inflammation also in the absence of activating FcgammaRI and III. To confirm the IC specificity of this finding, similar studies were done with ICs or zymosan as arthritogenic stimuli. Strongly elevated inflammation was found in FcgammaRI/II/III-/- mice with IC but not with zymosan. Clearance studies identified accumulation of IgG in the knee joint in the absence of FcgammaRII. Moreover, macrophages expressing only FcgammaRII showed prominent endocytosis of preformed soluble ICs not different from controls. In total absence of FcgammaR (FcgammaRI/II/III-/-), macrophages completely failed to endocytose ICs. Although joint inflammation was much higher in AIA arthritic knee joints of FcgammaRI/II/III-/- and the inflammatory cells still expressed an inflammatory phenotype, severe cartilage destruction (MMP-mediated neoepitopes in the matrix and chondrocyte death) was completely prevented in contrast to the marked destruction which was observed in the wild-type. Our study indicates that FcgammaRII reduces joint inflammation in the absence of activating FcgammaR by promoting endocytosis and clearance of ICs from the joint. Infiltrating cells, which fail to express activating FcgammaR although they still become stimulated are no longer capable of inducing severe cartilage destruction.

Effect of immune serum and role of individual Fcγ receptors on the intracellular distribution and survival of Salmonella enterica serovar Typhimurium in murine macrophages

Immune serum has a protective role against Salmonella infections in mice, domestic animals and humans. In this study, the effect of antibody on the interaction between murine macrophages and S. enterica serovar Typhimurium was examined. Detailed analysis at the single‐cell level demonstrated that opsonization of the bacteria with immune serum enhanced bacterial uptake and altered bacterial distribution within individual phagocytic cells. Using gene‐targeted mice deficient in individual Fc gamma receptors it was shown that immune serum enhanced bacterial internalization by macrophages via the high‐affinity immunoglobulin G (IgG) receptor, Fc gamma receptor I. Exposure of murine macrophages to S. enterica serovar Typhimurium opsonized with immune serum resulted in increased production of superoxide, leading to enhanced antibacterial functions of the infected cells. However, opsonization of bacteria with immune serum did not increase either nitric oxide production in response to S. enterica serovar Typhimurium or fusion of phagosomes with lysosomes.

Fcγ receptors are crucial for the expression of acquired resistance to virulent Salmonella enterica serovar Typhimurium in vivo but are not required for the induction of humoral or T‐cell‐mediated immunity

Antibodies play an important role in immunity to Salmonella enterica. Here we evaluated the requirement for Fcγ receptors in host resistance to S. enterica using an in vivo model of systemic infection. We show that mice lacking FcγRI, II and III can control and clear a primary infection with S. enterica micro‐organisms of low virulence, but are impaired in the expression of vaccine‐induced acquired immunity to oral challenge with virulent bacteria. We also show that, in vivo, FcγRI, II, III−/− mice were able to mount efficient T‐helper 1 type T‐cell responses and antibody responses specific for S. enterica. The work indicates that targeting S. enterica to FcγR is needed for the expression of vaccine‐induced acquired immunity, but is not essential for the engenderment of T‐ and B‐cell immunity to the bacterium in vivo.

IgG3-Mediated Enhancement of the Antibody Response is Normal in FcγRI-Deficient Mice

Antibodies, administered together with their specific antigen, can feedback‐regulate antibody responses to this antigen. IgG1, IgG2a and IgG2b enhance antibody responses to soluble protein antigens. This effect is primarily mediated by FcRs as enhancement is impaired in FcRγ–/– mice, reported to lack FcγRI and FcγRIII because of deletion of the common FcRγ chain. Also IgG3 can enhance antibody responses. However, this effect is unperturbed in FcRγ–/– mice but severely impaired in complement‐depleted animals and in animals lacking complement receptor 1 and 2. Although this argues against involvement of FcγRs, FcRγ–/– mice may express one‐fifth of the normal levels of FcγRI and, in addition, FcγRI has been suggested to bind IgG3. We re‐investigated the dependence of IgG3‐mediated enhancement on FcγRs using a mouse strain selectively lacking FcγRI and found that IgG3‐mediated enhancement is completely normal. Unlike IgE and IgG2a, which are both thought to enhance T‐cell proliferation via FcR‐mediated antigen presentation, IgG3 was a poor enhancer of T‐cell proliferation both in vivo and in vitro. These findings argue against a significant involvement of FcγRs in IgG3‐mediated enhancement of antibody responses and support our previous conclusion that complement plays a major role.

A dystrophic Duchenne mouse model for testing human antisense oligonucleotides

Duchenne muscular dystrophy (DMD) is a severe muscle-wasting disease generally caused by reading frame disrupting mutations in the DMD gene resulting in loss of functional dystrophin protein. The reading frame can be restored by antisense oligonucleotide (AON)-mediated exon skipping, allowing production of internally deleted, but partially functional dystrophin proteins as found in the less severe Becker muscular dystrophy. Due to genetic variation between species, mouse models with mutations in the murine genes are of limited use to test and further optimize human specific AONs in vivo. To address this we have generated the del52hDMD/mdx mouse. This model carries both murine and human DMD genes. However, mouse dystrophin expression is abolished due to a stop mutation in exon 23, while the expression of human dystrophin is abolished due to a deletion of exon 52. The del52hDMD/mdx model, like mdx, shows signs of muscle dystrophy on a histological level and phenotypically mild functional impairment. Local administration of human specific vivo morpholinos induces exon skipping and dystrophin restoration in these mice. Depending on the number of mismatches, occasional skipping of the murine Dmd gene, albeit at low levels, could be observed. Unlike previous models, the del52hDMD/mdx model enables the in vivo analysis of human specific AONs targeting exon 51 or exon 53 on RNA and protein level and muscle quality and function. Therefore, it will be a valuable tool for optimizing human specific AONs and genome editing approaches for DMD.

Identification of a novel CD40 ligand for targeted imaging of inflammatory plaques by phage display

The CD40/CD40L dyad is deemed to play a central role in several inflammatory processes, including atherosclerosis. As CD40 is overexpressed in atherosclerotic lesions, it constitutes a promising candidate for targeted imaging approaches. Here we describe the design of a novel, selective peptide ligand for CD40 by phage display. A synthetic peptide corresponding with the phage insert NP31 displayed nanomolar affinity for CD40. Affinity was further enhanced by mutimeric presentation of NP31. An essential 11‐mer peptide motif was identified by truncation and alanine scan studies. Enriched phage selectively bound human CD40 and homed to inflammatory joints in a murine model of rheumatoid arthritis. NP31 ablated VEGF and IL‐6 transcriptional activation and partially inhibited IL‐6 production by CD40L‐activated endothelial cells. Notably, NP31 did not only alter the biodistribution profile of a streptavidin scaffold but also markedly increased accumulation of the carrier in atherosclerotic aortic lesions of aged ApoE–/– mice in a CD40‐dependent manner. This potent and selective peptide ligand has potential for targeted imaging and drug delivery approaches in CD40‐dependent inflammatory disorders such as atherosclerosis.—Yu, H., Segers, F., Sliedregt‐Bol, K., Bot, I., Woltman, A. M. Boross, P., Verbeek, S., Overkleeft, H., van der Marel, G. A., van Kooten, C., van Berkel, T J. C., Biessen, E. A. L. Identification of a novel CD40 ligand for targeted imaging of inflammatory plaques by phage display. FASEBJ. 27, 4136‐4146 (2013). www.fasebj.org

FcγRI expression on macrophages is required for antibody-mediated tumor protection by cytomegalovirus-based vaccines

Cytomegalovirus (CMV)-based vaccine vectors are promising vaccine platforms because they induce strong and long-lasting immune responses. Recently it has been shown that vaccination with a mouse CMV (MCMV) vector expressing the melanoma-specific antigen TRP2 (MCMV-TRP2) protects mice against outgrowth of TRP2-positive B16 melanoma tumors, and this protection was dependent on the induction of IgG antibodies. Here we demonstrate that, although mice lacking all receptors for the Fc part of IgG (FcγRs) develop normal IgG responses after MCMV-TRP2 vaccination, the protection against B16 melanoma was completely abrogated, indicating that FcγRs are indispensable in the downstream effector pathway of the polyclonal anti-TRP2 antibody response. By investigating compound FcγR-deficient mouse strains and by using immune cell type-specific cell ablation we show that the IgG antibody-mediated tumor protection elicited by MCMV-TRP2 mainly depends on FcγRI expression on macrophages, whereas FcγRIV plays only a modest role. Thus, tumor-specific antibody therapy might benefit from combination therapy that recruits FcγRI-expressing pro-inflammatory macrophages to the tumor micro-environment.