Christine Sedlik

FcγRI (CD64) contributes substantially to severity of arthritis, hypersensitivity responses, and protection from bacterial infection

The high-affinity receptor for IgG, FcgammaRI, shares its capacity to bind IgG2a immune complexes (IgG2a-IC) with the low-affinity receptor FcgammaRIII and complement factors, hampering the definition of its biological role. Moreover, in vivo, FcgammaRI is occupied by monomeric IgG2a, reducing its accessibility to newly formed IgG2a-IC. By using a variety of FcgammaR(-/-) mice, we demonstrate that in the absence of FcgammaRI, the IgG2a-IC-induced cellular processes of phagocytosis, cytokine release, cellular cytotoxicity, and antigen presentation are impaired. FcgammaRI(-/-) mice showed impaired hypersensitivity responses, strongly reduced cartilage destruction in an arthritis model, and impaired protection from a bacterial infection. We conclude that FcgammaRI contributes substantially to a variety of IgG2a-IC-dependent immune functions and immunopathological responses.

Antigen-Antibody Immune Complexes Empower Dendritic Cells to Efficiently Prime Specific CD8+ CTL Responses In Vivo

Dendritic cells (DCs) require a maturation signal to acquire efficient CTL-priming capacity. In vitro FcγR-mediated internalization of Ag-Ab immune complexes (ICs) can induce maturation of DCs. In this study, we show that IC-induced DC maturation in vitro enables DCs to prime peptide-specific CD8+ CTLs in vivo, independently of CD4+ Th cells. Importantly, OVA/anti-OVA IC-treated DCs not only primed CD8+ CTLs to an exogenously loaded peptide nonrelated to OVA, but also efficiently primed CTLs against the dominant CTL epitope derived from the OVA Ag present in the ICs. Our studies show that ICs fulfill a dual role in priming of CD8+ CTL responses to exogenous Ags: enhancement of Ag uptake by DCs and activation of DCs, resulting in “license to kill.” These findings indicate that the presence of specific Abs can crucially affect the induction of cytotoxic cellular responses.

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.

In vivo, dendritic cells can cross-present virus-like particles using an endosome-to-cytosol pathway.

Recombinant parvovirus-like particles (PPV-VLPs) are particulate exogenous Ags that induce strong CTL response in the absence of adjuvant. In the present report to decipher the mechanisms responsible for CTL activation by such exogenous Ag, we analyzed ex vivo and in vitro the mechanisms of capture and processing of PPV-VLPs by dendritic cells (DCs). In vivo, PPV-VLPs are very efficiently captured by CD8α− and CD8α+ DCs and then localize in late endosomes of DCs. Macropinocytosis and lipid rafts participate in PPV-VLPs capture. Processing of PPV-VLPs does not depend upon recycling of MHC class I molecules, but requires vacuolar acidification as well as proteasome activity, TAP translocation, and neosynthesis of MHC class I molecules. This study therefore shows that in vivo DCs can cross-present PPV-VLPs using an endosome-to-cytosol processing pathway.

Acidosis Improves Uptake of Antigens and MHC Class I-Restricted Presentation by Dendritic Cells

It is widely appreciated that inflammatory responses in peripheral tissues are usually associated to the development of acidic microenvironments. Despite this, there are few studies aimed to analyze the effect of extracellular pH on immune cell functions. We analyzed the impact of acidosis on the behavior of dendritic cells (DCs) derived from murine bone marrow. We found that extracellular acidosis (pH 6.5) markedly stimulated the uptake of FITC-OVA, FITC-dextran, and HRP by DCs. In fact, to reach similar levels of endocytosis, DCs cultured at pH 7.3 required concentrations of Ag in the extracellular medium almost 10-fold higher compared with DCs cultured at pH 6.5. Not only the endocytic capacity of DCs was up-regulated by extracellular acidosis, but also the expression of CD11c, MHC class II, CD40, and CD86 as well as the acquisition of extracellular Ags by DCs for MHC class I-restricted presentation. Importantly, DCs pulsed with Ag under acidosis showed an improved efficacy to induce both specific CD8+ CTLs and specific Ab responses in vivo. Our results suggest that extracellular acidosis improves the Ag-presenting capacity of DCs.

Vesicular stomatitis virus and pseudorabies virus induce a vig1/cig5 homologue in mouse dendritic cells via different pathways

The homologous genes vig1 and cig5 were identified by differential display PCR as virus-induced genes in rainbow trout and humans, respectively. These genes are significantly related to sequences required for the biosynthesis of metal cofactors, but their function remains unknown. In this study, it is shown that the mouse homologue of vig1/cig5 was induced by vesicular stomatitis virus (VSV) and pseudorabies virus (PrV) in mouse spleen cells. Among a collection of cell lines from dendritic, myeloid, lymphoid or fibroblast lineages, only the dendritic cell line, D2SC1, showed expression of mvig after virus infection. This dendritic restriction was confirmed by our finding that mvig was also induced by both VSV and PrV in CD11c(++) spleen cells, separated by magnetic purification or derived from bone marrow precursor cells. Similar to the fish rhabdovirus viral haemorrhagic septicaemia virus in trout cells, VSV directly induced mvig in the dendritic cell line D2SC1, but the PrV-mediated induction required the integrity of the interferon pathway. This result indicates that mvig is interferon-inducible like its fish and human homologues. Furthermore, mvig was also induced by LPS in bone marrow-derived cells. Thus, mvig expression seems to correlate with an activated state of dendritic cells subjected to different pathogen-associated stimuli.

Long‐lasting cross‐presentation of tumor antigen in human DC

DC cross‐present exogenous antigens on MHC class I molecules, a process required for the onset of anti‐tumor immune responses. In order to study the cross‐presentation of tumor antigens by human DC, we compared the pathways of cross‐presentation of long peptides requiring internalization and intracellular processing with the direct presentation of short peptides, which does not require intracellular processing. We found that, after brief incubations with DC, short peptides were presented to CD8+ T cells with higher efficiencies than long peptides. After longer times of chase in the absence of peptide, however, the efficiency of presentation of the two types of peptides was reversed. After 2–3 days, DC pulsed with long peptides still activated T cells efficiently, while DC pulsed with short peptides failed to do so. Long‐lasting presentation of the long peptides was, at least in part, due to a stored persistent pool of antigen, which was still available for loading on MHC class I molecules after several days of chase. These results show that the use of long synthetic peptides allows the efficient, long‐lasting, presentation of tumor antigens, suggesting that long peptides represent an interesting approach for active anti‐tumor vaccination.

Universal Cancer Peptide-Based Therapeutic Vaccine Breaks Tolerance against Telomerase and Eradicates Established Tumor

Purpose: To evaluate CD4+ helper functions and antitumor effect of promiscuous universal cancer peptides (UCP) derived from telomerase reverse transcriptase (TERT). Experimental Design: To evaluate the widespread immunogenicity of UCPs in humans, spontaneous T-cell responses against UCPs were measured in various types of cancers using T-cell proliferation and ELISPOT assays. The humanized HLA-DRB1*0101/HLA-A*0201 transgenic mice were used to study the CD4+ helper effects of UCPs on antitumor CTL responses. UCP-based antitumor therapeutic vaccine was evaluated using HLA-A*0201–positive B16 melanoma that express TERT. Results: The presence of a high number of UCP-specific CD4+ T cells was found in the blood of patients with various types of cancer. These UCP-specific T cells mainly produce IFN-γ and TNF-α. In HLA transgenic mice, UCP vaccinations induced high avidity CD4+ TH1 cells and activated dendritic cells that produced interleukin-12. UCP-based vaccination breaks self-tolerance against TERT and enhances primary and memory CTL responses. Furthermore, the use of UCP strongly improves the efficacy of therapeutic vaccination against established B16-HLA-A*0201 melanoma and promotes tumor infiltration by TERT-specific CD8+ T cells. Conclusions: Our results showed that UCP-based vaccinations strongly stimulate antitumor immune responses and could be used to design efficient immunotherapies in multiple types of cancers. Clin Cancer Res; 18(22); 6284–95. ©2012 AACR.

Long Peptide Vaccination Can Lead to Lethality through CD4+ T Cell-Mediated Cytokine Storm

The optimization of anticancer therapeutic vaccines can lead to better efficacy but also to stronger adverse effects. In a mouse model of antitumor vaccination using a long peptide (LP), which included MHC class I- and II-restricted male (H-Y) epitopes, we observed unexpected mortality. Mice with an increased frequency of anti–H-Y CD4 T cells were primed with LP+CpG and boosted 10 d later. Within hours of boost, they displayed shock-like signs with high mortality. Serum cytokine levels were high. TNF-α secreted by the CD4 T cells was identified as the key effector molecule. Priming with a short peptide (SP), which included the MHC class II-restricted epitope, was a more efficient primer than LP, but did not lead to mortality when used as boost. The high mortality induced by LP compared with SP was probably related to its specific ability to be presented by B cells. Finally, targeting the LP sequence to dendritic cells allowed tumor protection without side effects. Our data: 1) confirm that the immune system can be very dangerous; 2) caution against the use of systemic activation of high-frequency Ag-specific T cells as induced by high doses of LP; and 3) underline the benefit of targeting Ag to dendritic cells.

Different immunogenicity but similar antitumor efficacy of two DNA vaccines coding for an antigen secreted in different membrane vesicle-associated forms

The induction of an active immune response to control or eliminate tumours is still an unfulfilled challenge. We focused on plasmid DNA vaccines using an innovative approach whereby the antigen is expressed in association with extracellular vesicles (EVs) to facilitate antigen cross-presentation and improve induced immunity. Our two groups had independently shown previously that DNA vaccines encoding EV-associated antigens are more efficient at inducing cytotoxic T-cell responses than vaccines encoding the non-EV-associated antigen. Here, we compared our two approaches to associate the ovalbumin (OVA) antigen to EVs: (a) by fusion to the lipid-binding domain C1C2 of MFGE8(=lactadherin), which is exposed on the surface of secreted membrane vesicles; and (b) by fusion to retroviral Gag capsid protein, which is incorporated inside membrane-enclosed virus-like particles. Plasmids encoding either form of modified OVA were used as DNA-based vaccines (i.e. injected into mice to allow in vivo expression of the antigen associated to EVs). We show that both DNA vaccines induced, with similar efficiency, OVA-specific CD8+ T cells and total IgG antibodies. By contrast, each vaccine preferentially stimulated different isotypes of immunoglobulins, and the OVA-C1C2-encoding vaccine favoured antigen-specific CD4+ T lymphocyte induction as compared to the Gag-OVA vaccine. Nevertheless, both OVA-C1C2 and Gag-OVA vaccines efficiently prevented in vivo outgrowth of OVA-expressing tumours and reduced tumour progression when administered to tumour-bearing mice, although with variable efficacies depending on the tumour models. DNA vaccines encoding EV-associated antigens are thus promising immunotherapy tools in cancer but also potentially other diseases.