Cross-presentation is a crucial mechanism in tumoral and microbial immunity because it allows internalized cell associated or exogenous antigens (Ags) to be delivered into the major histocompatibility complex I pathway. This pathway is important for the development of CD8 ؉ T-cell responses and for the induction of tolerance. In mice, cross-presentation is considered to be a unique property of CD8␣ ؉ conventional dendritic cells (DCs). Here we show that splenic plasmacytoid IntroductionCross-presentation is a mechanism that involves the uptake and processing of exogenous antigens (Ags) within the major histocompatibility complex (MHC) class I pathway. 1 This process differs from the classical MHC class I processing pathway in which MHC class I molecules present Ags that are synthesized within the cells. Cross-presentation occurs subsequent to transport of Ag from the endosome to cytosol and digestion by the proteasome and may concern different forms of exogenous Ags, such as soluble, 2 particulate, 3 or cell-associated Ags. 4 However, alternative mechanisms have been described, and the cellular and molecular pathways that lead to the presentation of endocytosed proteins on MHC class I molecules are not yet fully defined. 5 Cross-presentation is a crucial mechanism allowing induction of naive CD8 ϩ T-cell responses against exogenous Ag and has been implicated in the elicitation of protective cytotoxic T lymphocyte (CTL) responses against tumors as well as in the generation of immune responses against clinically relevant pathogens that do not infect tissues of hemopoietic origin. 6,7 In addition to cross-priming of naive CD8 ϩ T cells, cross-presentation appears to be central to the maintenance of peripheral tolerance to self-Ag, called "cross-tolerance." 8,9 With very few exceptions, 10,11 cross-presentation is restricted to dendritic cells (DCs) 2,12 and macrophages 13,14 but is not normally seen in other nucleated cells. DCs are considered the only Ag-presenting cell (APC) able to prime naive T cells and are thus involved in the induction of both adaptive immunity and tolerance. The outcome of T-cell responses depends on DC maturation state, as immature or semimature DCs have been described to induce tolerance. 15 Immature DCs are present in the periphery where they sample Ags. They are characterized by high expression of receptors involved in endocytosis and phagocytosis. 16 Maturation of DCs can be induced by pathogens or by inflammatory signals. Pathogens are recognized through pathogen-associated molecular patterns, which interact with pattern recognition receptors, including Toll-like receptors (TLRs), constitutively expressed by DCs. 17,18 Maturation involves the migration of DCs to lymphoid organs, the activation of proteolytic activity for Ag processing, and the up-regulation of MHC and costimulatory molecules. After maturation, DCs prime naive T cells, leading to their differentiation into effector T cells. 19 Several populations of DCs have been described, and it is unclear whether all these DC types...

Although much is described about the molecules involved in neutrophil migration from circulation into tissues, less is known about the molecular mechanisms that regulate neutrophil entry into lymph nodes (LNs) draining a local inflammatory site. In this study, we investigated neutrophil migration toward LNs in a context of inflammation induced by immunization of BALB/c mice with OVA emulsified in CFA. We demonstrated that neutrophils can enter LNs of OVA/CFA-immunized mice not only via lymphatic vessels but also from blood, across high endothelial venules. By adoptive transfer experiments, we showed that this influx was dependent on an inflammatory-state condition and previous neutrophil stimulation with OVA/anti-OVA immune complexes. Importantly, we have demonstrated that, in the migratory pattern to LNs, neutrophils used L-selectin and P-selectin glycoprotein ligand-1, macrophage-1 Ag and LFA-1 integrins, and CXCR4 to get access across high endothelial venules, whereas macrophage-1 Ag, LFA-1, and CXCR4 were involved in their trafficking through afferent lymphatics. Strikingly, we found that stimulation with immune complexes significantly upregulated the expression of sphingosine-1-phosphate receptor 4 on neutrophils, and that treatment with the sphingosine-1-phosphate agonist FTY720 altered neutrophil LN-homing ability. These findings summarized in this article disclose the molecular pattern that controls neutrophil recruitment to LNs.

Recombinant porcine parvovirus virus-like particles (PPV-VLPs) are particulate exogenous antigens that induce a strong, specific cytotoxic T lymphocyte (CTL) response in the absence of adjuvant. In the present report, we demonstrate in vivo that dendritic cells (DCs) present PPV-VLPs to CD8+ T cells after intracellular processing. PPV-VLPs are captured by DCs with a high efficacy, which results in the delivery of these exogenous antigens to 50% of the whole spleen DC population. In vivo, a few hours after injection, PPV-VLPs are presented exclusively to CD8+ T cells by CD8α− DCs, whereas 15 hours later they are presented mainly by CD8α+ DCs. After PPV-VLPs processing, a fraction of CD11b+ DCs undergo phenotypic changes, i.e., the up-regulation of CD8α and CD205 and the loss of CD4 molecules on their surface. The failure to detect mRNA coding for CD8α in CD11b+ DCs suggests that CD8α expression by these cells is not due to de novo synthesis. In recombination-activating gene knockout mice (Rag−/−), CD11b+ DCs did not express CD8α and PPV-VLPs presentation by CD8α+ DCs was severely diminished. These results indicate that both CD8α− and CD8α+ DCs play an important role in the induction of CTL responses by exogenous antigens, such as VLP.

No abstract

Baculoviruses (BV) are DNA viruses that are pathogenic for insects. Although BV infect a range of mammalian cell types, they do not replicate in these cells. Indeed, the potential effects of these insect viruses on the immune responses of mammals are only just beginning to be studied. We show in this paper that a recombinant Autographa californica multiple nuclear polyhedrosis virus carrying a fragment of ovalbumin (OVA) on the VP39 capsid protein (BV-OVA) has the capacity to act as an adjuvant and vector of antigens in mice, thereby promoting specific CD4 and cytotoxic T cell responses against OVA. BV also induced in vivo maturation of dendritic cells and the production of inflammatory cytokines, thus promoting innate and adaptive immune responses. The OVA-specific response induced by BV-OVA was strong enough to reject a challenge with OVA-expressing melanoma cells (MO5 cells) and effectively prolonged survival of MO5 bearing mice. All these findings, together with the absence of pre-existing immunity to BV in humans and the lack of viral gene expression in mammalian cells, make BV a candidate for vaccination.

BACKGROUND Owing to their minimal size, high production yield, versatility and robustness, the recombinant variable domain (nanobody) of camelid single chain antibodies are valued affinity reagents for research, diagnostic, and therapeutic applications. While their preparation against purified antigens is straightforward, the generation of nanobodies to difficult targets such as multi-pass or complex membrane cell receptors remains challenging. Here we devised a platform for high throughput identification of nanobodies to cell receptor based on the use of a biotin handle. METHODS Using a biotin-acceptor peptide tag, the in vivo biotinylation of nanobodies in 96 well culture blocks was optimized allowing their parallel analysis by flow cytometry and ELISA, and their direct used for pull-down/MS target identification. RESULTS The potential of this strategy was demonstrated by the selection and characterization of panels of nanobodies to Mac-1 (CD11b/CD18), MHC II and the mouse Ly-5 leukocyte common antigen (CD45) receptors, from a VHH library obtained from a llama immunized with mouse bone marrow derived dendritic cells. By on and off switching of the addition of biotin, the method also allowed the epitope binning of the selected Nbs directly on cells. CONCLUSIONS This strategy streamline the selection of potent nanobodies to complex antigens, and the selected nanobodies constitute ready-to-use biotinylated reagents. GENERAL SIGNIFICANCE This method will accelerate the discovery of nanobodies to cell membrane receptors which comprise the largest group of drug and analytical targets.

We have previously demonstrated that subcutaneously administered ovalbumin (OVA) plus synthetic oligodeoxynucleotides containing immunostimulatory CpG motifs (CpG-ODN) as adjuvant stimulate cellular and humoral immunity and promote T helper cell type 1 differentiation in aged mice. The present study assessed the ability of CpG-ODN to induce an OVA-specific immune response after oral immunization in young (3-month-old) and aged (18-month-old) BALB/c mice. Oral OVA/CpG-ODN immunization induces a similar OVA-specific T cell-proliferative response (in mucosal and systemic tissues), immunoglobulin G (IgG) in plasma, and IgA in intestinal washes in both groups of ages. The OVA-specific humoral immune response observed in aged mice was similar to the one observed in young mice, peaking at day 7 after the last oral immunization and was present over 40 days after the last oral immunization. The pattern of cytokines released in culture supernatants in both groups of mice was similar, with specific interferon-gamma secretion in the absence of interleukin-5 responses. These results provide evidence that orally administered OVA/CpG-ODN induces a young-like, specific, immune response against OVA in aged mice, showing that CpG-ODN might be used as a mucosal adjuvant during aging.

The development of antiviral vaccines has almost exclusively been based on live attenuated vaccines up until now. However, the efficacy of HBsAg particles as an antiHBV vaccine has clearly demonstrated that protective antiviral immunity can be achieved by other strategies. Virus-like particles formed by structural proteins were proven to be highly immunogenic and capable of inducing protective immunity against various viral infections in preclinical studies. Clinical trials using virus-like particles confirmed their safety and immunogenicity. Moreover, chimeric virus-like particles carrying foreign peptidic sequences were shown to elicit potent B- and T-cell responses. Virus-like particles formed by a fusion protein between the HBsAg and the circumsporozoïte surface protein are safe and immunogenic in volunteers and induce a partial protection against natural Plasmodium falciparum infection.