Stable isotope labeling by amino acids in cell culture (SILAC) has become a versatile tool for quantitative, mass spectrometry (MS)-based proteomics. Here, we completely label mice with a diet containing either the natural or the (13)C(6)-substituted version of lysine. Mice were labeled over four generations with the heavy diet, and development, growth, and behavior were not affected. MS analysis of incorporation levels allowed for the determination of incorporation rates of proteins from blood cells and organs. The F2 generation was completely labeled in all org...

We have tested the safety and feasibility of a synthetic long peptide-based HPV16-specific skin test to detect cellular immune responses to HPV16 E2, E6 and E7 in vivo. Women with cervical neoplasia (n = 11) and healthy individuals (n = 19) were intradermally challenged with 8 different pools of HPV16 E2, E6 and E7 peptides. The skin test was safe as the injections were perceived as mildly painful and no adverse events were observed. The majority of skin reactions appeared significantly earlier in HPV16+ patients (<8 days) than in healthy subjects (8-25 days). Th...

The tyrosine kinase inhibitors sorafenib and sunitinib are approved for the treatment of patients with malignant diseases. To analyze the possible use of these compounds in combination with immunotherapeutic approaches, we analyzed the effects of both inhibitors on the immunostimulatory capacity of human dendritic cells (DCs) and the induction of primary immune responses in vivo. Sorafenib, but not sunitinib, inhibits function of DCs, characterized by reduced secretion of cytokines and expression of CD1a, major histocompatibility complex, and costimulatory molecu...

The in vivo mechanisms of action of most vaccine adjuvants are poorly understood. In this study, we present data in mice that reveal a series of critical interactions between the cholera toxin (CT) adjuvant and the dendritic cells (DC) of the splenic marginal zone (MZ) that lead to effective priming of an immune response. For the first time, we have followed adjuvant targeting of MZ DC in vivo. We used CT-conjugated OVA and found that the Ag selectively accumulated in MZ DC following i.v. injections. The uptake of Ag into DC was GM1 ganglioside receptor dependent...

If the large collection of microarray-specific statistical tools was applicable to the analysis of quantitative shotgun proteomics datasets, it would certainly foster an important advancement of proteomics research. Here we analyze two large multidimensional protein identification technology datasets, one containing eight replicates of the soluble fraction of a yeast whole-cell lysate and one containing nine replicates of a human immunoprecipitate, to test whether normalized spectral abundance factor (NSAF) values share substantially similar statistical propertie...

Steady-state dendritic cells (DCs) migrating in the lymph from the intestine induce tolerance to harmless intestinal antigens, preventing inflammatory responses. To determine if such DCs are inherently tolerogenic we collected intestinal lymph DCs (L-DCs) by cannulation of the thoracic duct of rats after mesenteric lymphadenectomy, and examined their capacity to activate naive CD4+ lymphocytes in an allogeneic mixed leucocyte reaction. L-DCs stimulated strong proliferative responses, induced secretion of inflammatory cytokines including interferon-gamma, and indu...

The C-type lectin receptor dectin-1 functions as a pattern recognition receptor for beta-glucans and signals via Syk kinase but independently of the Toll-like receptor (TLR) pathway to regulate expression of innate response genes. Dectin-1 signaling can promote activation of dendritic cells (DCs), rendering them competent to prime Th1 and Th17 responses. Here we show that dectin-1-activated DCs can also prime cytotoxic T-lymphocyte (CTL) responses. DCs exposed to a dectin-1 agonist induced antigen-specific expansion of TCR transgenic CD8(+) T cells and their diff...

Over the past ten years, investigators have shown that T lymphocytes can recognize not only peptides in the context of MHC class I and class II molecules but also foreign and self-lipids in association with the nonclassical MHC class I molecules the CD1 proteins. We describe the events that have led to the discovery of the role of CD1 molecules, their pattern of intracellular trafficking, and their ability to sample different intracellular compartments for self- and foreign lipids. Structural and functional aspects of lipid presentation by CD1 molecules are prese...

Pathogenic microbes have evolved sophisticated molecular strategies to subvert host defenses. Here we show that virulent bacteria interfere directly with Toll-like receptor (TLR) function by secreting inhibitory homologs of the Toll/interleukin-1 receptor (TIR) domain. Genes encoding TIR domain containing-proteins (Tcps) were identified in Escherichia coli CFT073 (TcpC) and Brucella melitensis (TcpB). We found that TcpC is common in the most virulent uropathogenic E. coli strains and promotes bacterial survival and kidney pathology in vivo. In silico analysis pre...

We developed a nonradioactive fluorescence-activated cell sorting-based assay, called surface sensing of translation (SUnSET), which allows the monitoring and quantification of global protein synthesis in individual mammalian cells and in heterogeneous cell populations. We demonstrate here, using mouse dendritic and T cells as a model, that SUnSET offers a technical alternative to classical radioactive labeling methods for the study of mRNA translation and cellular activation.

Anti-tumor vaccines consisting of extended CTL peptides in combination with CpG-ODN were shown to be superior to those comprising minimal CTL epitopes and CpG-ODN, in that they elicit stronger effector CTL responses with greater tumoricidal potential. We now demonstrate that this improved performance is primarily due to the focusing of CTL epitope presentation onto activated DC in the inflamed lymph nodes draining the vaccination site. In the case of vaccination with minimal peptides, additional APC including T and B cells are also loaded with CTL epitopes. Our d...

Poxviruses such as the causative agent of smallpox have developed multiple strategies to suppress immune responses, including the suppression of DC activation. Since poxviruses are large DNA viruses, we hypothesized that their detection by DCs may involve the endosomal DNA recognition receptor TLR9. Indeed, we have shown here that DC recognition of ectromelia virus (ECTV), the causative agent of mousepox, completely depended on TLR9. The importance of TLR9 was highlighted by the fact that mice lacking TLR9 showed drastically increased susceptibility to infection ...

The C-type lectin dectin-1 binds to yeast and signals through the kinase Syk and the adaptor CARD9 to induce production of interleukin 10 (IL-10) and IL-2 in dendritic cells (DCs). However, whether this pathway promotes full DC activation remains unclear. Here we show that dectin-1-Syk-CARD9 signaling induced DC maturation and the secretion of proinflammatory cytokines, including IL-6, tumor necrosis factor and IL-23, but little IL-12. Dectin-1-activated DCs 'instructed' the differentiation of CD4+ IL-17-producing effector T cells (T(H)-17 cells) in vitro, and a d...

Antigen binding to the B-cell receptor (BCR) induces multiple signaling cascades that ultimately lead to B lymphocyte activation. In addition, the BCR regulates the key trafficking events that allow the antigen to reach endocytic compartments devoted to antigen processing, i.e., that are enriched for major histocompatibility factor class II (MHC II) and accessory molecules such as H2-DM. Here, we analyze the role in antigen processing and presentation of the tyrosine kinase Syk, which is activated upon BCR engagement. We show that convergence of MHC II- and H2-DM-...

Zymosan is a particulate yeast preparation that elicits high levels of IL-2 and IL-10 from dendritic cells (DC) and engages multiple innate receptors, including the Syk-coupled receptor dectin-1 and the MyD88-coupled receptor TLR2. Here, we show that induction of IL-2 and IL-10 by zymosan requires activation of ERK MAP kinase in murine DC. Surprisingly, ERK activation in response to zymosan is completely blocked in Syk-deficient DC and unaffected by MyD88 deficiency. Conversely, ERK activation in response to the TLR2 agonist Pam3Cys is completely MyD88 dependent a...

Dendritic cells (DCs) play a key role in antigen-specific immune regulation. DCs take up and process antigens and present these as peptides through MHC molecules to T cells. Recent pre-clinical and clinical studies have exploited DCs as a means to improve vaccine efficiency. In these studies, monocyte-derived autologous DCs are loaded ex vivo with antigens and re-administered to the patient. These tailor-made vaccines are costly and labor intensive, and therefore less suitable for large-scale immunization programs. As a next step in the development of DC vaccines,...

Expression of non-self antigens by tumors can induce activation of T cells in vivo, although this activation can lead to either immunity or tolerance. CD8+ T-cell activation can be direct (if the tumor expresses MHC class I molecules) or indirect (after the capture and cross-presentation of tumor antigens by dendritic cells). The modes of tumor antigen capture by dendritic cells in vivo remain unclear. Here we examine the immunogenicity of the same model antigen secreted by live tumors either in association with membrane vesicles (exosomes) or as a soluble protei...

The potential role of antibodies and T lymphocytes in the eradication of cancer has been demonstrated in numerous animal models and clinical trials. In the last decennia new strategies have been developed for the use of tumor-specific T cells and antibodies in cancer therapy. Effective anti-tumor immunotherapy requires the identification of suitable target antigens. The expression of tumor-specific antigens has been extensively studied for most types of adult tumors. Pediatric patients should be excellent candidates for immunotherapy since their immune system is ...

Antigen (Ag) capture and presentation onto major histocompatibility complex (MHC) class II molecules by B lymphocytes is mediated by their surface Ag receptor (B cell receptor [BCR]). Therefore, the transport of vesicles that carry MHC class II and BCR-Ag complexes must be coordinated for them to converge for processing. In this study, we identify the actin-associated motor protein myosin II as being essential for this process. Myosin II is activated upon BCR engagement and associates with MHC class II-invariant chain complexes. Myosin II inhibition or depletion c...

Although the impact of tumor immunology on the clinical management of most cancers is still negligible, there is increasing evidence that anticancer immune responses may contribute to the control of cancer after conventional chemotherapy. Thus, radiotherapy and some chemotherapeutic agents, in particular anthracyclines, can induce specific immune responses that result either in immunogenic cancer cell death or in immunostimulatory side effects. This anticancer immune response then helps to eliminate residual cancer cells (those that fail to be killed by chemother...