MHC-peptide complexes mediate key functions in adaptive immunity. In a classical view, MHC-I molecules present peptides from intracellular source proteins, whereas MHC-II molecules present antigenic peptides from exogenous and membrane proteins. Nevertheless, substantial crosstalk between these two pathways has been observed. We investigated the influence of autophagy on the MHC-II ligandome and demonstrated that peptide presentation is altered considerably upon induction of autophagy. The presentation of peptides from intracellular and lysosomal source proteins was strongly increased on MHC-II in contrast with peptides from membrane and secreted proteins. In addition, autophagy influenced the MHC-II antigen-processing machinery. Our study illustrates a profound influence of autophagy on the class II peptide repertoire and suggests that this finding has implications for the regulation of CD4 ؉ T cell-mediated processes.antigen processing ͉ lysosomal proteases ͉ T helper cells P eptides of foreign and self proteins are presented on MHC-I and MHC-II molecules at the cell surface and can be recognized by CD8ϩ and CD4 ϩ T lymphocytes, respectively (1, 2). From a classical point of view, MHC-I molecules present antigenic peptides derived from intracellular proteins, whereas MHC-II molecules do so for exogenous and membrane proteins (3). This phenomenon is reflected in the two major cellular breakdown pathways for proteins: proteasomal degradation, particularly relevant to the generation of MHC-I peptides (4), and degradation by the endosomal͞lysosomal system, which is responsible for the processing of MHC-II peptides (5). However, the separation of these distinct pools of source proteins is less stringent than originally believed. It is now well established that MHC-I molecules are able to present peptides derived from exogenous antigens (Ag) by a process known as cross presentation (6). On the other hand, intracellular proteins can be presented by MHC-II molecules (7,8), even though the underlying processes are less clear. It has been recently shown that peptides from cytosolic model proteins can be presented on MHC-II molecules through autophagy (9-11). Autophagy plays a role in the endosomal͞lysosomal degradation pathway and is responsible for feeding intracellular components into this pathway. It is thought to be required for normal turnover of cellular components, particularly in response to starvation (12). Against this background, we hypothesized that autophagy might mediate MHC-II presentation of intracellular Ag, meaning the contents of a cell contained within the plasma membrane, excluding large vacuoles and secretory or ingested material (Gene Ontology classifications), in general. Therefore, we performed a detailed characterization of the MHC-II ligand repertoire (ligandome) presented at the cell surface under normal conditions and after increased autophagy, leading to a comprehensive overall picture of changes in peptide processing and presentation. Materials and MethodsCell Culture and Autophagy Inducti...

Several environmental stresses have been demonstrated to increase polysaccharide intercellular adhesin (PIA) synthesis and biofilm formation by the human pathogens Staphylococcus aureus and Staphylococcus epidermidis. In this study we characterized an adaptive response of S. aureus SA113 to nitrite-induced stress and show that it involves concomitant impairment of PIA synthesis and biofilm formation. Transcriptional analysis provided evidence that nitrite, either as the endogenous product of respiratory nitrate reduction or after external addition, causes repression of the icaADBC gene cluster, mediated likely by IcaR. Comparative microarray analysis revealed a global change in gene expression during growth in the presence of 5 mM sodium nitrite and indicated a response to oxidative and nitrosative stress. Many nitrite-induced genes are involved in DNA repair, detoxification of reactive oxygen and nitrogen species, and iron homeostasis. Moreover, preformed biofilms could be eradicated by the addition of nitrite, likely the result of the formation of toxic acidified nitrite derivatives. Nitrite-mediated inhibition of S. aureus biofilm formation was abrogated by the addition of nitric oxide (NO) scavengers, suggesting that NO is directly or indirectly involved. Nitrite also repressed biofilm formation of S. epidermidis RP62A.Staphylococcus aureus and Staphylococcus epidermidis are the pathogens of nosocomial sepsis most frequently isolated, and especially those patients with indwelling medical devices are at risk for chronic staphylococcal foreign body-associated infections (39, 51, 52, 69), which are mediated by the organisms' ability to form biofilms on metal or polymeric surfaces (22, 73). Biofilm-embedded bacteria are more resistant to antimicrobial agents than their planktonic counterparts and often cause chronic infections and sepsis, particularly in immunocompromised patients (14,36,42,60,65). Staphylococcal biofilm formation is a multifactorial process. Primary attachment can be mediated by various cell surface-associated factors such as the major autolysin (5, 25), the teichoic acids (23), or the polysaccharide intercellular adhesin (PIA) (24), the product of the icaADBC gene cluster (26). The accumulation of cells into a multilayered community requires the synthesis of PIA, which consists of polymeric N-acetylglucosamine (40) and is also referred to as PNAG. Furthermore, PIA-independent mechanisms of intercellular adhesion and biofilm formation have been reported and are of overall importance (17, 58). PIA expression and biofilm formation are induced by a variety of environmental stresses, like low oxygen (16), high osmolarity (3% NaCl) (53), the presence of ethanol (34), subinhibitory concentrations of tetracycline and the streptogramin quinupristin-dalfopristin (54), and during the course of a devicerelated infection (20).Nitrate (NO 3 Ϫ ) and nitrite (NO 2 Ϫ ) can be used as terminal electron acceptors under anaerobic conditions. In Staphylococcus carnosus, the membrane-bound respiratory nitrate reduc...

Purpose: To elicit a long-lasting antitumor immune response, CD8+ and CD4 + Tcells should be activated. We attempted to isolate HLA-DR^presented peptides directly from dissected solid tumors, in particular from renal cell carcinoma, to identify MHC class II ligands from tumorassociated antigens (TAA) for their use in peptide-based immunotherapy. Experimental Design: Tumor specimens were analyzed by immunohistochemical staining for their HLA class II expression. HLA class II peptides were subsequently isolated and identified by mass spectrometry. Gene expression analysis was done to detect genes overexpressed in tumor tissue. Peptides from identifiedTAAs were used to induce peptide-specific CD4 + T-cell responses in healthy donors and in tumor patients. Results: In the absence of inflammation, expression of MHC class II molecules is mainly restricted to cells of the immune system. To our surprise, we were able to isolate and characterize hundreds of class II peptides directly from primary dissected solid tumors, especially from renal cell carcinomas, and from colorectal carcinomas and transitional cell carcinomas. Infiltrating leukocytes expressed MHC class II molecules and tumor cells, very likely under the influence of IFNg. Our list of identified peptides contains ligands from several TAAs, including insulin-like growth factor binding protein 3 and matrix metalloproteinase 7. The latter bound promiscuously to HLA-DR molecules and were able to elicit CD4 + T-cell responses. Conclusions: Thus, our direct approach will rapidly expand the limited number of T-helper epitopes fromTAAs for their use in clinical vaccination protocols. CD4+ helper T cells play an important role in orchestrating the effector function of antitumor T-cell responses (1), and for this reason, the identification of CD4 + T-cell epitopes derived from tumor-associated antigens (TAA) has recently been a major focus of attention (2, 3). Even in the absence of CTL effector cells, helper T cells in the mouse can inhibit tumor angiogenesis via IFNg (4) and counteract tumor progression via the induction of an antibody response (5). In contrast to HLA class I ligands, only a small number of class II ligands of TAA has been described. Because HLA class II molecules are constitutively presented on cells of the immune system alone (6), the possibility of isolating class II peptides directly from primary tumors as opposed to class I ligands (7) has not been considered viable. Therefore, numerous strategies to target antigens into the class II processing pathway of antigenpresenting cells have been described (e.g., the incubation of antigen-presenting cells with the antigen of interest to enable it to be taken up, processed, and presented; ref. 8).To identify HLA class II ligands from TAA for their use in peptide-based immunotherapy, we attempted to isolate HLA-DR -presented peptides directly from dissected solid tumors, in particular from renal cell carcinoma (RCC). Even if the majority of tumor cells were class II negative, with state-of-the-ar...

Vascular endothelial growth factor

Prothymosin alpha (ProTalpha) is an acidic polypeptide associated both with cell proliferation and immune regulation. Although ProTalpha's immunomodulating activity is well established at cellular level, limited information is available regarding the signaling pathways triggered by ProTalpha. Using 2-DE proteomic technology, we investigated changes in protein expression of ProTalpha-stimulated peripheral blood mononuclear cells (PBMC) in the course of a 3-day incubation. Using healthy donor- and cancer patient-derived PBMC, 12 gels were studied, identifying 53 differing protein spots via PMF comparison analysis. Among others, we identified interleukin-1 receptor-associated kinase 4, heat-shock protein 90, lipocalin 2, ribophorin 1, eukaryotic elongation factor 2, 14-3-3 protein, L-plastin, and MX2 protein, all of which were found to be overexpressed upon ProTalpha activation. Based on the physiological role of upregulated proteins, we propose the following model for ProTalpha's immunological mode of action: on day 1, ProTalpha triggers monocyte activation, possibly via toll-like receptor signaling, and enhances antigen presentation, consequently promoting and stabilizing monocyte-T-cell immune synapse; on day 2, activated monocytes produce interleukin (IL)-1, while T-cell receptor triggering promotes T-cell proliferation and IL-2 production; finally, on day 3, ProTalpha-activated PBMC express proteins related to adhesion and cytotoxic effector functions, both contributing to the increase of their lytic activity.