ovalbumin and lactacystin

Cross-presentation is one of the main features of dendritic cells (DCs), which is critically important for the development of spontaneous and therapy-inducible antitumor immune responses. Patients, at early stages of cancer, have normal presence of DCs. However, the difficulties in the development of antitumor responses in patients with low tumor burden raised the question of the mechanisms of DC dysfunction. In this study, we found that, in differentiated DCs, tumor-derived factors blocked the cross-presentation of exogenous Ags without inhibiting the Ag presentation of endogenous protein or peptides. This effect was caused by intracellular accumulation of different types of oxidized neutral lipids: triglycerides, cholesterol esters, and fatty acids. In contrast, the accumulation of nonoxidized lipids did not affect cross-presentation. Oxidized lipids blocked cross-presentation by reducing the expression of peptide-MHC class I complexes on the cell surface. Thus, this study suggests the novel role of oxidized lipids in the regulation of cross-presentation.

Topics: Acetylcysteine; Animals; Antigen Presentation; Cell Line, Tumor; Cells, Cultured; Cross-Priming; Culture Media, Conditioned; Cysteine Proteinase Inhibitors; Dendritic Cells; Flow Cytometry; Histocompatibility Antigens Class I; Histocompatibility Antigens Class II; Humans; Interferon-gamma; Lipids; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Transgenic; Microscopy, Confocal; Neoplasms; Ovalbumin; Oxidation-Reduction; Peptide Fragments

We demonstrate that uptake of oligomeric cognate antigen (OVA-hen egg lysozyme, OVA-HEL) alone or incorporated in immune-stimulating complexes (ISCOMS) facilitates presentation and simultaneous cross-presentation of OVA by HEL-specific B cells in vitro. HEL-specific B cells stimulated CD8(+) T cell responses in vitro to the same extent as bone marrow-derived dendritic cells. Cross-presentation by specific B cells required endosomal acidification, proteasomal processing and classical MHC class I/peptide transport. Specific B cells also acquired both antigens rapidly in vivo and presented them to CD4(+) T cells. However, only HEL-specific B cells from OVA-HEL ISCOMS-immunised mice could cross-present OVA to naive OVA-specific CD8(+) T cells. Antigen-specific B cells were also activated selectively by OVA-HEL ISCOMS in vitro and importantly, the presence of HEL-specific B cells promoted the persistence of clonal expansion of OVA-specific CD8(+) T cells after in vivo immunisation with OVA-HEL ISCOMS. These results demonstrate preferential MHC class I and class II processing of cognate antigen incorporated in ISCOMS by specific B cells in vitro and in vivo, highlighting the ability of ISCOMS to target B cells and offering novel insights into the role of B cells in cross-presentation to CD8(+) T cells.

Topics: Acetylcysteine; Adoptive Transfer; Animals; Antigen Presentation; Antigens; B-Lymphocytes; Brefeldin A; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Cell Proliferation; Chloroquine; Cysteine Proteinase Inhibitors; Epitopes, B-Lymphocyte; ISCOMs; Kinetics; Lymph Nodes; Mice; Mice, Inbred BALB C; Mice, Mutant Strains; Mice, Transgenic; Muramidase; Ovalbumin; Peptide Fragments; Receptors, Antigen, T-Cell; Vaccination

Biodegradable nanospheres generated from a biocompatible polymer, poly(D,L-lactide-co-glycolide) (PLGA), have been studied extensively as implantable reservoirs for sustained-release drug delivery. PLGA-nanospheres have also been studied as vehicles to deliver antigens to phagocytes. The intracellular processing pathway of antigens delivered to phagocytes by PLGA particles was studied in the present study. Ovalbumin (OVA) encapsulated with PLGA (OVA-nanosphere) was efficiently captured, processed and presented on class I major histocompatibility complex (MHC-I) by dendritic cells (DCs). The MHC-I processing of OVA-nanospheres was resistant to lactacystin, a proteosome inhibitor, and brefeldin A, which blocks anterograde transport from the endoplasmic reticulum (ER) through the Golgi apparatus. Chloroquine, which inhibits phagolysosomal enzymes by increasing phagolysosomal pH, inhibited MHC-I processing of OVA-nanospheres. In addition, DCs generated from TAP-/- mice were markedly suppressed in MHC-I processing of OVA-nanospheres. These results demonstrate that DCs process phagocytosed OVA-nanospheres via a vacuolar alternate MHC-I pathway for presentation of OVA peptides to T lymphocytes.

Topics: Acetylcysteine; Antigen Presentation; ATP Binding Cassette Transporter, Subfamily B, Member 2; ATP-Binding Cassette Transporters; Brefeldin A; Cell Line; Chloroquine; Dendritic Cells; Histocompatibility Antigens Class I; Humans; Lactic Acid; Nanospheres; Ovalbumin; Phagocytosis; Polyglycolic Acid; Polylactic Acid-Polyglycolic Acid Copolymer; Polymers

Exogenous proteins can be processed by antigen-presenting cells for the generation of MHC class I-restricted T cell responses. Where this occurs is not clear, although both transfer of internalized antigen into the cytosol and alternative processing in endolysosomes and phagosomes have been reported. Here we have studied the capacity of bone marrow-derived mouse myeloid dendritic cells (DC) to process the OVA protein for peptide presentation by H2-K(b). We have found that immature DC (iDC), both wild-type and transporter associated with antigen processing (TAP)-deficient cells, can transiently process OVA in a pathway which is resistant to inhibitors of the classical MHC class I pathway including the Golgi inhibitor Brefeldin A (BFA) and the proteasome inhibitor lactacystin. This alternative pathway is not found in subcultured DC with an intermediate maturity (imDC) or in resting, IL-3 expanded macrophages but can be re-expressed in imDC if these are activated by an immunostimulatory CpG oligonucleotide. Both iDC and CpG-activated DC were found to process OVA by regurgitation. In addition, we found that iDC secrete proteolytic enzymes into the supernatant, which can process OVA in the extracellular phase. These results suggest that multiple pathways exist for the processing of exogenous protein antigens into MHC class I-binding peptides.

Topics: Acetylcysteine; Animals; Antigen Presentation; Bone Marrow; Brefeldin A; Cells, Cultured; CpG Islands; Cysteine Proteinase Inhibitors; Dendritic Cells; Female; Flow Cytometry; Histocompatibility Antigens Class I; Mice; Ovalbumin; Protein Synthesis Inhibitors

The pharmacologic properties of ajoene, the major sulfur-containing compound purified from garlic, and its possible role in the prevention and treatment of cancer has received increasing attention. Several studies demonstrated that induction of apoptosis and cell cycle blockade are typical biologic effects observed in tumor cells after proteasome inhibition. The proteasome is responsible for the degradation of a variety of intracellular proteins and plays a key role in the regulation of many cellular processes. The aim of the present work was therefore to explore the effects of ajoene on the proteasome activities. In vitro activities of 20S proteasome purified from human erythrocytes on fluorogenic peptide substrates specific for trypsin-like, chymotrypsin-like and peptidylglutamyl peptide hydrolyzing activities revealed that ajoene inhibited the trypsin-like activity in a dose- and time-dependent manner. Further, the ability of 20S proteasome to degrade the OVA(51-71) peptide, a model proteasomal substrate, was partially but significantly inhibited by ajoene. In addition, when human leukemia cell line HL60 was treated with ajoene, both trypsin- and chymotrypsin-like activities were affected, cells arrested in G2/M phase and total amount of cytosolic proteasome increased. All these data clearly indicate that ajoene may affect proteasome function and activity both in vitro and in the living cell. This is a novel aspect in the biologic profile of this garlic compound giving new insights into the understanding of the molecular mechanisms of its potential antitumor action.

Topics: Acetylcysteine; Antineoplastic Agents; Cell Division; Chymotrypsin; Disulfides; Dose-Response Relationship, Drug; Enzyme Inhibitors; G2 Phase; Garlic; HL-60 Cells; Humans; Hybridomas; Ovalbumin; Peptides; Plant Extracts; Plant Stems; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Sulfoxides; Time Factors; Trypsin

"Cross-priming" describes the activation of naïve CD8+ T cells by professional antigen-presenting cells that have acquired viral or tumor antigens from "donor" cells. Antigen transfer is believed to be mediated by donor cell-derived molecular chaperones bearing short peptide ligands generated by proteasome degradation of protein antigens. We show here that cross-priming is based on the transfer of proteasome substrates rather than peptides. These findings are potentially important for the rational design of vaccines that elicit CD8+ T cell responses.

Topics: Acetylcysteine; Animals; Antigen Presentation; Antigens; Antigens, Viral; CD8-Positive T-Lymphocytes; Cell Line; Cross-Priming; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Endoplasmic Reticulum; Humans; Immunization; Influenza A virus; Lymphocyte Activation; Mice; Mice, Inbred C57BL; Mice, Transgenic; Molecular Chaperones; Multienzyme Complexes; Ovalbumin; Peptide Fragments; Proteasome Endopeptidase Complex; Recombinant Fusion Proteins; Vaccines; Vaccinia virus

We previously reported that exogenous antigens complexed with the cationic liposome lipofectin (LF) were efficiently presented via major histocompatibility complex (MHC) class I molecules on pulsed dendritic cells (DCs) in vitro. In the present study, we demonstrated that MHC class I-restricted antigen presentation on DC2.4 cells, a murine immature DC line, treated with LF-antigen complexes was remarkably suppressed through the inhibition of endocytosis, proteasome catalysis, and Golgi transport. We also found that LF did not influence expression of interleukin-12 p40 mRNA, MHC molecules, or co-stimulatory molecules in DC2.4 cells. These findings suggest that an antigen-loading procedure using LF could enhance delivery of exogenous antigens to the classical MHC class I pathway in DCs, but it does not initiate DC maturation.

Topics: Acetylcysteine; Animals; Antigen Presentation; Antigens; Brefeldin A; Cell Line; Cell Survival; Chloroquine; Cytochalasin B; Dendritic Cells; Histocompatibility Antigens Class I; Major Histocompatibility Complex; Mice; Ovalbumin; Phosphatidylethanolamines; Time Factors

Peptides from extracellular proteins presented on MHC class II are mostly generated and loaded in endolysosomal compartments, but the major pathways responsible for loading peptides from APC-endogenous sources on MHC class II are as yet unclear. In this study, we show that MHC class II molecules present peptides from proteins such as OVA or conalbumin introduced into the cytoplasm by hyperosmotic pinosome lysis, with efficiencies comparable to their presentation via extracellular fluid-phase endocytosis. This cytosolic presentation pathway is sensitive to proteasomal inhibitors, whereas the presentation of exogenous Ags taken up by endocytosis is not. Inhibitors of nonproteasomal cytosolic proteases can also inhibit MHC class II-restricted presentation of cytosolically delivered protein, without inhibiting MHC class I-restricted presentation from the same protein. Cytosolic processing of a soluble fusion protein containing the peptide epitope I-Ealpha(52-68) yields an epitope that is similar to the one generated during constitutive presentation of I-Ealpha as an endogenous transmembrane protein, but is subtly different from the one generated in the exogenous pathway. Constitutive MHC class II-mediated presentation of the endogenous transmembrane protein I-Ealpha is also specifically inhibited over time by inhibitors of cytosolic proteolysis. Thus, Ag processing in the cytoplasm appears to be essential for the efficient presentation of endogenous proteins, even transmembrane ones, on MHC class II, and the proteolytic pathways involved may differ from those used for MHC class I-mediated presentation.

Topics: Acetylcysteine; Amino Acid Chloromethyl Ketones; Animals; Antigen Presentation; Antigen-Presenting Cells; Cell Line; Conalbumin; Cysteine Endopeptidases; Cytosol; Endocytosis; Endosomes; Histocompatibility Antigens Class II; Membrane Proteins; Mice; Mice, Inbred C3H; Mice, Inbred C57BL; Multienzyme Complexes; Ovalbumin; Pinocytosis; Protease Inhibitors; Proteasome Endopeptidase Complex

Immunization with peptide or recombinant proteins generally fails to elicit CTL, which are thought to play a key role in the control of virus-infected cells and tumor growth. In this study we show that the nontoxic B subunit of Shiga toxin fused to a tumor peptide derived from the mouse mastocytoma P815 can induce specific CTL in mice without the use of adjuvant. The Shiga B subunit acts as a vector rather than as an adjuvant, because coinjection of the tumor peptide and the B subunit as separate entities does not lead to CTL induction. We also demonstrated that in vitro the B subunit mediates the delivery of various exogenous CD8 T cell epitopes into the conventional MHC class I-restricted pathway, as this process is inhibited by brefeldin A and lactacystin and requires a functional TAP system. In contrast to other nonviral methods for transport of exogenous Ags into the endogenous MHC class I pathway that involve macropinocytosis or phagocytosis, the Shiga B subunit targets this pathway in a receptor-dependent manner, namely via binding to the glycolipid Gb3. Because this receptor is highly expressed on various dendritic cells, it should allow preferential targeting of the Shiga B subunit to these professional APCs. Therefore, the Shiga B subunit appears to represent an attractive vector for vaccine development due to its ability to target dendritic cells and to induce specific CTL without the need for adjuvant.

Topics: Acetylcysteine; Animals; Antigen Presentation; Antigens, Neoplasm; ATP Binding Cassette Transporter, Subfamily B, Member 2; ATP-Binding Cassette Transporters; Bacterial Toxins; Brefeldin A; Cytotoxicity, Immunologic; Dendritic Cells; Female; Histocompatibility Antigens Class I; Injections, Intraperitoneal; Intracellular Fluid; Leukemia L1210; Lymphocyte Activation; Mice; Mice, Inbred C57BL; Mice, Inbred DBA; Mice, Knockout; Ovalbumin; Peptides; Protein Processing, Post-Translational; Recombinant Fusion Proteins; Sarcoma, Experimental; Shiga Toxins; Signal Transduction; T-Lymphocytes, Cytotoxic; Tumor Cells, Cultured

To study the role of proteasomes in Ag presentation, we analyzed the effects of proteasome inhibitors Cbz-Leu-Leu-Leucinal and lactacystin on the ability of mouse fibroblast cells to present recombinant vaccinia virus gene products to MHC class I-restricted T cells. The effects of the inhibitors depended on the determinant analyzed. For influenza virus nucleoprotein (NP), presentation of the immunodominant Kk-restricted determinant (NP(50-57)) was marginally inhibited, whereas presentation of the immunodominant Kd-restricted determinant (NP(147-155)) was enhanced, particularly by lactacystin. Biochemical purification of peptides confirmed that lactacystin enhanced the generation of Kd-NP(147-155) complexes fourfold. Lactacystin also enhanced the recovery of one Kd-restricted vaccinia virus determinant from HPLC fractions, while inhibiting recovery of another. The inhibitors were used at sufficient concentrations to block presentation of biosynthesized full-length OVA and to completely stabilize a rapidly degraded chimeric ubiquitin-NP fusion protein. Strikingly, presentation of antigenic peptides from this protein was unaffected by proteasome inhibitors. We also observed that proteasome inhibitors induced expression of cytosolic and endoplasmic reticulum stress-responsive proteins. These data demonstrate first that the processes of protein degradation and generation of antigenic peptides from cytosolic proteins can be dissociated, and second that effects of proteasome inhibitors on Ag presentation may reflect secondary effects on cellular metabolism.

Topics: Acetylcysteine; Animals; Antigen Presentation; Cysteine Endopeptidases; Histocompatibility Antigens Class I; Humans; Mice; Mice, Inbred BALB C; Mice, Inbred C3H; Multienzyme Complexes; Ovalbumin; Peptide Fragments; Proteasome Endopeptidase Complex; Recombinant Proteins; Tumor Cells, Cultured; Viral Proteins