muramidase and leupeptin

We have developed cell-based cancer vaccines that activate anti-tumor immunity by directly presenting endogenously synthesized tumor antigens to CD4+ T helper lymphocytes via MHC class II molecules. The vaccines are non-conventional antigen-presenting cells because they express MHC class II, do not express invariant chain or H-2M, and preferentially present endogenous antigen. To further improve therapeutic efficacy we have studied the intracellular trafficking pathway of MHC class II molecules in the vaccines using endoplasmic reticulumlocalized lysozyme as a model antigen. Experiments using endocytic and cytosolic pathway inhibitors (chloroquine, primaquine, and brefeldin A) and protease inhibitors (lactacystin, LLnL, E64, and leupeptin) indicate antigen presentation depends on the endocytic pathway, although antigen degradation is not mediated by endosomal or proteasomal proteases. Because H2-M facilitates presentation of exogenous antigen via the endocytic pathway, we investigated whether transfection of vaccine cells with H-2M could potentiate endogenous antigen presentation. In contrast to its role in conventional antigen presentation, H-2M had no effect on endogenous antigen presentation by vaccine cells or on vaccine efficacy. These results suggest that antigen/MHC class II complexes in the vaccines may follow a novel route for processing and presentation and may produce a repertoire of class II-restricted peptides different from those presented by professional APC. The therapeutic efficacy of the vaccines, therefore, may reside in their ability to present novel tumor peptides, consequently activating tumor-specific CD4+ T cells that would not otherwise be activated.

Topics: Acetylcysteine; Animals; Antigen Presentation; Antigen-Presenting Cells; Antigens, Neoplasm; Antimalarials; Brefeldin A; CD4-Positive T-Lymphocytes; Chloroquine; Coculture Techniques; Cysteine Proteinase Inhibitors; Cytosol; Dose-Response Relationship, Drug; Endocytosis; Endoplasmic Reticulum; Flow Cytometry; Fluorescent Antibody Technique, Indirect; Genes, MHC Class II; Humans; Hybridomas; Leupeptins; Major Histocompatibility Complex; Mice; Muramidase; Plasmids; Primaquine; Protein Synthesis Inhibitors; Protein Transport; Ribonucleases; Transfection; Tumor Cells, Cultured

To study the processing and presentation of endogenously synthesized Ag to class II MHC-restricted T cells, hen egg lysozyme (HEL), either tagged with a peptide that confers retention in the endoplasmic reticulum (HEL.KDEL), or in the secretory form (HELs), was stably expressed in LK-35.2 B hybridoma cells. Presentation of HEL peptides bound to class II molecules was assessed by activation of specific T cell hybridomas recognizing seven different epitopes derived from exogenous HEL. The presentation of endogenously synthesized HEL was not caused by reuptake of secreted of shed Ag. All the HEL epitopes examined were efficiently presented after processing of endogenous HEL by HELs-transfected LK-35.2 cells. Processing of HEL tagged with KDEL, however, gave rise to presentation of only six of the seven HEL epitopes. The epitope included in the HEL sequence 112-124 was not presented by HEL.KDEL-transfected B cells. In addition, two of the four T cell hybridomas recognizing HEL 116-129 together with I-Ak molecules were not activated by HEL.KDEL, and three other epitopes were presented with lower efficiency as compared with HELs. Thus, endogenously synthesized HEL in secretory form gives rise to a set of class II-binding epitopes indistinguishable from exogenous HEL, whereas endoplasmic reticulum-retained HEL generates a similar but not identical set of epitopes. The endosomal protease inhibitor leupeptin prevented presentation of the epitope 108-116, but not 46-61, both by HELs and HEL.KDEL transfected cells, indicating a requirement for endosomal processing in both cases. In addition, the presentation of peptides derived from endogenously synthesized, either secretory or endoplasmic reticulum-retained HEL, could be inhibited by lysosomotropic amines, further indicating that the intracellular route of class II molecules presenting peptides derived from endogenous Ag intersects the acidic endosomal compartment.

Topics: Amino Acid Sequence; Animals; Cells, Cultured; Chickens; Egg Proteins; Endoplasmic Reticulum; Epitopes; Histocompatibility Antigens Class II; Leupeptins; Mice; Molecular Sequence Data; Muramidase; Peptide Fragments; T-Lymphocytes; Transfection

The mechanism for binding of human erythrocyte calpain I to human erythrocyte inside-out vesicles was studied by immunoelectrophoretic blot analysis. Binding of calpain I to inside-out vesicles was observed both in the absence and presence of Ca2+. Moreover, in the absence of Ca2+, acidic proteins like casein, ovalbumin and calpastatin suppressed while basic proteins like arginase and lysozyme did not affect the binding of calpain I to inside-out vesicles. Here, we propose a model for the binding of calpain to the membrane.

Topics: Arginase; Calcium; Calcium-Binding Proteins; Calpain; Caseins; Erythrocyte Membrane; Humans; Immunoelectrophoresis; Immunoglobulins; Leupeptins; Models, Biological; Muramidase; Ovalbumin; Protease Inhibitors

The participation of lysosomal enzymes, hydroxyl radicals, and mitochondrial respiration in the cytocidal effect of TNF on tumor cells was investigated. The cytotoxicity of TNF on L-M cells was clearly reduced by lysosomotropic agents, DMSO (hydroxyl radical scavenger), NDGA (lipoxygenase inhibitor), and sodium azide (mitochondrial respiration inhibitor). The results suggest that lysosomal enzyme and hydroxyl radicals play an important triggering role in the destruction of tumor cells by TNF, and that the process of destruction might require ATP.

Topics: Ammonium Chloride; Animals; Chloroquine; Cytotoxicity, Immunologic; Dimethyl Sulfoxide; Free Radicals; Hydroxides; Hydroxyl Radical; Leupeptins; Masoprocol; Methylamines; Mice; Muramidase; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

The effect of a protease inhibitor, leupeptin, on the presentation of hen egg lysozyme (HEL) to cloned T cells was investigated. We found that leupeptin-sensitive thiol proteases are apparently less involved when HEL is presented by the I-Ad molecule, than when it is presented by the I-Ed molecule. This selectivity was more of a function of the antigen than that of the Ia molecule because presentation of denatured or fragmented HEL was not sensitive to leupeptin whereas antigen presentation to a number of I-A-restricted T cell clones specific to other antigens was sensitive to leupeptin. These data demonstrate that the particular combination of major histocompatibility complex/nominal antigen recognized by a certain T cell clone may require processing of the antigen molecule through a certain group of proteases and that other combinations are independent of that particular processing pathway. Furthermore, there is a preference for a certain type of processing depending on the Ia molecule involved.

Topics: Animals; Antigen-Presenting Cells; Antigens; Clone Cells; Epitopes; H-2 Antigens; Histocompatibility Antigens Class II; Leupeptins; Major Histocompatibility Complex; Mice; Mice, Inbred Strains; Muramidase; Peptide Hydrolases; T-Lymphocytes