sodium-dodecyl-sulfate and Lymphoma--B-Cell

Inside APCs, MHC class II molecules associate with antigenic peptides before reaching the cell surface. This association takes place in compartments of the endocytic pathway, more related to endosomes or lysosomes depending on the cell type. Here, we compared MHC class II transport from endosomal vs lysosomal compartments to the plasma membrane. We show that transport of MHC class II molecules to the cell surface does not depend on the cytosolic domains of the alpha- and beta-chains. In contrast, the stability of the alphabeta-peptide complexes determined the efficiency of transport to the cell surface from lysosomal, but not from endosomal, compartments. In murine B lymphoma cells, SDS-unstable and -stable complexes were transported to the cell surface at almost similar rates, whereas after lysosomal relocalization or in a cell line in which MHC class II molecules normally accumulate in lysosomal compartments, stable complexes were preferentially addressed to the cell surface. Our results suggest that when peptide loading occurs in lysosomal compartments, selective retention and lysosomal degradation of unstable dimers result in the expression of highly stable MHC class II-peptide complexes at the APC surface.

Topics: Amino Acid Sequence; Animals; Biological Transport; Cell Compartmentation; Cell Membrane; Cytosol; Dendritic Cells; Electrophoresis, Polyacrylamide Gel; Endosomes; Histocompatibility Antigens Class II; Lymphoma, B-Cell; Lysosomes; Mice; Molecular Sequence Data; Peptide Fragments; Protein Binding; Protein Structure, Tertiary; Sodium Dodecyl Sulfate; Tumor Cells, Cultured

X-ray crystallography of several MHC class II molecules revealed a structure described as a dimer of heterodimers, or a superdimer. This discovery led to the hypothesis that MHC class II molecules may interact with the TCR and CD4 as an (alpha beta)2 superdimer, potentially providing more stable and stimulatory interactions than can be provided by the simple alpha beta heterodimer alone. In this study, using chemical cross-linking, we provide evidence for the existence of the superdimers surface of B cells. We further characterize the superdimers and demonstrate that in lysates of B cells, I-Ek dimers and superdimers are derived from the same population of I-Ek molecules. Purified, I-Ek molecules in solution also exist as a mixture of 60-kDa dimers and 120-kDa superdimers, indicating that I-Ek has an intrinsic ability to form 120-kDa complexes in the absence of other cellular components. Peptide mapping showed that the alpha beta and (alpha beta)2 complexes are closely related and that the superdimers do not contain additional polypeptides not present in the dimers. The (alpha beta)2 complex displays thermal and pH stability similar to that of the alpha beta complex, both being denatured by SDS at temperatures above 50 degrees C and at a pH below 5. These data support the model that MHC class II has an intrinsic ability to assume the (alpha beta)2 superdimeric conformation, which may be important for interactions with the TCR and CD4 coreceptor.

Topics: Animals; B-Lymphocytes; Cell Membrane; Cross-Linking Reagents; Dimerization; Histocompatibility Antigens Class II; Hydrogen-Ion Concentration; Lymphoma, B-Cell; Macromolecular Substances; Mice; Molecular Weight; Protein Denaturation; Sodium Dodecyl Sulfate; Temperature; Tumor Cells, Cultured

Type II collagen (CII) is an arthritogenic self antigen in DBA/1 (H-2q) mice. To analyze the intracellular processing of this fibrillar protein in the context of I-Aq molecules, we have generated hybrid antigen-presenting cells (APC) by fusion of B lymphoma (A20 and M12) cells with CII-primed spleen cells from DBA/1 mice. Efficient presentation of CII by these APC to specific T cell hybridomas required prior cleavage of the antigen and intracellular handling of the peptides. Inhibition of protein transport by brefeldin A prevented the presentation of CII peptides to T cell hybridomas, indicating that the intracellular presentation of CII was dependent on neo-synthesis of I-Aq molecules. In contrast, exposure of hybrid B lymphomas to leupeptin, a protease inhibitor, induced a dose-dependent increase of CII-specific T cell response, while abrogating the I-Aq-restricted presentation of ovalbumin. The enhancing effect of leupeptin was also observed when immune B cells were used as APC. In contrast, leupeptin inhibited the presentation of CII peptides by macrophages or total spleen cells. Pulse-chase analysis of metabolically labeled hybrid APC and immunoprecipitation with antibodies specific for class II molecules or invariant (li) chain revealed that leupeptin did not affect the li chain processing or the formation of stable class II dimers. The stimulatory effect of leupeptin observed on CII presentation suggests that leupeptin protects CII epitopes by interfering with proteases involved in the intracellular degradation of CII.

Topics: Animals; Antigen Presentation; Antigen-Presenting Cells; Antigens, Differentiation, B-Lymphocyte; Brefeldin A; Collagen; Cyclopentanes; Dimerization; Epitopes; Histocompatibility Antigens Class II; Immunophenotyping; Leupeptins; Lymphoma, B-Cell; Mice; Mice, Inbred DBA; Protease Inhibitors; Protein Synthesis Inhibitors; Sodium Dodecyl Sulfate; Tumor Cells, Cultured

LPS is the most important antigen of Brucella bacteria which are gram-negative facultative intracellular pathogens infecting a large proportion of animals and humans in the world. In order to get insights into the immune response mechanisms monitored by Brucella, its LPS was used as a model antigen. S-LPS, R-LPS, lipid A and O-chain purified from Brucella abortus were tested in their capacity of inducing SDS-resistant MHC class II molecules after incubation with murine B lymphoma cells. S-LPS and O-chain gave a significant response suggesting that O-chain might induce an association with class II itself or might act as a carrier for antigens to bind MHC class II molecules.

Topics: Animals; Brucella abortus; CD4-Positive T-Lymphocytes; Chickens; Drug Resistance; Female; Histocompatibility Antigens Class II; Hot Temperature; Immunosorbent Techniques; Lipid A; Lipopolysaccharides; Lymphoma, B-Cell; Mice; Mice, Inbred CBA; Muramidase; Sodium Dodecyl Sulfate; T-Lymphocytes, Helper-Inducer; Tumor Cells, Cultured