Epitope-directed processing of specific antigen by B lymphocytes (original) (raw)
1989, The Journal of Cell Biology
Proteolytic processing of specific antigen was studied using Epstein Barr virus transformed B-lymphoblastoid cells expressing membrane IgG against tetanus toxin. As previously reported (Watts, C., and H.W. Davidson. 1988. EMBO (Eur. Mol. Biol. Organ.) J. 7:1937-1945), receptor-mediated endocytosis of monovalent antigen bound at 0 degrees C began immediately upon shifting the cells to 37 degrees C. In contrast, degradation of antigen, assessed either by the release of acid-soluble radiolabel into the incubation medium, or by SDS-PAGE analysis of total cell-associated antigen, proceeded after a lag of 10-20 min. Degradation was abolished by exposure of the cells to metabolic inhibitors, or by incubation at 20 degrees C, and inhibited in a dose-dependent fashion by chloroquine and by the lysosomal protease inhibitors leupeptin, E-64, and pepstatin A. Analysis of the cell-associated radiolabel by SDS-PAGE and autoradiography after incubations at 37 degrees C revealed the time-dependent ...
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Endocytosis and recycling of specific antigen by human B cell lines
The EMBO Journal, 1988
Communicated by M.J.Crumpton Human B cell lines expressing membrane inmmunoglobulin specific for tetanus toxoid/toxin were used to study the receptor-mediated endocytosis of antigen. Monovalent antigen, initially bound to cell surface immunoglobulin at 0°C, was rapidly endocytosed upon warming the cells to 37°C. The kinetics of endocytosis of antigen were independent of the number of occupied binding sites and indicated a half-life for antigen on the cell surface of 8.5 min. Endocytosis of antigen apparently ceased after 15 min at 37°C, although some 40-50% remained on the cell surface at this time. We show, using biotinylated antigen and an avidin detection assay, that this is due to recycling of antigen to the cell surface. By labelling the antigen on the cell surface with Fabs against different epitopes we show that antigen continues to be endocytosed for at least 1 h after the initial rapid phase of endocytosis, again indicating that there must be recycling of immunoglobulin/antigen complexes. As a consequence of the stable interaction between antigen and membrane immunoglobulin, the capacity of the cells to accumulate antigen was limited when the synthesis of membrane immunoglobulin was blocked; under these conditions only 2-3 times as much antigen was endocytosed and degraded when antigen was supplied continuously over a 4-h period at 37°C as could be bound to the cells at 0°C. These results reveal a rapid and efficient pathway for the endocytosis and recycling of monovalent antigen in B cells.
Immunology, 1996
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Journal of Experimental Medicine, 1995
S unll'l'lal'~ We have raised CD8 § cytotoxic T lymphocytes (CTL) from three Epstein-Barr virus-seropositive donors by incubating peripheral blood lymphocytes with irradiated autologous B95.8-strain EBV-transformed B lymphoblastoid cells (LCL). However, to detect lysis in a standard SlCr release assay of the LCL against which these CTL were raised, superinfection with recombinant vaccinia expressing the appropriate EBV protein or incubation with the peptide epitope was necessary. The untreated LCL were not lysed, even though Western blotting demonstrated that they expressed the EBV antigens containing the CTL epitopes. We have found CTL of this phenotype that are restricted by human leukocyte antigen-A2, -A3, -B7, or -B39, and which recognize the EBV latent proteins, EBV nuclear antigen (EBNA)-3A, EBNA-3C, or terminal protein. During these experiments, we identified a new human leukocyte antigen-A3-restricted EBNA-3A epitope, residues 603-611, RLtLAEAGVK. We raised a spontaneous LCL, transformed by endogenous EBV, from one donor, but this was also not lysed. For at least one of the epitopes, CTL from another donor lysed the LCL without superinfection or addition of peptides. We conclude that the CTL were unable to achieve a high enough avidity of interaction with untreated LCL to trigger effector function, although the LCL were able to stimulate them to grow in vitro for up to 4 too. To assess whether a small percentage of the LCL might possess a higher antigen density, we used an assay of tumor necrosis factor release from a CTL clone, which was able to detect antigen-bearing cells representing only 1% of a stimulating LCL population. Nevertheless, the untreated autologous LCL line failed to stimulate tumor necrosis factor release. nology,
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