Spleen cells from C57BL/6 mice sensitized t o the hapten (4-hydroxy-3-nitro-pheny1)acetyl (NP) were hybridized with myeloma cells, and a variety of hybrid cell lines was isolated which secreted homogeneous anti-NP antibodies. The antibodies were purified by affinity chromatography and their chain composition, affinity and fine specificity were determined. All antibodies recovered from the primary immune response carried A light and /J or y, heavy chains.Their variable portions were nonidentical but similar in terms of hapten-binding specificity with a higher affinity for the cross-reacting haptens (4-hydroxy-3,Sdinitro-phenyl) acetyl (NNP) and (4-hydroxy-5-iodo-3-nitro-phenyl)acetyl (NIP) than for the homologous hapten NP. This heteroclitic property as well as the presence of h , p and y1 chains are characteristic for primary anti-NP sera of C57BL/6 mice..In contrast, four families of anti-NP antibodies, each with a characteristic fine specificity pattern, were found among the hybrid cell antibodies derived from the hyperimmune anti-NP response. The antibodies of one of these families were related t o the antibodies recovered from the primary immune response in that they were heteroclitic and carried X light chains. All members of the other groups expressed K chains and were nonheteroclitic.The finding of well-defined antibody families in this system and the isolation of their members enable us to approach the problem of V gene expression and diversification in a new way.
MHC class I molecules present peptides derived from the ectodomains of endogenous transmembrane proteins; however, the processing of these Ags is incompletely understood. As model transmembrane Ags we investigated the processing of MHC-I-derived fusion proteins containing the N-terminally extended Kb-restricted OVA epitope SIINFEKL in the extracytoplasmic domain. In TAP-deficient, nonprofessional APCs, the epitope was cleaved out of various sequence contexts and presented to T cells. Ag presentation was inhibited by acidophilic amines and inhibitors of the vacuolar proton pump, indicating processing in endosomes. Endosomal aspartic-type cathepsins, and to some extent also the trans-Golgi network protease furin, were involved in processing. Clathrin-dependent and independent internalization from the cell surface targeted MHC-I fusion proteins to early and late endosomes, where SIINFEKL/Kb complexes were detected by immunofluorescence microscopy. Targeting of MHC-I fusion proteins to processing compartments was independent of sequence motifs in the cytoplasmic tail. Not only TAP-deficient cells, but also TAP-competent APCs used the vacuolar pathway for processing of MHC-I fusion proteins. Thus, endosomal processing of internalized endogenous transmembrane proteins represents a novel alternate pathway for the generation of MHC-I-binding peptides.
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