SP-A binding sites on bovine alveolar macrophages.

Surfactant protein A (SP-A) binding to bovine alveolar macrophages was examined in order to characterize SP-A binding proteins on the cell surface and to isolate putative receptors from these cells that could be obtained in large amounts. Human SP-A, unlabeled or labeled with gold particles, was bound to freshly isolated macrophages and analyzed with ELISA or the transmission electron microscope. Binding of SP-A was inhibited by Ca2+ chelation, by an excess of unlabeled SP-A, or by the presence of 20 mg/ml mannan. We conclude that bovine alveolar macrophages expose binding sites for SP-A that are specific and that depend on Ca2+ and on mannose residues. For isolation of SP-A receptors with homologous SP-A as ligand we isolated SP-A from bovine lung lavage. SDS-PAGE analysis of the purified SP-A showed a protein of 32-36 kDa. Functional integrity of the protein was demonstrated. Bovine SP-A bound to Dynabeads was used to isolate SP-A binding proteins. From the fractionated and blotted proteins of the receptor preparation two proteins bound SP-A in a Ca2+-dependent manner, a 40-kDa protein showing mannose dependency and a 210-kDa protein, showing no mannose sensitivity.

Analysis of major histocompatibility complex class I-restricted hapten recognition by mutation of the V-J joining of T cell receptor alpha chains.

Hapten-specific T cell responses are responsible for chemically induced immune disorders. However, the molecular details of hapten interactions with T cell receptors (TCR) are poorly understood. Recent studies of trinitrophenyl (TNP)-specific responses revealed major histocompatibility complex-associated TNP-peptides as dominant epitopes for CD8+ and CD4+ T cells. The present study is based on the observation that two H-2Kb/TNP-specific CTL clones (II/7 and III/1), differing exclusively in two amino acids of their TCR alpha chains, also differed in their carrier specificities for various TNP-peptides. The genes of the two alpha chains and the common beta chain were cloned into expression vectors. Transfection of the TCR alpha chain of clone III/1 into a hybridoma of clone II/7 also transferred the fine specificity of clone III/1, indicating that the small alpha chain variations were indeed responsible for the different carrier specificities. Point mutations bridging the difference between the alpha chains of clones II/7 and III/1 and functional studies of the respective TCR alpha beta transfectants into a TCR-negative hybridoma revealed an unexpected result: the two receptors did not represent examples of structural complementarity for different sets of hapten-peptide conjugates; rather, they resembled two structures of principally similar specificity but of significantly different overall affinity. This was demonstrated more directly by comparing the fine specificities of III/1 transfectants expressing or not expressing the co-receptor CD8: the CD8-negative III/1 transfectant assumed a specificity pattern indistinguishable from that of a CD8-expressing, II/7-derived transfectant. Hence, comparable alterations of antigen recognition may be induced either by subtle TCR alterations or by removal of CD8, i.e. by the presence or absence of a non-polymorphic adhesion molecule.

Purified MHC class I molecules present hapten-conjugated peptides to TNP/H-2Kb-specific T cell hybridomas.

Several trinitrophenyl (TNP)-specific mouse cytotoxic T cell (CTL) clones recognize TNP-conjugated peptides in association with class I MHC Kb-molecules. Here we show that CD8+ T cell hybridomas derived from these CTL exhibit the same pattern of antigen-specificity as their parent CTL-clones. These T cell hybridomas reacted with TNBS- or TNP-peptide modified syngeneic target cells, and also with affinity purified, immobilized Kb-molecules preloaded with TNP-peptides. These findings demonstrate most directly that MHC-associated, haptenated peptides create functional antigenic epitopes for TNP-specific CTL. Furthermore, using purified Kb-molecules and a panel of Kb-binding TNP-conjugated peptides, we demonstrated that the epitope density is a critical factor in triggering these T cell hybridomas. Chemical modification of immobilized Kb-layers resulted in poor antigenicity, implying low epitope density and therefore arguing against covalent MHC-haptenization as a major source of T cell antigenic determinants.