Mutations in CD8 that affect interactions with HLA class I and monoclonal anti-CD8 antibodies.
The T cell co-receptor, CD8, binds to the alpha 3 domain of HLA class I (Salter, R.D., R.J. Benjamin, P.K. Wesley, S.E. Buxton, T.P.J. Garrett, C. Clayberger, A.M. Krensky, A.M. Norman, D.R. Littman, and P. Parham. 1990. Nature [Lond.]. 345:41; Connolly, J.M., T.A. Potter, E.M. Wormstall, and T.H. Hansen. 1988. J. Exp. Med. 168:325; and Potter, T.A., T.V. Rajan, R.F. Dick II, and J.A. Bluestone. 1989. Nature [Lond.]. 337:73). To identify regions of CD8 that are important for binding to HLA class I, we performed a mutational analysis of the CD8 molecule in the immunoglobulin (Ig)-like variable domain. Our mutational analysis was based on our finding that using a cell-cell adhesion assay murine CD8 (Lyt-2) did not bind to human class I. Since the interaction of human CD8 with HLA class I is species specific, we substituted nonconservative amino acids from mouse CD8 and analyzed the ability of the mutated CD8 molecules expressed in COS 7 cells to bind HLA class I-bearing B lymphoblastoid cells, UC. Mutants with the greatest effect on binding were located in a portion of the molecule homologous to the first and second hypervariable regions of an antibody combining site. In addition, a panel of 12 anti-CD8 monoclonal antibodies were used to stain the 10 CD8 mutants, and amino acids that affected antibody binding were localized on the crystal structure of the Bence-Jones homodimer, REI. Support for an Ig-like structure of CD8 can be found in the pattern of substitutions affecting antibody binding. This work supports the similar tertiary structure of the CD8 alpha-terminal domain and an Ig variable domain.
Sanders
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Department of Laboratory Medicine, Yale University, New Haven, Connecticut 06510.
Fox
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Kavathas
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D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000945:Antigens, Differentiation, T-Lymphocyte / Q000235:genetics* / Q000276:immunology; D001402:B-Lymphocytes / Q000276:immunology; D016827:CD8 Antigens; D002448:Cell Adhesion / Q000276:immunology; D002460:Cell Line; D005434:Flow Cytometry; D015870:Gene Expression; D015395:Histocompatibility Antigens Class I / Q000276:immunology*; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000276:immunology; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016415:Sequence Alignment; D013045:Species Specificity; D014162:Transfection
D000911:Antibodies, Monoclonal; D000945:Antigens, Differentiation, T-Lymphocyte; D016827:CD8 Antigens; D015395:Histocompatibility Antigens Class I; D007135:Immunoglobulin Variable Region