title: Soluble major histocompatibility complex-peptide octamers with impaired CD8 binding selectively induce Fas-dependent apoptosis.
abstract: Fluorescence-labeled soluble major histocompatibility complex class I-peptide "tetramers" constitute a powerful tool to detect and isolate antigen-specific CD8(+) T cells by flow cytometry. Conventional "tetramers" are prepared by refolding of heavy and light chains with a specific peptide, enzymatic biotinylation at an added C-terminal biotinylation sequence, and "tetramerization" by reaction with phycoerythrin- or allophycocyanin-labeled avidin derivatives. We show here that such preparations are heterogeneous and describe a new procedure that allows the preparation of homogeneous tetra- or octameric major histocompatibility complex-peptide complexes. These compounds were tested on T1 cytotoxic T lymphocytes (CTLs), which recognize the Plasmodium berghei circumsporzoite peptide 252-260 (SYIPSAEKI) containing photoreactive 4-azidobenzoic acid on Lys(259) in the context of H-2K(d). We report that mutation of the CD8 binding site of K(d) greatly impairs the binding of tetrameric but not octameric or multimeric K(d)-PbCS(ABA) complexes to CTLs. This mutation abolishes the ability of the octamer to elicit significant phosphorylation of CD3, intracellular calcium mobilization, and CTL degranulation. Remarkably, however, this octamer efficiently activates CTLs for Fas (CD95)-dependent apoptosis.
authors.affiliation: Ludwig Institute for Cancer Research, Lausanne Branch, University of Lausanne, 1066 Epalinges, Switzerland.
authors.forename: Philippe
authors.initials: P
authors.lastname: Guillaume
authors.forename: Daniel F
authors.initials: DF
authors.lastname: Legler
authors.forename: Nicole
authors.initials: N
authors.lastname: Boucheron
authors.forename: Marie-Agnès
authors.initials: MA
authors.lastname: Doucey
authors.forename: Jean-Charles
authors.initials: JC
authors.lastname: Cerottini
authors.forename: Immanuel F
authors.initials: IF
authors.lastname: Luescher
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000276:immunology*; D001665:Binding Sites / Q000235:genetics; D016827:CD8 Antigens / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology / Q000473:pathology; D003602:Cytotoxicity, Immunologic; D015395:Histocompatibility Antigens Class I / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D010455:Peptides / Q000276:immunology; D011485:Protein Binding / Q000276:immunology; D019014:fas Receptor / Q000276:immunology
chemical_list: D016827:CD8 Antigens; D015395:Histocompatibility Antigens Class I; D010455:Peptides; D019014:fas Receptor