title: Low binding capacity of murine tetramers mutated at residue 227 does not preclude the ability to efficiently activate CD8+ T lymphocytes.
abstract: MHC tetramers are used to directly enumerate and visualize the antigen-specific T lymphocyte population of interest by flow cytometry, regardless of the T lymphocyte's functional capacity. Assay sensitivity can be hindered by non-specific binding activity, which is due to the inherent interactions of CD8 and MHC. Point mutations within the alpha3 loop of the HLA MHC class I heavy chain have been shown to reduce or abrogate MHC/CD8 interactions and also alleviate non-specific binding. This report compares the effects of two well-described mutations on the binding capacity and functional capacity of MHC tetramers in the H-2 MHC murine system. Tetramers folded with MHC mutated at either residue 227 or 245 of the class I heavy chain were compared to wild-type tetramer in binding studies using various antigen-specific, TCR-positive lymphocytes and cell lines. These experiments showed that the binding of wild-type and residue 245-mutated tetramer were comparable on CTL cultures, OT-1 splenocytes, and hybridomas. Both wild-type and 245-mutated tetramers' binding capacity was observed to be equally dependent on CD8 expression. Residue 227-mutated tetramer consistently bound antigen-specific CTL less efficiently, but in the absence of CD8 all three tetramers had similar binding capacity. In functional studies, 227-mutated tetramer had the greatest capacity to stimulate cytokine production in the absence of exogenous antigen addition. These experiments demonstrate that reduction of a tetramer's high avidity interaction with CD8 will not necessarily decrease the ability to stimulate the effector functions of activated T cells.
authors.affiliation: Beckman Coulter, Inc., 7330 Carroll Road, San Diego, CA 92121, USA. ctnugent@beckman.com
authors.forename: C Thomas
authors.initials: CT
authors.lastname: Nugent
authors.forename: Roberto O
authors.initials: RO
authors.lastname: Renteria
authors.forename: Kristine
authors.initials: K
authors.lastname: Kuus-Reichel
authors.forename: Abhay
authors.initials: A
authors.lastname: Kumar
mesh_terms: D000409:Alanine / Q000235:genetics / Q000378:metabolism; D000818:Animals; D001224:Aspartic Acid / Q000235:genetics / Q000378:metabolism; D016827:CD8 Antigens / Q000378:metabolism; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology*; D002478:Cells, Cultured; D015395:Histocompatibility Antigens Class I / Q000737:chemistry / Q000235:genetics* / Q000276:immunology*; D006825:Hybridomas / Q000276:immunology; D008213:Lymphocyte Activation / Q000276:immunology*; D051379:Mice; D008810:Mice, Inbred C57BL; D009154:Mutation / Q000235:genetics*; D011485:Protein Binding; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism; D013154:Spleen / Q000276:immunology; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology
chemical_list: D016827:CD8 Antigens; D015395:Histocompatibility Antigens Class I; D011948:Receptors, Antigen, T-Cell; D001224:Aspartic Acid; D000409:Alanine