Location of the epitope for an anti-CD8alpha antibody 53.6.7 which enhances CD8alpha-MHC class I interaction indicates antibody stabilization of a higher affinity CD8 conformation.
MHC class I tetramers are widely used, usually in combination with an antibody to CD8, to detect antigen specific T cells. Some anti-CD8alpha antibodies block the interaction of murine MHC class I tetramers with CD8 T cells, while others such as 53.6.7, enhance. To understand the molecular basis for this effect, we mapped the epitope for the enhancing antibody 53.6.7 and three other blocking antibodies using a panel of murine CD8alpha (Lyt-2) mutants expressed on COS-7 transfectants. Mutations in residues that contact MHC class I affected binding of the blocking antibodies. In contrast, antibody 53.6.7 was affected by a mutation in the residue T81A located on the D-E loop. In the cocrystal of CD8alphaalpha with MHC class I, two different complexes (A and B) were observed, indicating the existence of different CD8 conformations. The T81 residue does not make contact with MHC class I in either complex, however, neighboring residues in the D-E loop make very different contacts in the two different complexes. The most likely explanation for antibody enhancement of tetramer bindings is that binding of 53.6.7 to CD8alphabeta stabilizes a conformation with a higher affinity for interaction with MHC class I and suggests that the CD8 binding site is flexible.
Devine
Lesley
L
Department of Laboratory Medicine, Yale University School of Medicine, New Haven, CT 06520-8011, USA.
Hodsdon
Michael E
ME
Daniels
Mark A
MA
Jameson
Stephen C
SC
Kavathas
Paula B
PB
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000494:pharmacology; D000915:Antibody Affinity / Q000235:genetics / Q000276:immunology; D000918:Antibody Specificity / Q000235:genetics / Q000276:immunology; D019921:Biotinylation; D016827:CD8 Antigens / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D019556:COS Cells; D002522:Chlorocebus aethiops; D004527:Egg Proteins / Q000276:immunology; D018604:Epitope Mapping; D004926:Escherichia coli / Q000235:genetics; D005434:Flow Cytometry; D006183:H-2 Antigens / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D015395:Histocompatibility Antigens Class I / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D051379:Mice; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009842:Oligopeptides / Q000276:immunology; D010047:Ovalbumin / Q000276:immunology; D010446:Peptide Fragments; D011485:Protein Binding / Q000187:drug effects / Q000276:immunology; D011487:Protein Conformation / Q000187:drug effects; D014162:Transfection
D000911:Antibodies, Monoclonal; D016827:CD8 Antigens; C409469:CD8 antigen, alpha chain; C431698:CD8alpha antigen; C433839:CD8alphabeta antigen; C477867:CD8beta antigen; D004527:Egg Proteins; D006183:H-2 Antigens; C032876:H-2Kb protein, mouse; D015395:Histocompatibility Antigens Class I; C110366:OVA-8; D009842:Oligopeptides; D010446:Peptide Fragments; C406862:superagonist SIYR; D010047:Ovalbumin