title: MHC-peptide-specific antibodies reveal inefficient presentation of an HLA-A*0201-restricted, Melan-A-derived peptide after active intracellular processing.
abstract: MHC-peptide-specific Fab antibodies binding to HLA-A*0201 complexes presenting the wild-type EAAGIGILTV (EAA) or analogue Melan-A 10-mer ELAGIGILTV (ELA) peptide were generated to study efficacy of peptide processing and presentation. None of the selected Fab antibodies detected the naturally processed EAA/HLA-A*0201 complex on melanoma tumor cells, confirming the known low peptide number on the cell surface. To study the effect of peptide presentation and processing in more detail, genes coding for the A27L-mutated Melan-A protein or the processed ELA peptide were introduced into HLA-A*0201(+) B cells by infection with the respective recombinant vaccinia virus construct producing equimolar amounts of GFP-ubiquitin directly linked to the fragment of interest. Correlating GFP expression to actual numbers of peptide presented, 1100-2600 [corrected] ELA peptides had to be synthesized to be presented by a single MHC class I antigen-peptide complex. This number increased 10- to 20-fold when ELA peptide presentation from the A27L-mutated full length Melan-A protein was studied, since 16000-52000 [corrected] GFP molecules needed to be synthesized for the detection of one ELA peptide. Our results indicate that peptide processing rather than presentation is the rate-limiting step in our experimental setting and is much more ineffective for Melan-A than has been previously shown for other MHC class I-restricted epitopes.
authors.affiliation: I Med Klinik, Saarland University Medical School, Homburg/Saar, Germany. gerhard.held@uniklinikum-saarland.de
authors.forename: Gerhard
authors.initials: G
authors.lastname: Held
authors.forename: Andreas
authors.initials: A
authors.lastname: Wadle
authors.forename: Nina
authors.initials: N
authors.lastname: Dauth
authors.forename: Guillaume
authors.initials: G
authors.lastname: Stewart-Jones
authors.forename: Christine
authors.initials: C
authors.lastname: Sturm
authors.forename: Markus
authors.initials: M
authors.lastname: Thiel
authors.forename: Carsten
authors.initials: C
authors.lastname: Zwick
authors.forename: Detlef
authors.initials: D
authors.lastname: Dieckmann
authors.forename: Gerold
authors.initials: G
authors.lastname: Schuler
authors.forename: Hennie R
authors.initials: HR
authors.lastname: Hoogenboom
authors.forename: Frédéric
authors.initials: F
authors.lastname: Lévy
authors.forename: Vincenzo
authors.initials: V
authors.lastname: Cerundolo
authors.forename: Michael
authors.initials: M
authors.lastname: Pfreundschuh
authors.forename: Christoph
authors.initials: C
authors.lastname: Renner
mesh_terms: D000918:Antibody Specificity / Q000276:immunology*; D017951:Antigen Presentation / Q000276:immunology*; D000951:Antigens, Neoplasm / Q000276:immunology / Q000378:metabolism*; D015153:Blotting, Western; D005434:Flow Cytometry; D015234:HLA-A Antigens / Q000276:immunology / Q000378:metabolism*; D015789:HLA-A2 Antigen; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000276:immunology*; D058965:MART-1 Antigen; D009363:Neoplasm Proteins / Q000276:immunology / Q000378:metabolism*; D010455:Peptides / Q000276:immunology / Q000378:metabolism
chemical_list: D000951:Antigens, Neoplasm; D015234:HLA-A Antigens; C435939:HLA-A*02:01 antigen; D015789:HLA-A2 Antigen; D007140:Immunoglobulin Fab Fragments; D058965:MART-1 Antigen; C548667:MLANA protein, human; D009363:Neoplasm Proteins; D010455:Peptides