Analysis of mutant HLA-A2 molecules. Differential effects on peptide binding and CTL recognition.

Abstract

Previous studies have identified several residues lining the groove of the HLA-A2.1 molecule that are critical for Ag presentation. However, it is not clear whether these residues are critical for binding of the peptide epitope per se or for determining the appropriate conformation of bound peptide. To distinguish between these possibilities, mutations at eight of these residues have been tested for their effects on the ability of the molecule to bind and present two known peptide epitopes--one derived from the influenza A matrix protein, the other from HIV pol. With only one exception, the mutations were found to affect the binding of the two peptides similarly. Most of the mutations resulted in intermediate deleterious effects on binding, with the B pocket mutant F9Y having the most dramatic negative effect on binding for both peptides. Two of the mutations significantly enhanced binding of both peptides and a peptide-specific effect on binding was seen with the substitution, Y99H, which enhanced binding of the matrix peptide yet diminished binding of the pol peptide. In contrast to the effects on binding, the effects of the mutations on presentation differed considerably for the two peptides. The most striking difference was seen with two alpha 2 alpha helix mutants that are fully recognized by pol peptide-specific CTL but not recognized by matrix peptide-specific CTL even though levels of binding were comparably diminished for the two peptides. These results suggest that some interactions, although not critical for binding per se, are critical for functional binding and the importance of these interactions differs among peptide epitopes.

Effects of HLA single chain trimer design on peptide presentation and stability

“…SCTs were engineered based on the general template N–[ leader peptide ]–[ antigenic peptide ]–[ (G 4 S) 3 linker ]–[ β 2 m ]–[ (G 4 S) 3 linker ]–[ HLA heavy chain ]–[ 6 His tag ]–C and were expressed in HEK293F cells via the Daedalus lentiviral transduction system ( Supplementary Information ) ( 12 ). We evaluated the following SCT platforms based on the native HLA ectodomain with incorporated combinations of mutations: SCT Y84A which contains the groove-opening mutation Y84A facilitating linker clearance from the groove ( 8 , 13 ); SCT H74L/Y84C which contains both a stabilizing disulfide between residue 84 in the groove and a cysteine in the peptide/β 2 m linker (GCGGS(G 4 S) 3 ) ( 14 , 15 ) and the stabilizing H74L mutation ( 16 , 17 ); and SCT Y84C/A139C which contains a stabilizing engineered disulfide at the C-terminal end of the peptide binding cleft between the α1 and α2 helix ( 18 ). We also tested novel human A*24:02 α1/α2–murine H-2K d α3/β 2 m chimeric SCTs (χSCT) with the goal of focusing murine humoral responses to improve recovery of TCR-mimic monoclonal antibodies ( 19 ) during immunization.…”