POCKET-4 OF THE HLA-DR(ALPHA,BETA-1-ASTERISK-0401) MOLECULE IS A MAJOR DETERMINANT OF T-CELL RECOGNITION OF PEPTIDE

To investigate the functional roles of individual HLA-DR residues in T cell recognition, transfectants expressing wild-type or mutant DR(alp ha,beta 1 star 0401) molecules with single amino acid substitutions at 14 polymorphic positions of the DR beta 1 star 0401 chain or 19 posit ions of the DR alpha chain were used as antigen-presenting cells for f ive T cell clones specific for the influenza hemagglutinin peptide, HA 307-19. Of the six polymorphic positions in the DR beta floor that wer e examined, mutations at only two positions eliminated T cell recognit ion: positions 13 (four clones) and 28 (one clone). In contrast, indiv idual mutations at DR beta positions 70, 71, 78, and 86 on the alpha h elix eliminated recognition by each of the clones, and mutations at po sitions 74 and 67 eliminated recognition by four and two clones, respe ctively. Most of the DR alpha mutations had minimal or no effect on mo st of the clones, although one clone was very sensitive to changes in the DR alpha chain, with loss of recognition in response to 10 mutants . Mutants that abrogated recognition by all of the clones were assesse d for peptide binding, and only the beta 86 mutation drastically decre ased peptide binding. Single amino acid substitutions at Polymorphic p ositions in the central part of the DR beta alpha helix disrupted T ce ll recognition much more frequently than substitutions in the floor, s uggesting that DR beta residues on the alpha helix make relatively gre ater contributions than those in the floor to the ability of the DR(al pha,beta 1 star 0401) molecule to present HA307-19. The data indicate that DR beta residues 13, 70, 71, 74, and 78, which are located in poc ket 4 of the peptide binding site in the crystal structure of the DR1 molecule, exert a major and disproportionate influence on the outcome of T cell recognition, compared with other polymorphic residues.