POINT MUTATIONS IN THE ALPHA-2 DOMAIN OF HLA-A2.1 DEFINE A FUNCTIONALLY RELEVANT INTERACTION WITH TAP

Background: Glycoproteins encoded by the major histocompatibility comp lex class I region (MHC Glass I) present peptide antigens to cytotoxic T cells (CTLs). Peptides are delivered to the site of MHC class I ass embly by the transporter associated with antigen processing (TAP), and cell lines that lack this transporter are unable to present endogenou s antigens to CTLs. Although it has been shown that a fraction of newl y synthesized class I molecules are in physical association with TAP, it is not known whether this interaction is functionally relevant, or where on the class I molecule the TAP binding site might be. Results: C1R cells transfected with a mutant HLA-A2.1 heavy chain (HC), where t hreonine at position 134 in the alpha 2 domain is changed to lysine (T 134K), are unable to present endogenous antigens to CTLs. We have stud ied the biochemistry of this mutant in C1R cells, and found that a lar ge pool of unstable empty class I HC-beta(2)m (beta-2 microglobulin) h eterodimers exist that are rapidly transported to the cell surface. Th e T134K mutant seemed to bind peptide antigens and assemble with beta( 2)m as efficiently as wild-type HLA-A2.1. However, we show here that t he inefficiency with which T134K presents intracellular antigen is ass ociated with its inability to interact with the TAP heterodimer. Concl usions: These experiments establish that the class I-TAP interaction i s obligatory for the presentation of peptide epitopes delivered to the endoplasmic reticulum (ER) by TAP. Wild-type HLA-A2.1 molecules in TA P-deficient cells are retained in the ER, whereas T134K is rapidly rel eased to the cell surface, but is unstable, suggesting a role for the TAP complex as an intracellular checkpoint that only affects the relea se of class I molecules with stably bound peptide ligands.