Characteristics of heterologous beta 2-m exchange into H-2Db at the cell surface

W6/32 is a monomorphic anti-HLA class I Ab that cross-reacts with the product of the exchange of murine beta 2-microglobulin (beta 2-m) with human or bovine beta 2-m on H-2 Db. Using W6/32 we have developed a simple and rapid flow cytometric method to measure the beta 2-m exchange kinetics for Db at the surface of intact H-2b cell lines. We find that 10 to 25% of the Db heavy chains exchange bound beta 2-m for soluble beta 2-m with a t1/2 of 10 to 15 min. The kinetics of the exchange are consistent with a mechanism that includes a free heavy chain intermediate on the reaction coordinate. The remainder of Db molecules appear to be refractory to beta 2-m exchange even after long-term culture with an exogenous source of beta 2-m. The exchange process proceeds at a similar rate on wild-type cells that bear a diverse complement of autologous peptides on their class I molecules, and on mutant RMA-S cells having class I molecules primarily occupied with a single, defined, high affinity synthetic peptide Ag. The t1/2 for dissociation of a radiolabeled analogue of a high affinity naturally presented peptide Ag is 10 h or more, implying that the free heavy chain intermediate in the beta 2-m exchange mechanism retains bound peptides. Further, we find that synthetic peptide Ags added to non-mutant cells bind both to class I molecules that subsequently exchange bound beta 2-m, and to molecules that retain endogenous beta 2-m.