The ability to directly load cell surface major histocompatibility complex (MHC) class I molecules with peptides provides a potentially powerful approach toward the development of vaccines to generate cell-mediated immunity. We demonstrate that exogenous ␤ 2 -microglobulin (␤ 2 m) stabilizes human cell surface MHC I molecules and facilitates their loading with exogenous peptides. Additionally, using three-dimensional crystal structures and known interaction sites between MHC I heavy chains and ␤ 2 m, we engineered variants of human ␤ 2 m (h␤ 2 m) with a single serine substitution at residue 55. This alteration was predicted to promote hydrophobic interactions at the MHC I heavy chain/␤ 2 m interface and displace an ordered water molecule. Compared with h␤ 2 m, the serine to valine substitution at residue 55 had improved ability to bind to cell surface HLA-A1, HLA-A2, and HLA-A3 molecules, facilitate exogenous peptide loading, and promote recognition by peptide-specific T cells. The inclusion of h␤ 2 m or higher affinity variants when pulsing cells with MHC-restricted peptides increases the efficiency of peptide loading 50 -80-fold. Therefore, the inclusion of h␤ 2 m in peptide-based vaccines may increase cell surface antigen densities above thresholds that allow recognition of peptide antigens by the immune system, particularly for cryptic, subdominant, or marginally antigenic peptides.In the field of vaccine development, it has been relatively simple to induce humoral responses to injected antigens. However, one of the major challenges in the treatment of tumors and viral infections is the generation of vaccines that stimulate cell-mediated immune responses to these pathogens. Specific cytolytic responses are generally mediated by CD8ϩ T cell recognition of antigenic peptides in the context of major histocompatibility complex (MHC) 1 class I molecules. Recent advances in defining "supermotif" antigens capable of being presented by multiple MHC I alleles (1-4) and immunodominant epitopes (5-9) will undoubtedly have a significant impact on realizing these goals. However, beyond defining appropriate antigenic peptides, a second challenge lies in establishing effective methods with which to deliver these antigens to the MHC I loading pathway. Typically, MHC I molecules acquire peptides generated by the degradation of endogenous proteins by the proteasome. These peptides are transported into the endoplasmic reticulum, where they are bound by MHC I⅐␤ 2 -microglobulin (␤ 2 m) complexes and finally transit to the cell surface (10 -12). Although this pathway is the dominant means of loading MHC I molecules, other methods of delivering antigenic peptides to MHC I have been described including direct incorporation of DNA into cells (13-16), phagocytosis-dependent representation of antigens (17, 18), and infection by bacterial (19) or viral vectors (20, 21). All of these approaches attempt to introduce peptide into the endogenous loading pathway. Alternatively, direct cell surface loading of MHC I molecules in vitro has al...

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Human β2m (hβ2m) binds to murine MHC I molecules with higher affinity than does murine β2m and therefore can be used as a model system to define and dissect the interactions between β2m and MHC I heavy chains that promote the stability of the complex. In the present study we compare three-dimensional crystal structures of human and murine MHC I molecules and use functional studies of chimeric human:murine β2m variants to define a region of β2m that is involved in the higher affinity of hβ2m for murine MHC I heavy chains. Further examination of the three-dimensional structure in this region revealed conformational differences between human and murine β2m that affect the ability of an aspartic acid residue at position 53 (D53) conserved in both β2ms to form an ionic bond with arginine residues at positions 35 and 48 of the heavy chain. Mutation of residue D53 to either asparagine (D53N) or valine (D53V) largely abrogated the stabilizing effects of hβ2m on murine MHC I expression in a predictable manner. Based on this observation a variant of hβ2m was engineered to create an ionic bond between the heavy chain and β2m. This variant stabilizes cell surface H-2Dd heavy chains to a greater extent than wild-type hβ2m. Studying these interactions in light of the growing database of MHC I crystal structures should allow the rational design of higher affinity hβ2m variants for use in novel peptide-based vaccines capable of inducing cell-mediated immune responses to viruses and tumors.