Allele- and temperature-dependency of in vitro HLA class I assembly

Allelic variations of in vitro HLA class I assembly have been investigated in both the absence and the presence of binding peptides by flow cytometry using human leukocyte antigen (HLA) class I alpha chains isolated by alkali treatment from cultured HLA homozygous B cells and polystyrene beads coated with anti-HLA class I alpha chain antibodies specific to the C-terminal segment (anti-HLA class I beads). The specificity of assembly was temperature dependent, while the stability of the assembled complex depended on the bound peptide. The efficiency of assembly was allele dependent and primarily ruled by the binding affinity of alpha chains with beta(2)m. Thus, an allele hierarchy could be defined for the binding of HLA-B alpha chain with beta(2)-microglobulin: B7, B18 > B35, B62 > B27, B51. Allele and temperature dependency was found in HLA class I reassembly on acid treated B cells. The HLA class I proteins, reassembled with specific single peptides, could be efficiently transferred to anti-HLA class I beads. These findings would be used to produce microspheres coupled at high surface density with oriented single-peptide loaded HLA class I molecules and also to improve the preparation efficiency of HLA class I tetramers by the use of site-specific biotinylation.