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 a chains isolated by alkali tre atment from cultured HLA homozygous B cells and polystyrene beads coated wi th anti-HLA class I alpha chain antibodies specific to the C-terminal segme nt (anti-HLA class I beads). The specificity of assembly was temperature de pendent, while the stability of the assembled complex depended on the bound peptide. The efficiency of assembly was allele dependent and primarily rul ed by the binding affinity of alpha chains with beta M-2. Thus, an allele h ierarchy could be defined for the binding of HLA-B alpha chain with beta (2 )-microglobulin- B7, B18 > B35, B62 > B27, B51. Allele and temperature depe ndency was found in HLA class I reassembly on acid treated B cells. The HLA class I proteins, reassembled with specific single peptides, could be effi ciently transferred to anti-HLA class I beads. These findings would be used to produce microspheres coupled at high surface density with oriented sing le-peptide loaded HLA class I molecules and also to improve the preparation efficiency of HLA class I tetramers by the use of site-specific biotinylat ion. Human Immunology 62, 858-868 (2001). (C) American Society for Histocom patibility and Immunogenetics, 2001. Published by Elsevier Science Inc.