P022 Variations in surface expression and stability among human leukocyte antigens (HLA-B)

Abstract

Aim Among HLA class I (HLA-I) molecules, HLA-B allotypes are the most polymorphic and have dominant influences upon disease outcomes. HLA-B allotypes are known to differ markedly in their intracellular folding and peptidome characteristics. The Aim of this study was to assess whether intracellular assembly or peptidome variations influence the cell surface expression levels or stabilities of HLA-B allotypes. Methods Commercial HLA-Bw4 and HLA-Bw6 antibodies were assessed for HLA-I specificities using Luminex beads, and antibody binding differences were quantified. 244 healthy donors were recruited and HLA genotyping was performed using next generation sequencing (NGS). Donors were selected for further measurements based on heterozygosity for the Bw4 and Bw6 epitopes within their HLA-B antigens, and based on the absence of cross-reactive HLA-A antigens. Using quantitative flow cytometry, antibody binding capacity (ABC) values were measured on freshly isolated lymphocyte subsets using HLA-Bw4, HLA-Bw6 and total class I-specific antibodies. For each selected donor, HLA-I ABC measurements were undertaken at multiple time points. For HLA-B alleles with measurements from at least three donors, averaged ABC values for HLA-Bw4 and HLA-Bw6 were analyzed directly or as a ratio relative to total HLA-I, after taking into account relevant antibody recognition differences. For some HLA-Bw4 and HLA-Bw6 allotypes, cell surface stabilities were also assessed on freshly isolated lymphocytes in the presence of brefeldin A. Results Statistically significant allele-dependent differences are measured in cell surface HLA-B expression levels. For some HLA-B allotypes, lower expression could be related to reduced cell surface stability. Cell surface expression and stability differences appear to be related to the individual peptide-binding characteristics of HLA-B, rather than diversities (promiscuities) of their peptide repertoires. Conclusions Overall, these studies indicate allele-dependent variations in cell surface expression and stability of HLA-B, which in some cases correlate with previously known disease progression outcomes in human immunodeficiency virus (HIV) infections.