Measuring HLA-B allele expression across differential cell types.
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Background: The human leukocyte antigen (HLA) region has shown to have the strongest disease associations and recent studies have shown that expression levels of these HLA molecules play a major role in the clinical course of diseases. Differences in the expression levels of these molecules have been found to have a major effect on their ability to present specific peptide antigens. HLA molecules are critical to the interaction between diseases and components of the immune system. Expression of such molecules, namely HLA-C and HLA-A, have been shown to associate with HIV disease outcomes. An increase in expression of HLA-C leads to protection against HIV whereas an increase in HLA-A expression leads to rapid HIV progression. Furthermore, studies have shown the region with the strongest genetic effect falls within the HLA-B gene, as determined by genome wide association studies. However, limited information is available for HLA-B allelic expression levels and the variation across differential cell types. Materials and Methods: Allelic expression levels of HLA-B were measured using cryopreserved PBMC samples from HIV negative and positive cohorts with HLA typing. Antibodies specific to the HLA-B protein were identified. The affinity of the antibodies relative to class-I alleles were determined. Based on these affinities, donors with specific alleles were selected for HLA-B cell surface measurement using the flow cytometer. mRNA levels were measured across HLA-A, -B, -C and -E genes within the following cell types T-cells, B-cells, Monocytes and NK cells. These levels and a comparison of HIV infected and uninfected mRNA levels from the same donor were measured using droplet digital PCR (ddPCR). Conclusions: Contrary to HLA-B mRNA expression levels, we find cell surface expression levels vary in an allele-specific manner. We further observed differential mRNA expression patterns for HLA-A, HLA-B, HLA-C and HLA-E across cell types. We also observed no mRNA expression variation across pre- and post- HIV samples. When comparing HLA-B mRNA and surface expression across alleles and donors no significant correlation was found. However, at an donor level, some alleles may be differentially regulated at the cell surface. This study built existing knowledge and fills in some of the gaps in knowledge surrounding HLA-B expression. We also report, for the first-time, variation in allele specific expression, variation in expression across differential cell types and lack of expression variation across pre- and post- HIV infection at the mRNA level. ”