Strategies for the measurements of expression levels and half-lives of HLA class I allotypes

HLA class I molecules are highly polymorphic cell surface proteins that trigger immune responses by CD8⁺ T cells and natural killer (NK) cells. Most humans express six different HLA class I proteins encoded by the HLA-A, HLA-B and HLA-C genes. HLA class I molecules bind to peptide antigens and present these antigens to T cell receptors (TCR) of CD8⁺ T cells. HLA class I expression levels also regulate NK cell activation. The presence of individual HLA class I genes is linked to many different disease, transplantation and therapy outcomes. An understanding of HLA class I expression and stability patterns is fundamentally important towards a better understanding of the associations of HLA class I genes with disease and treatment outcomes, and towards HLA class I targeting for vaccine development. Quantitative flow cytometry allows for assessments of variations in expression levels of HLA class I molecules in cells from a single blood donor over time, as well as averaged measurements across donors for the same allotype. Since all HLA class I molecules are structurally-related, cellular measurements of the HLA class I expression levels and stabilities of individual variants in human cells require careful choices of donors and antibodies, which are discussed here.