Acquired aplastic anemia (aAA) is an autoimmune blood disease caused by the immune attack of early hematopoietic cells in the bone marrow. Because the mechanism behind aAA is not well understood, disease prognosis remains poor. Treatments target general immune processes and are not very effective. However, upon observing patients responding positively to T cell-directed immunosuppressive therapies, scientists hypothesize that the autoimmune attack may be mediated by cytotoxic T cells. Recent studies have identified recurrent somatic mutations in Human Leukocyte Antigen (HLA) class I alleles among the surviving hematopoietic stem cells of aAA patients. HLA molecules present intracellular peptides on the surface of cells for immune surveillance. The observed survival advantage of hematopoietic stem cells with mutated HLA class I alleles leads us to hypothesize that the mutated HLA allele may have a key role in mediating autoantigen presentation in the autoimmune attack, and that the mutations allow the cells to survive. While most mutations in patients with aAA cause complete loss of HLA expression, some missense mutations result in partial or reduced surface expression. Additionally, the mutations selected for this study all result in amino acid substitutions in the peptide binding region of the HLA molecule. These mutations have the potential to be informative about autoantigen binding in aplastic anemia. 

We compiled a list of mutations bearing these characteristics and sought to evaluate how each mutation affected HLA surface expression. To do this, we utilized lentiviral plasmids that we had previously constructed to introduce the mutated and wildtype alleles into stable cell lines via transfection and transduction. Then, we performed flow cytometry experiments with a HLA-PE antibody in order to check for differences in surface HLA expression. We found that some mutations led to loss of HLA expression or reduced expression, while other mutations resulted in relatively preserved expression. 

Moving forward, we will repeat the flow cytometry experiments with an alternative antibody to confirm our initial results. For the alleles of interest demonstrating preserved surface HLA expression, we will evaluate the mutated HLA molecules’ peptide binding capabilities. To do this, we will immunoprecipitate the HLA class-1 molecules and subsequently perform proteomic analysis of the eluted peptides. The collected data could potentially reveal features of the autoantigens that result in reduced binding to the mutated HLA molecules, which will help elucidate characteristics of candidate autoantigens in aplastic anemia and improve our understanding of the mechanism behind the disease.