Acquired aplastic anemia (AA) is caused by autoreactive T-cell-mediated destruction of early hematopoietic cells. Somatic loss of human leukocyte antigen (HLA) Class I alleles was identified as a mechanism of immune escape in surviving hematopoietic cells of some AA patients. However, pathogenicity, structural characteristics and clinical impact of specific HLA alleles in AA remain poorly understood. Here, we evaluated somatic HLA loss in 505 AA patients from two multi-institutional cohorts.Using a combination of HLA mutation frequencies, peptide-binding structures, and association with AA in an independent cohort of 6,323 patients from the National Marrow Donor Program, we identified 19 AA risk alleles and 12 non-risk alleles and established a novel AA HLA pathogenicity stratification.Our results define pathogenicity for the majority of common HLA-A/B alleles across diverse populations. Our study demonstrates that HLA alleles confer different risks of developing AA, but once AA develops, specific alleles are not associated with response to immunosuppression or transplant outcomes. However, higher pathogenicity alleles, particularly HLA-B*14:02, are associated with higher rates of clonal evolution in adult AA patients. Our study provides novel insights into the immune pathogenesis of AA, opening the door to future autoantigen identification and improved understanding of clonal evolution in AA.

Nitric oxide (NO) is an essential signaling molecule for neurotransmission, cardiovascular function, and cellular defense. However, NO overproduction from neuronal nitric oxide synthase (nNOS) increases infarct size after ischemic stroke and has been linked to the pathogenesis of Alzheimer's and Parkinson's disease. Despite fundamental roles of NO in cellular signaling and disease, intact nNOS holoenzyme structures have remained elusive, and the structural basis for the electron transfer cycle across the reductase and oxygenase domains is unclear. In previous studies, we used negative-stain electron microscopy (EM) to determine conformational states and the overall domain organization of nNOS during its catalytic cycle. In current work, we have interrogated the nNOS and nNOS:Calmodulin (nNOS:CaM) complexes by cryogenic-EM (cryo-EM). In these intact complexes, the oxygenase dimer structure was determined to $4Å , revealing its active-state architecture, while the nNOS reductase domains were identified to be flexibly linked in both CaM-free and CaM-bound states. Additional structural analysis of crosslinked-stabilized complexes, including 2D and 3D classification, will be presented that reveals the overall architecture and highlights the use of cryo-EM methods in elucidating different conformations of the nNOS:CaM complex.