Imaging Mass Cytometry Reveals the Order of Events in the Pathogenesis of Immune-Mediated Aplastic Anemia Free

• Development of AA is the result of a coordinated immune reaction that concentrates within immune hotspots in the bone marrow.

• During bone marrow destruction, the immune response gradually shifts from an activated to a more differentiated phenotype.

The autoimmune response driving hematopoietic stem and progenitor cell (HSPC) destruction in immune-mediated aplastic anemia (AA) remains incompletely understood. We previously identified a disease-specific immune cell network involving T-, B-, and myeloid cells. However, the interactions within this network, the interaction with the microenvironment and the chronological events in AA development, remain unclear. In this study, we aimed to characterize the changes occurring during disease development and to define the interactions between potential autoreactive cells and their target. Using imaging mass cytometry, we analyzed bone marrow (BM) biopsies from AA patients at diagnosis and after treatment with horse-derived anti-thymocyte globulin (hATG), and six controls. Within the hypocellular BM architecture, we identified lymphoid-dominant 'immune hotspots' with high densities of pro-inflammatory lymphocytes, and macrophage-enriched hotspots that additionally contained activated macrophages in proximity to progenitors. These immune hotspots potentially represent sites where the active immune response resulting in HSPC destruction takes place. In BM regions depleted of progenitors, effector cells with a differentiated phenotype remain. Our data indicate that HSPC destruction in AA is mediated by coordinated interactions among specific immune cell subpopulations. As the immune response progresses and HSPCs are depleted, the immune composition shifts, with activated T- and B-cells differentiating into terminally differentiated T-cells and plasma cells. In patients with normalizing BM post-hATG treatment, most immune hotspots were depleted, underscoring their potential pathogenic role. Collectively, our study visualizes the complex interactions among immune cell subpopulations and, for the first time, reveals the order of events in the immune-mediated pathogenesis of AA.