Establishing the immune structure of the human spleen with spatial profiling

Chronic red blood cell (RBC) transfusion therapy is essential for the survival of patients with hematological disorders including sickle cell disease (SCD). These patients’ abnormal RBCs do not survive well in circulation and can obstruct blood vessels, resulting in both chronic anemia and acute crises which can lead to severe pain, stroke, multiorgan failure, respiratory failure and even death. These patients require frequent RBC transfusions. Unfortunately, exposure to foreign proteins on transfused RBCs can lead to alloimmunization, a condition in which transfusion recipients generate antibodies against donated RBCs. The spleen is the site where immune responses are initiated against blood-borne antigens, such as the foreign molecules on transfused RBCs, and is the focus of the proposed study. We and others have described a complex system of cellular organization in the spleens of mice, the manipulation of which could potentially mitigate alloimmunization. Unfortunately, the cellular organization of the human spleen is poorly understood, in part due to the scarcity and fragility of human samples. Therefore, our textbook understanding of the function of cells in the human spleen is extrapolated from mice, even though significant cellular and architectural differences are thought to exist between rodents and humans. The lack of a comprehensive organizational map of immune cells in the human spleen limits the translatability of mouse studies. In Aim 1 we will address this issue by directly comparing the structures of human and mouse spleens with the limited set of markers available for fixed human spleen samples. In Aims 2 and 3 we will then apply a new technological approach (transcriptomic and proteomic spatial profiling and single-cell RNA-sequencing) that works on fixed, banked tissues to human and mouse spleen samples. This has not been done before in any type of spleen sample; we will adapt the protocols used in other tissues to make this approach work in mo