Project Summary/Abstract Individuals with sickle cell disease (SCD) have a significantly higher red blood cell (RBC) alloimmunization rate than other populations receiving transfused donor RBCs. Depending on the study, circumstances of transfusion, and environment, it has been reported that 18-47% of individuals with SCD become immunized to RBC allo- antigens following a transfusion and are also more likely to then make additional alloantibodies with subsequent transfusions. Transfusions in the setting of an acute SCD complication have an 8-12-fold higher odds ratio(1) of resulting in alloantibody formation compared to those given at steady-state. We hypothesize that we will identify key drivers of alloimmunization with a large-scale, unbiased human study that integrates recipient omics and environmental conditions (i.e., presence or absence of acute illness and high levels of inflammation) at the time of transfusion. We will test this through first identifying variants associated with alloimmunization through analysis of genome sequencing (WGS) from responders and non-responders. We hypothesize that SCD individuals with African heritage will have genetic variants associated with increased immunogenicity. To test this, we will analyze the WGS of our Emory SCD cohort (n=2000) who have or have not developed alloantibodies after 10 or more blood transfusions and compare to WGS from the REDSIII cohort in TOPMed, which has alloimmunization phenotypes on 2800 subjects with SCD from Brazil. Then, we will assess transcriptome changes of leukocyte subsets at baseline, at time of transfusion, and at one-month post-transfusion in individuals with SCD who did or did not develop alloimmunization. We hypothesize that we will identify alterations in gene expression (and affiliated pathways) that associate with alloimmunization. This will have the immediate benefit of allowing us to develop a transcriptional risk score (TRS) for alloimmunization, and the longer-term benefit if identifying pathways for rational development of therapeutic interventions. Finally, we will assess the impact of inflammation on alloimmunization. We hypothesize that alloimmunization in individuals with SCD that occur in the setting of acute inflammation differs mechanistically from that of alloimmunization that occurs in the setting of chronic transfusion or in healthy individuals. We will compare the transcriptomes obtained in aim 2 to scRNAseq data obtained from stored PBMCs from a cohort of 40 non-SCD individuals who developed alloantibodies when challenged by Rh mismatched RBCs. We predict our analysis will allow us to identify individuals at risk for alloimmunization before it occurs, and elucidate the role of inflammation in alloimmunization, both in patients with SCD and in healthy individuals.