Alloimmunization and Humoral Immune Response to Hemolysis and EPO

Blood transfusions remain a cornerstone treatment in sickle cell disease (SCD). However, 15-50% of these patients develop alloantibodies causing complications ranging from difficulty in finding matched units, increased risk of autoantibody production (~12% in our patient cohort) to potentially life-threatening delayed hemolytic transfusion reactions (DHTR). Elevated EPO levels and intravascular hemolysis are two hallmarks of SCD. Our preliminary data indicate that EPO leads to upregulation of plasma asparagine (Asn) in SCD. EPO-derived Asn increased anti-RBC immune responses by targeting key B cell subsets and T follicular regulatory cell (TFR) which was counteracted by hemolysis. Our overall hypothesis is that the balance between EPO/Asn and hemolysis activation pathways in SCD dictate the risk of allo- and autoimmunization and DHTR in SCD. To test our hypothesis, we will use our in vivo and in vitro models including studies of SCD patients receiving transfusions to establish whether: i) EPO/Asn increases whereas hemolysis lowers alloimmunization risk (Aim1) in naïve B cells through up- or downregulation of Src family kinase (SFK) activation, respectively; ii) heme-driven mitochondrial dysfunction in memory B cells modulates DHTR risk in alloimmunized SCD patients (Aim1); iii) EPO/Asn increases autoanti-RBC antibody production (Aim2) by inducing splenic pre-B cell expansion through SFK pathway; iv) EPO/Asn driven decrease in TFR numbers in SCD occurs by activation-induced apoptosis through HPK1 pathway but hemolysis increases TFR inhibitory activity through upregulation of HO-1 but only in non-alloimmunized SCD patients. Altogether, we believe that our proposed studies to dissect mechanistically the role of EPO/Asn and hemolysis in anti-RBC humoral immune response in SCD has potential to identify biomarkers and therapeutic strategies for diagnosis and reversal of pathologic transfusion responses in SCD.