TLR9 is required in donor blood for inflammation-induced immunization to an alloantigen on transfused RBCs in mice

Humoral immunization to alloantigens on transfused red blood cells (RBCs) remains a major barrier to safe and effective blood transfusion in chronically transfused patients. However, only some patients become alloimmunized following multiple transfusion. It was first observed in mice and then confirmed in humans that recipient inflammation at the time of transfusion increases rates of RBC alloimmunization. Viral double-stranded RNA has been extensively studied and shown to enhance alloimmunization through activation of recipient MAVS (mitochondrial antiviral-signaling protein) and subsequent induction of alpha-interferon. However, no one has reported testing the role of innate immune receptors on donor cells. Herein, we report that recipient exposure to another innate immune activator (CpG) enhances alloimmunization to transfused RBCs in a TLR9-dependent fashion. In addition, TLR9 is required both on the transfused donor cells and in the recipient. To the best of our knowledge, this is the first report of innate immune receptors on transfused donor cells regulating RBC alloimmunization. In addition to adding a new conceptual component to the mechanistic landscape of RBC alloimmunization, these findings raise the possibility of decreasing RBC alloimmunization either by selecting RBC units from donors with known hypomorphic TLR9 gene variants or by introducing TLR9 antagonists into donor blood.