Background: It remains unclear why certain transfusion recipients make clinically significant RBC alloantibody after antibody (“responders”), yet others are transfused hundreds of times without generating any detectable alloantibodies (“non-responders”). We have discovered that the recipient inflammatory status at the time of RBC transfusion, in combination with the ability of the recipients to sense type 1 interferons (IFN), are critical in determining responder/non-responder status in our reductionist murine models. Further, recent studies in humans suggest that the inflammation/alloimmunization connection transcends mice, though genetic variables must also be considered. Central Hypothesis: We hypothesize that inflammation (specifically type 1 IFN induction and sensing) and genetic factors together determine which transfusion recipients will form alloantibodies following RBC transfusion, with non-responders possibly being tolerant and not simply ignorant of RBC antigens. The proposed studies utilize reductionist murine models, with blood donors expressing the authentic human blood group antigens glycophorin A (hGPA) or KEL glycoprotein, and with genetically identical recipients generating RBC alloantibodies only when transfusion occurs in the presence of an adjuvant or an ability to sense IFNs. In depth in vivo studies of early immune activation events in responder and non-responder mice, in combination with ex vivo studies of CD4+ T-cell subset phenotype, function, and transcriptome in responder and non- responder humans, seek to increase knowledge of RBC alloimmunization. Specific Aim 1: To investigate which recipient cell population(s) in mice mediate responsiveness to transfused RBCs. Specific Aim 2: To investigate the mechanism(s) through which non-responsiveness in mice occurs following transfusion. Specific Aim 3: To investigate differences in CD4+ T-cells in responder and non-responder humans.