Type I IFN Is Necessary and Sufficient for Inflammation-Induced Red Blood Cell Alloimmunization in Mice.

During RBC transfusion, production of alloantibodies against RBC non-ABO Ags can cause hemolytic transfusion reactions and limit availability of compatible blood products, resulting in anemia-associated morbidity and mortality. Multiple studies have established that certain inflammatory disorders and inflammatory stimuli promote alloimmune responses to RBC Ags. However, the molecular mechanisms underlying these findings are poorly understood. Type I IFNs (IFN-α/ß) are induced in inflammatory conditions associated with increased alloimmunization. By developing a new transgenic murine model, we demonstrate that signaling through the IFN-α/ß receptor is required for inflammation-induced alloimmunization. Additionally, mitochondrial antiviral signaling protein-mediated signaling through cytosolic pattern recognition receptors was required for polyinosinic-polycytidylic acid-induced IFN-α/ß production and alloimmunization. We further report that IFN-α, in the absence of an adjuvant, is sufficient to induce RBC alloimmunization. These findings raise the possibility that patients with IFN-α/ß-mediated conditions, including autoimmunity and viral infections, may have an increased risk of RBC alloimmunization and may benefit from personalized transfusion protocols and/or targeted therapies.

B cells require Type 1 interferon to produce alloantibodies to transfused KEL-expressing red blood cells in mice.

BACKGROUND: Alloantibodies to red blood cell (RBC) antigens can cause significant hemolytic events. Prior studies have demonstrated that inflammatory stimuli in animal models and inflammatory states in humans, including autoimmunity and viremia, promote alloimmunization. However, molecular mechanisms underlying these findings are poorly understood. Given that Type 1 interferons (IFN-α/ß) regulate antiviral immunity and autoimmune pathology, the hypothesis that IFN-α/ß regulates RBC alloimmunization was tested in a murine model. STUDY DESIGN AND METHODS: Leukoreduced murine RBCs expressing the human KEL glycoprotein were transfused into control mice (WT), mice lacking the unique IFN-α/ß receptor (IFNAR1-/- ), or bone marrow chimeric mice lacking IFNAR1 on specific cell populations. Anti-KEL IgG production, expressed as mean fluorescence intensity (MFI), and B-cell differentiation were examined. RESULTS: Transfused WT mice produced anti-KEL IgG alloantibodies (peak response MFI, 50.4). However, the alloimmune response of IFNAR1-/- mice was almost completely abrogated (MFI, 4.2; p < 0.05). The response of bone marrow chimeric mice lacking IFNAR1 expression in all hematopoietic cells or specifically in B cells was also diminished (MFI, 3.8 and 5.4, respectively, compared to control chimeras, MFI, 65.6; p < 0.01). Accordingly, transfusion-induced differentiation of IFNAR1-/- B cells into germinal center B cells and plasma cells was significantly reduced, compared to WT B cells. CONCLUSIONS: This study demonstrates that B cells require signaling from IFN-α/ß to produce alloantibodies to the human KEL glycoprotein in mice. These findings provide a potential mechanistic basis for inflammation-induced alloimmunization. If these findings extend to human studies, patients with IFN-α/ß-associated conditions may have an elevated risk of alloimmunization and benefit from personalized transfusion protocols.

Transfused platelets enhance alloimmune responses to transfused KEL-expressing red blood cells in a murine model.

BACKGROUND: Factors influencing the development of alloantibodies against blood group antigens on transfused red blood cells are poorly defined. We hypothesised that transfused platelets may act as a danger signal to recipients and affect humoral immune responses to transfused red blood cells. MATERIALS AND METHODS: Platelet-rich plasma prepared from wild-type C57BL/6 or CD40L knock-out donors was transfused into wild-type or CD40L knock-out recipients. Leucoreduced red blood cells from transgenic donors expressing high levels of the human KEL glycoprotein in an erythrocyte-specific manner (KELhi donors) were transfused after the platelets, and anti-KEL responses were measured longitudinally. In some experiments, recipients were treated with poly (I:C), monoclonal CD40L-blocking antibody, or CD4-depleting antibody prior to transfusion. RESULTS: Transfusion of wild-type C57BL/6 platelets or treatment with poly (I:C) prior to KELhi red blood cell transfusion led to an anti-KEL alloimmune response in wild-type recipients. Transfusion of platelets from wild-type but not CD40L knock-out donors prior to KELhi red blood cell transfusion led to an IgG anti-KEL alloimmune response in CD40L knock-out recipients; unexpectedly, transfusion of platelets from CD40L knock-out donors prior to KELhi red blood cell transfusion led to a robust anti-KEL alloimmune response in wild-type recipients. Recipient treatment with MR1 CD40L-blocking antibody or CD4-depleting antibody prevented KEL alloimmunisation altogether. DISCUSSION: Transfused platelets serve as an adjuvant in this T-dependent murine model of anti-KEL red blood cell alloimmunisation, with CD40/CD40L interactions being involved to some degree but with additional mechanisms also playing a role. These findings raise questions about the role that transfused or endogenous platelets may play in other innate/adaptive immune responses.

CD4 Depletion or CD40L Blockade Results in Antigen-Specific Tolerance in a Red Blood Cell Alloimmunization Model.

Approximately 3-10% of human red blood cell (RBC) transfusion recipients form alloantibodies to non-self, non-ABO blood group antigens expressed on donor RBCs, with these alloantibodies having the potential to be clinically significant in transfusion and pregnancy settings. However, the majority of transfused individuals never form detectable alloantibodies. Expanding upon observations that children initially transfused with RBCs at a young age are less likely to form alloantibodies throughout their lives, we hypothesized that "non-responders" may not only be ignorant of antigens on RBCs but instead tolerized. We investigated this question in a reductionist murine model, in which transgenic donors express the human glycophorin A (hGPA) antigen in an RBC-specific manner. Although wild-type mice treated with poly IC and transfused with hGPA RBCs generated robust anti-hGPA IgG alloantibodies that led to rapid clearance of incompatible RBCs, those transfused in the absence of an adjuvant failed to become alloimmunized. Animals depleted of CD4+ cells or treated with CD40L blockade prior to initial hGPA RBC exposure, in the presence of poly IC, failed to generate detectable anti-hGPA IgG alloantibodies. These non-responders to a primary transfusion remained unable to generate anti-hGPA IgG alloantibodies upon secondary hGPA exposure and did not prematurely clear transfused hGPA RBCs even after their CD4 cells had returned or their CD40L blockade had resolved. This observed tolerance was antigen (hGPA) specific, as robust IgG responses to transfused RBCs expressing a third-party antigen occurred in all studied groups. Experiments completed in an RBC alloimmunization model that allowed evaluation of antigen-specific CD4+ T-cells (HOD (hen egg lysozyme, ovalbumin, and human duffyb)) demonstrated that CD40L blockade prevented the expansion of ovalbumin 323-339 specific T-cells after HOD RBC transfusion and also prevented germinal center formation. Taken together, our data suggest that recipients may indeed become tolerized to antigens expressed on RBCs, with the recipient's immune status upon initial RBC exposure dictating future responses. Although questions surrounding mechanism(s) and sustainability of tolerance remain, these data lay the groundwork for future work investigating RBC immunity versus tolerance in reductionist models and in humans.