Project Grant R01HL172760

INNATE IMMUNE TRIGGERS OF ALLOANTIBODY INDUCTION - PROJECT SUMMARY RED BLOOD CELL (RBC) TRANSFUSION IS A LIFE-SAVING THERAPEUTIC TOOL AND THE MOST COMMON FORM OF ALLOGENEIC TRANSPLANTATION IN CLINICAL PRACTICE. DESPITE ABO BLOOD TYPE MATCHING, TRANSFUSION RECIPIENTS CAN MAKE ALLOANTIBODIES AGAINST NON-ABO POLYMORPHIC RBC ANTIGENS. SUCH ANTIBODIES CAN RESULT IN SIGNIFICANT MORBIDITY AND EVEN MORTALITY, DUE TO DIFFICULTY LOCATING COMPATIBLE RBC UNITS, HEMOLYTIC TRANSFUSION REACTIONS, OR HEMOLYTIC DISEASE OF THE FETUS AND NEWBORN. THE BURDEN OF ALLOIMMUNIZATION IS HIGH IN PATIENTS REQUIRING LIFE-LONG CHRONIC TRANSFUSION THERAPY, SUCH AS THOSE WITH SICKLE CELL DISEASE. DESPITE ITS MEDICAL IMPORTANCE, LITTLE IS KNOWN ABOUT WHAT MAKES TRANSFUSED RBCS CAPABLE OF ACTIVATING THE IMMUNE SYSTEM IN ORDER TO GENERATE ALLOANTIBODIES. USING A PRE-CLINICAL MODEL, WE DISCOVERED THAT MOUSE RBCS, AFTER PROCESSING AND STORAGE IN CONDITIONS THAT REPLICATE CLINICALLY USED METHODS AND STORAGE SOLUTIONS, ACTIVATE SPLENIC DENDRITIC CELLS (DCS) IN THE SPLEEN OF TRANSFUSION RECIPIENTS. WE FURTHER SHOWED THAT ACTIVATION OF THESE DCS IS REQUIRED FOR ALLOIMMUNIZATION BY INITIATING CD4+ T CELL PRIMING. DC ACTIVATION DOES NOT OCCUR IF FRESH RBCS (WITHOUT STORAGE BUT IDENTICAL PROCESSING) ARE TRANSFUSED. FURTHER, DC ACTIVATION OCCURS EVEN WHEN STORED RBCS LACK AN ALLOANTIGEN. THIS LED US TO SEARCH FOR HOW STORED RBCS TRIGGER INNATE IMMUNE RECEPTORS AFTER TRANSFUSION. BY TESTING NUMEROUS FAMILIES OF PATTERN RECOGNITION RECEPTORS, WE DISCOVERED THAT RBC ALLOIMMUNIZATION REQUIRES THE TOLL-LIKE RECEPTOR (TLR) SIGNALING ADAPTOR MOLECULE MYD88. MYD88 HAS ALSO BEEN IMPLICATED IN THE ALLOIMMUNE RESPONSES IN SOLID ORGAN TRANSPLANTATION BY ACTIVATING DCS FOR T CELL PRIMING; HOWEVER, THE NATURE OF THE LIGANDS OR THE TLR RELEVANT FOR GRAFT REJECTION IN THESE SYSTEMS REMAINS UNKNOWN. USING CELL TYPE SPECIFIC MYD88 DELETION, WE FOUND THAT DC AND B CELL, BUT NOT MACROPHAGE INTRINSIC MYD88 SIGNALING IS REQUIRED TO MOUNT AN ALLOANTIBODY RESPONSE. WE RULED OUT A REQUIREMENT FOR MOST MYD88 DEPENDENT CYTOKINE PATHWAYS IN RBC ALLOIMMUNIZATION; INSTEAD, OUR PRELIMINARY DATA SUGGEST THAT PARTICULAR TLRS ARE NECESSARY FOR THE RESPONSE TO RBCS. GIVEN THE STERILITY OF RBC UNITS USED FOR TRANSFUSION IN PATIENTS AND ANIMAL MODELS, WE HYPOTHESIZE THAT MAMMALIAN DAMAGED SELF-MOLECULES ADHERE TO RBCS EITHER DURING STORAGE OR AFTER TRANSFUSION INTO THE RECIPIENT AND TRIGGER TLRS ON B CELLS AND DCS TO INDUCE ALLOIMMUNIZATION. TO TEST THIS HYPOTHESIS, WE WILL IDENTIFY THE IMMUNOSTIMULATORY LIGANDS ON STORED RBCS THAT ARE RESPONSIBLE FOR INITIATING THE ALLOANTIBODY RESPONSE. OUR PRELIMINARY DATA USING SCREENING TLR CELL REPORTER ASSAYS SUPPORT THAT HUMAN RBCS CAN ACTIVATE SELECT TLRS IN VITRO. WE WILL DETERMINE TLR-DEPENDENT ACTIVATION PATHWAYS IN DCS AND B CELLS NECESSARY FOR ALLOIMMUNIZATION AND WILL TRIAL VALIDATED TLR INHIBITORS TO SEE IF WE CAN AVERT ALLOIMMUNIZATION IN OUR MOUSE MODEL. SUCCESSFUL IDENTIFICATION OF RBC-DERIVED TLR TRIGGERS WOULD ENABLE SCREENING OF RBCS WITH IMMUNOSTIMULATORY POTENTIAL, AS WELL AS IDENTIFYING NEW THERAPEUTIC TARGETS TO MITIGATE RBC ALLOIMMUNIZATION.