Project Summary Transfusion of platelets is a vital life-sustaining therapy for numerous diseases that result in thrombocytopenia. However, platelet transfusion can also result in humoral alloimmunization, predominantly to human leukocyte antigens (HLA). With immunization to multiple alloantigens, patients can become refractory to platelet transfusion, resulting in difficulty supporting platelet transfusion needs, and in extreme cases can eliminate platelets as a viable therapy, leading to morbidity and/or mortality from hemorrhage. Anti-HLA alloantibodies can also be substantial barriers to transplantation, rendering patients ineligible for transplant in some cases, or if they do get transplanted, leading to an increased kinetics and/or severity of rejection. Multigravid females are particularly prone to alloimmunization – pregnancy appears to prime for subsequent alloimmunization to platelet transfusion. Thus, alloimmunization to HLA is a significant problem in a number of settings. Although leukoreduction of platelets has reduced rates of alloimmunization, residual immunity remains substantial. Importantly, there are provocative data that distinct leukocyte subsets affect immunity differently (some promoting immunity and others suppressing). Thus, the bulk removal of leukocytes may remove suppressing (as well as immunizing) populations. A detailed understanding of the differential effects of distinct leukocyte subsets would allow a more sophisticated engineering of platelet units with regards to selective modification of leukocyte composition, if immunizing subsets can be removed and suppressing subsets retained. Such information may also be of great utility in cellular therapies outside the context of platelet transfusion. This proposal utilizes an innovative, novel, and tractable murine model, that allows a detailed dissection of the relative contribution of different leukocyte subsets to alloimmunization. We have already used this platform to make the observation that there is cooperativity between MHC alloantigens in inducing an immune response; alloimmunization to the same alloantigen differs based upon the context of the mismatch, opening the door to sophisticated matching/mismatching strategies for transfusion and transplantation in an era of personalized medicine. We have also discovered that in mice, as in humans, pregnancy primes for a subsequent increased alloimmune response rate to transfusion. We propose two specific aims, focusing on the cellular mechanisms of alloimmune responses to different leukocyte subsets in naïve recipients and in pregnancy primed recipients, respectively. We have built into this approach a further analysis of how context of mismatch alters immunogenicity of a given alloantigen, to expand on our initial findings. The models generated for this proposal use naturally occurring MHC alloantigens in mice, but focus on specific alloantigens for which cutting edge tools are available to perform a detailed analysi...