SUMMARY: Despite being the first human polymorphisms described, ABO(H) blood group antigens and corresponding anti-ABO(H) antibodies continue to be the most common immunological barrier to transfusion and transplantation. Remarkably, however, the factors responsible for generating anti-ABO(H) antibodies capable of causing a hemolytic transfusion reaction (HTR) remain relatively unknown. Our long-term goal is to identify the key factors responsible for the development of anti-ABO(H) antibodies. Our central hypothesis is that a distinct developmental window exists in which host innate-like B1 B cells are uniquely sensitive to stimulation by ABO blood group decorated microbes (BG+ microbes) and that continual exposure to BG+ microbes is required for sustained anti-blood group antibody production. As ABO(H) blood group antigens (hereafter referred to as BG) are carbohydrate structures that, as polymorphisms, are largely confined to humans, preclinical models capable of defining factors that influence anti-BG antibody formation have not been available. To address this, we generated a novel preclinical model by genetic removal of the enzyme required for murine blood group B-like antigen (murine B or Bm) synthesis, to generate blood group O-like (murine O or Om) mice. Om mice spontaneously develop varying levels of anti-Bm antibodies, where antibodies levels correlate with their ability to induce hemolytic transfusion reactions (HTRs). Anti-Bm antibodies eluted from Bm RBCs recognize distinct microbiota and isolation of anti-Bm antibody reactive microbiota identified a strain of Klebsiella pneumoniae that specifically expresses the Bm antigen, suggesting a microbial influence on anti-Bm antibody formation. Consistent with this, exposure of Om recipients with undetectable anti-Bm antibodies to Bm+ K. pneumoniae induced anti-Bm antibodies that can cause HTRs. However, robust anti-Bm antibody formation only occurred following Bm+ K. pneumoniae exposure within the first month of life, while sustained anti-Bm antibody formation required continual microbial colonization. These results suggest that distinct B1 B cell populations, which are uniquely sensitive to early developmental cues, may be responsible for anti-Bm antibody formation. Consistent with this, Bm+ K. pneumoniae exposure increased Bm specific B1 B cells, a B cell population that undergoes unique developmental programs early in life. The possible role of B1 B cells is not unique to this model, as ABO(H) specific B1 B cells were likewise detected in human subjects. These results suggest that early exposure to distinct BG+ microbes drives proliferation of antigen specific B1 B cells, which then requires ongoing microbial input later in life to sustain antibody production. To test this, we will weld clinical correlative data with our preclinical model through the following specific aims. Aim 1. Define the role of early BG+ microbe exposure in the development of anti-BG antibodies. Aim 2. Define the r...