Project 2 SUMMARY: Anti-ABO(H) antibodies, which form spontaneously within the first few months of life, represent the most common immunological barrier to transfusion and transplantation. However, these antibodies can vary widely between individuals, with significant differences in the levels and specificity of anti-ABO(H) antibodies that have direct clinical consequence on ABO(H) incompatible outcomes. However, the factors that influence this variability remain incompletely understood. Our central hypothesis is that innate immune galectins differentially target blood group expressing microbes, which in turn shapes the specificity and hemolytic activity of naturally occurring anti-ABO(H) antibodies. Our hypothesis is formulated on the basis of our recent results utilizing a preclinical model of anti-ABO(H) antibody formation that demonstrates that microbes decorated with carbohydrate blood group (BG) antigens not only drive anti-BG antibody formation, but in doing so, raise fundamental questions regarding how BG+ individuals, who do not generate anti-BG antibodies, protect themselves against BG+ microbes. Our results demonstrate that a series of innate immune proteins called galectins possess the ability to specifically bind and kill BG+ microbes through engagement of the distinct carbohydrate BG antigens that decorate the surface of these microbes, thereby protecting BG+ individuals from these microbes. Consistent with this, inhibition of galectins in vivo results in an increase in BG+ microbes. Surprisingly, galectin inhibition also reduces anti-BG antibody formation following exposure to BG+ microbes. Galectins not only possess the ability to kill BG+ microbes, but also induce the release of bacterial-derived BG decorated lipopolysaccharide (LPS). Our data demonstrate that LPS from BG+, but not BG-, microbes, stimulates the formation of anti-BG antibodies. These results suggest that a dynamic interplay exists between the composition of distinct BG+ microbes in an individual’s microbiota and the ability of galectins to target these microbes, which ultimately shapes the specificity and overall levels of anti-BG antibodies, with distinct consequences on ABO(H) incompatibility. To test this, we will leverage preclinical and clinical studies to define the binding specificity and overall antimicrobial activity of galectins and define how these innate immune factors shape anti-BG antibody formation through the following specific aims. Aim 1: Define the role of galectins in shaping BG+ microbial communities. Aim 2: Define the role of galectins in shaping anti-BG antibody repertoire, levels, and hemolytic activity. Together, these aims will define the fundamental and previously unrecognized role of innate immune factors in shaping the most common immunological barrier to transfusion and transplantation. In doing so, these studies hold significant promise in providing important insight into the development of anti-BG antibodies and clinically relevant ...