Innate immune lectins shape anti-blood group antibody development and transfusion outcomes

Abstract Introduction Anti-ABO(H) antibodies are the primary immunological barrier in both transfusion and transplantation. Although these antibodies were identified more than a hundred years ago, the mechanisms controlling their formation, which naturally occurs within the first few months of life, are still not fully understood. Early research indicated that microbes capable of mimicking ABO blood group antigens might drive the development of anti-ABO antibodies. More recent studies have shown that innate immune proteins called galectins can specifically detect and eliminate such microbes, providing protection for blood group-positive individuals, who do not develop anti-blood group antibodies, from these microbes. However, the ability of galectins to target microbes that mimic blood group antigens also suggests a previously unrecognized interplay may exist between innate immunity, blood group molecular mimicry, and the production of anti-ABO antibodies. Methods Total microbiota composition was analyzed using 16S sequencing, while the presence of blood group B positive (B+) microbes was determined by flow cytometry. The specificity of galectins and antibodies was characterized through the use of mammalian and microbial glycan microarrays. Galectin antimicrobial activity was measured by colony-forming unit enumeration. To investigate the influence of galectins on B+ microbe abundance and anti-blood group B (anti-B) antibody production, blood group O mice were either treated with thiodigalactoside (TDG), a broad galectin inhibitor, or bred with galectin-8 (Gal-8) knockouts. Development of anti-B antibodies was assessed via ELISA and flow cross-match using B+red blood cells (RBCs). Anti-B antibody activity was assessed by transfusing B+ RBCs, followed by evaluating antibody binding, complement activation, RBC clearance, and hemolysis. Each group consisted of at least 10 mice, and statistical differences were evaluated by one-way ANOVA, considering p<0.05 as significant. Results Blood group O mice spontaneously developed anti-B antibodies that increased over time and correlated with the abundance of B+ microbes, irrespective of the composition of other microbes present (p<0.01). Anti-B antibodies specifically recognized the mammalian B antigen but also cross-reacted with microbial mimics of the same structure. Innate immune galectins, especially Gal-8, recognized the same microbial glycans as anti-B antibodies and interacted with B+ microbes in vivo; notably, one strain of Klebsiella pneumoniae in particular showed strong reactivity to both anti-B antibodies and Gal-8. Gal-8 bound to B+K. pneumoniae and, upon incubation, led to microbial death, whereas it did not bind to or affect the viability of K. pneumoniae strains lacking the B antigen (p<0.0001). Exposure to B+K. pneumoniaeresulted in robust anti-B antibody formation in O mice with undetectable anti-B antibody levels at baseline (p<0.001). Incubating Gal-8 with B+K. pneumoniae led to both microbial death and the release of B+ lipopolysaccharide (LPS) (p<0.01), while oral exposure to B+ LPS alone, but not an unrelated LPS, likewise induced a strong anti-B antibody response in O mice (p<0.01). Removing Gal-8, either genetically or pharmacologically, increased B+K. pneumoniaelevels in vivo (p<0.01), and prevented anti-B antibody formation (p<0.01). Transfusing B+ RBCs into O recipients resulted in robust antibody binding, complement activation, rapid RBC clearance, and hemolysis consistent with a hemolytic transfusion reaction (HTR); however, in galectin-deficient or inhibited O recipients exposed to B+K. pneumoniae, B+ RBC transfusion did not trigger an HTR or lead to altered RBC survival (p<0.001). Conclusions These findings uncover a direct and previously unrecognized link between innate immunity, the microbiota, and the formation of the most common immunological barrier in medicine. Our data demonstrate that galectin-mediated microbial killing results in the release of B⁺ LPS, which in turn stimulates the production of anti-blood group antibodies. Inhibiting galectin activity prevents the formation of anti-blood group antibodies, enabling transfusion of B+ RBCs into blood group O recipients without any evidence of an HTR. This provides a novel strategy to deliberately inhibit anti-ABO antibody formation in patients requiring repeated transfusions or transplants, with broad implications for modulating antibody repertoires in general.