Microbial Exposure Regulates the Development of Anti-Blood Group Antibodies
Authors: Arthur Connie M. , Sullivan Harold Clifford , Gerner-Smidt Christian , Ahmed-Kamili Nourine , Bennett Ashley , Patel Seema R. , Henry Steve , Stowell Sean R. 
Published: October 2019

Abstract
Introduction: Anti-ABO antibodies represent the earliest recognized immunological barrier to transfusion and transplantation. However, despite Landsteiner's discovery of ABO blood group antigens over a century ago, the factors that regulate anti-ABO antibody formation remain relatively unknown. Anti-ABO antibodies develop spontaneously within the first few months of life, in the absence of a known antigenic exposure. However, antibody levels vary considerably between individuals suggesting that differences in exposure to environmental triggers may regulate anti-ABO antibody formation. As distinct microbes can stimulate very specific anti-carbohydrate antibodies, we hypothesized that variation in exposure to microbes that decorate themselves in ABO carbohydrates may regulate anti-blood group antibody formation. However, no model currently exists to examine the potential impact of microbial exposure on the development of naturally occurring anti-blood group antibodies.
Methods: To examine the regulatory capacity of specific microbial exposure on naturally occurring antibody formation, we generated a model of ABO blood group antigens. As lower mammals do not express ABO antigens, we used recipients knocked out (KO) for the glycosyltransferase responsible for the synthesis of the carbohydrate B disaccharide (Bdis) antigen, an antigen very similar to the human blood group B antigen. Microbial flora was assessed in WT (Blood group B-like) or Bdis KO (Blood group O-like) by sequencing ribosomal DNA isolated from stool samples. Serum was assessed for Bdis reactivity at baseline and following Bdis+ microbial exposure using a glycan microarray. WT or Bdis KO recipients were transfused with Bdis+ RBCs followed by the evaluation of RBC clearance, antibody engagement and complement fixation by flow cytometry.
Results: Similar to blood group O individuals, Bdis KO recipients spontaneously develop varied amounts of anti-Bdis antibodies within the first few weeks of life capable of inducing an acute hemolytic transfusion reaction following incompatible Bdis+ RBC transfusion. To determine whether specific microbial exposure is the primary regulating factor in anti-Bdis antibody formation, we separately housed Bdis KO recipients with low titer anti-Bdis antibodies, yielding an entire Bdis KO colony that never developed detectable anti-Bdis antibodies. Exposure of Bdis KO recipients that lacked detectable anti-Bdis antibodies to specific microbes that express the Bdis antigen induced robust anti-Bdis antibodies. However, the timing of microbial exposure was critical in dictating the likelihood of anti-Bdis antibody formation as younger mice produced anti-Bdis antibodies much more readily than adult mice following Bdis+ microbial exposure. Consistent with this, only Bdis KO recipients that experienced early Bdis+ microbial exposure possessed anti-Bdis antibodies with the capacity to induce an acute hemolytic transfusion reaction following Bdis+ RBC transfusion.
Conclusions: These results demonstrate that Bdis KO recipients provide an attractive model to study naturally occurring antibody formation and suggest that anti-ABO antibodies are not an inevitable outcome of not expressing ABO blood group antigens. Instead, naturally occurring antibody formation appears to be temporally regulated by specific microbial exposure. As younger Bdis KO recipients developed antibody more readily than older Bdis KO recipients, in addition to Bdis+ microbial exposure, the actual timing of exposure appears to be a key regulator of anti-Bdis antibody formation. Thus variations in an individual's microbiota, particularly during early immunological development, likely dictate the level of anti-ABO antibody formation. As a result, intentional manipulation of an individual's microbial flora may provide a unique and previously unrecognized approach to prevent anti-ABO(H) antibody development in patients requiring transplantation or chronic transfusion.

Disclosures
No relevant conflicts of interest to declare.