Microbial Exposure Regulates the Development of Anti-Blood Group Antibodies

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 (B^dis) antigen, an antigen very similar to the human blood group B antigen. Microbial flora was assessed in WT (Blood group B-like) or B^dis KO (Blood group O-like) by sequencing ribosomal DNA isolated from stool samples. Serum was assessed for B^dis reactivity at baseline and following B^dis+ microbial exposure using a glycan microarray. WT or B^dis KO recipients were transfused with B^dis+ RBCs followed by the evaluation of RBC clearance, antibody engagement and complement fixation by flow cytometry.

Results: Similar to blood group O individuals, B^dis KO recipients spontaneously develop varied amounts of anti-B^dis antibodies within the first few weeks of life capable of inducing an acute hemolytic transfusion reaction following incompatible B^dis+ RBC transfusion. To determine whether specific microbial exposure is the primary regulating factor in anti-B^dis antibody formation, we separately housed B^dis KO recipients with low titer anti-B^dis antibodies, yielding an entire B^dis KO colony that never developed detectable anti-B^dis antibodies. Exposure of B^dis KO recipients that lacked detectable anti-B^dis antibodies to specific microbes that express the B^dis antigen induced robust anti-B^dis antibodies. However, the timing of microbial exposure was critical in dictating the likelihood of anti-B^dis antibody formation as younger mice produced anti-B^dis antibodies much more readily than adult mice following B^dis+ microbial exposure. Consistent with this, only B^dis KO recipients that experienced early B^dis+ microbial exposure possessed anti-B^dis antibodies with the capacity to induce an acute hemolytic transfusion reaction following B^dis+ RBC transfusion.

Conclusions: These results demonstrate that B^dis 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 B^dis KO recipients developed antibody more readily than older B^dis KO recipients, in addition to B^dis+ microbial exposure, the actual timing of exposure appears to be a key regulator of anti-B^dis 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.