Lack of Alloantibody Response to Red Blood Cell Antigens in Juvenile Mice Following Transfusion: Non-Immunogenic or Tolerogenic Response?.

Introduction: Alloimmunization to red blood cell (RBC) antigens occurs in approximately 6% of chronically transfused adults, with some patient populations having significantly higher rates of alloimmunization. Despite these numbers, multiple studies have failed to detect alloantibodies to RBC antigens in neonates following transfusion, even after repeat exposures from the same donor. This is consistent with two possible scenarios: RBC antigens are non-immunogenic in neonates, or RBC antigens are tolerogenic. If early exposure to RBC antigens is a tolerogenic stimulus, then the potential clinical implications are considerable. To allow a controlled testing of these hypotheses, we have constructed a murine model of alloimmunization to RBC antigens in pediatric populations. Methods: Weight-adjusted volumes of leukoreduced blood from mHEL donors, expressing membrane bound hen-egg lysozyme as a unique RBC antigen, were transfused into B10.BR recipients at 3 weeks (juveniles) or 3 months (adults) of age. To assess the effects of inflammation, which we have recently reported augments RBC alloimmunization in adult mice, half of the recipients were treated with Poly (I:C), a double-stranded RNA that mimics viral inflammation. Two weeks post-transfusion, RBC alloimmunization was determined by measuring serum anti-HEL IgG levels by ELISA. Results: In 2 out of 2 experiments, as part of an ongoing study, whereas all 10 adult mice mounted a low level alloantibody response by ELISA, only one out of 10 juvenile mice made a detectable alloantibody response. The non-responsiveness of juvenile mice was not significantly altered by inflammation, as only 2 out of 10 juvenile mice treated with Poly (I:C) mounted an alloantibody response. Conclusion: Our data demonstrate that transfused RBCs are weakly immunogenic in adult mice, but only rarely result in alloimmunization in juvenile mice. This is consistent with the pattern of alloimmunization seen in adult versus neonatal humans. Since our model system detects alloantibodies by ELISA, which is 2000 fold more sensitive than agglutination assays using mHEL RBCs, these data support the notion that the majority of juvenile mice do not make alloantibodies to the mHEL RBC antigen. Additionally, inflammation enhances alloimmunization in juvenile mice to a significantly lesser extent than in adult mice. Ongoing studies will utilize the ability of this model to assess if juvenile transfusion recipients are tolerized, by challenging with an immunogenic stimulus (transfusion in the presence of inflammation or subcutaneous injection of HEL/CFA ). If these studies demonstrate that tolerance to RBC antigens occurs after exposure in the neonatal or juvenile period, then the potential clinical implications are significant.