Antigen-Mediated Immune Suppression Is Antigen Specific
Authors: Maier Cheryl , Mener Amanda , Patel Seema R , Bennett Ashley L , Stowell Sean R 
Published: April 2021

Abstract
Background: Exposure to red blood cell (RBC) alloantigens during pregnancy or transfusion can lead to the development of alloantibodies and result in transfusion-related complications, like hemolytic transfusion reactions, or hemolytic disease of the fetus and newborn. Currently, antibody-mediated immune suppression (AMIS) is the only known methodology to actively inhibit RBC alloantibody formation. The best-known example of AMIS is Rh immune globulin, which is given to Rh(D) negative women to prevent the development of anti-D alloantibodies. Mouse models to elucidate the mechanism by which AMIS occurs have used mice genetically engineered to specifically express the human KEL antigen (KEL RBCs) or the Hel-Ova-Duffy antigen (HOD RBCs) on murine RBCs, and found that AMIS to these two antigens is dependent on antigen loss (also called antigen modulation). As RBCs express many antigens simultaneously, we sought to determine whether AMIS to one antigen can impact the immune response to a completely different antigen on the same RBC by generating mice that express both the KEL and HOD antigens (HOD x KEL RBCs).
Methods: WT recipients received polyclonal anti-KEL antibody, a cocktail of two anti-HEL monoclonal antibodies, or PBS, followed by transfusion with WT and HOD x KEL donor RBCs. HOD x KEL RBCs were labeled with the lipophilic dye DiI prior to transfusion, while WT RBCs were labeled with the lipophilic dye DiO and served as a RBC tracer population. RBC survival and levels of detectable IgG, Kel antigen, Hel antigen, Duffy antigen, and complement (C3) on transfused cells were measured by flow cytometry at 10min, 1hr, 2hr, and 4hr, and on days 1, 2, 3, and 5 post-transfusion. Serum was collected on day 5 and analyzed for IgM antibody development by flow-crossmatch using KEL or HOD RBCs.
Results: Anti-KEL and anti-HEL treated WT recipients demonstrated significant IgG deposition on transfused HOD x KEL RBCs at 10min post-transfusion. Levels of IgG on transfused RBCs decreased over time but did not correlate with complete RBC clearance. RBC survival was nearly 100% over 5 days in recipients passively immunized with anti-HEL antibodies, while a fraction of RBCs (~20%) were cleared within 4hr post-transfusion but not thereafter (survival plateaued at 80% thru day 5) in anti-KEL immunized recipients. Conversely, decreased bound IgG on transfused RBCs was found to correlate with a decrease in target antigen levels. KEL antigen levels decreased on transfused RBCs in the anti-KEL immunized recipients beginning at 1hr post transfusion, as compared to anti-HEL and PBS recipients. Similarly, HEL antigen expression decreased on transfused RBCs in the anti-HEL immunized recipients, but not in the anti-KEL or PBS recipients, demonstrating that passive immunization causes antigen modulation for the corresponding antigen only. C3 deposition was found to occur on transfused RBCs at 10min post-transfusion in the anti-KEL recipients that remained significantly increased at other time-points, but was not detected on RBCs in the anti-HEL or PBS recipients until day 3, when recipients develop IgM antibodies. Flow crossmatch of serum samples showed that anti-KEL treated recipients did not make anti-KEL IgM but did make anti-HOD IgM, while anti-HEL treated recipients did not make anti-HOD IgM but did make anti-KEL IgM.
Conclusions: Although AMIS serves as the basis of a widely successful clinical intervention, the mechanism by which it occurs, including any impact on additional antigens, remains incompletely understood. Here we demonstrate that AMIS is antigen specific - that is, passively acquired antibody can inhibit the immune response only to the corresponding antigen. Specifically, murine RBCs expressing both the human and model RBC antigen KEL and HOD, respectively, demonstrate similar clearance kinetics, antigen modulation, complement deposition, and antibody development after transfusion into WT mice as is observed when KEL-only or HOD-only RBCs are used. Although it is unclear whether these findings apply to all RBC antigens and what relevance this holds for human patients, our study provides insight into the specificity of AMIS that may enlighten development of additional therapeutics based on this mode of immune suppression.

Disclosures
No relevant conflicts of interest to declare.