Antibody-Mediated Antigen Loss Can Switch an Augmented Immune Response to Antibody-Mediated Immunosuppression

Background: Exposure to red blood cell (RBC) alloantigens during pregnancy or transfusion can lead to the development of alloantibodies that can cause significant complications, including hemolytic transfusion reactions and hemolytic disease of the fetus and newborn (HDFN). The only method currently available to inhibit RBC alloantibody formation following RBC alloantigen exposure is the administration of antigen-specific antibodies, a phenomenon termed antibody-mediated immune suppression (AMIS). However, the mechanism whereby antibodies induce AMIS remains incompletely defined. While polyclonal anti-RhD antibodies routinely induce AMIS, monoclonal antibody alternatives can have varied outcomes that range from AMIS to actual enhancement of alloimmunization. In addition, the RBC challenge dose in previous studies often varies, suggesting that a combination of the antibodies utilized and the RBC challenge dose employed may, in part, account for the disparate outcomes observed in prior AMIS studies.

Methods: WT C57BL/6 or Fc common gamma receptor (FcgR) knock out (KO) mice were challenged with three different doses (10⁷, 10⁸, and 10⁹) of HOD (HEL, OVA, and Duffy) RBCs in the presence or absence of distinct anti-HEL and anti-Duffy antibody combinations. Anti-HOD antibody formation was assessed by flow cytometry crossmatch. To determine the CD4 T cell response to HOD RBC transfusion, T cell receptor transgenics specific to ovalbumin (OTII) were labeled with CFSE, followed by adoptive transfer, transfusion of HOD RBCs and evaluation of T cell proliferation, activation and cytokine secretion. HOD RBC removal following transfusion in the presence or absence of different anti-HOD antibody combinations was determined by flow cytometric examination of DiO-labeled HOD RBCs. Finally, antigen levels on HOD RBCs were determined specifically for HEL, OVA and Duffy by flow cytometric examination. All groups were subjected to one-way ANOVA analysis with a Tukey’s post hoc analysis with a p value <0.05 considered significant.

Results: To define the impact of RBC dose on AMIS, recipient mice were challenged with 10⁷, 10⁸, and 10⁹ HOD RBCs to cover the breadth of doses used in prior studies. Passive immunization with anti-Duffy antibodies blunted anti-HOD antibody formation following challenge with 10⁷ HOD RBCs, consistent with previous results (p<0.01). In contrast, anti-Duffy antibodies had the opposite outcome following challenge with 10⁸ and 10⁹ HOD RBCs, resulting in nearly a 10-fold increase in anti-HOD antibody formation (p<0.0001). Despite inhibiting or enhancing the humoral immune response, anti-Duffy antibodies uniformly enhanced OTII proliferation and overall activation following exposure to extreme ends of the challenge dose spectrum, 10⁷ or 10⁹ HOD RBCs (p<0.01). Anti-Duffy antibodies induced rapid initial HOD RBC clearance following exposure to 10⁷ or 10⁹ HOD RBCs. However, following exposure to 10⁹ HOD RBCs, the clearance rate was significantly reduced after 4 hours, which resulted in continual exposure to HOD RBCs with persistent antigen levels (p<0.01). No impact on antibody formation, CD4 T cell proliferation, HOD RBC clearance, or changes in HOD antigen levels were observed following HOD RBC challenge in the presence or absence of anti-Duffy antibodies in FcgR KO recipients, demonstrating a key role for activating FcgRs in this process. Inclusion of anti-HEL antibodies, which fail to induce HOD RBC clearance, but induce rapid antigen removal, completely reversed anti-Duffy induced enhancement of HOD RBC alloimmunization.

Conclusions: These results demonstrate that the challenge dose and antibody combinations can impact AMIS outcomes. The ability of antibody combinations to reverse the augmentation observed with anti-Duffy antibodies alone suggests that antigen loss may represent a key feature of antibodies that can selectively induce AMIS regardless of the challenge dose. These results suggest that focusing on antibody activities that mediate antigen removal may provide a useful strategy when seeking to identify monoclonal antibodies or antibody mixtures that will be most effective at inducing AMIS following alloantigen exposure. As RBC alloimmunization can occur following exposure to RBC alloantigens beyond RhD, leveraging this understanding to actively prevent alloantibody formation against other alloantigens may prove useful in at-risk individuals.