Removing antigens but not cells: a key to AMIS?

In this issue of Blood, Jajosky et al demonstrate that passive immunization with red blood cell (RBC) antibodies that have the ability to remove target antigens from RBCs, without affecting RBC clearance or CD4⁺ T-cell proliferation, can convert an augmented RBC alloimmune response to antibody-mediated immunosuppression (AMIS) in an established murine model of RBC alloimmunization.¹ These findings shed light on the mechanisms of AMIS and have the potential to facilitate development of new strategies for AMIS against non-RhD RBC alloantigens to expand the prevention of hemolytic disease of the fetus and newborn (HDFN).

Development of maternal alloantibodies against paternally inherited fetal RBC antigens can lead to HDFN.² These maternal antibodies, most commonly anti-RhD, are actively transported across the placenta, where they bind to fetal RBCs carrying the corresponding antigen, which can result in RBC clearance via phagocytic Fcγ receptors (FcγRs).³ If untreated, this can result in perinatal mortality and morbidity, with the occurrence of fetal anemia, jaundice, hydrops, and stillbirth.² Polyclonal anti-RhD immunoprophylaxis administered to women at risk has immensely decreased the rate of alloimmunization and reduced the risk of HDFN,⁴ and it is widely considered to be among the major medical breakthroughs of the 20th century. Despite this major success, anti-RhD immunoprophylaxis targets a single RBC antigen and is the only clinical example of AMIS. Notably, alloimmunization toward non-RhD and non-ABO alloantigens accounts for 0.1% to 1.1% of live births,⁵ and for these alloantigens, no immunoprophylactic approaches exist.

The mechanisms involved in AMIS remain incompletely understood. RBC antibodies can elicit various immune responses that impact the development of alloantibodies or induction of AMIS, dependent on the target antigen and the antibody used.⁶ AMIS has previously been attributed to increased RBC clearance; however, murine studies have suggested that AMIS can also occur independent of RBC clearance or epitope masking⁷ and may result from RBC antigen loss unrelated to FcγRs.⁸ RBC challenge dosages may also differentially influence the outcomes⁹; however, different RBC challenge dosages have not been investigated in parallel and in relation to antibodies with different targets in AMIS studies.

In the current study, Jajosky et al employ a highly systematic approach examining the effects of RBC challenge dosages and antibody combinations on the outcome of AMIS, with evaluation of RBC alloantibody formation, RBC clearance, and RBC antigen loss and induction of CD4⁺ T-cell proliferation. They use an established murine model of HOD RBC alloimmunization, which consists of a chimeric triple-fusion protein containing hen egg lysozyme (HEL; contains B-cell epitopes), a portion of ovalbumin (contains CD4⁺ T-cell epitopes), and the Duffy antigen (anchors the protein using its transmembrane domain) expressed on murine RBCs. To investigate the occurrence of AMIS, different dosages of HOD RBCs (10⁷, 10⁸, and 10⁹) were transfused in the presence of anti-Duffy. A low dose of 10⁷ RBCs induced a detectable anti-HEL IgG response, which was blunted by passive immunization with anti-Duffy. In contrast, a high dose of 10⁸ or 10⁹ RBCs resulted in an opposite outcome, reflected by an increase in anti-HEL IgG on passive immunization with anti-Duffy. This illustrates that the HOD RBC dose can determine the occurrence of RBC alloimmunization in the presence of anti-Duffy. The anti-Duffy responses were shown to be related to induction of antigen loss on the RBC surface and increased RBC clearance at low and high RBC dosages. This suggests that at a high RBC dose, antibody-mediated RBC clearance may enhance de novo alloimmunization. Using FcγR-knockout mice as HOD RBC transfusion recipients, FcγRs were found to be required for both anti–Duffy-mediated RBC clearance and antigen removal. Similarly, FcγRs were found to be required for the anti–Duffy-induced suppression (low-dose 10⁷ RBCs) or augmentation (high-dose 10⁹ RBCs) of anti-HEL IgG formation. Anti-Duffy was also shown to enhance CD4⁺ T-cell proliferation on exposure to both 10⁷ and 10⁹ HOD RBCs, thus in the setting of AMIS and RBC alloimmunization, respectively. Next, the authors focused on the impact of the combination of anti-Duffy with anti-HEL on the immune response after transfusion with a high dose of 10⁹ RBCs. Interestingly, passive immunization with the combination of anti-Duffy with anti-HEL reduced the anti–Duffy-mediated anti-HEL IgG formation. Anti-HEL alone was shown to have no impact on RBC clearance or on CD4⁺ T-cell proliferation but could induce a loss of RBC surface antigens.

The study by Jajosky et al significantly increases our understanding of the complex mechanisms governing AMIS. Limitations of the study include the use of a murine model of RBC alloimmunization that, as with any model system, may not necessarily recapitulate all of the critical pathophysiological features culminating in clinical RBC alloimmunization. Because of the apparent importance of antibody-mediated loss of RBC surface antigens, determining the mechanism(s) involved (eg, due to antigen shedding or internalization) is clearly needed. FcγR engagement appears to be critical for RBC clearance, which is likely related to FcγR-mediated phagocytosis of opsonized RBCs, but also for removal of target RBC antigens by anti-Duffy. The mechanisms of the required FcγR involvement in the induction of anti–Duffy-induced antigen loss, AMIS (at low dose, transfused HOD RBCs), or augmentation of alloimmunization (at high dose, transfused HOD RBCs) should also be further investigated. The structural features of antibodies can determine their biological effector functions and could play a significant contributing role in driving AMIS, as prophylactic anti-RhD preparations were shown to display variable decreases in antibody-Fc fucosylation.¹⁰ The specific contribution of antibody-Fc glycan residues could also be further systematically researched in relation to the outcome of AMIS in the HOD RBC alloimmunization mouse model.

In sum, Jajosky et al convincingly demonstrate that, in a setting of a high HOD RBC challenge dose, passive immunization with anti-HEL can result in removal of RBC target antigens without affecting RBC clearance or CD4⁺ T-cell proliferation, and thereby overturn the anti–Duffy-induced alloimmunization toward AMIS (see figure). Key antibody features and activities that specifically and potently induce AMIS remain to be elucidated in future studies, particularly with a focus on non-RhD RBC alloantigens.

Conflict-of-interest disclosure: The author declares no competing financial interests.