Antibody-Mediated Immune Suppression of Erythrocyte Alloimmunization Can Occur Independently from Red Cell Clearance or Epitope Masking in a Murine Model

Abstract

Anti-D can prevent immunization to the RhD Ag on RBCs, a phenomenon commonly termed Ab-mediated immune suppression (AMIS). The most accepted theory to explain this effect has been the rapid clearance of RBCs. In mouse models using SRBC, these xenogeneic cells are always rapidly cleared even without Ab, and involvement of epitope masking of the SRBC Ags by the AMIS-inducing Ab (anti-SRBC) has been suggested. To address these hypotheses, we immunized mice with murine transgenic RBCs expressing the HOD Ag (hen egg lysozyme [HEL], in sequence with ovalbumin, and the human Duffy transmembrane protein) in the presence of polyclonal Abs or mAbs to the HOD molecule. The isotype, specificity, and ability to induce AMIS of these Abs were compared with accelerated clearance as well as steric hindrance of the HOD Ag. Mice made IgM and IgG reactive with the HEL portion of the molecule only. All six of the mAbs could inhibit the response. The HEL-specific Abs (4B7, IgG1; GD7, IgG2b; 2F4, IgG1) did not accelerate clearance of the HOD-RBCs and displayed partial epitope masking. The Duffy-specific Abs (MIMA 29, IgG2a; CBC-512, IgG1; K6, IgG1) all caused rapid clearance of HOD RBCs without steric hindrance. To our knowledge, this is the first demonstration of AMIS to erythrocytes in an all-murine model and shows that AMIS can occur in the absence of RBC clearance or epitope masking. The AMIS effect was also independent of IgG isotype and epitope specificity of the AMIS-inducing Ab.

IgG-Mediated Immune Suppression to Erythrocytes by Polyclonal Antibodies Can Occur in the Absence of Activating or Inhibitory Fcγ Receptors in a Full Mouse Model

“…The development of IgM and IgG Abs specific for HEL sequences was evaluated at day 7, 14, 21, and 28. Consistent with our previous paper (36), the transfusion of mice with HOD-RBC alone induced both IgM and IgG Abs to the HEL protein (Fig. 1).…”

“…However, we have previously demonstrated using HOD erythrocytes that Abs directed to the Duffy portion of the HOD molecule suppress the Ab response to the HEL Ag without interference in the binding between the anti-Duffy and anti-HEL Abs (36). Significant suppression of the anti-HEL Ab response by anti-Duffy Abs can still occur in the absence of activating FcgR function (unpublished data).…”

“…The polyclonal anti-HEL Ab was produced and purified in our laboratory as described previously (36). In brief, C57BL/6 mice were immunized with three doses of 200 mg HEL (Sigma-Aldrich, St Louis, MO) in Freund's adjuvant (complete Freund adjuvant for the first dose and incomplete for the others) (Chondrex, Redmond, WA), and IgG was purified using protein G affinity chromatography.…”

“…IgM and IgG subclasses specific for HEL were detected by ELISA as described previously (36). Briefly, ELISA plates were coated with 10 mg/ml HEL overnight at 4˚C and blocked with 2% BSA (Sigma-Aldrich) for 2 h at room temperature.…”

Anti-KEL sera prevents alloimmunization to transfused KEL RBCs in a murine model

“…In addition, intracellular RBC staining fails to haematologica 2015; 100:e396 [6][7][8][9] and murine RBC antigen models. [10][11][12] However, the mechanism(s) of modulation appears to vary by antigen.…”