Antigen‐specific IgG subclass composition in recipient mice can indicate the degree of red blood cell alloimmunization as well as discern between primary and secondary immunization

Abstract

Background: Despite the vast antigen disparity between donor and recipient red blood cells (RBCs), only 2%-6% of transfusion patients mount an alloantibody response. Recently, RBC antigen density has been proposed as one of the factors that can influence alloimmunization, however, there has been no characterization of the role of antigen density along with RBC dose in primary and secondary immunization. Study Design and Methods: To generate RBCs that express distinct antigen copy numbers, different quantities of hen egg lysozyme (HEL) were coupled to murine RBCs. The HEL-RBCs were subsequently transfused into recipient mice at different RBC doses and their HEL-specific IgM, IgG, and IgG subclass response was evaluated. Results: Productive immune responses could be generated through a high copy number antigen transfused at low RBC doses or a low copy number transfused at high RBC doses. Further, primary but submaximal humoral immunization predominantly induced the IgG2b and IgG3 subclasses. In contrast, a maximal primary immunization or a secondary immunization induced all four IgG subclasses.Discussion: Our results confirm the existence of an antigen threshold for productive immune responses but indicate that a high antigen copy number alone might not be enough to induce a response, but rather a combination of both antigen copy number and cell dosage may determine the outcome of immunization. Further, this study provides a proof of concept that the IgG subclass composition can be an indicator of the level of RBC alloimmunization as well as discern between primary and secondary immunization at least in this murine model.Abbreviations: ABC, antibody binding capacity; ANOVA, analysis of variance; BSA, bovine serum albumin; ELISA, enzyme-linked immunosorbent assay; FMH, fetomaternal hemorrhage; GPA, glycophorin A; GPB, glycophorin B; HDFN, hemolytic disease of the fetus and newborn; HEL, hen egg lysozyme; hGPA, human glycophorin A antigen; HOD, recombinant antigen comprised of hen egg lysozyme linked with ovalbumin peptide and human duffy protein;

Antigen copy number and antibody dose can determine the outcome of erythrocyte alloimmunization inducing either antibody‐mediated immune suppression or enhancement in a murine model

“…Interestingly, the lowest antibody dose of 0.2 μg was able to cause a trend toward enhancement for the HEL med ‐RBCs (Figure 1B), which was not seen for the HEL hi ‐RBCs (Figure 1D). This could potentially be related to the magnitude of the HEL hi ‐RBC IgG response, which as described previously by our laboratory, was maximal 15 or might indicate that an even lower quantity of AMIS‐inducing antibody is needed to induce enhancement.…”

“…We previously reported that antigen copy number not only influences the magnitude of the recipient's immune response but can also alter the composition of the IgG response with the induction of all four IgG subclasses requiring a maximal primary immunogenic stimulus or secondary exposure for submaximal stimuli. 15 Here, we further characterized the IgG subclass response under AMIS conditions. Due to the predominance of the IgG1 subclass in the anti-HEL polyclonal antibody used (Table S1), we did not analyze the AMIS effect for this subclass.…”

“…HEL‐specific IgM, IgG, and IgG subclass responses were detected by ELISA as described previously 15 . Briefly, ELISA plates coated with HEL protein were incubated with diluted serum (1:100 for IgM and IgG, 1:300 for the IgG subclasses) at RT for 1.5 h. After washing, the IgM and IgG plates were incubated with the indicated secondary antibodies as previously reported 15 .…”

“…The HEL-RBCs were generated as described previously. 15 Briefly, RBCs from C57BL/6 mice were incubated with 0.8 mg/mL of N-(3-Dimethylaminopropyl)-N 0 -ethylcarbodiimide hydrochloride (EDC) (Sigma-Aldrich) and either 5 or 25 mg/mL of HEL (Sigma-Aldrich) for 1 h at room temperature (RT), generating HEL med -RBCs (HEL copy number of approximately 3,600) and HEL hi -RBCs (HEL copy number of approximately 12,400), respectively. The antigen copy number on each HEL-RBC population was estimated based on their antibody binding capacity as described previously.…”

“…15 Briefly, ELISA plates coated with HEL protein were incubated with diluted serum (1:100 for IgM and IgG, 1:300 for the IgG subclasses) at RT for 1.5 h. After washing, the IgM and IgG plates were incubated with the indicated secondary antibodies as previously reported. 15 For the IgG subclass analysis, the plates were incubated with a 1:5000 dilution of either Biotin-SP (long spacer) AffiniPure Goat Anti-Mouse IgG, Fcγ subclass 1 specific, Fcγ subclass 2b specific, Fcγ subclass 2c specific, or Fcγ subclass 3 specific (all from Jackson ImmunoResearch Laboratories Inc., West Baltimore Pike, PA) for 1 h at RT. Following washing, a 1:10,000 dilution of HRP-conjugated streptavidin (Thermo Scientific™) was added for 1 h at RT.…”