FcγRIV is required for IgG2c mediated enhancement of RBC alloimmunization

Abstract

Passive immunization with anti-D can prevent maternal alloimmunization to RhD thereby preventing hemolytic disease of the fetus and newborn. Unexpectedly, anti-D fails in some cases and some monoclonal anti-D preparations paradoxically enhances alloimmunization. The underlying mechanisms modulating humoral alloimmunization by anti-D are unknown. We previously reported that IgG antibody subclasses differentially regulate alloimmunity in response to red blood cell (RBC) transfusions in a mouse model; in particular, IgG2c significantly enhanced RBC alloantibody responses. Initial mechanistic studies revealed that IgG2c:RBC immune complexes were preferentially consumed by the splenic dendritic cell (DC) subsets that play a role in RBC alloimmunization. The deletion of activating Fc-gamma receptors (FcγRs) (i.e., FcγRI, FcγRIII, and FcγRIV) on DCs abrogated IgG2c-mediated enhanced alloimmunization. Because DCs express high levels of FcγRIV, which has high affinity for the IgG2c subclass, we hypothesized that FcγRIV was required for enhanced alloimmunization. To test this hypothesis, knockout mice and blocking antibodies were used to manipulate FcγR expression. The data presented herein demonstrate that FcγRIV, but not FcγRI or FcγRIII, is required for IgG2c-mediated enhancement of RBC alloantibody production. Additionally, FcγRI is alone sufficient for IgG2c-mediated RBC clearance but not for increased alloimmunization, demonstrating that RBC clearance can occur without inducing alloimmunization. Together, these data, combined with prior observations, support the hypothesis that passive immunization with an RBC-specific IgG2c antibody increases RBC alloantibody production through FcγRIV ligation on splenic conventional DCs (cDCs). This raises the question of whether standardizing antibody subclasses in immunoprophylaxis preparations is desirable and suggests which subclasses may be optimal for generating monoclonal anti-D therapeutics.

Reticulocytes in donor RBC units enhance RBC alloimmunization

“…Erythrophagocytosis. Spleens were excised into complete RPMI, finely chopped, and filtered, as previously described(9, 27, 28). Single cells were washed with FACS buffer, RBCs were lysed, and the remaining cells stained with antibodies in FACS buffer to delineate leukocyte subsets ( Supplemental Table 1 )(8, 9, 28, 29).…”

“…PTR was measured as the ratio of biotin-positive RBCs to GFP-positive RBCs circulating 24 hours-post transfusion relative to the same ratio in the spiked blood unit. Sera were collected from recipients and total HOD alloantibodies (IgM + IgG + IgA) were quantified by flow crossmatch (27).…”

Characterization of a Novel Mouse Platelet Transfusion Model

“…Platelet activation and aggregation. Platelet activation was assessed by incubating 1 µL of the platelet unit with 0.5 U/mL thrombin (Sigma-Aldrich, 604980) diluted in FACS buffer (phosphate-buffered saline + 0.2mg/mL bovine serum albumin + 0.9mg/mL ethylenediaminetetraacetic acid) [18] at room temperature for 2 minutes or with 10mM ADP (Fisher, 22-515-225) at 37 C for 5 minutes. Platelets were washed with 1 mL FACS buffer, and then stained with t antibodies against CD41a, Ter119, CD45, and CD62P (P-selectin; 1:100, Psel.KO2.3, APC, ThermoFisher).…”

Reticulocytes in donor blood units enhance red blood cell alloimmunization

“…Blood samples were collected via tail puncture and PTR was measured as the ratio of biotin-positive RBCs to GFP-positive RBCs circulating 24 hours-post transfusion relative to the same ratio in the spiked blood unit. Sera were collected from recipients and total HOD alloantibodies (IgM + IgG + IgA) were quantified by flow crossmatch 27 .…”

“…Erythrophagocytosis. Spleens were processed into single cells 27 and splenocytes were stained with antibodies to delineate leukocyte subsets (Table 1S) 8,9,28,29 . Results were acquired on an Attune NxT flow cytometer (ThermoFisher) and data analyzed with FlowJo software.…”