IgG Subtype Affects Immunomodulation through FcγRs, on Non-Macrophage Populations in a Murine Model - Mechanistic Implications for Anti-D
Authors: Zimring James C. , Waterman Hayley , Wang Xiaohong , Kapp Linda , Lebedev Jenna , Howie Heather , Hay Ariel , Stegmann Tamara Carola , Vidarsson Gestur , Van der Schoot C. Ellen 
Published: October 2019

Abstract
Background: The administration of anti-D as an immune prophylaxis remains a great success in human immunotherapy; however, the mechanism of anti-D function remains unclear. Currently, anti-D is purified from plasma of D alloimmunized humans; 19 different monoclonal anti-D reagents have been tested but have not shown equivalent efficacy as polyclonal anti-D. While anti-D prevents alloimmunization in most cases, it causes paradoxical enhancement of alloimmunization in certain situations. Likewise, whereas some monoclonal anti-D decrease alloimmunization rates, others enhance. Further progress in refining monoclonals have been limited due to a lack of mechanistic understanding of anti-D.
Methods: KEL1 transgenic mice express the human KEL1 antigen selectively on RBCs. Transfusion of KEL1 RBCs into wild-type mice results in alloimmunization to the KEL1 glycoprotein. Polyclonal anti-KEL (from immunized mice) given to naïve mice prevents alloimmunization by KEL1 RBCs. Thus, the KEL1 mouse serves as a murine model of RhD and anti-D [similar to the RhD immune barrier in humans, KEL1 is present on KEL1 mouse RBCs and lacking in wild-type recipients]. We isolated a novel monoclonal anti-KEL1 antibody (PUMA1), sequenced the heavy and light chain variable regions, and generated a panel of recombinant variants of each of the known murine IgG subtypes (IgG1, IgG2a, IgG2b, IgG2c and IgG3) - this gives rise to a panel of antibodies in which IgG subtype is an independent variable, as they each have the same antigen-binding domain. In addition, we generated a novel conditional knockout mouse (Con-FcγR) in which the Fc-receptor common gamma chain (required for expression of all activating murine FcγRs) was deleted upon exposure to CRE recombinase. Con-FcγR mice were crossed with a monocyte/neutrophil specific CRE mouse, resulting in deletion of FcgRs from MØs and neutrophils, but not other tissues.
Results: Significant clearance of KEL1+ RBCs was observed with PUMA1 IgG1, IgG2a, and IgG2c, but not IgG2b or IgG3. IgG1 significantly decrease, whereas IgG2a and IgG2c increased anti-KEL alloimmunization (alloimmunization was unaffected by IgG2b or IgG3). When FcγRs were deleted in all tissues, no clearance was observed with any IgG subtype. Likewise, increased anti-KEL alloimmunization by IgG2a and IgG2c was eliminated in FcγR KO mice. In mice with a selective deletion of FcγRs from MØs and neutrophils, clearance of RBCs was eliminated, whereas immunomodulation persisted (decreased with IgG1, increased with IgG2a and IgG2c). Consistent with recent reports in other systems, suppression by IgG1 occurred normally in the FcγKO mice.
Conclusions: As in humans, some monoclonal anti-RBC antibodies prevented alloimmunization whereas others enhanced. Inhibiting vs. enhancing RBC alloimmunization was a function of IgG subtype. As both suppressing and enhancing antibodies caused clearance of RBCs, these data dissociate clearance as a mechanistic determinant of suppression vs. enhancement. While clearance and immune enhancement required FcγR dependent pathways, the use of the Con-FcγR mouse demonstrates that while macrophages/neutrophils are required for clearance, macrophages/neutrophils are not required for enhancement of alloimmunization.

Disclosures
Zimring: Immucor In.: Research Funding; BloodworksNW: Patents & Royalties; Rubius Therapeutics: Membership on an entity's Board of Directors or advisory committees; State of New Mexico: Consultancy.