Human IgG is the main antibody class used in antibody therapies because of its efficacy and longer halflife, which are completely or partly due to FcgR-mediated functions of the molecules. Preclinical testing in mouse models are frequently performed using human IgG, but no detailed information on binding of human IgG to mouse FcgRs is available. The orthologous mouse and human FcgRs share roughly 60-70% identity, suggesting some incompatibility. Here, we report binding affinities of all mouse and human IgG subclasses to mouse FcgR. Human IgGs bound to mouse FcgR with remarkably similar binding strengths as we know from binding to human ortholog receptors, with relative affinities IgG3>IgG1>IgG4>IgG2 and FcgRI>>FcgRIV>FcgRIII>FcgRIIb. This suggests human IgG subclasses to have similar relative FcgRmediated biological activities in mice.

Weak Vel expression levels are caused by multiple genetic factors in SMIM1 and probably also by other genetic or environmental factors. Due to the variation in Vel expression levels, serologic determination of the Vel- phenotype is difficult and a genotyping assay targeting the c.64_80del deletion in SMIM1 should be used to screen donors for the Vel- phenotype.

Background Human IgG exists in 4 different subtypes (IgG1, IgG2, IgG3, and IgG4), but it is also now appreciated that there is genetic variation within IgG subtypes (called isoallotypes). 29 different isoallotypes have been described, with 7, 4, 15, and 3 isoallotypes described for IgG1-IgG4, respectively. The reactivity of anti-IgG with different isoallotypes has not been characterized. Study Design and Methods A novel monoclonal anti-K antibody (PUMA1) was isolated, sequenced, and a panel of PUMA1 variants was expressed consisting of the 29 known IgG isoallotypes. The resulting panel of antibodies was pre-incubated with K+ RBCs and was then subjected to testing with currently approved anti-IgG, by flow cytometry, solid phase systems, gel card, and tube testing. Results/Findings An FDA approved monoclonal anti-IgG (Gamma-clone) failed to recognize 2 out of 15 IgG3 isoallotypes (IgG3-03 and IgG3-13) and 3 out of 3 IgG4 isoallotypes (IgG4-01, 02, 03). In contrast, an FDA approved rabbit polyclonal anti-IgG recognized each of the known human IgG isoallotypes. Conclusion These findings demonstrate “blind spots” in isoalloantibody detection by a monoclonal anti-IgG. Should a patient have anti-RBC antibodies predominantly of an IgG3 subtype of the IgG3-03 and/or IgG3-13 variety, it is possible that a clinically significant alloantibody would be missed. IgG-03 and IgG-13 are estimated at a frequency of 1–3% of Caucasian and 20–30% of certain African populations. The non-reactivity with IgG4 is a known characteristic of this monoclonal anti-IgG, but IgG4 isoallotypes have not been previously reported.

To guide anti-D prophylaxis, Dutch D- pregnant women are offered a quantitative fetal-RHD-genotyping assay to determine the RHD status of their fetus. This allowed us to determine the frequency of different maternal RHD variants in 37 782 serologically D- pregnant women. A variant allele is present in at least 0·96% of Dutch D- pregnant women The D- serology could be confirmed after further serological testing in only 54% of these women, which emphasizes the potential relevance of genotyping of blood donors. 43 different RHD variant alleles were detected, including 15 novel alleles (11 null-, 2 partial D- and 2 DEL-alleles). Of those novel null alleles, one allele contained a single missense mutation (RHD*443C>G) and one allele had a single amino acid deletion (RHD*424_426del). The D- phenotype was confirmed by transduction of human D- erythroblasts, consolidating that, for the first time, a single amino acid change or deletion causes the D- phenotype. Transduction also confirmed the phenotypes for the two new variant DEL-alleles (RHD*721A>C and RHD*884T>C) and the novel partial RHD*492C>A allele. Notably, in three additional cases the DEL phenotype was observed but sequencing of the coding sequence, flanking introns and promoter region revealed an apparently wild-type RHD allele without mutations.

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It has long been appreciated that immunoglobulins are not just the effector endpoint of humoral immunity, but rather have a complex role in regulating antibody responses themselves. Donor derived anti-RhD IgG has been used for over 50 years as an immunoprophylactic to prevent maternal alloimmunization to RhD. Although anti-RhD has dramatically decreased rates of hemolytic disease of the fetus and newborn (for the RhD alloantigen), anti-RhD also fails in some cases, and can even paradoxically enhance immune responses in some circumstances. Attempts to generate a monoclonal anti-RhD have largely failed, with some monoclonals suppressing less than donor derived anti-RhD and others enhancing immunity. These difficulties likely result, in part, because the mechanism of anti-RhD remains unclear. However, substantial evidence exists to reject the common explanations of simple clearance of RhD + RBCs or masking of antigen. Donor derived anti-RhD is a mixture of 4 different IgG subtypes. To the best of our knowledge an analysis of the role different IgG subtypes play in immunoregulation has not been carried out; and, only IgG1 and IgG3 have been tested as monoclonals. Multiple attempts to elicit alloimmune responses to human RhD epitopes in mice have failed. To circumvent this limitation, we utilize a tractable animal model of RBC alloimmunization using the human Kell glycoprotein as an antigen to test the effect of IgG subtype on immunoregulation by antibodies to RBC alloantigens. We report that the ability of an anti-RBC IgG to enhance, suppress (at the level of IgM responses), or have no effect is a function of the IgG subclass in this model system.

Genotyping of individuals carrying this variant by standard RHCE genotyping might falsely predict a C- phenotype or a c+ phenotype. This new variant should be taken into account in RHCE genotyping assays designed for the Chinese population.

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.