This concern was also addressed by including control groups that were injected with mAb anti-K1 but received no transfusion; baseline transmission was decided from these control groups at the same time points as experimental groups. Compared to control compatible mice injected with PBS, a statistically significant clearance of K1 RBCs was observed with mAb anti-K1 of the IgG1, IgG2a, IgG2b, and IgG2c subclasses (p< 0.001 for each subclass) (Figure 2A). 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 statement that the ability of an anti-RBC IgG to enhance, suppress (at the level of IgM responses), or have no effect is usually a function of the IgG subclass in this model system. Keywords:antibody, immune regulation, IgG subclass, reddish blood cell, alloimmunity == Introduction == The use of passively transferred antibodies to RhD (anti-D) as an immunoprophylaxis to prevent maternal alloimmunization represents a highly successful therapeutic intervention to avoid hemolytic disease of the fetus Amoxicillin Sodium and newborn (at least with regards to RhD). Moreover, use of anti-D is usually one of only a very few examples of successful immunomodulation preventing reactivity to a specific antigen without inducing general immunosuppression. However, despite its common success, the mechanisms by which anti-D prevents alloimmunization to RhD remains obscure. Although a number of popular theories have been put forward, there is substantial evidence to reject both of the most common explanations, that immunization is usually prevented by avoiding exposure of the immune system through clearance of RhD+ RBCs or masking of antigen (1). The lack of mechanistic understanding has precluded explanations for a number of phenomena surrounding the use of anti-RhD. Despite proper use, anti-D fails to protect some pregnant women from alloimmunization (2). Moreover, under certain circumstances, anti-D results in enhancement, rather than suppression, of alloimmunization to RhD (3). Finally, attempts to generate a monoclonal anti-D have largely failed, resulting in either decreased efficacy compared to donor derived anti-D, or in some cases, monoclonal anti-D has shown the same paradoxical enhancement of alloimmunization seen with certain preparations of donor derived anti-D (4,5); although a recent report indicates great progress in this area (6). Why anti-D suppresses alloimmunization in some cases and enhances alloimmunization in others remains an unsolved question. Humans express 4 different IgG subclasses, each with different effector functions regarding ligation of Fc Amoxicillin Sodium gamma receptors (FcRs), fixation of match, and integration with different biological systems (7). Polyclonal donor derived anti-D is usually a mixture of all 4 IgG subclasses each of which may have different functional effects. Only IgG1 and IgG3 have been tested as therapeutic monoclonal anti-D; and, to the best of our knowledge, the possibility that IgG subclass may be an independent variable affecting immunoregulatory effects Rabbit Polyclonal to C-RAF of anti-D has not been assessed. Like humans, mice express 4 different IgG subclasses, which differ in orthology but are analogous by function. Herein, we used a mouse model to test the hypothesis that IgG subclass of anti-RBC antibodies affects immunoregulatory function. Systems in Amoxicillin Sodium which mice make a humoral response to human RhD have remained elusive. As such, we utilized a murine system of humoral immunization to a human blood group antigen (K1 of the Kell system) in combination with a panel of anti-K1 IgG switch variants (i.e., antibodies with identical antigen binding domains but of different IgG subclasses). This model is not intended to exactly symbolize RhD; rather it serves to test how IgG subclass affects alloimmunity to RBC alloantigens in an analogous system. We statement that antibodies with the same antigen binding domain name have different immunoregulatory effects based upon IgG subclass. == Materials and Methods == == Mice == Wild-type (WT) C57BL/6J (B6), Fc–chain/(stock# 002847) and UbiC-GFP transgenic (stock #004353) were purchased from your Jackson laboratory (Bar Harbor, ME). K1 and K2 transgenic mice were generated as previously explained (8,9). K1 mice were crossed Amoxicillin Sodium with UbiC-GFP to allowin vivomonitoring by circulation cytometry Amoxicillin Sodium without staining (K1.GFP). All K1 RBCs transfused in this study were from K1.GFP mice; but are simply referred to as K1 in this paper for simplicity of nomenclature. All of these mice were housed and/or bred in Bloodworks Northwest Research Institute vivarium (Seattle, Washington) and all procedures were performed according to approved IACUC protocols. == Monoclonal Antibodies and Passive Immunization == PUMA1 and PUMA 6 and their switch variants were isolated, expressed, and purified to homogeneity as previously explained (10,11). B6 mice were passively immunized by tail vein injection with 0.25g of PUMA1 IgG1, IgG2a, IgG2b,.