Cellular Protection Against Antibodies Occurs Through a Complement-Independent Pathway

Although mechanisms exist whereby T cells differentiate self from non-self, occasionally tolerance breaks down and T cells target self-antigens. Under these circumstances, healthy cells possess unique receptors that serve to inhibit potential cell-mediated destruction. However, unlike cell-mediated tissue injury, antibody-secreting cells elicit their effects at remote sites, precluding direct feedback from target tissue to secreting cells. As a result, target cells appear to have evolved mechanisms of inhibiting antibody-mediated destruction. Indeed, patients with reduced levels of complement inhibitory factors experience severe hemolysis following antibody engagement. However, regulatory components responsible for protection against complement-independent antibody effecter functions remain poorly understood. RBCs provide a unique tool in the examination of cellular fate following antibody engagement as RBCs fail to synthesize new antigen and also fail to proliferate. As a result, we utilized a model of antibody-RBC binding to determine whether RBCs possess complement-independent mechanisms of cellular resistance to antibody-induced hemolysis.

B6, C3 KO or FcγR KO mice were passively immunized with an anti-Fy3 IgG2a monoclonal antibody. Fluorescent dye labeled RBCs that transgenically express a chimeric antigen including the Fy3 epitope from human Duffy (HOD mice) were then transfused into immunized or nonimmunized control mice. Over a timecourse, peripheral blood was obtained and transfused RBCs were visualized by flow cytometry, followed by examination of clearance, surface C3, bound IgG and Fy3 antigen. To discriminate old from new RBCs following transfusion, RBCs were harvested from mice 35 days post-biotinylation, transfused into immunized or nonimmunized mice and examined for differential clearance by strepavidin staining and flow cytometry. To further evaluate RBC resistance to antibody-induced clearance, HOD RBCs were harvested 2 days following transfusion and re-transfused into immunized or nonimmunized mice followed by examination for clearance, surface C3, bound IgG and Fy3 antigen.

Immunized mice rapidly cleared over half of HOD RBCs within 2 hours; however, a significant number of resistant cells remained in circulation despite the persistence of antibody levels capable of clearing additional RBCs following subsequent transfusion. No clearance was observed in FcγR KO mice but normal clearance and resistance was observed in C3 KO mice. Moreover, no complement degradation products were detected following transfusion into wt mice. RBCs 35 days and older displayed comparable antibody-induced clearance as younger RBCs, suggesting that resistance did not reflect preferential loss of older RBCs. Furthermore, HOD RBCs remained resistant to clearance following re-transfusion into recently immunized recipients. Examination of HOD antigen levels demonstrated a rapid decrease in HOD antigen that correlated with the development of resistance. Similar to clearance, HOD antigen levels decreased in C3 KO but not FcγR KO.

These results suggest that in addition to known complement-dependent mechanisms of cellular resistance to antibody-induced hemolysis, a novel pathway exists whereby Fcγ receptors not only participate in clearance, but also appear to facilitate specific loss of antibody reactive antigen, which may be responsible for a population of RBCs resistant to further clearance. Taken together, these results indicate that RBCs possess multiple mechanisms for cellular protection against antibodies that may be relevant in autoimmune hemolytic anemia and other antibody-mediated processes.

No relevant conflicts of interest to declare.