C3 Modulates RBC Antigen to Negatively Regulate Antibody Response

Background:Red blood cell (RBC) transfusion can result in the development of alloantibodies that can make it difficult to find compatible RBCs for future transfusions and increase the risk of hemolytic transfusion reactions. Despite the consequences of RBC alloimmunization, the factors that regulate this process remain relatively unknown. Recent studies suggest that complement deposition on an antigen surface can significantly enhance the immune response to foreign antigen. As many anti-RBC alloantibodies fix complement and RBCs otherwise lack known adjuvants, early antibody-mediated complement deposition may serve as a key regulator that enhances antibody production. To test this, we employed the KEL RBC model system, which employs RBCs that transgenically express the human KEL antigen specifically on RBCs (KEL RBCs). Using this system, we examined the immunological consequence of KEL RBC exposure following transfusion into C57BL/6 wild-type (WT) or complement component 3 (C3) knockout (KO) recipients.

Methods: KEL RBCs were transfused into WT or C3 KO recipients, followed by serum collection on days 3, 5, 7, 14, and 21 post-transfusion. Antibody development in WT or C3 KO recipients was examined by flow crossmatch, where serum was incubated with KEL RBCs followed by antibody detection with fluorescently-tagged secondary anti-IgM and anti-IgG antibodies using flow cytometry. To determine the impact of complement deposition on the level of detectable antigen on the RBC surface, RBCs were labeled with the lipophilic dye, DiI, prior to transfusion and then sampled 1, 2, 3, 5, 7 and 9 days post-transfusion. The level of detectable KEL antigen, complement deposition, KEL RBC survival and antibody bound to the RBC surface was measured by flow cytometry. To examine the effect of complement deposition on the level of KEL protein in the RBC membrane post-transfusion, RBCs stroma was isolated at various time points post transfusion, followed by western blot analysis for the KEL protein.

Results: While KEL RBCs induced robust anti-KEL antibody formation and C3 deposition in WT recipients, similar exposure to KEL RBCs in C3 KO recipients actually resulted in an unexpected increase in IgM and IgG anti-KEL antibodies when compared to WT recipients. To determine the consequence of C3 deposition, we examined the potential impact of antibody engagement and complement fixation on KEL antigen levels. Consistent with a potential role for complement in directly impacting KEL antigen availability to the immune system, KEL RBCs transferred into WT recipients experienced a decrease in the level of detectable KEL antigen over time that paralleled the development of anti-KEL antibodies and C3 deposition. In contrast, C3 KO recipients failed to experience the same degree of KEL antigen reduction despite the development of significant anti-KEL antibodies over this same time period. Western blot analysis of RBCs post-transfusion revealed that loss of detectable KEL antigen on the RBC surface paralleled a complete lack of detectable KEL antigen in RBC membranes, indicating that C3 may actually facilitate the removal of KEL from the RBC surface.

Conclusion: These results suggest an unexpected role for C3 in negatively regulating antibody responses following RBC transfusion. The impact of C3 on the developing alloantibody response strongly suggests that C3-mediated loss of antigen over time likely reduces antigen availability to the immune system, thereby facilitating the inhibition of antibody production over time. These results not only provide novel insight into potential impact of antigen modulation on the development of an immune response to a RBC alloantigen, but also suggest a completely unexpected role for complement in negatively regulating alloantibody production. In doing so, these results suggest that unique differences in complement activity and overall activation following RBC alloantigen exposure between individuals may represent a previously unrecognized factor that influences alloantibody formation following RBC transfusion.