Antibody-mediated in-vitro antigen removal from red blood cells prior to transfusion enhances survival and reduces alloimmunization

Abstract

Abstract Background: While transfusion can be beneficial for many patient populations, the formation of red blood cell (RBC) alloantibodies can make it challenging to obtain compatible RBCs for future transfusions, which may not only prolong anemia but also increase the risk of serious transfusion complications. Recent studies suggest that antibody-induced antigen modulation could help protect RBCs from further antibody-mediated destruction by reducing antibody engagement on the RBC surface. However, antigen modulation has been observed primarily in vivo after an initial phase of antibody-mediated RBC clearance, which often triggers a significant systemic inflammatory response. Although some RBCs remain in circulation, the immediate effects of incompatible RBC transfusion render this approach unsuitable for highly alloimmunized individuals. Given the substantial inflammatory response associated with the initial clearance of RBCs, we hypothesize that inducing antigen modulation in vitro, prior to transfusion, could circumvent this initial clearance, while allowing alloantigen modulated RBCs, which would otherwise be incompatible, to be successful transfused without experiencing the enhanced clearance that accompany a hemolytic transfusion reaction. Methods: To examine the potential for a prior antigen modulation to prevent rapid RBC clearance in an immunized recipient, we used the HOD model system, in which donor RBCs express a triple fusion protein comprising HEL, OVA, and Duffy antigens. HOD RBCs, with or without anti-Duffy antibody coating, were incubated with macrophages followed by assessment for RBC antigen levels by confocal microscopy, Western blotting, and flow cytometry. To assess the effect of antigen loss on post-transfusion RBC survival, these same HOD RBCs were labeled with the fluorescent dye DiI and transfused into non-immunized or anti-Duffy immunized recipient mice. In addition to monitoring RBC survival, the generation of alloantibodies in recipients not previously immunized with anti-Duffy was assessed by flow crossmatch. Each experimental group included at least 10 mice, and statistical analyses were conducted using the t-test and one-way ANOVA. p < 0.05 was considered significant. Results: Incubating HOD RBCs with both anti-Duffy antibodies and macrophages led to the selective removal of the HOD antigen, eliminating detectable levels of the HEL, OVA, and Duffy as determined by flow cytometry and confocal microscopy. In contrast, incubation of HOD RBCs with macrophages alone did not significantly affect HOD antigen levels (p < 0.0001). Further analysis by Western blot confirmed that the HOD antigen was not merely masked by anti-Duffy antibodies but was instead completely removed from the RBC membrane, while levels of unrelated RBC surface antigens remained unchanged. Transfusion of HOD RBCs that had been incubated with both anti-Duffy antibodies and macrophages into either anti-Duffy immunized or non-immunized recipients did not result in detectable change in RBC clearance when compared to transfusion of the same RBCs into non-immunized controls. These in vitro-modified HOD RBCs also circulated normally for weeks post-transfusion, displaying overall survival rates comparable to fully compatible RBCs that did not undergo antigen modulation. By contrast, HOD RBCs treated only with macrophages or not exposed in vitro to macrophages or antibodies were rapidly cleared following transfusion into immunized recipients (p < 0.0001). Macrophage- and anti-Duffy antibody–mediated antigen loss in vitro not only prevented anti-Duffy–mediated RBC clearance, but transfusion of these modified HOD RBCs into non-immunized recipients also failed to elicit alloantibody responses. Conclusions: These results show that in vitro antibody-induced antigen loss can protect RBCs from antibody-mediated clearance following what would otherwise be an incompatible transfusion, with no detectable impact on either the short- or long-term survival of the transfused RBCs. In vitro antigen loss also prevented the formation of new antibodies following transfusion of these same RBCs into non-immunized recipients. By selectively reducing RBC antigen levels in vitro while maintaining cell viability, in vitro antigen modulation suppresses alloimmunization and extends RBC survival in alloimmunized recipients, offering a potential strategy to transfuse alloimmunized patients without triggering rapid RBC clearance.