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Antigen modulation as a potential mechanism of anti-KEL immunoprophylaxis in mice

Jingchun Liu, Manjula Santhanakrishnan, Prabitha Natarajan, David R. Gibb, Stephanie C. Eisenbarth, Christopher A. Tormey, Alexa J. Siddon, Sean R. Stowell, Donald R. Branch and Jeanne E. Hendrickson

Key points

  • Despite immunoprophylaxis with anti-KEL sera, mice lacking both Fcγ receptors and C3 become alloimmunized to transfused KEL RBCs.

  • Antigen modulation may be important in the mechanism of action of immunoprophylaxis therapies against RBC antigens.

Abstract

RBC alloimmunization is a serious complication of transfusion or pregnancy. Despite the widespread use of RhIg to prevent pregnancy associated anti-D alloimmunization, its mechanism of action remains elusive. We have previously described a murine model in which immunoprophylaxis with polyclonal anti-KEL sera prevents alloimmunization in wild type recipients transfused with transgenic murine RBCs expressing the human KEL glycoprotein. To investigate the mechanism of action, we have now evaluated the outcome of immunoprophylaxis treatment in mice lacking Fcγ receptors (FcγR), complement (C3), both, or none. Whereas polyclonal anti-KEL sera completely prevented alloimmunization in wild type and single knock-out (KO) mice lacking FcγR or C3, double KO mice lacking both FcγR and C3 became alloimmunized despite immunoprophylaxis. Rapid clearance of essentially all transfused RBCs with detectable KEL glycoprotein antigen occurred within 24 hours in wild type and single KO recipients treated with immunoprophylaxis, with the transfused RBCs remaining in circulation having minimal KEL glycoprotein antigen detectable by flow cytometry or western blot. In contrast, transfused RBCs with the KEL glycoprotein antigen fully intact continued to circulate for days in double KO mice despite treatment with immunoprophylaxis. Further, in-vitro phagocytosis assays showed no consumption of opsonized murine RBCs by double KO splenocytes. Taken in combination, our data suggest that modulation of the KEL antigen (and potentially RBC clearance) by redundant recipient pathways involving both FcγR and C3 may be critical to the mechanism of action of polyclonal anti-KEL immunoprophylaxis. These findings could have implications for the development of immunoprophylaxis programs in humans.

  • Submitted June 27, 2016.
  • Accepted September 26, 2016.