Transfusion of murine red blood cells expressing the human KEL glycoprotein induces clinically significant alloantibodies

Abstract

Background Red blood cell (RBC) alloantibodies to non-self antigens may develop following transfusion or pregnancy, leading to morbidity and mortality in the form of hemolytic transfusion reactions or hemolytic disease of the newborn. A better understanding of the mechanisms of RBC alloantibody induction, or strategies to mitigate the consequences of such antibodies, may ultimately improve transfusion safety. However, such studies are inherently difficult in humans. Study Design and Methods We recently generated transgenic mice with RBC specific expression of the human KEL glycoprotein, with the KEL2 or KEL1 antigens. Herein, we investigate recipient alloimmune responses to transfused RBCs in this system. Results Transfusion of RBCs from KEL2 donors into wild type recipients (lacking the human KEL protein but expressing the murine KEL orthologue) resulted in dose dependent anti-KEL glycoprotein IgM and IgG antibody responses, enhanced by recipient inflammation with poly (I:C). Boostable responses were evident upon repeat transfusion, with morbid appearing alloimmunized recipients experiencing rapid clearance of transfused KEL2 but not control RBCs. Although KEL1 RBCs were also immunogenic following transfusion into wild type recipients, transfusion of KEL1 RBCs into KEL2 recipients or vice versa failed to lead to detectable anti-KEL1 or anti-KEL2 responses. Conclusions This murine model, with reproducible and clinically significant KEL glycoprotein alloantibody responses, provides a platform for future mechanistic studies of RBC alloantibody induction and consequences. Long term translational goals of these studies include improving transfusion safety for at risk patients.

A novel role for C3 in antibody-induced red blood cell clearance and antigen modulation

“…These studies describe a murine model in which polyclonal anti-KEL immunoglobulins are generated through transfusion 24 and are used to examine clearance following passive immunization. The RBC biology of incompatible transfusion in this model is multifaceted, with rapid intravascular clearance of a subset of incompatible RBCs, yet persistent circulation and apparent antigen modulation of other circulating transgenic RBCs.…”

“…23 We have recently reported that KEL2 RBCs transfused into wild-type (WT) recipients induce an anti-KEL glycoprotein response. 24 Herein, we report that anti-KEL immunoglobulins induce antigen modulation on incompatible KEL2 RBCs, with C3 playing a role not only in this antigen modulation but also in cellular clearance. FcgRs likewise contribute to RBC clearance in this Kell model but are not required for antigen modulation.…”

Alloantibodies to a paternally derived RBC KEL antigen lead to hemolytic disease of the fetus/newborn in a murine model

“…The distribution of anti-KEL IgG subtypes was similar but not identical between animals immunized through transfusion compared with pregnancy ( Figure 7A); recent studies in our laboratory have more fully characterized the time course of IgG and IgM antibody responses post-transfusion. 30 In 3 of 3 experiments (n 5 30 recipients total), 100% of recipients made detectable anti-KEL glycoprotein immunoglobulins after an initial transfusion, which increased further in response to subsequent transfusions ( Figure 7B). This boostable response was not simply due to a continued increase in response after the initial transfusion, For personal use only.…”

“…by guest www.bloodjournal.org From as antibody responses peaked between 14 and 21 days after the initial transfusion; furthermore, this boostable response was not dependent on poly (I:C) pretreatment, as animals transfused in the absence of poly (I:C) also had a boostable anti-KEL response. 30 To determine whether transfusion induced anti-KEL alloantibodies were detrimental to KEL-positive fetuses, wild-type C57BL/6 females who had previously been transfused 3 times with KEL RBCs in the presence of poly (I:C) were bred with KEL-positive males, and pregnancy outcomes were compared with pregnancy outcomes in control females never previously transfused or pregnant. Females immunized through transfusion had smaller litters compared with nonimmunized females ( Figure 7C) (5.5 vs 6.3 pups), fewer total pups alive at weaning per litter ( Figure 7D) (2.6 vs 5.9 pups; P , .05), and fewer KEL-positive pups ( Figure 7E) (22.6 vs 53.1%; P , .05).…”

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

“…This amount of RBCs was selected for these experiments, given the robust anti-KEL response known to occur following transfusion. 22 Survival of the transfused RBCs was determined by comparing the ratio of circulating KEL to control RBCs in recipients at select time points after transfusion.…”