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

Abstract

Background: Red blood cell (RBC) alloantibodies to nonself antigens may develop after 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, specifically 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 ortholog) resulted in dose-dependent anti-KEL glycoprotein immunoglobulin (Ig)M 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 after 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.

Figure 1. Transgenic murine RBCs express the human KEL2 antigen, and C57BL/6 recipients of KEL2 RBC transfusion make alloantibodies with KEL specificity

(A) Murine RBCs from KEL2 transgenic donors were collected and stained with anti-KEL2 prior to transfusion. (B) Serum from C57BL/6 recipients transfused with KEL2 RBCs was crossmatched with murine KEL2 RBCs (solid line) or wild type C57BL/6 RBCs (shaded histogram). (C) Serum from C57BL/6 recipients transfused with KEL2 RBCs was crossmatched with murine KEL2 RBCs (solid line) or with DTT treated murine KEL2 RBCs (shaded histogram). (D) Serum from C57BL/6 recipients transfused with KEL2 RBCs was crossmatched with human RBCs expressing KEL2 (solid line) or with DTT treated human RBCs (shaded histogram); representative results are shown.

Figure 2. C57BL/6 recipients lacking human KEL have a dose dependent anti-KEL glycoprotein antibody response

(A) Control (KEL2) or wild type C57BL/6 recipients were transfused with 0.5, 5, or 50 μL of KEL2 RBCs, with serum anti-KEL glycoprotein IgG evaluated 2 weeks post-transfusion. Serial evaluations of serum anti-KEL glycoprotein IgM (B) or IgG (C) were completed in KEL2 or wild type C57BL/6 recipients after a single transfusion of 50 μL of KEL2 RBCs. Results are representative of 2–3 independent experiments with at least 3–5 mice/group; *p<0.05.

Figure 3. C57BL/6 recipients have “boostable” responses to repeat KEL2 exposure, with a proinflammatory serum cytokine storm

(A) C57BL/6 recipients were transfused every 2 weeks with KEL2 RBCs, with serum anti-KEL glycoprotein Igs evaluated on day 14 after each transfusion. (B–F) Serum cytokine responses in alloimmunized animals, 90–120 minutes after a 4th KEL2 RBC transfusion. Results are representative of 2–3 independent experiments with at least 3–5 mice/group; *p<0.05.

Figure 4. Recipient inflammation with poly (I:C) enhances anti-KEL responses, with “boostable” responses

(A) Serum anti-KEL glycoprotein IgG responses in C57BL/6 animals transfused with KEL2 RBCs in the presence or absence of recipient poly (I:C) pretreatment; straight serum, 14 days post-transfusion. (B) Serum anti-KEL glycoprotein IgG responses after 1, 2, or 3 KEL2 RBC transfusions in the presence of poly (I:C); sera diluted 1:10, tested every 14 days. Results are representative of 2–3 experiments with 3–5 mice/group; *p<0.05

Figure 5. Circulating anti-KEL glycoprotein Igs bind to transfused KEL2 RBCs and are associated with KEL2 RBC clearance

KEL2 and C57BL/6 RBCs were labeled with DiI and DiO, respectively, prior to transfusion. RBC bound IgM (A) and IgG (B) were evaluated serially post-transfusion on DiI positive KEL RBCs; shaded histograms are control antigen negative cells. Post-transfusion survival and recovery of KEL2 RBCs was determined by comparing a ratio of DiI KEL to DiO C57BL/6 RBCs (C). These studies were completed KEL2 and wild type C57BL/6 recipients (D); similar studies were also completed following a 2^nd KEL2 transfusion in recipients initially transfused with or without poly (I:C) (E). Error bars represent standard deviation and results represent at least 3 independent experiments with 3–5 mice/group.

Figure 6. No detectable alloimmune responses to KEL2 RBCs occur in KEL1 recipients (or vice versa), whose KEL RBC antigens differ by a single amino acid polymorphism

(A) Serum responses of KEL1 or C57BL/6 recipients after 3 transfusions of KEL2 RBCs RBCs. (B) KEL1 RBCs were stained with monoclonal anti-KEL1 (Mima 23) prior to transfusion. Representative serum response of KEL2 (C) or C57BL/6 (D) recipients after 3 transfusions with KEL1 RBCs. Results are representative of 2–3 independent experiments with 2–5 mice/group; *p<0.05.