Clodronate inhibits alloimmunization against distinct red blood cell alloantigens in mice

Abstract

Background: Alloimmunization can be a significant barrier to red blood cell (RBC) transfusion. While alloantigen matching protocols hold promise in reducing alloantibody formation, transfusion-dependent patients can still experience RBC alloimmunization and associated complications even when matching protocols are employed. As a result, complementary strategies capable of actively preventing alloantibody formation following alloantigen exposure are warranted.

Study design and methods: We examined whether pharmacological removal of macrophages using clodronate may provide an additional strategy to actively inhibit RBC alloimmunization using two preclinical models of RBC alloimmunization. To accomplish this, mice were treated with clodronate, followed by transfusion of RBCs expressing the HOD (HEL, OVA, and Duffy) or KEL antigens. On days 5 and 14 post transfusion, anti-HOD or anti-KEL IgM and IgG antibodies were evaluated.

Results: Low dose clodronate effectively eliminated key marginal zone macrophage populations from the marginal sinus. Prior treatment with clodronate, but not empty liposomes, also significantly inhibited IgM and IgG anti-HOD alloantibody formation following transfusion of HOD RBCs. Similar exposure to clodronate inhibited IgM and IgG antibody formation following KEL RBC transfusion.

Conclusions: Clodronate can inhibit anti-HOD and anti-KEL antibody formation following RBC transfusion in preclinical models. These results suggest that clodronate may provide an alternative approach to actively inhibit or prevent the development of alloantibodies following RBC transfusion, although future studies will certainly be needed to fully explore this possibility.

Keywords: RBC; alloimmunization; marginal zone macrophages; prevention.

Figure 1:. Experimental overview.

A) WT C57BL/6 recipients were pre-treated or not with clodronate, followed by transfusion of HOD or KEL RBCs and evaluation of anti-HOD or anti-KEL antibody formation. B) Evaluation of marginal zone macrophages (MZM) 24 hours following treatment or no treatment with clodronate. Spleens were stained for MARCO (red), SIGNR1 (blue) and IgD (yellow). Images shown were acquired at 10x using a Leica SP8 multiphoton confocal microscope. The results are representative of two independent experiments.

Figure 2:. Clodronate significantly inhibits antibody formation following RBC transfusion.

A-B) Representative histograms of a flow cross-match using KEL RBC targets. Serum collected from recipients pre-treated with PBS (PBS, black), clodronate (clod., red) or empty liposomes (lipo., blue) was collected on days 5 or 14 post transfusion to examine IgM (A) or IgG (B) anti-KEL antibody formation, respectively. C-D) Quantitation of anti-HOD IgM (C) or IgG (D) formation following HOD RBC flow cross-match on days 5 or 14 following HOD RBC transfusion into recipients pre-treated with PBS (black), clodronate (red) or empty liposomes (blue). **p < 0.01, ***p < 0.001. The results are representative of two independent experiments.