Transfusion of Soluble Target Antigens to Pre-Immunized Recipients: A Previously Overlooked Mechanism in Transfusion-Related Acute Lung Injury
Authors: Bayat Behnaz , Nielsen Kaspar , Burg-Roderfeld Monika , Bein Gregor , Sachs Ulrich J 
Published: October 2019

Abstract
Transfusion related lung injury (TRALI) is a leading cause for transfusion related morbidity and mortality in industrialized countries. The majority of cases is induced by antibodies present in donor plasma, and exclusion of antibody carriers from the donor pool has decreased the number of TRALI cases significantly. However, TRALI is still reported. We have identified a new and previously overlooked mechanism in which soluble antigens present in the blood component become the target of pre-existing antibodies present in the recipient.
Some of us have recently reported a case of TRALI in a female patient precipitating after the transfusion of 120 mL of leuko-reduced packed red blood cells (PRBC). Immediate serologic work-up revealed no antibody in the donor material; but anti-CD177 was detected in the recipient's blood. The following transfusions with two units of PRBCs from CD177 negative donors were uneventful. CD177 is a glycosylphosphatidylinositol-anchored protein exclusively expressed on neutrophils. Only a small minority of Caucasians (<5%) do not express CD177 and can form antibodies when challenged with CD177 during pregnancy or blood transfusions. CD177 functions as a major surface carrier protein for proteinase-3 (PR3), one key contributor to PECAM-1 dependent neutrophil extravasation.
Using a sandwich ELISA, we could demonstrate the presence of soluble CD177 in complex with PR3 (sCD177/PR3) in human plasma. As demonstrated by surface plasmon resonance analysis (SPR), PR3 (KD 4.98e-8 M) and the CD177/PR3 complex (KD 7.39e-9 M) specifically bind to immobilized PECAM-1, whereas CD177 does not interact with PECAM-1. In vitro, TNF-alpha pre-treated endothelial cells were negative with anti-CD177 or anti-PR3 antibodies by flow cytometry, but became positive after incubation with sCD177/PR3. Subsequent stimulation with monoclonal anti-CD177 or anti-PR3 induced the production of reactive oxygen species (ROS) in sCD177/PR3 positive (+EC), but not negative (-EC), endothelial cells. The same results were obtained after incubation with human anti-CD177 IgG (hIgG) isolated from plasma. If pre-treated ECs were incubated with recombinant PR3, only monoclonal anti-PR3, but not anti-CD177 or anti-CD177 hIgG induced ROS production. When experiments were repeated with F(ab)2 fragments, overall effects were still present, but diminished. To study the effects of this interaction on EC barrier integrity, ECs were grown on a transwell system, and pre-treated as outlined above. Monoclonal anti-CD177 or anti-PR3 as well as anti-CD177 hIgG significantly increased the albumin-FITC flux through +EC, but not -EC. In the presence of recombinant PR3 only, anti-PR3, but not anti-CD177, increased the albumin flux. Finally, we could demonstrate that the filtration process of buffy coat-reduced red blood cells by standard blood bank technology enhances the concentration of sCD177/PR3 in the storage solution almost 6-fold.
In summary, our data demonstrate the presence of sCD177/PR3 in plasma and PRBC storage solution following filtration. When sCD177/PR3 is transfused, it has the capacity to bind to PECAM-1 on the EC surface of the transfusion recipient. The exposure of sCD177/PR3 positive endothelial cells to antibodies against CD177 or PR3 induces endothelial activation and barrier dysfunction in a partially Fcgamma-receptor dependent mechanism, comparable to the mechanism described for "classical" antibody-dependent TRALI. The relevance of soluble target proteins in transfused blood components has previously been overlooked. Furthermore, we assume that the identified new pathway might also be involved in endothelial dysfunction present in Wegener's granulomatosis.

Disclosures
No relevant conflicts of interest to declare.