Abstract
Physiological erythrocyte removal is associated with a selective increase in expression of neoantigens on erythrocytes and their vesicles, and subsequent autologous antibody binding and phagocytosis. Chronic erythrocyte transfusion often leads to immunization and the formation of alloantibodies and autoantibodies. We investigated whether erythrocyte storage leads to the increased expression of non-physiological antigens. Immunoprecipitations were performed with erythrocytes and vesicles from blood bank erythrocyte concentrates of increasing storage periods, using patient plasma containing erythrocyte autoantibodies. Immunoprecipitate composition was identified using proteomics. Patient plasma antibody binding increased with erythrocyte storage time, while the opposite was observed for healthy volunteer plasma, showing that pathology-associated antigenicity changes during erythrocyte storage. Several membrane proteins were identified as candidate antigens. The protein complexes that were precipitated by the patient antibodies in erythrocytes were different from the ones in the vesicles formed during erythrocyte storage, indicating that the storage-associated vesicles have a different immunization potential. Soluble immune mediators including complement factors were present in the patient plasma immunoprecipitates, but not in the allogeneic control immunoprecipitates. The results support the theory that disturbed erythrocyte aging during storage of erythrocyte concentrates contributes to transfusion-induced alloantibody and autoantibody formation.
Highlights
Physiological, age-dependent removal of erythrocytes is an efficient and well-regulated process, consisting of controlled exposure of molecules that induce recognition of old erythrocytes by the immune system
In order to test the hypothesis that storage of erythrocytes under blood bank conditions leads to the formation of non-physiological neoantigens, a modified indirect immunoprecipitation was performed using plasma of six patients with erythrocyte autoantibodies (Table 1) or of healthy donors, in combination with erythrocytes sampled at different time points from a stored erythrocyte unit
Protein quantification of complex protein mixtures using silver staining can be problematic due to a limited dynamic range of the technique, [16] silver staining proved to work well with the highly purified immunoprecipitates we obtained (Figure 1A). Both the patient and the allogeneic control plasmas showed a decrease in signal after the first week of erythrocyte storage
Summary
Physiological, age-dependent removal of erythrocytes is an efficient and well-regulated process, consisting of controlled exposure of molecules that induce recognition of old erythrocytes by the immune system This process includes senescent cell antigen formation on band 3, possibly in combination with phosphatidylserine (PS) exposure on the outer leaflet of the membrane and/or decreased CD47 expression, resulting in binding of autologous IgG and subsequent phagocytosis by macrophages of the reticulo-endothelial system. [1] During aging, the erythrocyte produces numerous vesicles, most of which expose PS, and that are enriched for IgG and age-related band 3 breakdown products These vesicles are rapidly removed from the circulation, probably by the same mechanism that is responsible for erythrocyte removal. The molecular details, triggers and cross-talk between these pathways are largely unknown [1]
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