Reticulocytes Are an Unappreciated Risk Factor for RBC Alloimmunization at the Donor and Recipient Levels

Production of red blood cell (RBC) alloantibodies can be clinically significant and lead to hemolytic transfusion reactions and hyperhemolysis, pose barriers to transplantation, and make it difficult to obtain compatible blood for transfusion. Currently, there are no strategies to prevent RBC alloimmunization, other than extended phenotypic matching, and only a few treatments available (with limited efficacy) once alloimmunization occurs. Thus, there is a clinical need to identify additional risk factors to minimize potential alloimmunization.

Alloimmunization risk exists at 2 levels: (1) the donor RBC unit and (2) the recipient’s immune system. In recent studies, mitochondria stimulated the immune system, eliciting an inflammatory signature that correlated with increased alloantibody production. Reticulocytes, particularly those produced during stress erythropoiesis, contain high numbers of mitochondria. Thus, high frequencies of reticulocytes may enhance the immunogenicity of donor RBC units. Likewise, high reticulocyte frequencies (induced in response to anemia) may prime a transfusion recipient’s immune system for alloimmunization. Here, we test the hypothesis, in a mouse model, that reticulocytes are an unappreciated risk factor for RBC alloimmunization at both the donor and recipient levels.

To test whether reticulocytes make RBC units more immunogenic, we generated leukoreduced RBC units with defined reticulocyte frequencies (i.e., <2, 5, 10, 33, 40, 45%). Reticulocyte-rich RBC units were collected from HOD mice (which express the RBC-specific HOD alloantigen) who were treated with phenylhydrazine (PHZ, 2 intraperitoneal [i.p.] injections, one day apart, of 50mg/kg) or after iron dextran administration (5mg i.p. to HOD mice with iron-deficiency anemia); both treatments induce stress erythropoiesis and reticulocytosis. RBC units were refrigerator stored for 1 or 6 days before transfusion into C57BL/6 (B6) mice, which do not express HOD. HOD alloantibodies were significantly increased after transfusion with PHZ-generated RBC units (p<0.0001), as compared to control RBC units with <2% reticulocytes Reticulocyte-rich RBC units stored 1 and 6 days similarly enhanced alloantibody responses. There was a direct correlation between the RBC unit reticulocyte frequency and alloantibody production (R²=0.47, p<0.001); thus, even at a 5% reticulocyte concentration, the RBC unit induced more HOD alloantibodies. Likewise, in a separate model of reticulocytosis, RBC units from iron dextran-treated anemic mice significantly enhanced RBC alloantibody production (p<0.001). RBC units containing reticulocytes had an exacerbated RBC storage lesion, as there was markedly increased post-transfusion pro-inflammatory cytokine production and decreased RBC recovery (p<0.0001). Unbiased omics analysis revealed an enrichment of mitochondrial-containing proteins and metabolites in high reticulocyte RBC units relative to control units. Together, these data demonstrate that reticulocytes, which contain high numbers of mitochondria, make RBC units more immunogenic, as evidenced by increased recipient inflammation and alloantibody production.

Transfused patients, particularly when chronically transfused (e.g., patients with sickle cell disease [SCD]), have high alloimmunization rates and often have high endogenous reticulocyte frequencies. To test whether recipient reticulocytes modulate RBC alloimmune responses, we induced reticulocytosis with PHZ in B6 mice. At peak reticulocytosis, B6 mice received a fresh allogeneic HOD RBC transfusion. Compared to controls, mice with ongoing reticulocytosis had significantly higher alloantibody production 2 weeks post-transfusion (p<0.05). These data demonstrate that endogenous stress erythropoiesis and reticulocytosis in a transfusion recipient can prime its immune system towards alloimmunization. Thus, recipient reticulocyte frequency is a risk factor for RBC alloimmunization and provide one explanation for higher alloimmunization risk post-transfusion in patients with diseases associated with reticulocytosis (i.e., SCD).

The novel finding that reticulocytes per se (in either donor RBC units or transfusion recipients) are a risk factor for RBC alloimmunization may explain why some RBC units are more immunogenic than others and/or why some patients are more at risk of becoming alloimmunized.