Sickle cell disease (SCD) is an inherited blood disorder characterized by sickled red blood cells (RBCs), which are more sensitive to haemolysis and can contribute to disease pathophysiology. Although treatment of SCD can include RBC transfusion, patients with SCD have high rates of alloimmunization. We hypothesized that RBCs from patients with SCD have functionally active mitochondria and can elicit a type 1 interferon response. We evaluated blood samples from more than 100 patients with SCD and found elevated frequencies of mitochondria in reticulocytes and mature RBCs, as compared to healthy blood donors. The presence of mitochondria in mature RBCs was confirmed by flow cytometry, electron microscopy, and proteomic analysis. The mitochondria in mature RBCs were metabolically competent, as determined by enzymatic activities and elevated levels of mitochondria-derived metabolites.Metabolically-active mitochondria in RBCs may increase oxidative stress, which could facilitate and/or exacerbate SCD complications. Coculture of mitochondriapositive RBCs with neutrophils induced production of type 1 interferons, which are known to increase RBC alloimmunization rates. These data demonstrate that mitochondria retained in mature RBCs are functional and can elicit immune responses, suggesting that inappropriate retention of mitochondria in RBCs may play an underappreciated role in SCD complications and be an RBC alloimmunization risk factor.

Red blood cells expressing alloantigens are well known to be capable of inducing robust humoral alloantibody responses both in transfusion and pregnancy. However, the majority of transfusion recipients and pregnant women never make alloantibodies, even after repeat exposure to foreign RBCs. More recently, RBCs have been used as a cellular therapeutic—very much like transfusion, engineered RBCs are highly immunogenic in some cases but not others. In animal models of both transfusion and RBC based therapeutics, RBCs that do not induce an immune response also cause tolerance. Despite a robust phenomenology, the mechanisms of what regulates immunity vs. tolerance to RBCs remains unclear. However, it has been reported that copy number of alloantigens on the RBCs is a critical factor, with a very low copy number causing non-responsiveness (in both humans and mice) and also leading to tolerance in mice. Recently, we reported that an IgG2c specific for an RBC antigen can substantially enhance the humoral immune response upon transfusion of RBCs expressing that antigen. Herein, we report that an IgG2c converts RBCs with low antigen copy number from a tolerogenic to an immunogenic stimulus. These findings report the first known stimulus that induces humoral alloimmunization to a low copy number RBC alloantigen and identify a previously undescribed molecular switch that has the ability to affect responder vs. non-responder phenotypes of transfusion recipients.

Passive immunization with anti-D can prevent maternal alloimmunization to RhD thereby preventing hemolytic disease of the fetus and newborn. Unexpectedly, anti-D fails in some cases and some monoclonal anti-D preparations paradoxically enhances alloimmunization. The underlying mechanisms modulating humoral alloimmunization by anti-D are unknown. We previously reported that IgG antibody subclasses differentially regulate alloimmunity in response to red blood cell (RBC) transfusions in a mouse model; in particular, IgG2c significantly enhanced RBC alloantibody responses. Initial mechanistic studies revealed that IgG2c:RBC immune complexes were preferentially consumed by the splenic dendritic cell (DC) subsets that play a role in RBC alloimmunization. The deletion of activating Fc-gamma receptors (FcγRs) (i.e., FcγRI, FcγRIII, and FcγRIV) on DCs abrogated IgG2c-mediated enhanced alloimmunization. Because DCs express high levels of FcγRIV, which has high affinity for the IgG2c subclass, we hypothesized that FcγRIV was required for enhanced alloimmunization. To test this hypothesis, knockout mice and blocking antibodies were used to manipulate FcγR expression. The data presented herein demonstrate that FcγRIV, but not FcγRI or FcγRIII, is required for IgG2c-mediated enhancement of RBC alloantibody production. Additionally, FcγRI is alone sufficient for IgG2c-mediated RBC clearance but not for increased alloimmunization, demonstrating that RBC clearance can occur without inducing alloimmunization. Together, these data, combined with prior observations, support the hypothesis that passive immunization with an RBC-specific IgG2c antibody increases RBC alloantibody production through FcγRIV ligation on splenic conventional DCs (cDCs). This raises the question of whether standardizing antibody subclasses in immunoprophylaxis preparations is desirable and suggests which subclasses may be optimal for generating monoclonal anti-D therapeutics.

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Background: Failure of humoral tolerance to red blood cell (RBC) antigens may lead to autoimmune hemolytic anemia (AIHA), a severe and sometimes fatal disease. Previous studies have shown that although tolerance is robust in HOD mice, autoantibodies are generated upon adoptive transfer of OTII CD4 + T cells, which are specific for an epitope contained within the HOD antigen. These data imply that antigen-presenting cells (APCs) are presenting RBCderived autoantigen(s) and are capable of driving T-cell activation. Given that multiple APCs participate in erythrophagocytosis, we used a transgenic approach to determine which cellular subsets were required for autoantigen presentation and subsequent autoreactive T-cell activation. Study Design and Methods: HOD mice, which express an RBC-specific antigen consisting of hen egg lysozyme, ovalbumin, and human blood group molecule Duffy, were bred with IAb fl/fl and Cre-expressing transgenic animals to generate mice that lack I-A b expression on particular cell subsets. OTII CD4 + T cell proliferation was assessed in vivo in HOD + I-Ab fl/fl xCre + mice and in vitro upon coculture with sorted APCs. Results: Analysis of HOD + I-Ab fl/fl xCre + mice demonstrated that splenic conventional dendritic cells (DCs), but not macrophages or monocytes, were required for autoantigen presentation to OTII CD4 + T cells. Subsequent in vitro coculture experiments revealed that both CD8 + and CD8 − DC subsets participate in erythrophagocytosis, present RBC-derived autoantigen and stimulate autoreactive T-cell proliferation. Conclusion: These data suggest that if erythrocyte T-cell tolerance fails, DCs are capable of initiating autoimmune responses. As such, targeting DCs may be a fruitful strategy for AIHA therapies.