Introduction: The impact of blood storage on red blood cell (RBC) alloimmunization remains controversial, with some studies suggesting enhancement of RBC-induced alloantibody production and others failing to observe any impact of storage on alloantibody formation. Since evaluation of storage on RBC alloimmunization in patients has examined antibody formation against a broad range of alloantigens, it remains possible that different clinical outcomes reflect a variable impact of storage on alloimmunization to specific antigens. Methods: RBCs expressing two distinct model antigens, HEL-OVA-Duffy (HOD) and KEL, separately or together (HOD × KEL), were stored for 0, 8, or 14 days, followed by detection of antigen levels prior to transfusion. Transfused donor RBC survival was assessed within 24 h of transfusion, while IgM and IgG antibody production were assessed 5 and 14 days after transfusion. Results: Stored HOD or KEL RBCs retained similar HEL or KEL antigen levels, respectively, as fresh RBCs, but did exhibit enhanced RBC clearance with increased storage age. Storage enhanced IgG antibody formation against HOD, while the oppositive outcome occurred following transfusion of stored KEL RBCs. The distinct impact of storage on HOD or KEL alloimmunization did not appear to reflect intrinsic differences between HOD or KEL RBCs, as transfusion of stored HOD × KEL RBCs resulted in increased IgG anti-HOD antibody development and reduced IgG anti-KEL antibody formation. Conclusions: These data demonstrate a dichotomous impact of storage on immunization to distinct RBC antigens, offering a possible explanation for inconsistent clinical experience and the need for additional studies on the relationship between RBC storage and alloimmunization.

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.

Despite being the first genetic disease described, sickle cell disease (SCD) continues to afflict patients with immense pain, significant comorbidities and premature death. SCD has only recently benefited from new interventions with L-glutamine (2017), voxelotor (2019) and crizanlizumab (2019) representing the first Food and Drug Administration approved medications for SCD since hydroxyurea in 1997. These interventions have demonstrated some ability to reduce vaso-occlusive pain crisis episodes, improve hemoglobin (HGB), or reduce markers of hemolysis and have largely been used as preventative care measures. While these and additional approaches, such as hematopoietic stem cell transplant and gene therapy, can improve SCD care, many patients with SCD continue to suffer from severe acute SCD complications that can result in organ damage and early death.1,2 Unfortunately, in these situations, supportive care remains the primary approach to alleviate complications. The lack of more targeted approaches in part reflects an incomplete understanding of the pathophysiology and accompanying pharmacological targets that could specifically mitigate acute disease complications. We present a summary of three cases of children with SCD who developed significant acute complications that demonstrate underlying complement-mediated thrombotic microangiopathy (CM-TMA). These cases include a delayed hemolytic transfusion reaction (DHTR), vasoocclusive crisis (VOC) and drug-induced immune hemolytic anemia (DIIHA).

Anti-factor VIII (fVIII) alloantibodies, which can develop in patients with hemophilia A, limit the therapeutic options and increase morbidity and mortality of these patients. However, the factors that influence anti-fVIII antibody development remain incompletely understood. Recent studies suggest that Fc gamma receptors (FcγRs) may facilitate recognition and uptake of fVIII by recently developed or pre-existing naturally occurring anti-fVIII antibodies, providing a mechanism whereby the immune system may recognize fVIII following infusion. However, the role of FcγRs in anti-fVIII antibody formation remains unknown. In order to define the influence of FcγRs on the development of anti-fVIII antibodies, fVIII was injected into WT or FcγR knockout recipients, followed by evaluation of anti-fVIII antibodies. Anti-fVIII antibodies were readily observed following fVIII injection into FcγR knockouts, with similar anti-fVIII antibody levels occurring in FcγR knockouts as detected in WT mice injected in parallel. As antibodies can also fix complement, providing a potential mechanism whereby anti-fVIII antibodies may influence anti-fVIII antibody formation independent of FcγRs, fVIII was also injected into complement component 3 (C3) knockout recipients in parallel. Similar to FcγR knockouts, C3 knockout recipients developed a robust response to fVIII, which was likewise similar to that observed in WT recipients. As FcγRs or C3 may compensate for each other in recipients only deficient in FcγRs or C3 alone, we generated mice deficient in both FcγRs and C3 to test for potential antibody effector redundancy in anti-fVIII antibody formation. Infusion of fVIII into FcγRs and C3 (FcγR × C3) double knockouts likewise induced anti-fVIII antibodies. However, unlike individual knockouts, anti-fVIII antibodies in FcγRs × C3 knockouts were initially lower than WT recipients, although anti-fVIII antibodies increased to WT levels following additional fVIII exposure. In contrast, infusion of RBCs expressing distinct alloantigens into FcγRs, C3 or FcγR × C3 knockout recipients either failed to change anti-RBC levels when compared to WT recipients or actually increased antibody responses, depending on the target antigen. Taken together, these results suggest FcγRs and C3 can differentially impact antibody formation following exposure to distinct alloantigens and that FcγRs and C3 work in concert to facilitate early anti-fVIII antibody formation.

Red blood cell (RBC) alloimmunization represents a significant immunological challenge for some patients. While a variety of immune constituents likely contribute to the initiation and orchestration of alloantibodies to RBC antigens, identification of key immune factors that initiate alloantibody formation may aid in the development of a therapeutic modality to minimize or prevent this process. To define the immune factors that may be important in driving alloimmunization to an RBC antigen, we determined the specific immune compartment and distinct cells that may initially engage transfused RBCs and facilitate subsequent alloimmunization. Our findings demonstrate that the splenic compartment is essential for formation of anti-KEL antibodies following KEL RBC transfusion. Within the spleen, transfused KEL RBCs are found within the marginal sinus, where they appear to specifically co-localize with marginal zone (MZ) B cells. Consistent with this, removal of MZ B cells completely prevented alloantibody formation following KEL RBC transfusion. While MZ B cells can mediate a variety of key downstream immune pathways, depletion of follicular B cells or CD4 T cells failed to similarly impact the anti-KEL antibody response, suggesting that MZ B cells may play a key role in the development of anti-KEL IgM and IgG following KEL RBC transfusion. These findings highlight a key contributor to KEL RBC-induced antibody formation, wherein MZ B cells facilitate antibody formation following RBC transfusion.

The development of anti-drug antibodies represents a significant barrier to the utilization of protein-based therapies for a wide variety of diseases. While the rate of antibody formation can vary depending on the therapeutic employed and the target patient population receiving the drug, the antigen-specific immune response underlying the development of anti-drug antibodies often remains difficult to define. This is especially true for patients with hemophilia A who, following exposure, develop antibodies against the coagulation factor, factor VIII (FVIII). Models capable of studying this response in an antigen-specific manner have been lacking. To overcome this challenge, we engineered FVIII to contain a peptide (323–339) from the model antigen ovalbumin (OVA), a very common tool used to study antigen-specific immunity. FVIII with an OVA peptide (FVIII-OVA) retained clotting activity and possessed the ability to activate CD4 T cells specific to OVA323–339 in vitro. When compared to FVIII alone, FVIII-OVA also exhibited a similar level of immunogenicity, suggesting that the presence of OVA323–339 does not substantially alter the anti-FVIII immune response. Intriguingly, while little CD4 T cell response could be observed following exposure to FVIII-OVA alone, inclusion of anti-FVIII antibodies, recently shown to favorably modulate anti-FVIII immune responses, significantly enhanced CD4 T cell activation following FVIII-OVA exposure. These results demonstrate that model antigens can be incorporated into a therapeutic protein to study antigen-specific responses and more specifically that the CD4 T cell response to FVIII-OVA can be augmented by pre-existing anti-FVIII antibodies.

Transfusion of red blood cells, platelets, and fresh frozen plasma in neonatal patients has not been well characterized in the literature, with guidelines varying greatly between institutions. However, anemia and thrombocytopenia are highly prevalent, especially in preterm neonates. When transfusing a neonatal patient, clinicians must take into consideration physiologic differences, gestational and postnatal age, congenital disorders, and maternal factors while weighing the risks and benefits of transfusion. This review of existing literature summarizes current evidence-based neonatal transfusion guidelines and highlights areas of current ongoing research and those in need of future studies.

Background: Chronic automated red cell exchange (RCE) is increasingly employed for sickle cell disease (SCD). There is a paucity of data on the incidence of RCE adverse events (AEs) and potential patient and procedural risk factors for AEs. Methods: A retrospective review of pediatric SCD patients receiving chronic RCE over 3 years was performed to determine the frequency of AEs and identify procedural and patient AE risk factors. AE incidence, AE rate, incidence rate ratios (IRRs), and relative risks (RRs) were calculated based on various procedural and patient characteristics by univariable (UV) and multivariable (MV) analyses. Results: In 38 patients receiving 760 procedures, there were 150 (19.7%) AEs, 36 (4.7%) were symptomatic AEs. AE rates were 20.2 [95% CI 17.2, 23.6] and 4.8 [95% CI 3.49, 6.70] per 100 person months for AEs and symptomatic AEs, respectively. AE incidences were: hypocalcemia (117; 15.4%), dizziness (22; 3.0%), hypotension (15; 2.0%), and nausea (14; 1.8%). Patients with baseline Hct ≥30% experienced more total AEs and symptomatic AEs. Patients with pre-procedure systolic BP <50th percentile, severe CNS vasculopathy, and non-SCA genotype (HbSC or Sβ+ thalassemia) exhibited more total AEs. IHD depletion was not associated with an increased incidence of AEs or symptomatic AEs. Conclusion: SCD patients with Hct ≥30%, systolic BP <50th percentile, severe CNS vasculopathy, and possibly non-SCA genotype may be at higher risk for RCE-related AEs. The effect of IHD on AE risk is likely minimal. Individualized AE risk assessment should be performed in all SCD patients undergoing chronic automated RCE.

Humoral immunity to factor VIII (FVIII) represents a significant challenge for the treatment of patients with hemophilia A. Current paradigms indicate that neutralizing antibodies against FVIII (inhibitors) occur through a classical CD4 T cell, germinal center (GC) dependent process. However, clinical observations suggest that the nature of the immune response to FVIII may differ between patients. While some patients produce persistent low or high inhibitor titers, others generate a transient response. Moreover, FVIII reactive memory B cells are only detectable in some patients with sustained inhibitor titers. The determinants regulating the type of immune response a patient develops, let alone how the immune response differs in these patients remains incompletely understood. One hypothesis is that polymorphisms within immunoregulatory genes alter the underlying immune response to FVIII, and thereby the inhibitor response. Consistent with this, studies report that inhibitor titers to FVIII differ in animals with the same F8 pathogenic variant but completely distinct backgrounds; though, how these genetic disparities affect the immune response to FVIII remains to be investigated. Given this, we sought to mechanistically dissect how genetics impact the underlying immune response to FVIII. In particular, as the risk of producing inhibitors is weakly associated with differences in HLA, we hypothesized that genetic factors other than HLA influence the immune response to FVIII and downstream inhibitor formation. Our data demonstrate that FVIII deficient mice encoding the same MHC and F8 variant produce disparate inhibitor titers, and that the type of inhibitor response formed associates with the ability to generate GCs. Interestingly, the formation of antibodies through a GC or non-GC pathway does not appear to be due to differences in CD4 T cell immunity, as the CD4 T cell response to an immunodominant epitope in FVIII was similar in these mice. These results indicate that genetics can impact the process by which inhibitors develop and may in part explain the apparent propensity of patients to form distinct inhibitor responses. Moreover, these data highlight an underappreciated immunological pathway of humoral immunity to FVIII and lay the groundwork for identification of biomarkers for the development of approaches to tolerize against FVIII.

Incompatible red blood cell (RBC) transfusion can result in life-threatening transfusion complications that can be challenging to manage in patients with transfusion-dependent anemia. However, not all incompatible RBC transfusions result in significant RBC removal. One factor that may regulate the outcome of incompatible RBC transfusion is the density of the incompatible antigen. Despite the potential influence of target antigen levels during incompatible RBC transfusion, a model system capable of defining the role of antigen density in this process has not been developed. In this study, we describe a novel model system of incompatible transfusion using donor mice that express different levels of the KEL antigen and recipients with varying anti-KEL antibody concentrations. Transfusion of KEL1 RBCs that express high or moderate KEL antigen levels results in rapid antibody-mediated RBC clearance. In contrast, relatively little RBC clearance was observed following the transfusion of KEL RBCs that express low KEL antigen levels. Intriguingly, unlike RBC clearance, loss of the KEL antigen from the transfused RBCs occurred at a similar rate regardless of the KEL antigen density following an incompatible transfusion. In addition to antigen density, anti-KEL antibody levels also regulated RBC removal and KEL antigen loss, suggesting that antigen density and antibody levels dictate incompatible RBC transfusion outcomes. These results demonstrate that antibody-induced antigen loss and RBC clearance can occur at distinct antigen density thresholds, providing important insight into factors that may dictate the outcome of an incompatible RBC transfusion.