Antibody-mediated antigen loss switches augmented immunity to antibody-mediated immunosuppression.

Antibodies against fetal red blood cell (RBC) antigens can cause hemolytic disease of the fetus and newborn (HDFN). Reductions in HDFN due to anti-RhD antibodies have been achieved through use of Rh immune globulin (RhIg), a polyclonal antibody preparation that causes antibody-mediated immunosuppression (AMIS), thereby preventing maternal immune responses against fetal RBCs. Despite the success of RhIg, it is only effective against 1 alloantigen. The lack of similar interventions that mitigate immune responses toward other RBC alloantigens reflects an incomplete understanding of AMIS mechanisms. AMIS has been previously attributed to rapid antibody-mediated RBC removal, resulting in B-cell ignorance of the RBC alloantigen. However, our data demonstrate that antibody-mediated RBC removal can enhance de novo alloimmunization. In contrast, inclusion of antibodies that possess the ability to rapidly remove the target antigen in the absence of detectable RBC clearance can convert an augmented antibody response to AMIS. These results suggest that the ability of antibodies to remove target antigens from the RBC surface can trigger AMIS in situations in which enhanced immunity may otherwise occur. In doing so, these results hold promise in identifying key antibody characteristics that can drive AMIS, thereby facilitating the design of AMIS approaches toward other RBC antigens to eliminate all forms of HDFN.

Storage differentially impacts alloimmunization to distinct red cell antigens following transfusion in mice.

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.

Neutralizing Antibodies Against Factor VIII Can Occur Through a Non-Germinal Center Pathway.

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.

Clodronate inhibits alloimmunization against distinct red blood cell alloantigens in mice.

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.

Antigen density dictates RBC clearance, but not antigen modulation, following incompatible RBC transfusion in mice.

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 KEL+ 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.

Fc Gamma Receptors and Complement Component 3 Facilitate Anti-fVIII Antibody Formation.

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.

Challenges in preventing and treating hemolytic complications associated with red blood cell transfusion.

Red blood cell (RBC) transfusion support represents a critical component of sickle cell disease (SCD) management. However, as with any therapeutic intervention, RBC transfusion is not without risk. Repeat exposure to allogeneic RBCs can result in the development of RBC alloantibodies that can make it difficult to find compatible RBCs for future transfusions and can directly increase the risk of developing acute or delayed hemolytic transfusion reactions, which can be further complicated by hyperhemolysis. Several prophylactic and treatment strategies have been employed in an effort to reduce or prevent hemolytic transfusion reactions. However, conflicting data exist regarding the efficacy of many of these approaches. We will explore the challenges associated with predicting, preventing and treating different types of hemolytic transfusion reactions in patients with SCD in addition to describing future strategies that may aid in the management of the complex transfusion requirements of SCD patients.

Marginal Zone B Cells Induce Alloantibody Formation Following RBC Transfusion.

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.

Antibody-mediated immune suppression by antigen modulation is antigen-specific.

Alloantibodies developing after exposure to red blood cell (RBC) alloantigens can complicate pregnancy and transfusion therapy. The only method currently available to actively inhibit RBC alloantibody formation is administration of antigen-specific antibodies, a phenomenon termed antibody-mediated immune suppression (AMIS). A well-known example of AMIS is RhD immune globulin prophylaxis to prevent anti-D formation in RhD- individuals. However, whether AMIS is specific or impacts alloimmunization to other antigens on the same RBC remains unclear. To evaluate the specificity of AMIS, we passively immunized antigen-negative recipients with anti-KEL or anti-hen egg lysozyme (HEL) antibodies, followed by transfusion of murine RBC expressing both the HEL-ovalbumin-Duffy (HOD) and human KEL antigens (HOD × KEL RBC). Significant immunoglobulin G deposition on transfused HOD × KEL RBC occurred in all passively immunized recipients. Complement deposition and antigen modulation of the KEL antigen occurred on transfused RBC only in anti-KEL-treated recipients, whereas HEL antigen levels decreased only in the presence of anti-HEL antibodies. Western blot analysis confirmed the specificity of antigen loss, which was not attributable to RBC endocytosis and appears distinct for the 2 antigens. Specifically, removal of KEL was attenuated by clodronate treatment, whereas loss of HEL was unaffected by clodronate in vivo but sensitive to protease treatment in vitro. Antigen-specific modulation correlated with antigen-specific AMIS, with anti-KEL treated recipients forming antibodies to the HOD antigen and anti-HEL-treated recipients developing antibodies to the KEL antigen. Together, these results demonstrate that passively administered antibodies can selectively inhibit the immune response to a specific antigen.

Complement serves as a switch between CD4+ T cell-independent and -dependent RBC antibody responses.

RBC alloimmunization represents a significant immunological challenge for patients requiring lifelong transfusion support. The majority of clinically relevant non-ABO(H) blood group antigens have been thought to drive antibody formation through T cell-dependent immune pathways. Thus, we initially sought to define the role of CD4+ T cells in formation of alloantibodies to KEL, one of the leading causes of hemolytic transfusion reactions. Unexpectedly, our findings demonstrated that KEL RBCs actually possess the ability to induce antibody formation independent of CD4+ T cells or complement component 3 (C3), two common regulators of antibody formation. However, despite the ability of KEL RBCs to induce anti-KEL antibodies in the absence of complement, removal of C3 or complement receptors 1 and 2 (CR1/2) rendered recipients completely reliant on CD4+ T cells for IgG anti-KEL antibody formation. Together, these findings suggest that C3 may serve as a novel molecular switch that regulates the type of immunological pathway engaged following RBC transfusion.

Hematologic complications in a patient with Glycine soja polyagglutination following fresh frozen plasma transfusion.

CONCLUSIONS: Polyagglutination is a rare and underdiagnosed condition, characterized by agglutination of red blood cells(RBCs) with almost all ABO-compatible adult sera. Polyagglutination can occur when a cryptantigen is exposed on RBCs via microbial enzyme activity. Becausenearly all adults naturally produce antibodies against cryptantigens, transfusion of plasma can cause unexpected hemolysis and hematologic complications, such as thrombocytopenia and disseminated intravascular coagulation, in patients whose cryptantigens are exposed. We report a case of Glycine soja polyagglutination occurring in a 60-year-old African-American man with disseminated methicillin-resistant Staphylococcus aureus (MRSA) infection. Prior to transfusion, the patient developed severe anemia of unknown etiology. Following transfusion of 3 units of fresh frozen plasma (FFP), his RBC count could not be determined for 24 days because of RBC agglutination in his blood sample. In addition, the FFP transfusion correlated with the rapid development of severe, transfusionrefractory thrombocytopenia and anemia. The perplexed clinical team consulted the blood bank. A direct antiglobulin test demonstrated 1+ mixed-field reactivity with both monoclonal anti-IgG and anti-C3d. Lectin panel testing showed reactivity with only Glycine soja, confirming the condition. Subsequently, plasma components were avoided, and RBC and platelet (PLT) components were washed prior to transfusion. After a 44-day hospitalization involving the transfusion of 22 units of RBCs and 13 units of PLTs, the patient was discharged to a long-term care facility. The patient's confounding hematologic complications can best be explained by polyagglutination, which developed secondary to the severe MRSA infection. The FFP transfusion likely passively transferred antibodies that bound to the patient's RBC cryptantigens, leading to RBC agglutination and anemia. The development of severe thrombocytopenia may be related to cryptantigen exposure on the patient's PLTs. Although difficult to identify, polyagglutination needs to be recognized to appropriately manage hemotherapy. The purpose of this case study is to report hematologic complications following FFP transfusion in a patient with Glycine soja polyagglutination, a rarely described condition.

A suspected delayed hemolytic transfusion reaction mediated by anti-Joa.

CONCLUSIONS: A 32-year-old African-American woman with a history of sickle cell disease presented for surgical evaluation of left total hip arthroplasty due to avascular necrosis of the femoral head. In anticipation of a complex orthopedic procedure, pre-surgical blood work was ordered. The patient's Fenwal blood sample typed as group O, D+. Although the patient had a history of anti-Fya, the antibody identification was inconclusive, so the workup was sent to a reference laboratory. The patient was last transfused with red blood cells (RBCs) 2 years earlier, but had no history of transfusion reactions. Due to surgery, the patient's hemoglobin (Hb) decreased from 10.2 g/dL (preoperative) to 8.6 g/dL (postoperative). One unit of weakly crossmatch-incompatible Fy(a-), C-, E-, K-, and sickle cell hemoglobin S (HbS)-negative RBCs was transfused without incident, and the patient was discharged. Several days later, the reference lab reported two new specificities, anti-Joa and anti-Jkb. Fortunately, the transfused RBC unit was Jk(b-). Therefore, the crossmatch incompatibility was attributed to anti-Joa, which targets a high-prevalence antigen found in 100 percent of most populations. Two weeks after discharge, the patient returned in sickle vaso-occlusive pain crisis. The patient was clinically stable, but her Hb was 6.7 g/dL. One unit of Fy(a-), Jk(b-), C-, E-, K-, HbS- RBCs, which was weakly crossmatch-incompatible, was transfused. The following day, her Hb was unchanged, lactic acid dehydrogenase increased from 951 to 2464 U/L, potassium increased from 3.7 to 4.6 mEq/L, creatinine increased from 0.60 to 0.98 mg/dL, and the patient developed a 38.4°C fever. These findings are consistent with a delayed hemolytic transfusion reaction (DHTR), mediated by anti-Joa, occurring 2 weeks after the first RBC transfusion. Further care could not be provided because the patient left the hospital against medical advice. The purpose of this case study is to report findings consistent with a DHTR attributed to anti-Joa, an antibody with relatively unknown clinical significance.

Splenic sarcoidosis without focal nodularity: a case of 1,25-dihydroxyvitamin D-mediated hypercalcemia localized with FDG PET/CT.

OBJECTIVE: To report an uncommon cause of 1,25-dihydroxyvitamin D (1,25[OH]2D)-mediated hypercalcemia associated with splenic sarcoidosis and illustrate the evaluation and potential role of fluorodeoxyglucose (FDG) positron emission tomography (PET)/computed tomography (CT) in such patients. METHODS: We present detailed clinical features, laboratory results, imaging results, and pathology results for this rare entity, discuss evaluation and management options, and review previous literature. RESULTS: A 65-year-old male presented with symptomatic hypercalcemia, with a serum calcium level of 14.1 mg/dL 3 months after being initiated on ergocalciferol for vitamin D deficiency. He was found to have a suppressed parathyroid hormone level, normal 25-hydroxyvitamin D (25[OH]D) level, and elevated 1,25(OH)2D level. Extensive evaluation did not yield a definitive diagnosis. His calcium levels normalized and symptoms resolved on prednisone then recurred when prednisone was discontinued. FDG PET/CT showed intense uptake in the spleen. Splenectomy was performed, which resulted in resolution of hypercalcemia and yielded a diagnosis of splenic sarcoidosis. CONCLUSION: Splenic sarcoidosis causing hypercalcemia has been rarely reported. Our case is unique in that the spleen lacked typical focal nodularity on cross-sectional CT imaging, which is expected in sarcoid involvement of the spleen. Our case adds to an emerging literature documenting the potential value of FDG PET/CT in localizing otherwise occult 1,25(OH)2D-mediated hypercalcemia.