RESUMO
RBC alloimmunization remains a significant barrier to ongoing transfusion therapy leading to morbidity, and in extreme cases mortality, due to delayed or insufficient units of compatible RBCs. In addition, the monitoring and characterization of alloantibodies, often with multiple specificities in a single patient, consumes substantial health care resources. Extended phenotypic matching has mitigated, but not eliminated, RBC alloimmunization and is only logistically available for specialized populations. Thus, RBC alloimmunization remains a substantial problem. In recent decades it has become clear that mechanisms of RBC alloimmunization are distinct from other antigens and lack of mechanistic understanding likely contributes to the fact that there are no approved interventions to prevent RBC alloimmunization from transfusion. The combination of human studies and murine modeling have identified several key factors in RBC alloimmunization. In both humans and mice, immunogenicity is a function of alloantigen copy number on RBCs. Murine studies have further shown that copy number not only changes rates of immunization but the mechanisms of antibody formation. This review summarizes the current understanding of quantitative and qualitative effects of alloantigen copy number on RBC alloimmunization.
Assuntos
Variações do Número de Cópias de DNA , Isoantígenos , Humanos , Camundongos , Animais , Eritrócitos , Transfusão de Sangue , IsoanticorposRESUMO
Antibodies against red blood cell (RBC) alloantigens can increase morbidity and mortality among transfusion recipients. However, alloimmunization rates can vary dramatically, as some patients never generate alloantibodies after transfusion, whereas others not only become alloimmunized but may also be prone to generating additional alloantibodies after subsequent transfusion. Previous studies suggested that CD4 T-cell responses that drive alloantibody formation recognize the same alloantigen engaged by B cells. However, because RBCs express numerous antigens, both internally and externally, it is possible that CD4 T-cell responses directed against intracellular antigens may facilitate subsequent alloimmunization against a surface RBC antigen. Here, we show that B cells can acquire intracellular antigens from RBCs. Using a mouse model of donor RBCs expressing 2 distinct alloantigens, we demonstrate that immune priming to an intracellular antigen, which would not be detected by any currently used RBC compatibility assays, can directly influence alloantibody formation after exposure to a subsequent distinct surface RBC alloantigen. These findings suggest a previously underappreciated mechanism whereby transfusion recipient responders may exhibit an increased rate of alloimmunization because of prior immune priming toward intracellular antigens.
Assuntos
Transfusão de Eritrócitos , Isoanticorpos , Transfusão de Eritrócitos/efeitos adversos , Eritrócitos , Antígenos , Isoantígenos , ImunizaçãoRESUMO
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.
Assuntos
Antígenos , Transfusão de Eritrócitos , Camundongos , Animais , Transfusão de Eritrócitos/efeitos adversos , Eritrócitos , Isoantígenos , Isoanticorpos , Imunoglobulina GRESUMO
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.
RESUMO
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.
Assuntos
Hemofilia A , Hemostáticos , Animais , Anticorpos Neutralizantes , Fator VIII , Centro Germinativo/metabolismo , Humanos , CamundongosRESUMO
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.
Assuntos
Ácido Clodrônico , Isoantígenos , Animais , Ácido Clodrônico/farmacologia , Eritrócitos , Humanos , Imunoglobulina G , Imunoglobulina M , Isoanticorpos , CamundongosRESUMO
Red blood cell (RBC) transfusions can result in alloimmunization toward RBC alloantigens that can increase the probability of complications following subsequent transfusion. An improved understanding of the immune mechanisms that underlie RBC alloimmunization is critical if future strategies capable of preventing or even reducing this process are to be realized. Using the HOD (hen egg lysozyme [HEL] and ovalbumin [OVA] fused with the human RBC antigen Duffy) model system, we aimed to identify initiating immune factors that may govern early anti-HOD alloantibody formation. Our findings demonstrate that HOD RBCs continuously localize to the marginal sinus following transfusion, where they colocalize with marginal zone (MZ) B cells. Depletion of MZ B cells inhibited immunoglobulin M (IgM) and IgG anti-HOD antibody formation, whereas CD4 T-cell depletion only prevented IgG anti-HOD antibody development. HOD-specific CD4 T cells displayed similar proliferation and activation following transfusion of HOD RBCs into wild-type or MZ B-cell-deficient recipients, suggesting that IgG formation is not dependent on MZ B-cell-mediated CD4 T-cell activation. Moreover, depletion of follicular B cells failed to substantially impact the anti-HOD antibody response, and no increase in antigen-specific germinal center B cells was detected following HOD RBC transfusion, suggesting that antibody formation is not dependent on the splenic follicle. Despite this, anti-HOD antibodies persisted for several months following HOD RBC transfusion. Overall, these data suggest that MZ B cells can initiate and then contribute to RBC alloantibody formation, highlighting a unique immune pathway that can be engaged following RBC transfusion.
Assuntos
Linfócitos B/imunologia , Sistema do Grupo Sanguíneo Duffy/imunologia , Transfusão de Eritrócitos , Centro Germinativo/imunologia , Isoanticorpos/imunologia , Isoantígenos/imunologia , Receptores de Superfície Celular/imunologia , Animais , Sistema do Grupo Sanguíneo Duffy/genética , Feminino , Humanos , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Imunoglobulina M/genética , Imunoglobulina M/imunologia , Isoanticorpos/genética , Isoantígenos/genética , Camundongos , Camundongos Knockout , Receptores de Superfície Celular/genéticaRESUMO
Neutralizing antibodies to factor VIII (fVIII), referred to as "inhibitors," remain the most challenging complication post-fVIII replacement therapy. Preclinical development of novel fVIII products involves studies incorporating hemophilia A (HA) and wild-type animal models. Though immunogenicity is a critical aspect of preclinical pharmacology studies, gene therapy studies tend to focus on fVIII expression levels without major consideration for immunogenicity. Therefore, little clarity exists on whether preclinical testing can be predictive of clinical immunogenicity risk. Despite this, but perhaps due to the potential for transformative benefits, clinical gene therapy trials have progressed rapidly. In more than two decades, no inhibitors have been observed. However, all trials are conducted in previously treated patients without a history of inhibitors. The current review thus focuses on our understanding of preclinical immunogenicity for HA gene therapy candidates and the potential indication for inhibitor treatment, with a focus on product- and platform-specific determinants, including fVIII transgene sequence composition and tissue/vector biodistribution. Currently, the two leading clinical gene therapy vectors are adeno-associated viral (AAV) and lentiviral (LV) vectors. For HA applications, AAV vectors are liver-tropic and employ synthetic, high-expressing, liver-specific promoters. Factors including vector serotype and biodistribution, transcriptional regulatory elements, transgene sequence, dosing, liver immunoprivilege, and host immune status may contribute to tipping the scale between immunogenicity and tolerance. Many of these factors can also be important in delivery of LV-fVIII gene therapy, especially when delivered intravenously for liver-directed fVIII expression. However, ex vivo LV-fVIII targeting and transplantation of hematopoietic stem and progenitor cells (HSPC) has been demonstrated to achieve durable and curative fVIII production without inhibitor development in preclinical models. A critical variable appears to be pre-transplantation conditioning regimens that suppress and/or ablate T cells. Additionally, we and others have demonstrated the potential of LV-fVIII HSPC and liver-directed AAV-fVIII gene therapy to eradicate pre-existing inhibitors in murine and canine models of HA, respectively. Future preclinical studies will be essential to elucidate immune mechanism(s) at play in the context of gene therapy for HA, as well as strategies for preventing adverse immune responses and promoting immune tolerance even in the setting of pre-existing inhibitors.
Assuntos
Anticorpos Neutralizantes/metabolismo , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/prevenção & controle , Fator VIII/imunologia , Vetores Genéticos/genética , Hemofilia A/terapia , Animais , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Fator VIII/genética , Fator VIII/uso terapêutico , Terapia Genética , HumanosRESUMO
BACKGROUND: Anti-RhD administration can prevent de novo anti-RhD formation following RhD+ red blood cell (RBC) exposure, termed antibody-mediated immunosuppression (AMIS). Recent studies suggest that AMIS may occur through target antigen alterations, known as antigen modulation. However, studies suggest that AMIS may occur independent of antigen modulation. In particular, AMIS to RBCs that transgenically express the fusion hen egg lysozyme-ovalbumin-Duffy (HOD) antigen have been shown to occur independent of activating Fcγ receptors (FcγRs) thought to be required for antigen modulation. Therefore, we sought to determine the mechanism behind AMIS following HOD RBC exposure. STUDY DESIGN AND METHODS: Following transfer of HOD RBCs into wild-type or FcγR-chain knockout recipients in the presence or absence of monoclonal anti-hen egg lysozyme (HEL) antibody, individually or in combination, HOD antigen levels and anti-HOD antibody formation were examined. RESULTS: Our results demonstrate that anti-HEL antibodies individually or in combination suppressed anti-HOD IgM, which correlated with the rate of detectable decrease in HEL on HOD RBCs. Furthermore, exposure to anti-HEL antibodies alone or in combination equally suppressed anti-HOD IgG formation. Unexpectedly, combination or individual anti-HEL antibodies induced AMIS and antigen modulation in an FcγR-independent manner. Pre-exposure of HOD RBCs to anti-HEL antibodies reduced antigen levels and suppressed anti-HOD antibody formation following HOD RBC exposure. CONCLUSION: These results suggest that antibody-mediated antigen modulation may reflect a mechanism of AMIS that can occur independent of activating FcγRs and may provide a surrogate to identify antibodies capable of inducing AMIS against different RBC antigens.
Assuntos
Eritrócitos/metabolismo , Animais , Anticorpos Monoclonais/metabolismo , Biotinilação , Western Blotting , Feminino , Citometria de Fluxo , Terapia de Imunossupressão , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de IgG/metabolismoRESUMO
BACKGROUND: Factors influencing the development of alloantibodies against blood group antigens on transfused red blood cells are poorly defined. We hypothesised that transfused platelets may act as a danger signal to recipients and affect humoral immune responses to transfused red blood cells. MATERIALS AND METHODS: Platelet-rich plasma prepared from wild-type C57BL/6 or CD40L knock-out donors was transfused into wild-type or CD40L knock-out recipients. Leucoreduced red blood cells from transgenic donors expressing high levels of the human KEL glycoprotein in an erythrocyte-specific manner (KELhi donors) were transfused after the platelets, and anti-KEL responses were measured longitudinally. In some experiments, recipients were treated with poly (I:C), monoclonal CD40L-blocking antibody, or CD4-depleting antibody prior to transfusion. RESULTS: Transfusion of wild-type C57BL/6 platelets or treatment with poly (I:C) prior to KELhi red blood cell transfusion led to an anti-KEL alloimmune response in wild-type recipients. Transfusion of platelets from wild-type but not CD40L knock-out donors prior to KELhi red blood cell transfusion led to an IgG anti-KEL alloimmune response in CD40L knock-out recipients; unexpectedly, transfusion of platelets from CD40L knock-out donors prior to KELhi red blood cell transfusion led to a robust anti-KEL alloimmune response in wild-type recipients. Recipient treatment with MR1 CD40L-blocking antibody or CD4-depleting antibody prevented KEL alloimmunisation altogether. DISCUSSION: Transfused platelets serve as an adjuvant in this T-dependent murine model of anti-KEL red blood cell alloimmunisation, with CD40/CD40L interactions being involved to some degree but with additional mechanisms also playing a role. These findings raise questions about the role that transfused or endogenous platelets may play in other innate/adaptive immune responses.
Assuntos
Plaquetas/imunologia , Transfusão de Eritrócitos , Eritrócitos/imunologia , Glicoproteínas de Membrana/imunologia , Metaloendopeptidases/imunologia , Transfusão de Plaquetas , Animais , Modelos Animais de Doenças , Humanos , Imunidade Humoral , Isoanticorpos/imunologia , Camundongos Endogâmicos C57BLRESUMO
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.
Assuntos
Formação de Anticorpos/imunologia , Linfócitos B/imunologia , Eritrócitos/imunologia , Isoanticorpos/imunologia , Animais , Antígenos/imunologia , Linfócitos T CD4-Positivos/imunologia , Transfusão de Eritrócitos/métodos , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Baço/imunologiaRESUMO
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.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Complemento C3/imunologia , Eritrócitos/imunologia , Animais , Formação de Anticorpos , Complemento C5/imunologia , Transfusão de Eritrócitos , Imunidade Humoral , Isoanticorpos/imunologia , Glicoproteínas de Membrana/imunologia , Metaloendopeptidases/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Receptores de Complemento 3b/imunologiaRESUMO
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.
Assuntos
Anticorpos/imunologia , Modulação Antigênica , Isoantígenos/imunologia , Animais , Anticorpos/administração & dosagem , Plaquetas , Proteínas do Sistema Complemento/metabolismo , Transfusão de Eritrócitos , Eritrócitos/metabolismo , Proteínas de Homeodomínio/imunologia , Sistema do Grupo Sanguíneo de Kell , Glicoproteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos TransgênicosRESUMO
Red blood cell (RBC) alloimmunization can make it difficult to procure compatible RBCs for future transfusion, directly leading to increased morbidity and mortality in transfusion-dependent patients. However, the factors that regulate RBC alloimmunization remain incompletely understood. As complement has been shown to serve as a key adjuvant in the development of antibody (Ab) responses against microbes, we examined the impact of complement on RBC alloimmunization. In contrast to the impact of complement component 3 (C3) in the development of an immune response following microbial exposure, transfusion of C3 knockout (C3 KO) recipients with RBCs expressing KEL (KEL RBCs) actually resulted in an enhanced anti-KEL Ab response. The impact of C3 appeared to be specific to KEL, as transfusion of RBCs bearing another model antigen, the chimeric HOD antigen (hen egg lysozyme, ovalbumin and Duffy), into C3 KO recipients failed to result in a similar increase in Ab formation. KEL RBCs experienced enhanced C3 deposition and loss of detectable target antigen over time when compared to HOD RBCs, suggesting that C3 may inhibit Ab formation by impacting the accessibility of the target KEL antigen. Loss of detectable KEL on the RBC surface did not reflect antigen masking by C3, but instead appeared to result from actual removal of the KEL antigen, as western blot analysis demonstrated complete loss of detectable KEL protein. Consistent with this, exposure of wild-type B6 or C3 KO recipients to KEL RBCs with reduced levels of detectable KEL antigen resulted in a significantly reduced anti-KEL Ab response. These results suggest that C3 possesses a unique ability to actually suppress Ab formation following transfusion by reducing the availability of the target antigen on the RBC surface.
Assuntos
Antígenos/imunologia , Complemento C3/imunologia , Transfusão de Eritrócitos , Eritrócitos/imunologia , Animais , Formação de Anticorpos , Complemento C3/genética , Feminino , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
Individuals that become immunized to red blood cell (RBC) alloantigens can experience an increased rate of antibody formation to additional RBC alloantigens following subsequent transfusion. Despite this, how an immune response to one RBC immunogen may impact subsequent alloimmunization to a completely different RBC alloantigen remains unknown. Our studies demonstrate that Kell blood group antigen (KEL) RBC transfusion in the presence of inflammation induced by poly (I:C) (PIC) not only enhances anti-KEL antibody production through a CD4+ T-cell-dependent process but also directly facilitates anti-HOD antibody formation following subsequent exposure to the disparate HOD (hen egg lysozyme, ovalbumin, fused to human blood group antigen Duffy b) antigen. PIC/KEL priming of the anti-HOD antibody response required that RBCs express both the KEL and HOD antigens (HOD × KEL RBCs), as transfusion of HOD RBCs plus KEL RBCs or HOD RBCs alone failed to impact anti-HOD antibody formation in recipients previously primed with PIC/KEL. Transfer of CD4+ T cells from PIC/KEL-primed recipients directly facilitated anti-HOD antibody formation following (HOD × KEL) RBC transfusion. RBC alloantigen priming was not limited to PIC/KEL enhancement of anti-HOD alloantibody formation, as HOD-reactive CD4+ T cells enhanced anti-glycophorin A (anti-GPA) antibody formation in the absence of inflammation following transfusion of RBCs coexpressing GPA and HOD. These results demonstrate that immune priming to one RBC alloantigen can directly enhance a humoral response to a completely different RBC alloantigen, providing a potential explanation for why alloantibody responders may exhibit increased immune responsiveness to additional RBC alloantigens following subsequent transfusion.
Assuntos
Formação de Anticorpos/imunologia , Eritrócitos/imunologia , Imunidade Humoral , Isoantígenos/imunologia , Animais , Linfócitos T CD4-Positivos/imunologia , Transfusão de Eritrócitos , Isoanticorpos/imunologia , CamundongosRESUMO
Although factor VIII (FVIII) replacement therapy can be lifesaving for patients with hemophilia A, neutralizing alloantibodies to FVIII, known as inhibitors, develop in a significant number of patients and actively block FVIII activity, making bleeding difficult to control and prevent. Although a variety of downstream immune factors likely regulate inhibitor formation, the identification and subsequent targeting of key initiators in inhibitor development may provide an attractive approach to prevent inhibitor formation before amplification of the FVIII immune response occurs. As the initial steps in FVIII inhibitor development remain incompletely understood, we sought to define early regulators of FVIII inhibitor formation. Our results demonstrate that FVIII localizes in the marginal sinus of the spleen of FVIII-deficient mice shortly after injection, with significant colocalization with marginal zone (MZ) B cells. FVIII not only colocalizes with MZ B cells, but specific removal of MZ B cells also completely prevented inhibitor development following FVIII infusion. Subsequent rechallenge with FVIII following MZ B-cell reconstitution resulted in a primary antibody response, demonstrating that MZ B-cell depletion did not result in FVIII tolerance. Although recipient exposure to the viral-like adjuvant polyinosinic:polycytidylic acid enhanced anti-FVIII antibody formation, MZ B-cell depletion continued to display similar effectiveness in preventing inhibitor formation following FVIII infusion in this inflammatory setting. These data strongly suggest that MZ B cells play a critical role in initiating FVIII inhibitor formation and suggest a potential strategy to prevent anti-FVIII alloantibody formation in patients with hemophilia A.
Assuntos
Linfócitos B/imunologia , Fator VIII/imunologia , Hemofilia A/sangue , Hemofilia A/imunologia , Isoanticorpos/sangue , Isoanticorpos/imunologia , Animais , Linfócitos B/metabolismo , Modelos Animais de Doenças , Fator VIII/genética , Fator VIII/metabolismo , Feminino , Hemofilia A/genética , Depleção Linfocítica , Masculino , Camundongos , Camundongos Knockout , Transporte Proteico , Baço/imunologia , Baço/metabolismoRESUMO
Approximately 3-10% of human red blood cell (RBC) transfusion recipients form alloantibodies to non-self, non-ABO blood group antigens expressed on donor RBCs, with these alloantibodies having the potential to be clinically significant in transfusion and pregnancy settings. However, the majority of transfused individuals never form detectable alloantibodies. Expanding upon observations that children initially transfused with RBCs at a young age are less likely to form alloantibodies throughout their lives, we hypothesized that "non-responders" may not only be ignorant of antigens on RBCs but instead tolerized. We investigated this question in a reductionist murine model, in which transgenic donors express the human glycophorin A (hGPA) antigen in an RBC-specific manner. Although wild-type mice treated with poly IC and transfused with hGPA RBCs generated robust anti-hGPA IgG alloantibodies that led to rapid clearance of incompatible RBCs, those transfused in the absence of an adjuvant failed to become alloimmunized. Animals depleted of CD4+ cells or treated with CD40L blockade prior to initial hGPA RBC exposure, in the presence of poly IC, failed to generate detectable anti-hGPA IgG alloantibodies. These non-responders to a primary transfusion remained unable to generate anti-hGPA IgG alloantibodies upon secondary hGPA exposure and did not prematurely clear transfused hGPA RBCs even after their CD4 cells had returned or their CD40L blockade had resolved. This observed tolerance was antigen (hGPA) specific, as robust IgG responses to transfused RBCs expressing a third-party antigen occurred in all studied groups. Experiments completed in an RBC alloimmunization model that allowed evaluation of antigen-specific CD4+ T-cells (HOD (hen egg lysozyme, ovalbumin, and human duffyb)) demonstrated that CD40L blockade prevented the expansion of ovalbumin 323-339 specific T-cells after HOD RBC transfusion and also prevented germinal center formation. Taken together, our data suggest that recipients may indeed become tolerized to antigens expressed on RBCs, with the recipient's immune status upon initial RBC exposure dictating future responses. Although questions surrounding mechanism(s) and sustainability of tolerance remain, these data lay the groundwork for future work investigating RBC immunity versus tolerance in reductionist models and in humans.