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1.
Haematologica ; 104(5): 1046-1054, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30545924

RESUMO

Hemophilia A is a rare hemorrhagic disorder caused by the lack of functional pro-coagulant factor VIII. Factor VIII replacement therapy in patients with severe hemophilia A results in the development of inhibitory anti-factor VIII IgG in up to 30% of cases. To date, immune tolerance induction, with daily injection of large amounts of factor VIII, is the only strategy to eradicate factor VIII inhibitors. This strategy is, however, efficient in only 60-80% of patients. We investigated whether blocking B-cell receptor signaling upon inhibition of Bruton tyrosine kinase prevents anti-factor VIII immune responses in a mouse model of severe hemophilia A. Factor VIII-naïve and factor VIII-sensitized factor VIII-deficient mice were fed with the selective inhibitor of Bruton tyrosine kinase, (R)-5-amino-1-(1-cyanopiperidin-3-yl)-3-(4-[2,4-difluorophenoxyl] phenyl)-1H pyrazole-4-carboxamide (PF-06250112), to inhibit B-cell receptor signaling prior to challenge with exogenous factor VIII. The consequences on the anti-factor VIII immune response were studied. Inhibition of Bruton tyrosine kinase during the primary anti-factor VIII immune response in factor VIII-naïve mice did not prevent the development of inhibitory anti-factor VIII IgG. In contrast, the anti-factor VIII memory B-cell response was consistently reduced upon treatment of factor VIII-sensitized mice with the Bruton tyrosine kinase inhibitor. The Bruton tyrosine kinase inhibitor reduced the differentiation of memory B cells ex vivo and in vivo following adoptive transfer to factor VIII-naïve animals. Taken together, our data identify inhibition of Bruton tyrosine kinase using PF-06250112 as a strategy to limit the reactivation of factor VIII-specific memory B cells upon re-challenge with therapeutic factor VIII.


Assuntos
Tirosina Quinase da Agamaglobulinemia/antagonistas & inibidores , Linfócitos B/imunologia , Modelos Animais de Doenças , Fator VIII/fisiologia , Hemofilia A/imunologia , Memória Imunológica/imunologia , Piperidinas/farmacologia , Pirazóis/farmacologia , Animais , Formação de Anticorpos , Linfócitos B/efeitos dos fármacos , Linfócitos B/metabolismo , Fator VIII/administração & dosagem , Fator VIII/antagonistas & inibidores , Hemofilia A/tratamento farmacológico , Hemofilia A/metabolismo , Tolerância Imunológica/efeitos dos fármacos , Tolerância Imunológica/imunologia , Imunoglobulina G/efeitos dos fármacos , Imunoglobulina G/imunologia , Memória Imunológica/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout
2.
Mol Immunol ; 97: 1-7, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29525557

RESUMO

Cobra venom factor (CVF) is the complement-activating protein in cobra venom. Humanized CVF (hCVF) is a human C3 derivative where the C-terminal 168 amino acid residues were replaced with the homologous sequence from CVF. hCVF has been shown in multiple models of disease with complement pathology to be a promising therapeutic agent, with no observed adverse effects. Here we describe the antibody response to hCVF in two different strains of mice. hCVF was able to repeatedly decomplement the mice after four injections in weekly intervals, demonstrating the absence of a neutralizing antibody response. In contrast, natural CVF caused decomplementation in all mice only after the first administration. After two additional administrations of natural CVF, decomplementation was inconsistent and varied tremendously from mouse to mouse. After the fourth administration, natural CVF was essentially unable to deplete complement, consistent with the known generation of a neutralizing antibody response. We also analyzed the IgG antibody response to hCVF. There was great variation, with approximately one quarter of the mice exhibiting non-detectable levels of anti-hCVF IgG, and another quarter very low levels. The levels of anti-hCVF IgG did not correlate with the levels of remaining C3. The anti-hCVF antibodies cross-reacted with natural CVF, recombinant CVF, and human C3. Whereas overall the level of anti-hCVF IgG cross-reacting with human C3 was lower compared to rCVF or nCVF, mice with higher levels of anti-hCVF IgG exhibited higher binding to CVF and human C3, excluding the possibility that higher antibody levels reflect preferential immunogenicity of CVF-specific or human C3-specific epitopes.


Assuntos
Anticorpos Neutralizantes/metabolismo , Formação de Anticorpos , Venenos Elapídicos/imunologia , Proteínas Recombinantes de Fusão/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Células CHO , Células Cultivadas , Cricetinae , Cricetulus , Drosophila melanogaster , Venenos Elapídicos/química , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Recombinantes de Fusão/química
3.
Haematologica ; 103(2): 351-360, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29146705

RESUMO

Development of neutralizing antibodies against therapeutic Factor VIII (FVIII) is the most serious complication of the treatment of hemophilia A. There is growing evidence to show the multifactorial origin of the anti-FVIII immune response, combining both genetic and environmental factors. While a role for the complement system on innate as well as adaptive immunity has been documented, the implication of complement activation on the onset of the anti-FVIII immune response is unknown. Here, using in vitro assays for FVIII endocytosis by human monocyte-derived dendritic cells and presentation to T cells, as well as in vivo complement depletion in FVIII-deficient mice, we show a novel role for complement C3 in enhancing the immune response against therapeutic FVIII. In vitro, complement C3 and its cleavage product C3b enhanced FVIII endocytosis by dendritic cells and presentation to a FVIII-specific CD4+ T-cell hybridoma. The C1 domain of FVIII had previously been shown to play an important role in FVIII endocytosis, and alanine substitutions of the K2092, F2093 and R2090 C1 residues drastically reduce FVIII uptake in vitro Interestingly, complement activation rescued the endocytosis of the FVIII C1 domain triple mutant. In a mouse model of severe hemophilia A, transient complement C3 depletion by humanized cobra venom factor, which does not generate anaphylatoxin C5a, significantly reduced the primary anti-FVIII immune response, but did not affect anti-FVIII recall immune responses. Taken together, our results suggest an important adjuvant role for the complement cascade in the initiation of the immune response to therapeutic FVIII.


Assuntos
Anticorpos Neutralizantes/imunologia , Complemento C3/farmacologia , Fator VIII/imunologia , Animais , Apresentação de Antígeno/imunologia , Ativação do Complemento , Células Dendríticas/fisiologia , Endocitose/efeitos dos fármacos , Humanos , Imunidade/efeitos dos fármacos , Camundongos
4.
Haematologica ; 102(11): 1833-1841, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28751567

RESUMO

Acquired thrombotic thrombocytopenic purpura is a rare and severe disease characterized by auto-antibodies directed against "A Disintegrin And Metalloproteinase with Thrombospondin type 1 repeats, 13th member" (ADAMTS13), a plasma protein involved in hemostasis. Involvement of CD4+ T cells in the pathogenesis of the disease is suggested by the IgG isotype of the antibodies. However, the nature of the CD4+ T-cell epitopes remains poorly characterized. Here, we determined the HLA-DR-restricted CD4+ T-cell epitopes of ADAMTS13. Candidate T-cell epitopes were predicted in silico and binding affinities were confirmed in competitive enzyme-linked immunosorbent assays. ADAMTS13-reactive CD4+ T-cell hybridomas were generated following immunization of HLA-DR1 transgenic mice (Sure-L1 strain) and used to screen the candidate epitopes. We identified the ADAMTS131239-1253 peptide as the single immunodominant HLA-DR1-restricted CD4+ T-cell epitope. This peptide is located in the CUB2 domain of ADAMTS13. It was processed by dendritic cells, stimulated CD4+ T cells from Sure-L1 mice and was recognized by CD4+ T cells from an HLA-DR1-positive patient with acute thrombotic thrombocytopenic purpura. Interestingly, the ADAMTS131239-1253 peptide demonstrated promiscuity towards HLA-DR11 and HLA-DR15. Our work paves the way towards the characterization of the ADAMTS13-specific CD4+ T-cell response in patients with thrombotic thrombocytopenic purpura using ADAMTS131239-1253-loaded HLA-DR tetramers.


Assuntos
Proteína ADAMTS13/imunologia , Linfócitos T CD4-Positivos/imunologia , Epitopos de Linfócito T/imunologia , Antígeno HLA-DR1/imunologia , Epitopos Imunodominantes/imunologia , Fragmentos de Peptídeos/imunologia , Proteína ADAMTS13/química , Alelos , Sequência de Aminoácidos , Animais , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Epitopos de Linfócito T/química , Antígeno HLA-DR1/química , Antígeno HLA-DR1/metabolismo , Humanos , Imunização , Epitopos Imunodominantes/química , Imunoglobulina G/imunologia , Camundongos , Camundongos Transgênicos , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Ligação Proteica/imunologia , Púrpura Trombocitopênica Trombótica/genética , Púrpura Trombocitopênica Trombótica/imunologia , Púrpura Trombocitopênica Trombótica/metabolismo
5.
Haematologica ; 102(2): 271-281, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-27758819

RESUMO

The development of inhibitory antibodies to therapeutic factor VIII is the major complication of replacement therapy in patients with hemophilia A. The first step in the initiation of the anti-factor VIII immune response is factor VIII interaction with receptor(s) on antigen-presenting cells, followed by endocytosis and presentation to naïve CD4+ T cells. Recent studies indicate a role for the C1 domain in factor VIII uptake. We investigated whether charged residues in the C2 domain participate in immunogenic factor VIII uptake. Co-incubation of factor VIII with BO2C11, a monoclonal C2-specific immunoglobulin G, reduced factor VIII endocytosis by dendritic cells and presentation to CD4+ T cells, and diminished factor VIII immunogenicity in factor VIII-deficient mice. The mutation of basic residues within the BO2C11 epitope of C2 replicated reduced in vitro immunogenic uptake, but failed to prevent factor VIII immunogenicity in mice. BO2C11 prevents factor VIII binding to von Willebrand factor, thus potentially biasing factor VIII immunogenicity by perturbing its half-life. Interestingly, a factor VIIIY1680C mutant, that does not bind von Willebrand factor, demonstrated unaltered endocytosis by dendritic cells as well as immunogenicity in factor VIII-deficient mice. Co-incubation of factor VIIIY1680C with BO2C11, however, resulted in decreased factor VIII immunogenicity in vivo In addition, a previously described triple C1 mutant showed decreased uptake in vitro, and reduced immunogenicity in vivo, but only in the absence of endogenous von Willebrand factor. Taken together, the results indicate that residues in the C1 and/or C2 domains of factor VIII are implicated in immunogenic factor VIII uptake, at least in vitro Conversely, in vivo, the binding to endogenous von Willebrand factor masks the reducing effect of mutations in the C domains on factor VIII immunogenicity.


Assuntos
Domínios C2 , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Endocitose/imunologia , Fator VIII/imunologia , Fator VIII/metabolismo , Domínios Proteicos , Animais , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/metabolismo , Fator VIII/química , Fator VIII/genética , Técnicas de Inativação de Genes , Hemofilia A/genética , Hemofilia A/imunologia , Hemofilia A/metabolismo , Humanos , Ativação Linfocitária/imunologia , Camundongos , Mutação , Ligação Proteica , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Fator de von Willebrand/metabolismo
6.
Cell Immunol ; 301: 40-8, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26723503

RESUMO

Replacement therapy for patients with hemophilia A using plasma-derived or recombinant factor VIII (FVIII) is complicated by the short half-life of the FVIII products and by the occurrence of neutralizing antibodies in a substantial number of patients. In the recent years, enormous efforts have been invested to develop new generations of coagulation factors with extended half-lives. Presumably, the use of long-lasting FVIII products should reduce the frequency of administration to the patients and drastically improve their quality of life. The question of their immunogenicity remains however unanswered as yet. The present review proposes a summary of the different strategies developed to enhance the half-life of FVIII, including fusion of FVIII to the Fc fragment of the human IgG1 or to human serum albumin, or attachment of polyethylene glycol. Based on the available literature, we hypothesize on the potential benefits or risks associated with each of the latter strategies in terms of immunogenicity of the newly derived hemostatic drugs.


Assuntos
Fator VIII/imunologia , Fator VIII/farmacocinética , Hemofilia A/imunologia , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/farmacocinética , Fator VIII/metabolismo , Meia-Vida , Humanos , Proteínas Recombinantes de Fusão/metabolismo
7.
Blood ; 123(1): 121-5, 2014 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-24014239

RESUMO

Vascular endothelial cells (ECs) link hemostasis, thrombosis, and complement. ECs synthesize both the clotting initiator von Willebrand factor (VWF) and the complement regulator factor H (FH). VWF is stored in EC Weibel-Palade bodies (WPBs), but the intracellular location of FH is not well defined. We found that FH colocalizes with VWF in WPBs of human umbilical vein ECs. Moreover, FH bound to VWF with an apparent nanomolar affinity and the complex was present in normal plasma. The binding of VWF to FH enhanced FH cofactor activity toward factor I-mediated downregulation of complement activation. Besides, this interaction inhibited ADAMTS13-mediated proteolysis of VWF and promoted platelet aggregation. Here, we describe a novel interaction between complement and hemostasis. The simultaneous secretion of VWF and FH by activated ECs may promote adhesion of platelets to endothelial injury sites to assure wound healing, simultaneously dampening the proinflammatory effect of complement to limit bystander tissue damage.


Assuntos
Fator H do Complemento/química , Trombose , Fator de von Willebrand/química , Proteínas ADAM/metabolismo , Proteína ADAMTS13 , Ativação do Complemento , Fator H do Complemento/metabolismo , Endotélio Vascular/citologia , Ensaio de Imunoadsorção Enzimática , Hemostasia , Heterozigoto , Células Endoteliais da Veia Umbilical Humana/citologia , Humanos , Imunoprecipitação , Inflamação , Ligação Proteica , Mapeamento de Interação de Proteínas , Ressonância de Plasmônio de Superfície , Corpos de Weibel-Palade/metabolismo , Fator de von Willebrand/metabolismo
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