RESUMO
M3258 is an orally bioavailable, potent, selective, reversible inhibitor of the large multifunctional peptidase 7 (LMP7, ß5i, PSMB8) proteolytic subunit of the immunoproteasome, a component of the cellular protein degradation machinery, highly expressed in malignant hematopoietic cells including multiple myeloma. Here we describe the fit-for-purpose pharmacokinetic (PK)/pharmacodynamic (PD)/efficacy modeling of M3258 based on preclinical data from several species. The inhibition of LMP7 activity (PD) and tumor growth (efficacy) were tested in human multiple myeloma xenografts in mice. PK and efficacy data were correlated yielding a free M3258 concentration of 45 nM for half-maximal tumor growth inhibition (KC50). As M3258 only weakly inhibits LMP7 in mouse cells, both in vitro and in vivo bridging studies were performed in rats, monkeys, and dogs for translational modeling. These data indicated that the PD response in human xenograft models was closely reflected in dog PBMCs. A PK/PD model was established, predicting a free IC50 value of 9 nM for M3258 in dogs in vivo, in close agreement with in vitro measurements. In parallel, the human PK parameters of M3258 were predicted by various approaches including in vitro extrapolation and allometric scaling. Using PK/PD/efficacy simulations, the efficacious dose range and corresponding PD response in human were predicted. Taken together, these efforts supported the design of a phase Ia study of M3258 in multiple myeloma patients (NCT04075721). At the lowest tested dose level, the predicted exposure matched well with the observed exposure while the duration of LMP7 inhibition was underpredicted by the model. SIGNIFICANCE STATEMENT: M3258 is a novel inhibitor of the immunoproteasome subunit LMP7. The human PK and human efficacious dose range of M3258 were predicted using in vitro-in vivo extrapolation and allometric scaling methods together with a fit-for-purpose PK/PD and efficacy model based on data from several species. A comparison with data from the Phase Ia clinical study showed that the human PK was accurately predicted, while the extent and duration of PD response were more pronounced than estimated.
Assuntos
Mieloma Múltiplo , Humanos , Ratos , Camundongos , Animais , Cães , Mieloma Múltiplo/tratamento farmacológico , Modelos BiológicosRESUMO
CTLA-4 and CD28 exemplify a co-inhibitory and co-stimulatory signaling axis that dynamically sculpts the interaction of antigen-specific T cells with antigen-presenting cells. Anti-CTLA-4 antibodies enhance tumor-specific immunity through a variety of mechanisms including: blockade of CD80 or CD86 binding to CTLA-4, repressing regulatory T cell function and selective elimination of intratumoral regulatory T cells via an Fcγ receptor-dependent mechanism. AGEN1884 is a novel IgG1 antibody targeting CTLA-4. It potently enhanced antigen-specific T cell responsiveness that could be potentiated in combination with other immunomodulatory antibodies. AGEN1884 was well-tolerated in non-human primates and enhanced vaccine-mediated antigen-specific immunity. AGEN1884 combined effectively with PD-1 blockade to elicit a T cell proliferative response in the periphery. Interestingly, an IgG2 variant of AGEN1884 revealed distinct functional differences that may have implications for optimal dosing regimens in patients. Taken together, the pharmacological properties of AGEN1884 support its clinical investigation as a single therapeutic and combination agent.
Assuntos
Adjuvantes Imunológicos/farmacologia , Antineoplásicos Imunológicos/farmacologia , Antígeno CTLA-4/imunologia , Imunoglobulina G/farmacologia , Neoplasias/terapia , Adjuvantes Imunológicos/química , Adjuvantes Imunológicos/farmacocinética , Adjuvantes Imunológicos/toxicidade , Sequência de Aminoácidos , Animais , Formação de Anticorpos/efeitos dos fármacos , Antineoplásicos Imunológicos/química , Antineoplásicos Imunológicos/farmacocinética , Antineoplásicos Imunológicos/toxicidade , Células CHO , Antígeno CTLA-4/antagonistas & inibidores , Vacinas Anticâncer/farmacologia , Células Cultivadas , Cricetulus , Mapeamento de Epitopos , Humanos , Imunidade Celular/efeitos dos fármacos , Imunoglobulina G/química , Imunoglobulina G/toxicidade , Ativação Linfocitária/efeitos dos fármacos , Macaca fascicularis , Modelos Moleculares , Neoplasias/imunologia , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/imunologiaRESUMO
Human leukocyte antigen-antigen D related (HLA-DR) molecules are highly expressed in synovial tissue (ST), the target of the immune response in chronic inflammatory forms of arthritis. Here, we used LC-MS/MS to identify HLA-DR-presented self-peptides in cells taken directly from clinical samples: ST, synovial fluid mononuclear cells (SFMC), or peripheral blood mononuclear cells (PBMC) from five patients with rheumatoid arthritis (RA) and eight with Lyme arthritis (LA). We identified 1593 non-redundant HLA-DR-presented peptides, derived from 870 source proteins. A total of 67% of the peptides identified in SFMC and 55% of those found in PBMC were found in ST, but analysis of SFMC/PBMC also revealed new antigen-presented peptides. Peptides were synthesized and examined for reactivity with the patients' PBMC. To date, three autoantigens in RA and four novel autoantigens in LA, presented in ST and/or PBMC, were shown to be targets of T- and B-cell responses in these diseases; ongoing analyses may add to this list. Thus, immunoprecipitation and LC-MS/MS can now identify hundreds of HLA-DR-presented self-peptides from individual patients' tissues or fluids with mixed cell populations. Importantly, identification of HLA-DR-presented peptides from SFMC or PBMC allows testing of more patients, including those early in the disease. Direct analysis of clinical samples facilitates identification of novel immunogenic T-cell epitopes.
Assuntos
Artrite Reumatoide/imunologia , Antígenos HLA-DR/imunologia , Doença de Lyme/imunologia , Peptídeos/imunologia , Líquido Sinovial/imunologia , Membrana Sinovial/imunologia , Adolescente , Adulto , Idoso , Apresentação de Antígeno , Artrite Reumatoide/genética , Artrite Reumatoide/patologia , Autoantígenos/química , Autoantígenos/genética , Autoantígenos/imunologia , Linfócitos B/imunologia , Linfócitos B/patologia , Expressão Gênica , Ontologia Genética , Antígenos HLA-DR/química , Antígenos HLA-DR/genética , Humanos , Doença de Lyme/genética , Doença de Lyme/patologia , Pessoa de Meia-Idade , Anotação de Sequência Molecular , Peptídeos/síntese química , Peptídeos/isolamento & purificação , Líquido Sinovial/química , Membrana Sinovial/química , Membrana Sinovial/patologia , Linfócitos T/imunologia , Linfócitos T/patologiaRESUMO
OBJECTIVE: Prevotella copri, an intestinal microbe, may overexpand in stool samples from patients with new-onset rheumatoid arthritis (RA), but it is not yet clear whether the organism has immune relevance in RA pathogenesis. METHODS: HLA-DR-presented peptides (T cell epitopes) from P copri were sought directly in the patients' synovial tissue or peripheral blood mononuclear cell (PBMC) samples using tandem mass spectrometry. The antigenicity of peptides or their source proteins was examined in samples from the RA patients or comparison groups. T cell reactivity was determined by enzyme-linked immunospot assay; antibody responses were measured by enzyme-linked immunosorbent assay, and cytokine/chemokine determinations were made by bead-based assays. Serum and synovial fluid samples were examined for 16S ribosomal DNA for P copri using nested polymerase chain reaction analysis. RESULTS: In PBMCs, we identified an HLA-DR-presented peptide from a 27-kd protein of P copri (Pc-p27), which stimulated Th1 responses in 42% of patients with new-onset RA. In both new-onset RA patients and chronic RA patients, 1 subgroup had IgA antibody responses to either Pc-p27 or the whole organism, which correlated with Th17 cytokine responses and frequent anti-citrullinated protein antibodies (ACPAs). The other subgroup had IgG P copri antibodies, which were associated with Prevotella DNA in synovial fluid, P copri-specific Th1 responses, and less frequent ACPAs. In contrast, P copri antibody responses were rarely found in patients with other rheumatic diseases or in healthy controls. CONCLUSION: Subgroups of RA patients have differential IgG or IgA immune reactivity with P copri, which appears to be specific for this disease. These observations provide evidence that P copri is immune-relevant in RA pathogenesis.
Assuntos
Artrite Reumatoide/imunologia , Proteínas de Bactérias/imunologia , Epitopos de Linfócito T/imunologia , Microbioma Gastrointestinal/imunologia , Leucócitos Mononucleares/imunologia , Prevotella/imunologia , Membrana Sinovial/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Artrite Reumatoide/microbiologia , DNA Ribossômico/genética , DNA Ribossômico/metabolismo , Ensaio de Imunoadsorção Enzimática , ELISPOT , Feminino , Microbioma Gastrointestinal/genética , Humanos , Imunoglobulina A/imunologia , Imunoglobulina G/imunologia , Masculino , Pessoa de Meia-Idade , Compostos Organotiofosforados/imunologia , Peptídeos/imunologia , Reação em Cadeia da Polimerase , Prevotella/genética , Líquido Sinovial/metabolismo , Espectrometria de Massas em Tandem , Células Th1/imunologia , Adulto JovemRESUMO
Infection-induced autoimmunity is thought to be a contributing factor in antibiotic-refractory Lyme arthritis, but studies of autoimmunity have been hindered by difficulty in identifying relevant autoantigens. We developed a novel approach that begins with the identification of T cell epitopes in synovial tissue using tandem mass spectrometry. Herein, we identified an immunogenic HLA-DR-presented peptide (T cell epitope) derived from the source protein matrix metalloproteinase-10 (MMP-10) from the synovium of a patient with antibiotic-refractory arthritis. This finding provided a bridge for the identification of autoantibody responses to MMP-10, the "first autoimmune hit" in a subgroup of patients with erythema migrans, the initial skin lesion of the infection. Months later, after priming of the immune response to MMP-10 in early infection, a subset of patients with antibiotic-responsive or antibiotic-refractory arthritis had MMP-10 autoantibodies, but only patients with antibiotic-refractory arthritis had both T and B cell responses to the protein, providing evidence for a "second autoimmune hit". Further support for a biologically relevant autoimmune event was observed by the positive correlation of anti-MMP-10 autoantibodies with distinct synovial pathology. This experience demonstrates the power of new, discovery-based methods to identify relevant autoimmune responses in chronic inflammatory forms of arthritis.
Assuntos
Linfócitos B/imunologia , Doença de Lyme/etiologia , Doença de Lyme/patologia , Metaloproteinase 10 da Matriz/imunologia , Membrana Sinovial/imunologia , Membrana Sinovial/patologia , Linfócitos T/imunologia , Antibacterianos/uso terapêutico , Apresentação de Antígeno , Autoanticorpos/imunologia , Autoantígenos/imunologia , Autoimunidade , Linfócitos B/metabolismo , Borrelia burgdorferi/imunologia , Resistência a Medicamentos , Epitopos de Linfócito T/imunologia , Antígenos HLA-DR/genética , Antígenos HLA-DR/imunologia , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Imunoglobulina M/sangue , Imunoglobulina M/imunologia , Imuno-Histoquímica , Doença de Lyme/tratamento farmacológico , Doença de Lyme/metabolismo , Metaloproteinase 10 da Matriz/química , Metaloproteinase 10 da Matriz/metabolismo , Metaloproteinase 3 da Matriz/imunologia , Peptídeos/imunologia , Líquido Sinovial/imunologia , Líquido Sinovial/metabolismo , Membrana Sinovial/metabolismo , Linfócitos T/metabolismoRESUMO
OBJECTIVE: Endothelial cell growth factor (ECGF) was recently identified as the first autoantigen known to be a target of T cell and B cell responses in ~20% of patients with antibiotic-refractory Lyme arthritis. The goal of the current study was to look for a pathologic correlate between ECGF autoantibody responses and histologic findings in synovial tissue. METHODS: Synovial tissue was examined from 14 patients with antibiotic-refractory Lyme arthritis and 6 patients with other forms of chronic inflammatory arthritis, primarily rheumatoid arthritis. The tissue sections were subjected to chemical and immunostaining, and IgG antibody responses to ECGF were determined by enzyme-linked immunosorbent assay (ELISA). Each finding was ranked for statistical analysis. RESULTS: In each disease, synovial tissue showed synovial hypertrophy, vascular proliferation, immune cell infiltrates, and fibrosis. However, among the 14 patients with antibiotic-refractory arthritis, 8 (57%) had obliterative microvascular lesions in the tissue, compared with none of the 6 patients with other forms of chronic inflammatory arthritis (P = 0.04). Among the patients with Lyme arthritis, 5 (36%) had autoantibody responses to ECGF, and all 5 had obliterative lesions, as compared with only 3 of 9 patients who lacked ECGF antibody responses (P = 0.009). Moreover, the magnitude of ECGF antibody responses correlated directly with the extent of obliterative lesions (P = 0.02) and with greater vascularity in the tissue (P = 0.05). CONCLUSION: The correlations of ECGF autoantibody reactivity with obliterative microvascular lesions imply that these autoantibodies may be involved in the obliterative process, suggesting that anti-ECGF antibodies have specific pathologic consequences in the synovial tissue of patients with antibiotic-refractory Lyme arthritis.
Assuntos
Autoanticorpos/imunologia , Fatores de Crescimento Endotelial/imunologia , Doença de Lyme/imunologia , Membrana Sinovial/imunologia , Adolescente , Adulto , Idoso , Antibacterianos/uso terapêutico , Criança , Feminino , Humanos , Doença de Lyme/tratamento farmacológico , Masculino , Pessoa de Meia-Idade , Falha de Tratamento , Adulto JovemRESUMO
OBJECTIVE: To examine the role of immune dysregulation in antibiotic-refractory Lyme arthritis by comparing the phenotype, frequency, and function of CD4+ Teff cells and Treg cells in patients with antibiotic- responsive arthritis and patients with antibiotic-refractory arthritis. METHODS: Matched peripheral blood and synovial fluid samples from 15 patients with antibiotic-responsive arthritis were compared with those from 16 patients with antibiotic-refractory arthritis, using flow cytometry, suppression assays, and cytokine assays. RESULTS: Critical differences between the 2 patient groups were observed in the synovial fluid CD4+CD25(high) population, a cell subset usually composed of FoxP3-positive Treg cells. In patients with antibiotic-refractory arthritis, this cell population often had fewer FoxP3-positive cells and a greater frequency of FoxP3-negative (Teff) cells compared with patients with antibiotic-responsive arthritis. Moreover, the expression of glucocorticoid-induced tumor necrosis factor receptor and OX40 on CD4+CD25(high) cells was significantly higher in the antibody-refractory group. Suppression assays showed that CD4+CD25(high) cells in patients with antibiotic-refractory arthritis did not effectively suppress proliferation of CD4+CD25- cells or secretion of interferon-γ and tumor necrosis factor α, whereas those cells in patients with antibiotic-responsive arthritis did suppress proliferation of CD4+CD25- cells and secretion of interferon-γ and tumor necrosis factor α. Finally, in the antibiotic-refractory group, higher ratios of CD25(high) FoxP3-negative cells to CD25(high) FoxP3-positive cells correlated directly with a longer duration of arthritis after antibiotic treatment. CONCLUSION: Patients with antibiotic-refractory Lyme arthritis often have lower frequencies of Treg cells, higher expression of activation coreceptors, and less effective inhibition of proinflammatory cytokines. This suggests that immune responses in these patients are excessively amplified, leading to immune dysregulation and antibiotic-refractory arthritis.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Citocinas/análise , Doença de Lyme/imunologia , Líquido Sinovial/imunologia , Linfócitos T Reguladores/imunologia , Adolescente , Adulto , Criança , Resistência Microbiana a Medicamentos , Feminino , Citometria de Fluxo , Humanos , Subunidade alfa de Receptor de Interleucina-2 , Masculino , Pessoa de Meia-Idade , Fenótipo , Adulto JovemRESUMO
OBJECTIVE: Autoantigen presentation by HLA-DR molecules is thought to be a central component of many autoimmune diseases, but identifying disease-relevant autoantigens has been a difficult challenge. In this study we aimed to identify autoantigens in patients with antibiotic-refractory Lyme arthritis, in which infection-induced autoimmunity is thought to play an important role. METHODS: Using tandem mass spectrometry, naturally presented HLA-DR self peptides from a patient's synovium were identified, synthesized, and reacted with his peripheral blood mononuclear cells (PBMCs). Immunoreactive peptides and their source proteins were then tested for T and B cell responses using large numbers of patient cells or sera. RESULTS: Of 120 HLA-DR-presented self peptides identified from one patient, one peptide derived from endothelial cell growth factor (ECGF) caused his PBMCs to proliferate. T and B cell responses to ECGF occurred systemically in â¼10-30% of patients with early or late manifestations of Lyme disease, primarily in those with refractory arthritis-associated HLA-DR alleles, such as DRB1*0101 and 0401. Compared with patients with antibiotic-responsive arthritis, those with antibiotic-refractory arthritis had significantly higher concentrations of ECGF in synovial fluid (P<0.0001) and more often had ECGF antibody reactivity. Among non-antibiotic-treated historical patients who developed arthritis, 26% had ECGF reactivity, which often developed before the onset of arthritis and was associated with significantly longer courses of arthritis. CONCLUSION: T and B cell responses to ECGF occur in a subset of patients with Lyme disease, particularly in those with antibiotic-refractory arthritis, providing the first direct evidence of autoimmune T and B cell responses in this illness.
Assuntos
Autoantígenos/imunologia , Linfócitos B/imunologia , Fatores de Crescimento Endotelial/imunologia , Antígenos HLA-DR/imunologia , Doença de Lyme/imunologia , Linfócitos B/metabolismo , Farmacorresistência Bacteriana , Ensaio de Imunoadsorção Enzimática , ELISPOT , Antígenos HLA-DR/metabolismo , Humanos , Immunoblotting , Imuno-Histoquímica , Doença de Lyme/tratamento farmacológico , Doença de Lyme/metabolismo , Proteômica , Linfócitos T , Espectrometria de Massas em TandemRESUMO
Evidence is emerging for differential pathogenicity among Borrelia burgdorferi genotypes in the United States. By using two linked genotyping systems, ribosomal RNA intergenic spacer type (RST) and outer surface protein C (OspC), we studied the inflammatory potential of B. burgdorferi genotypes in cells and patients with erythema migrans or Lyme arthritis. When macrophages were stimulated with 10 isolates of each RST1, RST2, or RST3 strain, RST1 (OspC type A)-stimulated cells expressed significantly higher levels of IL-6, IL-8, chemokine ligand (CCL) 3, CCL4, tumor necrosis factor, and IL-1ß, factors associated with innate immune responses. In peripheral blood mononuclear cells, RST1 strains again stimulated significantly higher levels of these mediators. Moreover, compared with RST2, RST1 isolates induced significantly more interferon (IFN)-α, IFN-γ, and CXCL10, which are needed for adaptive immune responses; however, OspC type I (RST3) approached RST1 (OspC type A) in stimulating these adaptive immune mediators. Similarly, serum samples from patients with erythema migrans who were infected with the RST1 genotype had significantly higher levels of almost all of these mediators, including exceptionally high levels of IFN-γ-inducible chemokines, CCL2, CXCL9, and CXCL10; and this pronounced inflammatory response was associated with more symptomatic infection. Differences among genotypes were not as great in patients with Lyme arthritis, but those infected with RST1 strains more often had antibiotic-refractory arthritis. Thus, the B. burgdorferi RST1 (OspC type A) genotype, followed by the RST3 (OspC type I) genotype, causes greater inflammation and more severe disease, establishing a link between spirochetal virulence and host inflammation.
Assuntos
Borrelia burgdorferi/genética , Inflamação/complicações , Inflamação/microbiologia , Doença de Lyme/complicações , Doença de Lyme/microbiologia , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Borrelia burgdorferi/efeitos dos fármacos , Borrelia burgdorferi/isolamento & purificação , Quimiocinas/sangue , Quimiocinas/metabolismo , Farmacorresistência Bacteriana/efeitos dos fármacos , Eritema/sangue , Eritema/complicações , Eritema/microbiologia , Eritema/patologia , Humanos , Inflamação/sangue , Inflamação/patologia , Articulações/efeitos dos fármacos , Articulações/patologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/microbiologia , Doença de Lyme/sangue , Doença de Lyme/tratamento farmacológico , Doença de Lyme/patologia , Ativação de Macrófagos/efeitos dos fármacos , Ativação de Macrófagos/imunologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Macrófagos/microbiologia , Líquido Sinovial/efeitos dos fármacos , Líquido Sinovial/metabolismoRESUMO
Disease-associated HLA-DR molecules, which may present autoantigens, constitute the greatest genetic risk factor for rheumatoid arthritis (RA) and antibiotic-refractory Lyme arthritis (LA). The peptides presented by HLA-DR molecules in synovia have not previously been defined. Using tandem mass spectrometry, rigorous database searches, and manual spectral interpretation, we identified 1,427 HLA-DR-presented peptides (220-464 per patient) from the synovia of four patients, two diagnosed with RA and two diagnosed with LA. The peptides were derived from 166 source proteins, including a wide range of intracellular and plasma proteins. A few epitopes were found only in RA or LA patients. However, two patients with different diseases who had the same HLA allele had the largest number of epitopes in common. In one RA patient, peptides were identified as originating from source proteins that have been reported to undergo citrullination under other circumstances, yet neither this post-translational modification nor anti-cyclic citrullinated peptide antibodies were detected. Instead, peptides with the post-translational modification of S-cysteinylation were identified. We conclude that a wide range of proteins enter the HLA-DR pathway of antigen-presenting cells in the patients' synovial tissue, and their HLA-DR genotype, not the disease type, appears to be the primary determinant of their HLA-DR-peptide repertoire. New insights into the naturally presented HLA-DR epitope repertoire in target tissues may allow the identification of pathogenic T cell epitopes, and this could lead to innovative therapeutic interventions.
Assuntos
Antibacterianos/uso terapêutico , Apresentação de Antígeno/imunologia , Artrite Reumatoide/imunologia , Antígenos HLA-DR/imunologia , Doença de Lyme/imunologia , Peptídeos/imunologia , Líquido Sinovial/imunologia , Adulto , Idoso , Sequência de Aminoácidos , Antibacterianos/farmacologia , Apresentação de Antígeno/efeitos dos fármacos , Artrite Reumatoide/patologia , Criança , Farmacorresistência Bacteriana/efeitos dos fármacos , Epitopos/química , Epitopos/imunologia , Feminino , Humanos , Doença de Lyme/tratamento farmacológico , Masculino , Dados de Sequência Molecular , Peptídeos/química , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Líquido Sinovial/efeitos dos fármacos , Espectrometria de Massas em TandemRESUMO
To delineate the inflammatory potential of the 3 pathogenic species of Borrelia burgdorferi sensu lato, we stimulated monocyte-derived macrophages from healthy human donors with 10 isolates each of B. burgdorferi, Borrelia afzelii, or Borrelia garinii recovered from erythema migrans skin lesions of patients with Lyme borreliosis from the United States or Slovenia. B. burgdorferi isolates from the United States induced macrophages to secrete significantly higher levels of interleukin (IL)-8, CCL3, CCL4, IL-6, IL-10, and tumor necrosis factor than B. garinii or B. afzelii isolates. Consistent with this response in cultured macrophages, chemokine and cytokine levels in serum samples of patients from whom the isolates were obtained were significantly greater in B. burgdorferi-infected patients than in B. afzelii- or B. garinii-infected patients. These results demonstrate in vitro and in vivo that B. burgdorferi has greater inflammatory potential than B. afzelii and B. garinii, which may account in part for variations in the clinical manifestations of Lyme borreliosis.
Assuntos
Grupo Borrelia Burgdorferi/imunologia , Citocinas/metabolismo , Doença de Lyme/imunologia , Doença de Lyme/microbiologia , Macrófagos/imunologia , Grupo Borrelia Burgdorferi/isolamento & purificação , Células Cultivadas , Citocinas/sangue , Humanos , Eslovênia , Estados UnidosRESUMO
Antibiotic-refractory Lyme arthritis is believed to result from an infection-induced autoimmune response triggered by the spirochete Borrelia burgdorferi (Bb). Disease susceptibility is associated with the HLA alleles DRB1*0101, 0401, 0402, 0404, 0405 and DRB5*0101, and all these MHC molecules bind the Bb epitope OspA(163-175.) However, not all patients have a proliferative response to this epitope. To identify other possible Bb epitopes involved in this disease process, the algorithm TEPITOPE was used to scan 17 immunogenic Bb proteins for potential T cell epitopes with a refractory arthritis-associated MHC binding profile, and the Bb proteome was searched for peptides with sequence homology to OspA(165-173). Sixteen promising T epitopes were identified and their MHC binding profiles to 13 MHC molecules were verified using in vitro MHC/peptide binding assays. One peptide, BBK32(392-404), had a strong refractory arthritis-associated MHC binding profile, and another GK(297-306) shared sequence homology to OspA(165-173). However, patient cells did not proliferate in response to either peptide making it highly unlikely they were involved in a refractory course. A comparison of the in silico and in vitro results revealed that TEPITOPE correctly predicted 74% of the in vitro binding peptides, but it incorrectly predicted that 44% of the in vitrononbinding peptides would bind. For a particular MHC molecule, concordance between the in silico and in vitro results varied anywhere between 33% and 100%. Therefore, while additional Bb epitopes may be involved in the development of antibiotic-refractory Lyme arthritis, recognition of OspA(163-175) remains the only known Bb epitope associated with this disease course.
Assuntos
Borrelia burgdorferi/imunologia , Resistência Microbiana a Medicamentos/imunologia , Epitopos de Linfócito T/imunologia , Doença de Lyme/imunologia , Doença de Lyme/microbiologia , Proteínas de Bactérias/imunologia , Biologia Computacional , Computadores , Antígenos HLA-DR/imunologia , Humanos , Peptídeos/imunologia , Homologia de Sequência de Aminoácidos , Linfócitos T/microbiologiaRESUMO
Antibiotic-refractory Lyme arthritis, which may result from infection-induced autoimmunity, is associated with HLA-DR molecules that bind an epitope of Borrelia burgdorferi (Bb) outer-surface protein A (OspA(165-173)) and with T cell reactivity with this epitope. One potential mechanism to explain these associations is molecular mimicry between OspA(165-173) and a self-peptide. Here, we searched the published human genome for peptides with sequence homology with OspA(165-173). The two peptides identified with the greatest sequence homology with the OspA epitope were MAWD-BP(276-288), which had identity at eight of the nine core amino acid residues, and T-span7(58-70), which had identity at six residues. MAWD-BP mRNA was expressed by synoviocytes, while T-span7 mRNA was not. However, neither peptide bound all of the HLA-DR molecules associated with antibiotic-refractory Lyme arthritis. Among 11 patients, 9 had T cell reactivity with OspA(161-170), 6 had responses to MAWD-BP(276-288), and 3 had reactivity with T-span7(58-70), but reactivity with the self-peptides was lower than that induced by the spirochetal epitope. Thus, there remains an association between OspA(165-173) and antibiotic-refractory Lyme arthritis, and infection-induced autoimmunity is an attractive hypothesis to explain this outcome. However, molecular mimicry due to sequence homology between OspA(165-173) and a human peptide seems unlikely to be the critical mechanism.
Assuntos
Borrelia burgdorferi/imunologia , Farmacorresistência Bacteriana/imunologia , Epitopos de Linfócito T/química , Doença de Lyme/imunologia , Homologia de Sequência de Aminoácidos , Sequência de Aminoácidos , Antígenos de Superfície/química , Antígenos de Superfície/metabolismo , Autoanticorpos/imunologia , Autoantígenos/genética , Autoantígenos/metabolismo , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/metabolismo , Vacinas Bacterianas/química , Vacinas Bacterianas/metabolismo , Borrelia burgdorferi/química , Proliferação de Células , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Antígenos HLA-DR/imunologia , Humanos , Lipoproteínas/química , Lipoproteínas/metabolismo , Dados de Sequência Molecular , Peptídeos/química , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Linfócitos T/citologiaRESUMO
Ig heavy chain class switch recombination (CSR) determines the expression of Ig isotypes. The molecular mechanism of CSR and the factors regulating this process have remained elusive. Recombination occurs primarily within switch (S) regions, located upstream of each heavy chain gene (except Cdelta). These repetitive sequences contain consensus DNA-binding sites for the DNA-binding protein late SV40 factor (LSF) (CP2/leader-binding protein-1c). In this study, we demonstrate by EMSA that purified rLSF, as well as LSF within B cell extracts, directly binds both Smu and Salpha sequences. To determine whether LSF is involved in regulating CSR, two different LSF dominant negative variants were stably expressed in the mouse B cell line I.29 mu, which can be induced to switch from IgM to IgA. Overexpression of these dominant negative LSF proteins results in decreased levels of endogenous LSF DNA-binding activity and an increase in cells undergoing CSR. Thus, LSF represses class switching to IgA. In agreement, LSF DNA-binding activity was found to decrease in whole cell extracts from splenic B cells induced to undergo class switching. To elucidate the mechanism of CSR regulation by LSF, the interactions of LSF with proteins involved in chromatin modification were tested in vitro. LSF interacts with both histone deacetylases and the corepressor Sin3A. We propose that LSF represses CSR by histone deacetylation of chromatin within S regions, thereby limiting accessibility to the switch recombination machinery.