RESUMO
Disturbed expression of microRNAs (miRNAs) in regulatory T cells (Tregs) leads to development of autoimmunity in experimental mouse models. However, the miRNA expression signature characterizing Tregs of autoimmune diseases, such as rheumatoid arthritis (RA) has not been determined yet. In this study, we have used a microarray approach to comprehensively analyze miRNA expression signatures of both naive Tregs (CD4+CD45RO-CD25++) and memory Tregs (CD4+CD45RO+CD25+++), as well as conventional naive (CD4+CD45RO-CD25-) and memory (CD4+CD45RO+CD25-) T cells (Tconvs) derived from peripheral blood of RA patients and matched healthy controls. Differential expression of selected miRNAs was validated by TaqMan-based quantitative reverse transcription-PCR. We found a positive correlation between increased expression of miR-451 in T cells of RA patients and disease activity score (DAS28), erythrocyte sedimentation rate levels and serum levels of interleukin-6. Moreover, we found characteristic, disease- and treatment-independent, global miRNA expression signatures defining naive Tregs, memory Tregs, naive Tconvs and memory Tconvs. The analysis allowed us to define miRNAs characteristic for a general naive phenotype (for example, miR-92a) and a general memory phenotype (for example, miR-21, miR-155). Importantly, the analysis allowed us to define miRNAs that are specifically expressed in both naive and memory Tregs, defining as such miRNA signature characterizing the Treg phenotype (that is, miR-146a, miR-3162, miR-1202, miR-1246 and miR-4281).
Assuntos
Artrite Reumatoide/genética , MicroRNAs/genética , Linfócitos T Reguladores/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Anti-Inflamatórios não Esteroides/uso terapêutico , Artrite Reumatoide/tratamento farmacológico , Sedimentação Sanguínea , Antígenos CD4/genética , Feminino , Humanos , Subunidade alfa de Receptor de Interleucina-2/genética , Interleucina-6/sangue , Antígenos Comuns de Leucócito/genética , Masculino , MicroRNAs/biossíntese , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , Líquido Sinovial/citologia , Líquido Sinovial/imunologiaRESUMO
Mycophenolic acid is the active ingredient of the immunosuppressant mycophenolate mofetil that is widely used in transplantation medicine and autoimmunity. Mycophenolic acid inhibits inosine monophosphate dehydrogenase, an enzyme involved in biosynthesis of guanine nucleotides required for lymphocyte clonal expansion. Here, we present novel insights into the mechanisms underlying mycophenolic acid-mediated suppression of human CD4+ T cells. Upon CD3/CD28 stimulation, mycophenolic acid inhibited T cell IL-17, IFN-γ and TNF-α production but not IL-2 production. Phenotypic analysis showed that drug treatment enhanced the expression of negative co-stimulators PD-1, CTLA-4 and the transcription factor FoxP3 and decreased the expression of positive co-stimulators CD27 and CD28, whereas CD25 was unaffected. Mycophenolic acid-treated cells were anergic, but not suppressive, and at the same time proved hyperblastoid with high metabolic activity. Moreover, a reduced Akt/mTOR and STAT5 signaling was observed. Interestingly, the co-stimulatory molecule CD70 was uniquely and dose-dependently upregulated on mycophenolic acid-treated T cells and found to be directly linked to target enzyme inhibition. CD70 on mycophenolic acid-treated cells proved functional: an anti-CD70 agonist was found to restore both STAT5 and Akt/mTOR signaling and may thereby prevent apoptosis and promote survival. These novel insights may contribute to optimization of protocols for MPA-based immunosuppressive regimens.
Assuntos
Antibióticos Antineoplásicos/farmacologia , Ligante CD27/metabolismo , Linfócitos T CD4-Positivos/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Nucleotídeos de Guanina/metabolismo , Ácido Micofenólico/farmacologia , Transdução de Sinais/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Western Blotting , Antígenos CD28/metabolismo , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Células Cultivadas , Citometria de Fluxo , Humanos , IMP Desidrogenase/metabolismo , Interferon gama/metabolismo , Interleucina-17/metabolismo , Interleucina-2/metabolismo , Transdução de Sinais/imunologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
BACKGROUND AND PURPOSE: The p38 kinase regulates the release of proinflammatory cytokines including tumour-necrosis factor-alpha (TNFalpha) and is regarded as a potential therapeutic target in rheumatoid arthritis (RA). Using the novel p38 inhibitor Org 48762-0, we investigated the therapeutic potential of p38 inhibition and compared this to anti-mouse (m)TNFalpha antibody treatment in murine collagen-induced arthritis (CIA). EXPERIMENTAL APPROACH: Pharmacological profiles of Org 48762-0 were characterized in kinase assays, cellular assays and in lipopolysaccharide (LPS)-induced inflammation in mice. The effects of Org 48762-0 and of mTNFalpha-neutralization on established arthritis were examined in murine CIA. KEY RESULTS: Org 48762-0 potently inhibited p38alpha kinase with a high degree of kinase selectivity. In cellular assays, Org 48762-0 reduced LPS-induced TNFalpha release. Oral administration of Org 48762-0 in mice showed drug-like pharmacokinetic properties and inhibited LPS-induced cytokine production. These pharmacological characteristics of Org 48762-0 prompted a comparison of therapeutic efficacy with mTNFalpha-neutralization in CIA. Org 48762-0 and anti-mTNFalpha antibody treatment equally inhibited development of arthritis when evaluated macroscopically. Radiological analyses revealed protection against bone damage for both treatments, although statistical difference was reached with Org 48762-0 treatment only. Further, micro-computed tomographical and histopathological analyses confirmed the protective effects of Org 48762-0 on joint damage. CONCLUSIONS AND IMPLICATIONS: Pharmacological targeting of p38 kinase provided good protection against joint tissue damage in CIA. In our experiments, neutralization of mTNFalpha produced less prominent suppression of bone damage. Our data suggest a therapeutic potential for selective and potent p38 inhibitors in RA.
Assuntos
Anticorpos Bloqueadores/uso terapêutico , Artrite Experimental/tratamento farmacológico , Inibidores Enzimáticos/uso terapêutico , Piridinas/farmacologia , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Animais , Anti-Inflamatórios/uso terapêutico , Artrite Experimental/patologia , Western Blotting , Cartilagem/patologia , Endotoxemia/tratamento farmacológico , Inibidores Enzimáticos/farmacocinética , Feminino , Inflamação/induzido quimicamente , Lipopolissacarídeos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos DBA , Prednisolona/uso terapêutico , Especificidade por Substrato , Tomografia Computadorizada por Raios XAssuntos
Fármacos Dermatológicos/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Psoríase/prevenção & controle , Transplante de Pele , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Animais , Modelos Animais de Doenças , Feminino , Humanos , Camundongos , Camundongos SCIDRESUMO
Monoclonal antibodies (mAb) directed against the clonotypic structure of the T-cell receptor (TCR) may be useful reagents in the study and therapy of T-cell-mediated diseases. In contrast to several reports concerning the generation of anti-clonotype mAb to mouse TCR, only very limited numbers of anti-clonotype mAb to human TCR have been described. So far, a suitable method for the generation of anti-clonotype mAb to a given TCR has not been available and in this report we describe a novel strategy for the generation of such mAb. Mice were immunized with intact human T-cells. Then. spleen cell populations were precleared from B-cells reactive to CD3 and the constant region of the TCR by adsorption to TCR/CD3 complexes derived from an irrelevant T-cell clone. Subsequently, clonotype-specific B-cells were selected with TCR/CD3 complexes from the T-cell clone of interest. The small number of B-cells resulting from this selection were clonally expanded in a B-cell culture system and then immortalized by mini-electrofusion. Ten clonotype-specific mAb were generated against a DRB1*0401-restricted T-cell clone recognizing an epitope of the human cartilage glycoprotein 39 (HC gp-39). All mAb immunoprecipitated a heterodimeric 85 kDa protein. Absolute specificity was demonstrated in a T-cell agglutination test with the T-cell clone of interest compared to a set of 16 defined, irrelevant T-cell clones or lines. In functional assays, the mAb were found to inhibit or block antigen-specific T-cell stimulation. In addition, crosslinked mAb were found to stimulate proliferation of the specific clone in the absence of antigen and antigen presenting cells (APC). Such mAb may have clinical relevance in deleting or modulating autoreactive T-cells in a clonotype-specific manner.
Assuntos
Anticorpos Monoclonais/biossíntese , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T/imunologia , Adipocinas , Animais , Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos , Linfócitos B/imunologia , Ligante de CD40 , Células Cultivadas , Proteína 1 Semelhante à Quitinase-3 , Células Clonais , Epitopos de Linfócito T/imunologia , Glicoproteínas/imunologia , Humanos , Lectinas , Ativação Linfocitária/imunologia , Glicoproteínas de Membrana/imunologia , Camundongos , VacinaçãoRESUMO
Previously, fusion of established T cell lines or clones has been claimed to be difficult. We now report our experiences in the fusion of both long term cultures of rat T cell clones and mouse T cell lines to rat W/Fu (C58NT)D. Upon fusion of rat T cell clones the hybrids obtained expressed antigen specificities identical to those of the parent clones. In addition, C58 was used for interspecies hybridisation of murine T cell lines. The specificity of intra- and inter-species hybrids was maintained by subcloning. We conclude that the C58 cell line can be used to generate continuously growing monoclonal T-cell reagents of sufficient stability using both intra- and inter-species hybridisation.
Assuntos
Hibridomas , Linfócitos T/imunologia , Animais , Antígenos CD4/análise , Fusão Celular , Linhagem Celular , Células Clonais , Antígenos de Histocompatibilidade Classe II/análise , Antígenos de Histocompatibilidade Classe II/fisiologia , Camundongos , Fenótipo , RatosRESUMO
In Lymnaea stagnalis injections with dead Escherichia coli or Staphylococcus saprophyticus bacteria resulted in an enhanced clearance of both live S. saprophyticus and E. coli injected 4 days later. A non-specific activation of the internal defence system was concluded from these findings. The activation was dose-dependent: pre-injections with high doses resulted in a higher increase in the clearance capacity of the snails than pre-injections with low doses of bacteria. The state of increased activity of the defence system, induced with injection of dead E. coli, lasted at least 64 days. The heightened responses of the defence system were probably due to an activation of the blood cells (amoebocytes) since: 1) amoebocyte numbers increased faster in bacteria pretreated snails than in control animals; 2) ultrastructural observations revealed that the amoebocytes of bacteria pre-treated animals had a more ruffled outline than those of control snails; 3) amoebocytes from sensitized snails showed a higher phagocytic activity in vitro; 4) mitotic activity of amoebocytes increased after snails had been injected with bacteria.
Assuntos
Lymnaea/imunologia , Animais , Atividade Bactericida do Sangue , Relação Dose-Resposta Imunológica , Escherichia coli/imunologia , Imunidade Celular , Memória Imunológica , Lymnaea/citologia , Microscopia Eletrônica , Mitose , Fagocitose , Staphylococcus/imunologiaAssuntos
Anticorpos Monoclonais/isolamento & purificação , Capsídeo/imunologia , Epitopos/imunologia , Vírus da Bronquite Infecciosa/imunologia , Ativação Linfocitária , Linfócitos T/imunologia , Proteínas do Core Viral/imunologia , Animais , Anticorpos Monoclonais/imunologia , Capsídeo/genética , Embrião de Galinha , Clonagem Molecular , Citometria de Fluxo , Biblioteca Gênica , Hibridomas/imunologia , Vírus da Bronquite Infecciosa/genética , Linfonodos , Camundongos , Camundongos Endogâmicos BALB C/imunologia , Vírus da Doença de Newcastle/imunologia , Proteínas do Core Viral/genéticaRESUMO
OBJECTIVE: The cartilage proteins melanoma inhibitory activity (MIA) and human cartilage gp-39 (HC gp-39) are candidate autoantigens in rheumatoid arthritis (RA). The present study was undertaken to investigate the endogenous HLA-DR4-restricted presentation of these self proteins, in order to seek in vivo evidence in support of their potential immunologic role. METHODS: MIA and HC gp-39 were assessed in synovial fluid (SF) by enzyme-linked immunosorbent assay and in synovial tissue (ST) by immunohistochemistry. Presentation by SF cells was investigated using specific, HLA-DR-restricted T cell hybridomas. RESULTS: MIA and HC gp-39 were detected in RA SF and ST, as well as in specimens from patients with other forms of arthritis. When HC gp-39-specific and MIA-specific HLA-DR4-restricted T cell hybridomas raised in HLA-DR4-transgenic mice were incubated with RA SF cells as antigen-presenting cells in the presence of HC gp-39 or MIA peptides, the corresponding T cell hybridomas showed strong responses, which were blocked by anti-HLA-DR antibodies. Weaker but qualitatively similar responses were observed with exogenous protein, indicating uptake and processing of these antigens by SF cells. More importantly, without addition of peptide or protein, endogenous presentation of MIA and HC gp-39 was detected in SF cells from 53% and 80% of HLA-DRB1*0401-positive RA patients, respectively. In addition, SF cells from 3 of 10 patients with spondylarthritis exhibited endogenous HC gp-39 presentation. CONCLUSION: These data indicate that immunodominant epitopes of MIA and HC gp-39 are actively presented in an HLA-DR-restricted manner in the inflamed RA joint. The question remains as to whether this leads to activation of autoreactive T cells, which could play a role in either the immunopathology or the immunomodulation of arthritis.
Assuntos
Artrite Reumatoide/imunologia , Proteínas da Matriz Extracelular/análise , Glicoproteínas/análise , Antígenos HLA-DR/imunologia , Inflamação/imunologia , Articulações/imunologia , Proteínas de Neoplasias/análise , Adipocinas , Animais , Proteína 1 Semelhante à Quitinase-3 , Ensaio de Imunoadsorção Enzimática , Antígeno HLA-DR4/imunologia , Cadeias HLA-DRB1 , Teste de Histocompatibilidade , Humanos , Hibridomas/imunologia , Articulações/fisiopatologia , Lectinas , Camundongos , Camundongos Transgênicos , Líquido Sinovial/química , Linfócitos T/imunologiaRESUMO
OBJECTIVES: To determine the impact on synovial histopathology of changes in clinical disease activity in the absence of effective treatment. METHODS: Twelve patients with active RA not receiving effective treatment were studied over a 14 week period. Synovial biopsy specimens obtained at baseline and week 14 were analysed by histology and immunohistochemistry. RESULTS: Over the course of 14 weeks, there was a trend towards a decrease of the DAS28, with 7/12 patients being good or moderate DAS28 responders despite the absence of effective treatment. Patients' assessment of global disease activity and swollen joint count both decreased significantly. Histologically, there was a decrease of lining layer hyperplasia and lymphoid aggregates, a similar trend for vascularity, but there was no effect on global synovial infiltration. Accordingly, there was no decrease of the cellular infiltration with T lymphocytes (CD3, CD4, CD8), B lymphocytes (CD20), plasma cells (CD38), dendritic cells (CD1a, CD83), and even an increase of CD163+ sublining macrophages, with a similar trend for CD68+ sublining macrophages. The changes in DAS28 scores in these patients did not correlate with changes in histological variables, with the exception of an inverse correlation with plasma cells. Remarkably, even in the DAS28 responders, no significant changes in synovial inflammatory infiltration were noted. CONCLUSIONS: Despite variations in global disease activity, synovial inflammatory infiltration did not change significantly in the absence of effective treatment. The lack of a placebo effect on synovial markers of treatment response such as sublining macrophages can facilitate conclusive early phase trials with small numbers of patients with RA.
Assuntos
Artrite Reumatoide/imunologia , Seleção de Pacientes , Membrana Sinovial/imunologia , Artrite Reumatoide/diagnóstico , Artrite Reumatoide/metabolismo , Artroscopia , Biomarcadores/análise , Moléculas de Adesão Celular/análise , Ensaios Clínicos como Assunto , Células Dendríticas/imunologia , Feminino , Humanos , Imunidade Celular , Imuno-Histoquímica/métodos , Articulação do Joelho , Linfócitos/imunologia , Macrófagos/imunologia , Masculino , Neutrófilos/patologia , Plasmócitos/patologia , Estatísticas não Paramétricas , Membrana Sinovial/metabolismo , Membrana Sinovial/patologiaRESUMO
Monoclonal antibodies (mAb) specific for the clonotype of an autoreactive T cell may be useful reagents in the modulation of autoimmune disease. We have previously reported the generation of a set of mAb specific for the clonotypic structure of a human T-cell clone recognizing an epitope of human cartilage gp-39. This glycoprotein was recently identified as a candidate autoantigen in rheumatoid arthritis. Here, we demonstrate for the first time that small amounts of immobilized anticlonotype mAb can induce anergy in the autoreactive clone. Following the anergic stimulus, T cells failed to proliferate upon restimulation as a result of a lack of interleukin-2 (IL-2) gene transcription. In addition, a diminished interferon-gamma (IFN-gamma) production was found. Our data indicate that anergy was not a result of T-cell receptor (TCR) downmodulation or the absence of free TCR. The anergic state was induced independent of costimulation or the presence of IL-2 and no protein synthesis was required for the induction of anergy. Anticlonotype mAb-induced anergy was prevented by cyclosporin A, suggesting that active signalling via the calcium/calcineurin pathway was required for the induction of anergy. In coculture experiments, anergic T cells were found to suppress the response of reactive cells from the same clone. This bystander suppression led to 90% inhibition of peptide-induced proliferation. Together, these findings suggest that mAb to the clonotypic structure of autoreactive T cells may be suitable reagents for the functional inactivation of these T cells in autoimmune diseases.
Assuntos
Anticorpos Monoclonais/imunologia , Anergia Clonal/imunologia , Linfócitos T/imunologia , Células Apresentadoras de Antígenos/imunologia , Técnicas de Cultura de Células , Sobrevivência Celular/imunologia , Anergia Clonal/efeitos dos fármacos , Cicloeximida/farmacologia , Ciclosporina/farmacologia , Humanos , Imunossupressores/farmacologia , Interferon gama/biossíntese , Interleucina-2/genética , Receptores de Antígenos de Linfócitos T/imunologia , Fatores de Tempo , Transcrição GênicaRESUMO
Mother and daughter sporocysts of Tricholbilharzia ocellata, developing in the snail host Lymnaea stagnalis, were searched for substances with antigenic similarities to the snail's haemolymph. Antisera to cell-free snail haemolymph and fractions thereof were used in three different immunocytochemical staining methods, applied on sections of parasitized snails. Snail tissue was consistently stained; cercariae were stained, indicating that the applied methods were successful. Most sections through mother and daughter sporocysts were completely unstained. It is concluded that neither mother nor daughter sporocysts are masked by the antigens studied or substances mimicking these. The relevance of the present observations is discussed.
Assuntos
Antígenos de Helmintos/análise , Antígenos/análise , Hemolinfa/imunologia , Lymnaea/imunologia , Schistosomatidae/imunologia , Animais , Patos/parasitologia , Epitopos , Soros Imunes , Imunodifusão , Técnicas Imunoenzimáticas , Coelhos/imunologiaRESUMO
In normal, healthy joints, synovial fibroblasts do not express major histocompatibility complex (MHC) class II molecules. However, in inflamed joints of rheumatoid arthritis (RA) patients, synovial fibroblasts show an abundant expression of MHC class II. Does this increase in expression have functional consequences for antigen presentation to T cells? To date, the precise role of synovial fibroblasts in antigen presentation has not been documented. Here, we show by three different examples that cultured synovial fibroblasts with interferon-gamma (IFN-gamma)-induced MHC class II expression are capable of processing soluble protein for presentation to CD4+ T cells. First, the antigen-presenting cell (APC) function of synovial fibroblasts was studied in an autologous model. From synovial tissue of a RA patient both a fibroblast cell line and a tetanus toxoid (TT)-specific CD4+ T-cell line were generated. A dose-dependent TT response was observed only when TT was presented by IFN-gamma-pretreated synovial fibroblasts. As more direct evidence for MHC class II-restricted antigen presentation, the response of a Mycobacterium tuberculosis-specific CD4+ T-cell clone isolated from rheumatoid synovial fluid was demonstrated in the presence of synovial fibroblasts. The response was DR4Dw4-restricted and could be inhibited by monoclonal antibody (mAb) to HLA-DR. In addition, the lymphokine secretion pattern of the synovial T-cell clone did not differ qualitatively upon antigen-specific stimulation using peripheral blood mononuclear cells (PBMC) or synovial fibroblasts as APC. In order to provide evidence for intracellular antigen processing we next examined the response of a M. leprae-specific T-cell clone with known epitope specificity. Our data suggest that synovial fibroblasts are not passive bystanders, but can become active participants in the development and maintenance of chronic inflammation.
Assuntos
Células Apresentadoras de Antígenos/imunologia , Artrite Reumatoide/imunologia , Membrana Sinovial/imunologia , Antígenos de Bactérias/imunologia , Antígenos de Superfície/análise , Linhagem Celular , Células Cultivadas , Relação Dose-Resposta Imunológica , Fibroblastos/imunologia , Antígenos HLA-DR/análise , Humanos , Interferon gama/imunologia , Proteínas Recombinantes , Toxoide Tetânico/imunologiaRESUMO
Immune responses to the infectious bronchitis virus (IBV) nucleocapsid protein were studied using a recombinant-DNA expression product. In mice, a lymphocyte proliferative response and a delayed-type hypersensitivity reaction to IBV were induced upon immunization with this nucleocapsid protein. Next, we studied the role of the expressed nucleocapsid protein in induction of a protective immune response to IBV in chickens. Chickens were primed with nucleocapsid protein and subsequently boosted with inactivated IBV, strain M41. Proliferative responses of blood mononuclear cells corresponded with increased mean haemagglutination inhibition and virus neutralization titres. Finally, an increased tracheal protection against challenge with live IBV was observed. These results indicate that infectious bronchitis virus nucleocapsid protein is a relevant target for immune recognition in both the mouse and the chicken.
Assuntos
Anticorpos Antivirais/biossíntese , Vírus da Bronquite Infecciosa/imunologia , Vacinas Virais/imunologia , Animais , Capsídeo/genética , Capsídeo/imunologia , Galinhas , DNA Recombinante , Feminino , Imunização , Vírus da Bronquite Infecciosa/genética , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos BALB C , Linfócitos T/imunologia , Vacinas Sintéticas/imunologiaRESUMO
Mice were immunized with purified infectious bronchitis virus (IBV), strain M41. Spleen cells, expanded in vitro by stimulation with M41, were immortalized by fusion to obtain T-cell hybridomas, and two major histocompatability complex (MHC) class II (I-E)-restricted T-cell hybridomas were selected with specificity for IBV. Both hybridomas selectively recognized the internal nucleocapsid protein. The responses to 12 different strains of IBV varied markedly. This demonstrates antigenic variation of the nucleocapsid protein in addition to the known variation of the surface glycoprotein S.
Assuntos
Antígenos Virais/imunologia , Capsídeo/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Vírus da Bronquite Infecciosa/imunologia , Linfócitos T/imunologia , Proteínas do Core Viral/imunologia , Animais , Hibridomas/imunologia , Interleucina-2/biossíntese , Camundongos , Camundongos Endogâmicos BALB CRESUMO
This study reports an identification of the major processing products of an exogenous protein antigen, viz, sperm-whale myoglobin, as obtained after cell-free processing with partially purified macrophage endosomes. It is demonstrated that such a system yields fragments that are indistinguishable by high performance liquid chromatography analysis from those generated after uptake of myoglobin inside live macrophages. The concerted action of the endosomal proteases cathepsin D and cathepsin B can account for nearly all cleavages observed. Cathepsin D appears to be mainly responsible for the initial cleavage of myoglobin, while cathepsin B catalyzes the C-terminal trimming of initially released fragments. The fragments released by cathepsin D contain most, if not all, major epitopes for murine myoglobin-specific helper T cells. Interestingly, each known T cell epitope of myoglobin is located at the very N terminus of a different myoglobin fragment released upon processing. In order to explain this correspondence, noted also in several other protein antigens, a structural relationship is proposed between antigen processing by cathepsin D and antigen recognition by major histocompatibility complex (MHC) class II products. As is demonstrated here, this relationship may be used as a predictive tool for the identification of MHC-binding sequences as well as of T cell epitopes in their naturally occurring form.
Assuntos
Endopeptidases/fisiologia , Mioglobina/imunologia , Linfócitos T/imunologia , Sequência de Aminoácidos , Animais , Sítios de Ligação de Anticorpos , Catepsina B/farmacologia , Catepsina D/farmacologia , Bovinos , Cromatografia Líquida de Alta Pressão , Epitopos , Antígenos de Histocompatibilidade Classe II/imunologia , Técnicas In Vitro , Ativação Linfocitária , Macrófagos/metabolismo , Dados de Sequência MolecularRESUMO
In a previous study, two murine T-cell hybridomas generated after immunization with infectious bronchitis virus (IBV) were shown to be responsive to the internally localized viral nucleocapsid protein. In the present study, the antigenic determinants were mapped using recombinant expression products and synthetic peptides. Both hybridomas recognized the region spanning amino acid residues 71 to 78 of the nucleocapsid protein. The experimentally determined epitope corresponded with predicted motifs. Both an I-Ed binding motif and a predicted cleavage site for the aspartyl protease cathepsin D were contained within the sequence. The epitope was shown to prime cellular immune responses to IBV in the chicken.
Assuntos
Capsídeo/imunologia , Epitopos/análise , Vírus da Bronquite Infecciosa/imunologia , Linfócitos T/imunologia , Sequência de Aminoácidos , Animais , Células Apresentadoras de Antígenos/imunologia , Galinhas , Feminino , Leucócitos Mononucleares/imunologia , Mitose , Dados de Sequência MolecularRESUMO
OBJECTIVE: Human cartilage glycoprotein 39 (HC gp-39) was recently identified as a candidate autoantigen in the pathogenesis of rheumatoid arthritis. In the present studies, we investigated the capacity of HC gp-39 to interfere in clinical disease induced by an unrelated autoantigen, type II collagen (CII), by the induction of cross-tolerance. METHODS: DBA-1j/Bom mice were immunized with bovine CII/complete Freund's adjuvant and were given intraperitoneal booster injections of CII on day 21. Tolerance was induced via the intranasal pathway with either the disease-inducing antigen (CII), a control antigen (ovalbumin), or HC gp-39 either before priming with CII or near the day of the booster injection. Arthritis was monitored visually, and joint pathology was examined histologically and radiologically. In addition, CII antibody levels in serum were analyzed by enzyme-linked immunosorbent assay. RESULTS: In contrast to treatment before priming, intranasal application of HC gp-39 after immunization markedly suppressed disease activity and prevented joint destruction, whereas application of ovalbumin or CII was ineffective. Interference of HC gp-39 with the immune response to CII was demonstrated by decreased anti-CII antibody levels. The combined data indicate that intranasal treatment with HC gp-39 may trigger modulatory or regulatory mechanisms that interfere with the expression of disease in murine collagen-induced arthritis. CONCLUSION: HC gp-39 is the first cross-tolerance-inducing protein in arthritis that down-modulates a spectrum of disease features when given in a semitherapeutic protocol.
Assuntos
Artrite/tratamento farmacológico , Glicoproteínas/administração & dosagem , Adipocinas , Administração Intranasal , Animais , Anticorpos/efeitos dos fármacos , Anticorpos/metabolismo , Artrite/induzido quimicamente , Proteína 1 Semelhante à Quitinase-3 , Colágeno/imunologia , Regulação para Baixo/efeitos dos fármacos , Tolerância a Medicamentos , Feminino , Glicoproteínas/uso terapêutico , Humanos , Articulações/patologia , Lectinas , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos DBARESUMO
OBJECTIVE: To analyze the CD4+ T cell responses to the human cartilage antigen glycoprotein-39 (HCgp-39) in the context of rheumatoid arthritis (RA)-associated (DRalphabeta1*0401) and nonassociated (DRalphabeta1*0402) HLA class II molecules. METHODS: Large numbers of HCgp-39-specific T cell hybridomas were generated following immunization of HLA-DR4/human CD4 transgenic, murine major histocompatibility complex class II deficient mice with native HCgp-39. Fine epitope mapping of DRalphabeta1*0401-and DRalphabeta1*0402-restricted T cell hybridomas was performed using overlapping synthetic peptides. Antigen-specific cytokine production by lymph node T cells was evaluated after immunization with native antigen. Proliferative T cell responses of healthy human subjects were compared with the T cell responses of patients with active RA using HCgp-39 epitopes defined in HLA-DR4 transgenic mice. RESULTS: CD4+ T cells from DRalphabeta1*0401 and DRalphabeta1*0402 transgenic mice identified completely different immunodominant peptide epitopes of HCgp-39, and this was not explained by known DR4-binding motifs or direct peptide-binding studies. DRalphabeta1*0401-restricted, antigen-specific T cells produced significantly more interferon-gamma and tumor necrosis factor a in response to HCgp-39 than did T cells from DRalphabeta1*0402 transgenic mice. Finally, HCgp-39 peptides defined in DRalphabeta1*0401 transgenic mice stimulated T cells from HLA-DR4 positive human subjects and RA patients, but not T cells from HLA-DR4 negative individuals. CONCLUSION: T cell epitopes of HCgp-39 that were defined in HLA-DR4 transgenic mice stimulated T cells from human subjects carrying RA-associated HLA-DR4 alleles. HLA-DR4 molecules may influence the disease process in RA both by presentation of selected peptide epitopes and by promoting the production of proinflammatory cytokines in synovial joints.
Assuntos
Artrite Reumatoide/imunologia , Cartilagem/imunologia , Antígeno HLA-DR4/imunologia , Linfócitos T/imunologia , Adipocinas , Alelos , Animais , Autoantígenos , Proteína 1 Semelhante à Quitinase-3 , Citocinas/biossíntese , Mapeamento de Epitopos , Epitopos de Linfócito T/química , Feminino , Glicoproteínas/imunologia , Humanos , Epitopos Imunodominantes/imunologia , Lectinas , Masculino , Camundongos , Camundongos TransgênicosRESUMO
The pathogenesis of joint destruction in rheumatoid arthritis remains ill-defined. Joint destruction is thought to be the result of tissue damage mediated by T cells. The mere presence of articular cartilage appears responsible for sustaining chronic synovitis and thereby forwards a role for cartilage-responsive T cells in RA. Taking advantage of the positive DRB1*0401 association with RA susceptibility, we reasoned that T-cell recognition of autoantigens in RA would be restricted by DRB1*0401-encoded molecules. A DR4 (B1*0401) peptide binding motif was used for the identification of putative T-cell epitopes within human aggrecan, a candidate autoantigen. Thirteen peptides were synthesized and tested for binding DRB1*0401 or 0404-encoded molecules. Selected binders were tested for induction of proliferative responses in peripheral blood mononuclear cells from donors carrying the DR4 or DR1 specificity. Both healthy and RA donors responded to human aggrecan-derived peptides, thereby identifying these sequences as T-cell epitopes. Interestingly, responses to aggrecan-derived epitopes were significantly decreased in RA patients compared to controls. This was not due to an overall hyporesponsiveness of RA patients since responses to a recall antigen or mitogen did not differ from controls. The data suggest that in RA, aggrecan-specific T cells may exist in a different stage of activation or may have left the periphery to home to the joint.