RESUMEN
The presence of the PTPN22 risk allele (1858T) is associated with several autoimmune diseases including rheumatoid arthritis (RA). Despite a number of studies exploring the function of PTPN22 in T cells, the exact impact of the PTPN22 risk allele on T-cell function in humans is still unclear. In this study, using RNA sequencing, we show that, upon TCR-activation, naïve human CD4+ T cells homozygous for the PTPN22 risk allele overexpress a set of genes including CFLAR and 4-1BB, which are important for cytotoxic T-cell differentiation. Moreover, the protein expression of the T-box transcription factor Eomesodermin (EOMES) was increased in T cells from healthy donors homozygous for the PTPN22 risk allele and correlated with a decreased number of naïve CD4+ T cells. There was no difference in the frequency of other CD4+ T-cell subsets (Th1, Th17, Tfh, Treg). Finally, an accumulation of EOMES+ CD4+ T cells was observed in synovial fluid of RA patients with a more pronounced production of Perforin-1 in PTPN22 risk allele carriers. Altogether, we propose a novel mechanism of action of PTPN22 risk allele through the generation of cytotoxic CD4+ T cells and identify EOMES+ CD4+ T cells as a relevant T-cell subset in RA pathogenesis.
Asunto(s)
Artritis Reumatoide/patología , Linfocitos T CD4-Positivos/inmunología , Proteína Tirosina Fosfatasa no Receptora Tipo 22/genética , Proteínas de Dominio T Box/metabolismo , Linfocitos T Citotóxicos/inmunología , Ligando 4-1BB/biosíntesis , Artritis Reumatoide/genética , Secuencia de Bases , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/biosíntesis , Diferenciación Celular/inmunología , Humanos , Perforina/biosíntesis , Receptores de Antígenos de Linfocitos T/inmunología , Análisis de Secuencia de ARN , Líquido Sinovial/citología , Linfocitos T Citotóxicos/citologíaRESUMEN
Treg cells are important for the maintenance of self-tolerance and are implicated in autoimmunity. Despite enrichment of Treg cells in joints of rheumatoid arthritis (RA) patients, local inflammation persists. As expression of the ATP-hydrolyzing enzymes CD39 and CD73 and the resulting anti-inflammatory adenosine production have been implicated as an important mechanism of suppression, we characterized FOXP3(+) Treg cells in blood and synovial fluid samples of RA patients in the context of CD39 and CD73 expression. Synovial FOXP3(+) Treg cells displayed high expression levels of rate-limiting CD39, whereas CD73 was diminished. FOXP3(+) CD39(+) Treg cells were also abundant in synovial tissue. Furthermore, FOXP3(+) CD39(+) Treg cells did not secrete the proinflammatory cytokines IFN-γ and TNF after in vitro stimulation in contrast to FOXP3(+) CD39(-) T cells. FOXP3(+) CD39(+) Treg cells could be isolated by CD39 and CD25 coexpression, displayed a demethylated Treg-specific demethylated region and coculture assays confirmed that CD25(+) CD39(+) T cells have suppressive capacity, while their CD39(-) counterparts do not. Overall, our data show that FOXP3(+) CD39(+) Treg cells are enriched at the site of inflammation, do not produce proinflammatory cytokines, and are good suppressors of many effector T-cell functions including production of IFN-γ, TNF, and IL-17F but do not limit IL-17A secretion.
Asunto(s)
Antígenos CD/biosíntesis , Apirasa/biosíntesis , Artritis Reumatoide/inmunología , Interleucina-17/metabolismo , Subgrupos de Linfocitos T/inmunología , Adulto , Anciano , Anciano de 80 o más Años , Antígenos CD/inmunología , Apirasa/inmunología , Artritis Reumatoide/metabolismo , Separación Celular , Femenino , Citometría de Flujo , Humanos , Interleucina-17/inmunología , Masculino , Microscopía Confocal , Persona de Mediana Edad , Líquido Sinovial/inmunología , Subgrupos de Linfocitos T/metabolismoRESUMEN
BACKGROUND: Rheumatoid arthritis is a chronic inflammatory disease with a strong MHC class II component and where many patients develop characteristic autoantibodies towards the noncoding amino acid citrulline. Such anti-citrullinated protein antibodies (ACPA) have recently been put forward as an independent predictive factor for treatment response by co-stimulation blockade by CTLA4-Ig (abatacept). We have performed a mechanism of action study to dissect T cell functionality in RA patients with long-standing disease undergoing abatacept treatment and the influence of ACPA status. RESULTS: Peripheral blood samples were collected from RA patients as they started CTLA4-Ig treatment and 3 and 6 months later. A general decrease of regulatory T cell subsets was observed in the cohort. Additionally within the ACPA-positive group significant down-regulation of all key T cell effector subsets including Th1, Th2, and Th17 was observed by analyzing cytokines by intracellular flow cytometry and in cell culture supernatants.RA synovial fluid samples were cultured in vitro in the presence or absence of CTLA4-Ig (abatacept). T cell cytokine production was diminished, but without increasing the functional capacity of CD4+CD25hi regulatory T cells as previously demonstrated in the context of TNF-blockade and anti-IL6R therapy. CONCLUSIONS: Our immunological study of T cell functionality in RA patients, both ACPA-positive and ACPA-negative starting biological therapy with the co-stimulation blockade abatacept (CTLA4-Ig) supports the recently published registry study implicating ACPA seropositivity as an independent predictive factor to treatment response as we observed the most striking effect on T cell subset modulation in ACPA-positive patients. These data further support the notion of RA as a disease with several sub-entities, where the ACPA-positive fraction represents a classical HLA-associated autoimmune disorder while ACPA-negative patients may have other driving forces apart from classical adaptive immune responses.
Asunto(s)
Artritis Reumatoide/tratamiento farmacológico , Artritis Reumatoide/inmunología , Autoanticuerpos/inmunología , Inmunoconjugados/uso terapéutico , Linfocitos T/inmunología , Abatacept , Adolescente , Adulto , Anciano , Artritis Reumatoide/sangre , Autoanticuerpos/sangre , Proliferación Celular/efectos de los fármacos , Citrulina/inmunología , Estudios de Cohortes , Citocinas/metabolismo , Femenino , Citometría de Flujo , Humanos , Inmunoconjugados/farmacología , Recuento de Linfocitos , Masculino , Persona de Mediana Edad , Membrana Sinovial/efectos de los fármacos , Membrana Sinovial/inmunología , Membrana Sinovial/patología , Subgrupos de Linfocitos T/efectos de los fármacos , Subgrupos de Linfocitos T/inmunología , Linfocitos T/efectos de los fármacos , Adulto JovenRESUMEN
Regulatory T cells (Tregs) are important for maintaining immune homeostasis, but many studies suggest that Tregs are functionally impaired in autoimmune and chronic inflammatory disorders. In addition, effector T cells may vary in sensitivity toward Treg suppression. Herein, we have studied the interplay between T effectors and Tregs in the rheumatic joint. Synovial Tregs demonstrated a high degree of FOXP3 demethylation and displayed only marginal IL-17 and virtually no IFN-γ production following in vitro stimulation, altogether indicating suppressive capacity. Still, the frequency of FOXP3 expression could not predict the degree of suppression. Instead, the inflammatory milieu in the joint, i.e. proliferative capacity of effector T cells and in situ levels of pro-inflammatory cytokines influenced Treg function. Indeed, blocking IL-6 or TNF increased the suppression by Tregs in co-cultures. Additionally, approximately 30% of the synovial FOXP3(+) T cells were Ki67(+) and hence actively dividing, but proliferation did not overlap with cytokine production, suggesting that these cells represent functional Tregs having met their cognate antigen and expanded in an attempt to alleviate joint inflammation. Overall, our data argue against a general functional deficit in joint-derived Tregs and instead emphasize the importance of the inflammatory milieu to set the threshold for immune regulation.
Asunto(s)
Inflamación/inmunología , Artropatías/inmunología , Enfermedades Reumáticas/inmunología , Linfocitos T Reguladores/inmunología , Adulto , Anciano , Antígenos CD2/inmunología , Antígenos CD2/metabolismo , Células Cultivadas , Técnicas de Cocultivo , Metilación de ADN , Femenino , Citometría de Flujo , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/inmunología , Factores de Transcripción Forkhead/metabolismo , Humanos , Inflamación/metabolismo , Inflamación/patología , Interferón gamma/inmunología , Interferón gamma/metabolismo , Subunidad alfa del Receptor de Interleucina-2/inmunología , Subunidad alfa del Receptor de Interleucina-2/metabolismo , Interleucina-6/inmunología , Interleucina-6/metabolismo , Artropatías/metabolismo , Artropatías/patología , Antígeno Ki-67/inmunología , Antígeno Ki-67/metabolismo , Masculino , Persona de Mediana Edad , Regiones Promotoras Genéticas/genética , Enfermedades Reumáticas/metabolismo , Enfermedades Reumáticas/patología , Líquido Sinovial/inmunología , Líquido Sinovial/metabolismo , Linfocitos T/inmunología , Linfocitos T/metabolismo , Linfocitos T Reguladores/metabolismo , Factor de Necrosis Tumoral alfa/inmunología , Factor de Necrosis Tumoral alfa/metabolismo , Adulto JovenRESUMEN
OBJECTIVE: Autoantibodies targeting histidyl-transfer RNA synthetase (HisRS; anti-Jo-1) are common in the idiopathic inflammatory myopathies (IIMs) and antisynthetase syndrome. This study was undertaken to investigate immunity against HisRS in the blood and lungs of patients with IIM/antisynthetase syndrome. METHODS: Bronchoalveolar lavage (BAL) fluid, BAL fluid cells, and peripheral blood mononuclear cells (PBMCs) from patients with IIM/antisynthetase syndrome (n = 24) were stimulated with full-length HisRS protein or a HisRS-derived peptide (HisRS11-23 ). BAL fluid and PBMCs from patients with sarcoidosis (n = 7) and healthy subjects (n = 12) were included as controls. The CD4+ T cell response was determined according to levels of CD40L up-regulation and cytokine expression using flow cytometry. Anti-Jo-1 autoantibody responses in the serum and BAL fluid were assessed by enzyme-linked immunosorbent assay. Lung biopsy samples from patients with IIM/antisynthetase syndrome (n = 14) were investigated by immunohistochemistry. RESULTS: In BAL fluid, CD4+ T cells from 3 of 4 patients with IIM/antisynthetase syndrome responded to stimulation with HisRS protein, as measured by the median fold change in CD40L expresssion in stimulated cells compared to unstimulated cells (median fold change 3.6, interquartile range [IQR] 2.7-14.7), and 2 of 3 patients with IIM/antisynthetase syndrome had the highest responses to HisRS11-23 (median fold change 88, IQR 27-149). In PBMCs, CD4+ T cells from 14 of 18 patients with IIM/antisynthetase syndrome responded to HisRS protein (median fold change 7.38, IQR 2.69-31.86; P < 0.001), whereas a HisRS11-23 response was present in 11 of 14 patients with IIM/antisynthetase syndrome (median fold change 3.4, IQR 1.87-10.9; P < 0.001). In the control group, there was a HisRS11-23 response in 3 of 7 patients with sarcoidosis (median fold change 2.09, IQR 1.45-3.29) and in 5 of 12 healthy controls (median fold change 2, IQR 1.89-2.42). CD4+ T cells from patients with IIM/antisynthetase syndrome displayed a pronounced Th1 phenotype in the BAL fluid when compared to the PBMCs (P < 0.001), producing high amounts of interferon-γ and interleukin-2 following stimulation. Anti-Jo-1 autoantibodies were detected in BAL fluid and germinal center (GC)-like structures were seen in the lung biopsy samples from patients with IIM/antisynthetase syndrome. CONCLUSION: The results of this study demonstrate a pronounced presence of HisRS-reactive CD4+ T cells in PBMCs and BAL fluid cells from patients with IIM/antisynthetase syndrome as compared to patients with sarcoidosis and healthy controls. These findings, combined with the presence of anti-Jo-1 autoantibodies in BAL fluid and GC-like structures in the lungs, suggest that immune activation against HisRS might take place within the lungs of patients with IIM/antisynthetase syndrome.
Asunto(s)
Anticuerpos Antinucleares/inmunología , Linfocitos T CD4-Positivos/inmunología , Enfermedades Pulmonares Intersticiales/inmunología , Pulmón/inmunología , Monocitos/inmunología , Miositis/inmunología , Adulto , Anciano , Anciano de 80 o más Años , Anticuerpos Antinucleares/sangre , Líquido del Lavado Bronquioalveolar/citología , Líquido del Lavado Bronquioalveolar/inmunología , Femenino , Histidina-ARNt Ligasa/inmunología , Humanos , Interferón gamma/inmunología , Interleucina-2/inmunología , Pulmón/citología , Pulmón/patología , Enfermedades Pulmonares Intersticiales/patología , Masculino , Persona de Mediana Edad , Miositis/sangre , Células TH1RESUMEN
OBJECTIVE: Antibodies against citrullinated type II collagen (Cit-CII) are common in the sera and synovial fluid of patients with rheumatoid arthritis (RA); however, the known T cell epitope of CII is not dependent on citrullination. The aim of this study was to identify and functionally characterize the Cit-CII-restricted T cell epitopes that are relevant to RA. METHODS: Peripheral blood mononuclear cells (PBMCs) from HLA-DRB1*10:01-positive patients with RA and healthy donors were stimulated in vitro with candidate CII peptides. CD154 up-regulation was measured as a marker of antigen-specific activation, and anti-HLA-DR-blocking experiments confirmed HLA restriction. Cytokine production was measured using a Luminex technique. Direct peptide-binding assays using HLA-DRB1*10:01 and HLA-DRB1*04:01 monomeric proteins were performed. The T cell receptor (TCR) ß-chain of CD154-enriched antigen-specific T cells was analyzed using high-throughput sequencing. RESULTS: A novel Cit-CII peptide was identified based on its ability to activate CD4+ T cells from HLA-DRB1*10:01-positive individuals. When stimulated in vitro, Cit-CII autoreactive T cells produced proinflammatory cytokines. Cit-CII(311-325) bound (with low affinity) to HLA-DRB1*10:01 but not to HLA-DRB1*04:01, while the native form was unable to bind either protein. In addition, highly expanded clones were identified in the TCRß repertoire of Cit-CII(311-325) -stimulated PBMCs. CONCLUSION: These results illustrate the ability of the citrullination process to create T cell epitopes from CII, a cartilage-restricted protein that is relevant to RA pathogenesis. The exclusive binding of Cit-CII(311-325) to HLA-DRB1*10:01 suggests that recognition of citrullinated epitopes might vary between individuals carrying different RA-associated HLA-DR molecules.
Asunto(s)
Artritis Reumatoide/inmunología , Citrulina/inmunología , Colágeno Tipo II/inmunología , Epítopos de Linfocito T/inmunología , Cadenas HLA-DRB1/inmunología , Adulto , Anciano , Estudios de Casos y Controles , Citrulina/metabolismo , Colágeno Tipo II/metabolismo , Citocinas/inmunología , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Técnicas In Vitro , Leucocitos Mononucleares/inmunología , Masculino , Persona de Mediana Edad , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Análisis de Secuencia de ADNRESUMEN
Mutations of the gene encoding four-and-a-half LIM domain 1 (FHL1) are the causative factor of several X-linked hereditary myopathies that are collectively termed FHL1-related myopathies. These disorders are characterized by severe muscle dysfunction and damage. Here, we have shown that patients with idiopathic inflammatory myopathies (IIMs) develop autoimmunity to FHL1, which is a muscle-specific protein. Anti-FHL1 autoantibodies were detected in 25% of IIM patients, while patients with other autoimmune diseases or muscular dystrophies were largely anti-FHL1 negative. Anti-FHL1 reactivity was predictive for muscle atrophy, dysphagia, pronounced muscle fiber damage, and vasculitis. FHL1 showed an altered expression pattern, with focal accumulation in the muscle fibers of autoantibody-positive patients compared with a homogeneous expression in anti-FHL1-negative patients and healthy controls. We determined that FHL1 is a target of the cytotoxic protease granzyme B, indicating that the generation of FHL1 fragments may initiate FHL1 autoimmunity. Moreover, immunization of myositis-prone mice with FHL1 aggravated muscle weakness and increased mortality, suggesting a direct link between anti-FHL1 responses and muscle damage. Together, our findings provide evidence that FHL1 may be involved in the pathogenesis not only of genetic FHL1-related myopathies but also of autoimmune IIM. Importantly, these results indicate that anti-FHL1 autoantibodies in peripheral blood have promising potential as a biomarker to identify a subset of severe IIM.