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Disturbances in immune regulation, intestinal dysbiosis and inflammation characterize ankylosing spondylitis (AS), which is associated with RUNX3 loss-of-function variants. ZAP70W163C mutant (SKG) mice have reduced ZAP70 signaling, spondyloarthritis and ileitis. In small intestine, Foxp3+ regulatory T cells (Treg) and CD4+CD8αα+TCRαß+ intraepithelial lymphocytes (CD4-IEL) control inflammation. TGF-ß and retinoic acid (RA)-producing dendritic cells and MHC-class II+ intestinal epithelial cells (IEC) are required for Treg and CD4-IEL differentiation from CD4+ conventional or Treg precursors, with upregulation of Runx3 and suppression of ThPOK. We show in SKG mouse ileum, that ZAP70W163C or ZAP70 inhibition prevented CD4-IEL but not Treg differentiation, dysregulating Runx3 and ThPOK. TGF-ß/RA-mediated CD4-IEL development, T-cell IFN-γ production, MHC class-II+ IEC, tissue-resident memory T-cell and Runx3-regulated genes were reduced. In AS intestine, CD4-IEL were decreased, while in AS blood CD4+CD8+ T cells were reduced and Treg increased. Thus, genetically-encoded TCR signaling dysfunction links intestinal T-cell immunodeficiency in mouse and human spondyloarthropathy.
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Linfócitos T CD8-Positivos , Subunidade alfa 3 de Fator de Ligação ao Core , Espondiloartropatias , Animais , Humanos , Camundongos , Linfócitos T CD4-Positivos , Subunidade alfa 3 de Fator de Ligação ao Core/genética , Inflamação , Mucosa Intestinal , Intestinos , Receptores de Antígenos de Linfócitos T alfa-beta , Espondiloartropatias/genética , Fator de Crescimento Transformador betaRESUMO
The complexity of the cellular and acellular players within the tumor microenvironment (TME) allows for significant variation in TME constitution and role in anticancer treatment response. Spatial alterations in populations of tumor cells and adjacent non-malignant cells, including endothelial cells, fibroblasts and tissue-infiltrating immune cells, often have a major role in determining disease progression and treatment response in cancer. Many current standard systemic antineoplastic treatments target the cancer cells and could be further refined to directly target commonly dysregulated cell populations of the TME. Recent developments in immuno-oncology and bioengineering have created an attractive potential to model these complexities at the level of the individual patient. These developments, along with the increasing momentum in precision medicine research and application, have catalysed exciting new discoveries in understanding drug-TME interactions, target identification, and improved efficacy of therapies. While rapid progress has been made, there are still many challenges to overcome in the development of accurate in vitro, in vivo and ex vivo models incorporating the cellular interactions that take place in the TME. In this review, we describe how advances in immuno-oncology and patient-derived models, such as patient-derived organoids and explant cultures, have enhanced the landscape of personalised immunotherapy prediction and treatment of solid organ malignancies. We describe and compare different immunological targets and perspectives on two-dimensional and three-dimensional modelling approaches that may be used to better rationalise immunotherapy use, ultimately providing a knowledge base for the integration of the autologous TME into these predictive models.
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BACKGROUND: The human enthesis conventional T cells are poorly characterised. OBJECTIVES: To study the biology of the conventional T cells in human enthesis. METHODS: CD4+ and CD8+ T cells were investigated in 25 enthesis samples using immunofluorescence, cytometrically, bulk RNAseq and quantitative real-time PCR following anti-CD3/CD28 bead stimulation to determine interleukin (IL)-17A and tumour necrosis factor (TNF) levels. T-cell receptor (TCR) repertoires were characterised and a search for putative T-cell reactivity was carried out using TCR3 database. The impact of pharmacological antagonism with retinoic acid receptor-related orphan nuclear receptor gamma t inhibitor (RORγti), methotrexate and phosphodiesterase type 4 inhibitor (PDE4i) was investigated. RESULTS: Immunofluorescence and cytometry suggested entheseal resident CD4+ and CD8+ T cells with a resident memory phenotype (CD69+/CD45RA-) and tissue residency gene transcripts (higher NR4A1/AhR and lower KLF2/T-bet transcripts). Both CD4+ and CD8+ T cells showed increased expression of immunomodulatory genes including IL-10 and TGF-ß compared with peripheral blood T cells with entheseal CD8+ T cells having higher CD103, CD49a and lower SIPR1 transcript that matched CD4+ T cells. Following stimulation, CD4+ T cells produced more TNF than CD8+ T cells and IL-17A was produced exclusively by CD4+ T cells. RNAseq suggested both Cytomegalovirus and influenza A virus entheseal resident T-cell clonotype reactivity. TNF and IL-17A production from CD4+ T cells was effectively inhibited by PDE4i, while RORγti only reduced IL-17A secretion. CONCLUSIONS: Healthy human entheseal CD4+ and CD8+ T cells exhibit regulatory characteristics and are predicted to exhibit antiviral reactivity with CD8+ T cells expressing higher levels of transcripts suggestive of tissue residency. Inducible IL-17A and TNF production can be robustly inhibited in vitro.
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Linfócitos T CD8-Positivos/imunologia , Ligamentos Articulares/imunologia , Linfócitos T Reguladores/imunologia , Tendões/imunologia , Adulto , Linfócitos T CD4-Positivos/imunologia , Feminino , Humanos , Interleucina-17/imunologia , Masculino , Pessoa de Meia-Idade , Fator de Necrose Tumoral alfa/imunologiaRESUMO
OBJECTIVE: Ankylosing spondylitis (AS) is a common spondyloarthropathy primarily affecting the axial skeleton and strongly associated with HLA-B*27 carriage. Genetic evidence implicates both autoinflammatory processes and autoimmunity against an HLA-B*27-restricted autoantigen in immunopathology. In addition to articular symptoms, up to 70% of AS patients present with concurrent bowel inflammation, suggesting that adverse interactions between a genetically primed host immune system and the gut microbiome contribute to the disease. Accordingly, this study aimed to characterize adaptive immune responses to antigenic stimuli in AS. METHODS: The peripheral CD4 and CD8 T cell receptor (TCR) repertoire was profiled in AS patients (n = 47) and HLA-B*27-matched healthy controls (n = 38). Repertoire diversity was estimated using the Normalized Shannon Diversity Entropy (NSDE) index, and univariate and multivariate statistical analyses were performed to characterize AS-associated clonal signatures. Furthermore, T cell proliferation and cytokine production in response to immunogenic antigen exposure were investigated in vitro in peripheral blood mononuclear cells from AS patients (n = 19) and HLA-B*27-matched healthy controls (n = 14). RESULTS: Based on the NSDE measure of sample diversity across CD4 and CD8 T cell repertoires, AS patients showed increased TCR diversity compared to healthy controls (for CD4 T cells, P = 7.8 × 10-6 ; for CD8 T cells, P = 9.3 × 10-4 ), which was attributed to a significant reduction in the magnitude of peripheral T cell expansions globally. Upon in vitro stimulation, fewer T cells from AS patients than from healthy controls expressed interferon-γ (for CD8 T cells, P = 0.03) and tumor necrosis factor (for CD4 T cells, P = 0.01; for CD8 T cells, P = 0.002). In addition, the CD8 TCR signature was altered in HLA-B*27+ AS patients compared to healthy controls, with significantly expanded Epstein-Barr virus-specific clonotypes (P = 0.03) and cytomegalovirus-specific clonotypes (P = 0.02). HLA-B*27+ AS patients also showed an increased incidence of "public" CD8 TCRs, representing identical clonotypes emerging in response to common antigen encounters, including homologous clonotypes matching those previously isolated from individuals with bacterial-induced reactive arthritis. CONCLUSION: The dynamics of peripheral T cell responses in AS patients are altered, suggesting that differential antigen exposure and disrupted adaptive immunity are underlying features of the disease.
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Imunidade Adaptativa/genética , Variação Antigênica/genética , Receptores de Antígenos de Linfócitos T/imunologia , Espondilite Anquilosante/genética , Espondilite Anquilosante/imunologia , Adulto , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Estudos de Casos e Controles , Entropia , Feminino , Antígeno HLA-B27/genética , Antígeno HLA-B27/imunologia , Humanos , Leucócitos Mononucleares , MasculinoRESUMO
Susceptibility to ankylosing spondylitis (AS) is polygenic with more than 100 genes identified to date. These include HLA-B27 and the aminopeptidases (ERAP1, ERAP2, and LNPEPS), which are involved in antigen processing and presentation to T-cells, and several genes (IL23R, IL6R, STAT3, JAK2, IL1R1/2, IL12B, and IL7R) involved in IL23 driven pathways of inflammation. AS is also strongly associated with polymorphisms in two transcription factors, RUNX3 and T-bet (encoded by TBX21), which are important in T-cell development and function. The influence of these genes on the pathogenesis of AS and their potential for identifying drug targets is discussed here.
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Subunidade alfa 3 de Fator de Ligação ao Core/genética , Regulação da Expressão Gênica/imunologia , Interleucina-23/metabolismo , Espondilite Anquilosante/imunologia , Proteínas com Domínio T/genética , Aminopeptidases/genética , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Subunidade alfa 3 de Fator de Ligação ao Core/antagonistas & inibidores , Subunidade alfa 3 de Fator de Ligação ao Core/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Antígeno HLA-B27/genética , Humanos , Fatores Imunológicos/farmacologia , Fatores Imunológicos/uso terapêutico , Interleucina-23/imunologia , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Terapia de Alvo Molecular/métodos , Polimorfismo de Nucleotídeo Único , Receptores de Interleucina/imunologia , Receptores de Interleucina/metabolismo , Espondilite Anquilosante/genética , Proteínas com Domínio T/antagonistas & inibidores , Proteínas com Domínio T/metabolismoRESUMO
Viral respiratory infections are usually mild and self-limiting; still they exceptionally result in life-threatening infections in previously healthy children. To investigate a potential genetic cause, we recruited 120 previously healthy children requiring support in intensive care because of a severe illness caused by a respiratory virus. Using exome and transcriptome sequencing, we identified and characterized three rare loss-of-function variants in IFIH1, which encodes an RIG-I-like receptor involved in the sensing of viral RNA. Functional testing of the variants IFIH1 alleles demonstrated that the resulting proteins are unable to induce IFN-ß, are intrinsically less stable than wild-type IFIH1, and lack ATPase activity. In vitro assays showed that IFIH1 effectively restricts replication of human respiratory syncytial virus and rhinoviruses. We conclude that IFIH1 deficiency causes a primary immunodeficiency manifested in extreme susceptibility to common respiratory RNA viruses.
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Predisposição Genética para Doença/genética , Síndromes de Imunodeficiência/genética , Helicase IFIH1 Induzida por Interferon/genética , Interferon beta/biossíntese , Vírus Sinciciais Respiratórios/imunologia , Infecções Respiratórias/virologia , Rhinovirus/imunologia , Adenosina Trifosfatases/genética , Pré-Escolar , Cuidados Críticos , Feminino , Variação Genética/genética , Humanos , Síndromes de Imunodeficiência/imunologia , Lactente , Recém-Nascido , Interferon beta/imunologia , Masculino , Estudos Prospectivos , Isoformas de Proteínas/genética , Infecções Respiratórias/imunologia , Replicação Viral/imunologiaRESUMO
BACKGROUND: Acromelic dysplasias are a group of disorders characterised by short stature, brachydactyly, limited joint extension and thickened skin and comprises acromicric dysplasia (AD), geleophysic dysplasia (GD), Myhre syndrome and Weill-Marchesani syndrome. Mutations in several genes have been identified for these disorders (including latent transforming growth factor ß (TGF-ß)-binding protein-2 (LTBP2), ADAMTS10, ADAMSTS17 and fibrillin-1 (FBN1) for Weill-Marchesani syndrome, ADAMTSL2 for recessive GD and FBN1 for AD and dominant GD), encoding proteins involved in the microfibrillar network. However, not all cases have mutations in these genes. METHODS: Individuals negative for mutations in known acromelic dysplasia genes underwent whole exome sequencing. RESULTS: A heterozygous missense mutation (exon 14: c.2087C>G: p.Ser696Cys) in latent transforming growth factor ß (TGF-ß)-binding protein-3 (LTBP3) was identified in a dominant AD family. Two distinct de novo heterozygous LTPB3 mutations were also identified in two unrelated GD individuals who had died in early childhood from respiratory failure-a donor splice site mutation (exon 12 c.1846+5G>A) and a stop-loss mutation (exon 28: c.3912A>T: p.1304*Cysext*12). CONCLUSIONS: The constellation of features in these AD and GD cases, including postnatal growth retardation of long bones and lung involvement, is reminiscent of the null ltbp3 mice phenotype. We conclude that LTBP3 is a novel component of the microfibrillar network involved in the acromelic dysplasia spectrum.
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Doenças do Desenvolvimento Ósseo/genética , Proteínas de Ligação a TGF-beta Latente/genética , Deformidades Congênitas dos Membros/genética , Mutação de Sentido Incorreto/genética , Exoma/genética , Éxons/genética , Fibrilina-1/genética , Heterozigoto , Humanos , Proteínas dos Microfilamentos/genética , Mutação , Fenótipo , Fator de Crescimento Transformador beta/genética , Síndrome de Weill-Marchesani/genéticaRESUMO
BACKGROUND: Ankylosing spondylitis (AS) is an immune-mediated arthritis particularly targeting the spine and pelvis and is characterised by inflammation, osteoproliferation and frequently ankylosis. Current treatments that predominately target inflammatory pathways have disappointing efficacy in slowing disease progression. Thus, a better understanding of the causal association and pathological progression from inflammation to bone formation, particularly whether inflammation directly initiates osteoproliferation, is required. METHODS: The proteoglycan-induced spondylitis (PGISp) mouse model of AS was used to histopathologically map the progressive axial disease events, assess molecular changes during disease progression and define disease progression using unbiased clustering of semi-quantitative histology. PGISp mice were followed over a 24-week time course. Spinal disease was assessed using a novel semi-quantitative histological scoring system that independently evaluated the breadth of pathological features associated with PGISp axial disease, including inflammation, joint destruction and excessive tissue formation (osteoproliferation). Matrix components were identified using immunohistochemistry. RESULTS: Disease initiated with inflammation at the periphery of the intervertebral disc (IVD) adjacent to the longitudinal ligament, reminiscent of enthesitis, and was associated with upregulated tumor necrosis factor and metalloproteinases. After a lag phase, established inflammation was temporospatially associated with destruction of IVDs, cartilage and bone. At later time points, advanced disease was characterised by substantially reduced inflammation, excessive tissue formation and ectopic chondrocyte expansion. These distinct features differentiated affected mice into early, intermediate and advanced disease stages. Excessive tissue formation was observed in vertebral joints only if the IVD was destroyed as a consequence of the early inflammation. Ectopic excessive tissue was predominantly chondroidal with chondrocyte-like cells embedded within collagen type II- and X-rich matrix. This corresponded with upregulation of mRNA for cartilage markers Col2a1, sox9 and Comp. Osteophytes, though infrequent, were more prevalent in later disease. CONCLUSIONS: The inflammation-driven IVD destruction was shown to be a prerequisite for axial disease progression to osteoproliferation in the PGISp mouse. Osteoproliferation led to vertebral body deformity and fusion but was never seen concurrent with persistent inflammation, suggesting a sequential process. The findings support that early intervention with anti-inflammatory therapies will be needed to limit destructive processes and consequently prevent progression of AS.
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Modelos Animais de Doenças , Progressão da Doença , Osteogênese/fisiologia , Espondilite Anquilosante/etiologia , Espondilite Anquilosante/patologia , Animais , Feminino , Inflamação/complicações , Inflamação/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos KnockoutRESUMO
INTRODUCTION: Single nucleotide polymorphisms in ERAP2 are strongly associated with ankylosing spondylitis (AS). One AS-associated single nucleotide polymorphism, rs2248374, causes a truncated ERAP2 protein that is degraded by nonsense-mediated decay. Approximately 25% of the populations of European ancestry are therefore natural ERAP2 knockouts. We investigated the effect of this associated variant on HLA class I allele presentation, surface heavy chains, endoplasmic reticulum (ER) stress markers and cytokine gene transcription in AS. METHODS: Patients with AS and healthy controls with either AA or GG homozygous status for rs2248374 were studied. Antibodies to CD14, CD19-ECD, HLA-A-B-C, Valpha7.2, CD161, anti-HC10 and anti-HLA-B27 were used to analyse peripheral blood mononuclear cells. Expression levels of ER stress markers (GRP78 and CHOP) and proinflammatory genes (tumour necrosis factor (TNF), IL6, IL17 and IL22) were assessed by qPCR. RESULTS: There was no significant difference in HLA-class I allele presentation or major histocompatibility class I heavy chains or ER stress markers GRP78 and CHOP or proinflammatory gene expression between genotypes for rs2248374 either between cases, between cases and controls, and between controls. DISCUSSION: Large differences were not seen in HLA-B27 expression or cytokine levels between subjects with and without ERAP2 in AS cases and controls. This suggests that ERAP2 is more likely to influence AS risk through other mechanisms.
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Aminopeptidases/genética , Citocinas/genética , Estresse do Retículo Endoplasmático/genética , Antígeno HLA-B27/genética , Leucócitos Mononucleares/imunologia , RNA Mensageiro/metabolismo , Espondilite Anquilosante/genética , Aminopeptidases/imunologia , Estudos de Casos e Controles , Citocinas/imunologia , Chaperona BiP do Retículo Endoplasmático , Estresse do Retículo Endoplasmático/imunologia , Expressão Gênica , Antígeno HLA-B27/imunologia , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/imunologia , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/imunologia , Interleucina-17/genética , Interleucina-17/imunologia , Interleucina-6/genética , Interleucina-6/imunologia , Interleucinas/genética , Interleucinas/imunologia , Reação em Cadeia da Polimerase , Polimorfismo de Nucleotídeo Único , Receptores de Antígenos de Linfócitos B/genética , Receptores de Antígenos de Linfócitos B/imunologia , Espondilite Anquilosante/imunologia , Fator de Transcrição CHOP/genética , Fator de Transcrição CHOP/imunologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia , Interleucina 22RESUMO
Adoptive cellular immunotherapy using in vitro expanded CD8+ T cells shows promise for tumour immunotherapy but is limited by eventual loss of function of the transferred T cells through factors that likely include inactivation by tolerogenic dendritic cells (DC). The co-inhibitory receptor programmed death-1 (PD-1), in addition to controlling T-cell responsiveness at effector sites in malignancies and chronic viral diseases is an important modulator of dendritic cell-induced tolerance in naive T cell populations. The most potent therapeutic capacity amongst CD8+ T cells appears to lie within Tcm or Tcm-like cells but memory T cells express elevated levels of PD-1. Based on established trafficking patterns for Tcm it is likely Tcm-like cells interact with lymphoid-tissue DC that present tumour-derived antigens and may be inherently tolerogenic to develop therapeutic effector function. As little is understood of the effect of PD-1/PD-L1 blockade on Tcm-like CD8+ T cells, particularly in relation to inactivation by DC, we explored the effects of PD-1/PD-L1 blockade in a mouse model where resting DC tolerise effector and memory CD8+ T cells. Blockade of PD-1/PD-L1 promoted effector differentiation of adoptively-transferred Tcm-phenotype cells interacting with tolerising DC. In tumour-bearing mice with tolerising DC, effector activity was increased in both lymphoid tissues and the tumour-site and anti-tumour activity was promoted. Our findings suggest PD-1/PD-L1 blockade may be a useful adjunct for adoptive immunotherapy by promoting effector differentiation in the host of transferred Tcm-like cells.
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Antígeno B7-H1/imunologia , Imunoterapia Adotiva , Receptor de Morte Celular Programada 1/imunologia , Linfócitos T/imunologia , Animais , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
OBJECTIVE: Ankylosing spondylitis (AS) is a common, highly heritable immune-mediated arthropathy that occurs in genetically susceptible individuals exposed to an unknown but likely ubiquitous environmental trigger. There is a close relationship between the gut and spondyloarthritis, as exemplified in patients with reactive arthritis, in whom a typically self-limiting arthropathy follows either a gastrointestinal or urogenital infection. Microbial involvement in AS has been suggested; however, no definitive link has been established. The aim of this study was to determine whether the gut in patients with AS carries a distinct microbial signature compared with that in the gut of healthy control subjects. METHODS: Microbial profiles for terminal ileum biopsy specimens obtained from patients with recent-onset tumor necrosis factor antagonist-naive AS and from healthy control subjects were generated using culture-independent 16S ribosomal RNA gene sequencing and analysis techniques. RESULTS: Our results showed that the terminal ileum microbial communities in patients with AS differ significantly (P < 0.001) from those in healthy control subjects, driven by a higher abundance of 5 families of bacteria (Lachnospiraceae [P = 0.001], Ruminococcaceae [P = 0.012], Rikenellaceae [P = 0.004], Porphyromonadaceae [P = 0.001], and Bacteroidaceae [P = 0.001]) and a decrease in the abundance of 2 families of bacteria (Veillonellaceae [P = 0.01] and Prevotellaceae [P = 0.004]). CONCLUSION: We show evidence for a discrete microbial signature in the terminal ileum of patients with AS compared with healthy control subjects. The microbial composition was demonstrated to correlate with disease status, and greater differences were observed between disease groups than within disease groups. These results are consistent with the hypothesis that genes associated with AS act, at least in part, through effects on the gut microbiome.
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Adoptive T cell therapy uses the specificity of the adaptive immune system to target cancer and virally infected cells. Yet the mechanism and means by which to enhance T cell function are incompletely described, especially in the skin. In this study, we use a murine model of immunotherapy to optimize cell-mediated immunity in the skin. We show that in vitro-derived central but not effector memory-like T cells bring about rapid regression of skin-expressing cognate Ag as a transgene in keratinocytes. Local inflammation induced by the TLR7 receptor agonist imiquimod subtly yet reproducibly decreases time to skin graft rejection elicited by central but not effector memory T cells in an immunodeficient mouse model. Local CCL4, a chemokine liberated by TLR7 agonism, similarly enhances central memory T cell function. In this model, IL-2 facilitates the development in vivo of effector function from central memory but not effector memory T cells. In a model of T cell tolerogenesis, we further show that adoptively transferred central but not effector memory T cells can give rise to successful cutaneous immunity, which is dependent on a local inflammatory cue in the target tissue at the time of adoptive T cell transfer. Thus, adoptive T cell therapy efficacy can be enhanced if CD8(+) T cells with a central memory T cell phenotype are transferred, and IL-2 is present with contemporaneous local inflammation.
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Memória Imunológica/imunologia , Pele/imunologia , Pele/patologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/patologia , Transferência Adotiva/métodos , Animais , Comunicação Celular/imunologia , Células Cultivadas , Células Epidérmicas , Epiderme/imunologia , Epiderme/transplante , Tolerância Imunológica , Imunidade Celular , Inflamação/imunologia , Inflamação/cirurgia , Inflamação/terapia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Pele/citologia , Transplante de Pele/imunologia , Transplante de Pele/métodos , Transplante de Pele/patologia , Subpopulações de Linfócitos T/transplanteRESUMO
Ankylosing spondylitis is a common form of inflammatory arthritis predominantly affecting the spine and pelvis that occurs in approximately 5 out of 1,000 adults of European descent. Here we report the identification of three variants in the RUNX3, LTBR-TNFRSF1A and IL12B regions convincingly associated with ankylosing spondylitis (P < 5 × 10(-8) in the combined discovery and replication datasets) and a further four loci at PTGER4, TBKBP1, ANTXR2 and CARD9 that show strong association across all our datasets (P < 5 × 10(-6) overall, with support in each of the three datasets studied). We also show that polymorphisms of ERAP1, which encodes an endoplasmic reticulum aminopeptidase involved in peptide trimming before HLA class I presentation, only affect ankylosing spondylitis risk in HLA-B27-positive individuals. These findings provide strong evidence that HLA-B27 operates in ankylosing spondylitis through a mechanism involving aberrant processing of antigenic peptides.
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Aminopeptidases/genética , Aminopeptidases/metabolismo , Antígeno HLA-B27/genética , Fragmentos de Peptídeos/metabolismo , Polimorfismo Genético/genética , Espondilite Anquilosante/genética , Proteínas Adaptadoras de Sinalização CARD/genética , Linfócitos T CD8-Positivos/metabolismo , Estudos de Casos e Controles , Subunidade alfa 3 de Fator de Ligação ao Core/genética , Suscetibilidade a Doenças , Estudo de Associação Genômica Ampla , Humanos , Subunidade p40 da Interleucina-12/genética , Proteínas de Ligação a TGF-beta Latente/genética , Proteínas de Membrana/genética , Metanálise como Assunto , Antígenos de Histocompatibilidade Menor , Receptores de Peptídeos , Receptores de Prostaglandina E Subtipo EP4/genética , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Espondilite Anquilosante/metabolismo , População BrancaRESUMO
Recent advances in DNA sequencing have enabled mapping of genes for monogenic traits in families with small pedigrees and even in unrelated cases. We report the identification of disease-causing mutations in a rare, severe, skeletal dysplasia, studying a family of two healthy unrelated parents and two affected children using whole-exome sequencing. The two affected daughters have clinical and radiographic features suggestive of anauxetic dysplasia (OMIM 607095), a rare form of dwarfism caused by mutations of RMRP. However, mutations of RMRP were excluded in this family by direct sequencing. Our studies identified two novel compound heterozygous loss-of-function mutations in POP1, which encodes a core component of the RNase mitochondrial RNA processing (RNase MRP) complex that directly interacts with the RMRP RNA domains that are affected in anauxetic dysplasia. We demonstrate that these mutations impair the integrity and activity of this complex and that they impair cell proliferation, providing likely molecular and cellular mechanisms by which POP1 mutations cause this severe skeletal dysplasia.
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Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Doenças do Desenvolvimento Ósseo/genética , Mutação , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Análise de Sequência de DNA , Doenças do Desenvolvimento Ósseo/patologia , Proliferação de Células , Éxons/genética , Feminino , Humanos , Lactente , Leucócitos Mononucleares/metabolismo , Masculino , Dados de Sequência Molecular , Linhagem , Polimorfismo de Nucleotídeo Único/genética , RNA Ribossômico 5,8S/genéticaRESUMO
Keratinocytes expressing tumor or viral antigens can be eliminated by antigen-primed CD8 cytotoxic T cells. CD4 T-helper cells help induction of CD8 cytotoxic T cells from naive precursors and generation of CD8 T-cell memory. In this study, we show, unexpectedly, that CD4 cells are also required to assist primed CD8 effector T cells in rejection of skin expressing human growth hormone, a neo-self-antigen, in keratinocytes. The requirement for CD4 cells can be substituted by CD40 costimulation. Rejection of skin expressing ovalbumin (OVA), a non-self-antigen, by primed CD8 cytotoxic T cells can in contrast occur without help from antigen-specific CD4 T cells. However, rejection of OVA expressing keratinocytes is helped by antigen-specific CD4 T cells if only low numbers of primed or naive OVA-specific CD8 T cells are available. Effective immunotherapy directed at antigens expressed in squamous cancer may therefore be facilitated by induction of tumor antigen-specific CD4 helper T cells, as well as cytotoxic CD8 T cells.
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Antígenos/imunologia , Queratinócitos/patologia , Transplante de Pele/patologia , Linfócitos T Citotóxicos/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Animais , Antígenos CD40/farmacologia , Modelos Animais de Doenças , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/patologia , Rejeição de Enxerto/fisiopatologia , Hormônio do Crescimento Humano/imunologia , Hormônio do Crescimento Humano/metabolismo , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Ovalbumina/imunologia , Ovalbumina/metabolismo , Transplante de Pele/imunologia , Transplante de Pele/fisiologia , Linfócitos T Citotóxicos/efeitos dos fármacos , Linfócitos T Citotóxicos/fisiologia , Linfócitos T Auxiliares-Indutores/efeitos dos fármacos , Linfócitos T Auxiliares-Indutores/fisiologiaRESUMO
Memory T cells develop early during the preclinical stages of autoimmune diseases and have traditionally been considered resistant to tolerance induction. As such, they may represent a potent barrier to the successful immunotherapy of established autoimmune diseases. It was recently shown that memory CD8+ T cell responses are terminated when Ag is genetically targeted to steady-state dendritic cells. However, under these conditions, inactivation of memory CD8+ T cells is slow, allowing transiently expanded memory CD8+ T cells to exert tissue-destructive effector function. In this study, we compared different Ag-targeting strategies and show, using an MHC class II promoter to drive Ag expression in a diverse range of APCs, that CD8+ memory T cells can be rapidly inactivated by MHC class II+ hematopoietic APCs through a mechanism that involves a rapid and sustained downregulation of TCR, in which the effector response of CD8+ memory cells is rapidly truncated and Ag-expressing target tissue destruction is prevented. Our data provide the first demonstration that genetically targeting Ag to a broad range of MHC class II+ APC types is a highly efficient way to terminate memory CD8+ T cell responses to prevent tissue-destructive effector function and potentially established autoimmune diseases.
Assuntos
Células Apresentadoras de Antígenos/imunologia , Antígenos/imunologia , Linfócitos T CD8-Positivos/imunologia , Memória Imunológica , Animais , Apresentação de Antígeno , Doenças Autoimunes/prevenção & controle , Antígenos de Histocompatibilidade Classe II/genética , Camundongos , Regiões Promotoras GenéticasRESUMO
Combinations of cellular immune-based therapies with chemotherapy and other antitumour agents may be of significant clinical benefit in the treatment of many forms of cancer. Gamma delta (gammadelta) T cells are of particular interest for use in such combined therapies due to their potent antitumour cytotoxicity and relative ease of generation in vitro. Here, we demonstrate high levels of cytotoxicity against solid tumour-derived cell lines with combination treatment utilizing Vgamma9Vdelta2 T cells, chemotherapeutic agents and the bisphosphonate, zoledronate. Pre-treatment with low concentrations of chemotherapeutic agents or zoledronate sensitized tumour cells to rapid killing by Vgamma9Vdelta2 T cells with levels of cytotoxicity approaching 90%. In addition, zoledronate enhanced the chemotherapy-induced sensitization of tumour cells to Vgamma9Vdelta2 T cell cytotoxicity resulting in almost 100% lysis of tumour targets in some cases. Vgamma9Vdelta2 T cell cytotoxicity was mediated by perforin following TCR-dependent and isoprenoid-mediated recognition of tumour cells. Production of IFN-gamma by Vgamma9Vdelta2 T cells was also induced after exposure to sensitized targets. We conclude that administration of Vgamma9Vdelta2 T cells at suitable intervals after chemotherapy and zoledronate may substantially increase antitumour activities in a range of malignancies.
Assuntos
Antineoplásicos/farmacologia , Difosfonatos/farmacologia , Imidazóis/farmacologia , Neoplasias/patologia , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Subpopulações de Linfócitos T/imunologia , Adenocarcinoma/imunologia , Adenocarcinoma/patologia , Apoptose/efeitos dos fármacos , Neoplasias da Mama/imunologia , Neoplasias da Mama/patologia , Linfoma de Burkitt/imunologia , Linfoma de Burkitt/patologia , Carcinoma/imunologia , Carcinoma/patologia , Linhagem Celular Tumoral/efeitos dos fármacos , Linhagem Celular Tumoral/imunologia , Cisplatino/farmacologia , Neoplasias Colorretais/imunologia , Neoplasias Colorretais/patologia , Concanavalina A/farmacologia , Citotoxicidade Imunológica/efeitos dos fármacos , Doxorrubicina/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais , Sinergismo Farmacológico , Etoposídeo/farmacologia , Feminino , Genes Codificadores da Cadeia delta de Receptores de Linfócitos T , Genes Codificadores da Cadeia gama de Receptores de Linfócitos T , Humanos , Interferon gama/biossíntese , Interferon gama/metabolismo , Lovastatina/análogos & derivados , Lovastatina/farmacologia , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/patologia , Masculino , Glicoproteínas de Membrana/análise , Neoplasias/imunologia , Perforina , Proteínas Citotóxicas Formadoras de Poros/análise , Neoplasias da Próstata/imunologia , Neoplasias da Próstata/patologia , Subpopulações de Linfócitos T/metabolismo , Neoplasias da Bexiga Urinária/imunologia , Neoplasias da Bexiga Urinária/patologia , Vincristina/farmacologia , Ácido ZoledrônicoRESUMO
CD1d-restricted natural killer T (NKT) cells expressing invariant Valpha14Jalpha18 T cell receptor alpha-chains are abundant in murine liver and are implicated in the control of malignancy, infection and autoimmunity. Invariant NKT cells have potent anti-metastatic effects in mice and phase I clinical trials involving their homologues in humans are ongoing. However, invariant NKT cells are less abundant in human liver ( approximately 0.5% of hepatic T cells) than in murine liver (up to 50%) and it is not known if other hepatic T cells are CD1-restricted. We have examined expression of CD1a, CD1b, CD1c and CD1d mRNA and protein in human liver and evaluated the reactivity of mononuclear cells (MNC) from histologically normal and tumour-bearing human liver specimens against these CD1 isoforms. Messenger RNA for all CD1 isotypes was detectable in all liver samples. CD1c and CD1d were expressed at the protein level by hepatic MNC. CD1d, only, was detectable at the cell surface, but CD1c and CD1d were found at an intracellular location in significant numbers of liver MNC. CD1b was not expressed by MNC from healthy livers but was detectable within MNC in all tumour samples tested. Hepatic T cells exhibited reactivity against C1R cells expressing transfected CD1c and CD1d, but neither CD1a nor CD1b. These cells secreted interferon-gamma (IFN-gamma) but not interleukin-4 (IL-4) upon stimulation. In contrast, similar numbers of peripheral T cells released 13- and 16-fold less IFN-gamma in response to CD1c and CD1d, respectively. CD1c and CD1d expression and T cell reactivity were not altered in tumour-bearing liver specimens compared to histologically normal livers. These data suggest that, in addition to invariant CD1d-restricted NKT cells, autoreactive T cells that recognise CD1c and CD1d and release inflammatory cytokines are abundant in human liver.
Assuntos
Antígenos CD1/metabolismo , Células Matadoras Naturais/imunologia , Neoplasias Hepáticas/imunologia , Fígado/imunologia , Linfócitos T Citotóxicos/imunologia , Linfócitos T/imunologia , Autoantígenos/imunologia , Autoantígenos/metabolismo , Citometria de Fluxo , Humanos , Células Matadoras Naturais/metabolismo , Fígado/metabolismo , Neoplasias Hepáticas/metabolismo , Isoformas de Proteínas/metabolismo , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Linfócitos T/metabolismo , Linfócitos T Citotóxicos/metabolismoRESUMO
There is an increasing awareness of the therapeutic potential for combining immune-based therapies with chemotherapy in the treatment of malignant diseases, but few published studies evaluate possible cytotoxic synergies between chemotherapy and cytotoxic immune cells. Human V alpha 24+/V beta 11+ NKT cells are being evaluated for use in cell-based immunotherapy of malignancy because of their immune regulatory functions and potent cytotoxic potential. In this study, we evaluated the cytotoxicity of combinations of chemotherapy and NKT cells to determine whether there is a potential to combine these treatment modalities for human cancer therapy. The cytotoxicity of NKT cells was tested against solid-tumor derived cell lines NCI-H358, DLD-1, HT-29, DU-145, TSU-Pr1 and MDA-MB231, with or without prior treatment of these target cells, with a range of chemotherapy agents. Low concentrations of chemotherapeutic agents led to sensitization of cell lines to NKT-mediated cytotoxicity, with the greatest effect being observed for prostate cancer cells. Synergistic cytotoxicity occurred in an NKT cell in a dose-dependent manner. Chemotherapy agents induced upregulation of cell surface TRAIL-R2 (DR5) and Fas (CD95) expression, increasing the capacity for NKT cells to recognize and kill via TRAIL- and FasL-mediated pathways. We conclude that administration of cytotoxic immune cells after chemotherapy may increase antitumor activities in comparison with the use of either treatment alone.
Assuntos
Antineoplásicos/farmacologia , Citotoxicidade Imunológica/imunologia , Neoplasias/tratamento farmacológico , Neoplasias/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T Reguladores/imunologia , Antineoplásicos/uso terapêutico , Proteínas Reguladoras de Apoptose/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Proteína Ligante Fas , Humanos , Glicoproteínas de Membrana/metabolismo , Neoplasias/patologia , Receptores do Fator de Necrose Tumoral/metabolismo , Linfócitos T Reguladores/citologia , Ligante Indutor de Apoptose Relacionado a TNF , Fator de Necrose Tumoral alfa/metabolismo , Fatores de Necrose Tumoral/metabolismo , Regulação para CimaRESUMO
Gamma delta T cells are thought to mediate immune responses at epithelial surfaces. We have quantified and characterized hepatic and peripheral blood gamma delta T cells from 11 normal and 13 unresolved tumor-bearing human liver specimens. gamma delta T cells are enriched in normal liver (6.6% of T cells) relative to matched blood (0.9%; P = 0.008). The majority express CD4(-)CD8(-) phenotypes and many express CD56 and/or CD161. In vitro, hepatic gamma delta T cells can be induced to kill tumor cell lines and release interferon-gamma, tumor necrosis factor-alpha, interleukin-2 and interleukin-4. Analysis of V gamma and V delta chain usage indicated that V delta 3(+) cells are expanded in normal livers (21.2% of gamma delta T cells) compared to blood (0.5%; P = 0.001). Tumor-bearing livers had significant expansions and depletions of gamma delta T cell subsets but normal cytolytic activity. This study identifies novel populations of liver T cells that may play a role in immunity against tumors.