Lucy R. Wedderburn

Th17 plasticity in human autoimmune arthritis is driven by the inflammatory environment

In several murine models of autoimmune arthritis, Th17 cells are the dominant initiators of inflammation. In human arthritis the majority of IL-17–secreting cells within the joint express a cytokine phenotype intermediate between Th17 and Th1. Here we show that Th17/1 cells from the joints of children with inflammatory arthritis express high levels of both Th17 and Th1 lineage-specific transcription factors, RORC2 and T-bet. Modeling the generation of Th17/1 in vitro, we show that Th17 cells “convert” to Th17/1 under conditions that mimic the disease site, namely low TGFβ and high IL-12 levels, whereas Th1 cells cannot convert to Th17. Th17/1 cells from the inflamed joint share T-cell receptor (TCR) clonality with Th17 cells, suggesting a shared clonal origin between Th17 and Th17/1 cells in arthritis. Using CD161, a lectin-like receptor that is a marker of human Th17, we show synovial Th17 and Th17/1 cells, and unexpectedly, a large proportion of Th1 cells express CD161. We provide evidence to support a Th17 origin for Th1 cells expressing CD161. In vitro, Th17 cells that convert to a Th1 phenotype maintain CD161 expression. In the joint CD161+ Th1 cells share features with Th17 cells, with shared TCR clonality, expression of RORC2 and CCR6 and response to IL-23, although they are IL-17 negative. We propose that the Th17 phenotype may be unstable and that Th17 cells may convert to Th17/1 and Th1 cells in human arthritis. Therefore therapies targeting the induction of Th17 cells could also attenuate Th17/1 and Th1 effector populations within the inflamed joint.

Interleukin‐17–producing T cells are enriched in the joints of children with arthritis, but have a reciprocal relationship to regulatory T cell numbers

Objective To identify interleukin-17 (IL-17)–producing T cells from patients with juvenile idiopathic arthritis (JIA), and investigate their cytokine production, migratory capacity, and relationship to Treg cells at sites of inflammation, as well as to test the hypothesis that IL-17+ T cell numbers correlate with clinical phenotype in childhood arthritis. Methods Flow cytometry was used to analyze the phenotype, cytokine production, and chemokine receptor expression of IL-17–producing T cells in peripheral blood and synovial fluid mononuclear cells from 36 children with JIA, in parallel with analysis of forkhead box P3 (FoxP3)–positive Treg cells. Migration of IL-17+ T cells toward CCL20 was assessed by a Transwell assay. Synovial tissue was analyzed by immunohistochemistry for IL-17 and IL-22. Results IL-17+ T cells were enriched in the joints of children with JIA as compared with the blood of JIA patients (P = 0.0001) and controls (P = 0.018) and were demonstrated in synovial tissue. IL-17+ T cell numbers were higher in patients with extended oligoarthritis, the more severe subtype of JIA, as compared with patients with persistent oligoarthritis, the milder subtype (P = 0.046). Within the joint, there was an inverse relationship between IL-17+ T cells and FoxP3+ Treg cells (r = 0.61, P = 0.016). IL-17+,CD4+ T cells were uniformly CCR6+ and migrated toward CCL20, but synovial IL-17+ T cells had variable CCR4 expression. A proportion of IL-17+ synovial T cells produced IL-22 and interferon-γ. Conclusion This study is the first to define the frequency and characteristics of “Th17” cells in JIA. We suggest that these highly proinflammatory cells contribute to joint pathology, as indicated by relationships with clinical phenotypes, and that the balance between IL-17+ T cells and Treg cells may be critical to outcome.

Blood and synovial fluid cytokine signatures in patients with juvenile idiopathic arthritis: a cross-sectional study

Background: Juvenile idiopathic arthritis (JIA) consists of a heterogeneous group of disorders with, for the most part, an unknown immunopathogenesis. Although onset and disease course differ, the subtypes of JIA share the occurrence of chronic inflammation of the joints, with infiltrations of immunocompetent cells that secrete inflammatory mediators. Objective: To identify a panel of cytokines specifically related to the inflammatory process in JIA. Methods: Using a new technology, the multiplex immunoassay, 30 cytokines were measured in plasma of 65 patients with JIA, of which 34 were paired with synovial fluid. These data were compared with plasma of 20 healthy controls and 9 patients with type I diabetes, a chronic inflammatory disease. Results: Patients with JIA had, irrespective of their subclassification, significantly higher levels of tumour necrosis factor α, macrophage inhibitory factor (MIF), CCL2, CCL3, CCL11, CCL22 and CXCL9 in plasma than controls. In paired plasma and synovial fluid samples of patients with JIA, significantly higher levels of interleukin (IL)6, IL15, CCL2, CCL3, CXCL8, CXCL9 and CXCL10 were present in synovial fluid. Cluster analysis in all patients with JIA revealed a predominant pro-inflammatory cytokine cluster during active disease and a regulatory/anti-inflammatory-related cytokine cluster during remission. Whether a discrimination profile of various cytokines could help in the determination of disease classification was tested. Conclusion: It is suggested that several cytokines (IL18, MIF, CCL2, CCL3, CCL11, CXCL9 and CXCL10) may correspond to the activation status during inflammation in JIA and could be instrumental in monitoring disease activity and outcomes of (new) immunotherapies.

Dense genotyping of immune-related disease regions identifies 14 new susceptibility loci for juvenile idiopathic arthritis

We used the Immunochip array to analyze 2,816 individuals with juvenile idiopathic arthritis (JIA), comprising the most common subtypes (oligoarticular and rheumatoid factor–negative polyarticular JIA), and 13,056 controls. We confirmed association of 3 known JIA risk loci (the human leukocyte antigen (HLA) region, PTPN22 and PTPN2) and identified 14 loci reaching genome-wide significance (P < 5 × 10−8) for the first time. Eleven additional new regions showed suggestive evidence of association with JIA (P < 1 × 10−6). Dense mapping of loci along with bioinformatics analysis refined the associations to one gene in each of eight regions, highlighting crucial pathways, including the interleukin (IL)-2 pathway, in JIA disease pathogenesis. The entire Immunochip content, the HLA region and the top 27 loci (P < 1 × 10−6) explain an estimated 18, 13 and 6% of the risk of JIA, respectively. In summary, this is the largest collection of JIA cases investigated so far and provides new insight into the genetic basis of this childhood autoimmune disease.

Methotrexate withdrawal at 6 vs 12 months in juvenile idiopathic arthritis in remission: a randomized clinical trial.

CONTEXT Novel therapies have improved the remission rate in chronic inflammatory disorders including juvenile idiopathic arthritis (JIA). Therefore, strategies of tapering therapy and reliable parameters for detecting subclinical inflammation have now become challenging questions. OBJECTIVES To analyze whether longer methotrexate treatment during remission of JIA prevents flares after withdrawal of medication and whether specific biomarkers identify patients at risk for flares. DESIGN, SETTING, AND PATIENTS Prospective, open, multicenter, medication-withdrawal randomized clinical trial including 364 patients (median age, 11.0 years) with JIA recruited in 61 centers from 29 countries between February 2005 and June 2006. Patients were included at first confirmation of clinical remission while continuing medication. At the time of therapy withdrawal, levels of the phagocyte activation marker myeloid-related proteins 8 and 14 heterocomplex (MRP8/14) were determined. INTERVENTION Patients were randomly assigned to continue with methotrexate therapy for either 6 months (group 1 [n = 183]) or 12 months (group 2 [n = 181]) after induction of disease remission. MAIN OUTCOME MEASURES Primary outcome was relapse rate in the 2 treatment groups; secondary outcome was time to relapse. In a prespecified cohort analysis, the prognostic accuracy of MRP8/14 concentrations for the risk of flares was assessed. RESULTS Intention-to-treat analysis of the primary outcome revealed relapse within 24 months after the inclusion into the study in 98 of 183 patients (relapse rate, 56.7%) in group 1 and 94 of 181 (55.6%) in group 2. The odds ratio for group 1 vs group 2 was 1.02 (95% CI, 0.82-1.27; P = .86). The median relapse-free interval after inclusion was 21.0 months in group 1 and 23.0 months in group 2. The hazard ratio for group 1 vs group 2 was 1.07 (95% CI, 0.82-1.41; P = .61). Median follow-up duration after inclusion was 34.2 and 34.3 months in groups 1 and 2, respectively. Levels of MRP8/14 during remission were significantly higher in patients who subsequently developed flares (median, 715 [IQR, 320-1 110] ng/mL) compared with patients maintaining stable remission (400 [IQR, 220-800] ng/mL; P = .003). Low MRP8/14 levels indicated a low risk of flares within the next 3 months following the biomarker test (area under the receiver operating characteristic curve, 0.76; 95% CI, 0.62-0.90). CONCLUSIONS In patients with JIA in remission, a 12-month vs 6-month withdrawal of methotrexate did not reduce the relapse rate. Higher MRP8/14 concentrations were associated with risk of relapse after discontinuing methotrexate. TRIAL REGISTRATION isrctn.org Identifier: ISRCTN18186313.

Selective recruitment of polarized T cells expressing CCR5 and CXCR3 to the inflamed joints of children with juvenile idiopathic arthritis

OBJECTIVE To study the expression of chemokine receptors CCR5 and CXCR3 and the Th1/Th2 cytokine balance in children with oligoarticular or polyarticular juvenile idiopathic arthritis (JIA). METHODS Using 3-color immunofluorescence, we studied the expression of CCR5 and CXCR3 on, and T cell cytokine production by, paired samples of synovial fluid (SF) and peripheral blood (PB) T cells from 20 patients with oligoarticular- or polyarticular-onset JIA. Chemokine and cytokine phenotypes were also compared within the CD45RO+,CD3+ subsets. CCR5 genotypes were confirmed by polymerase chain reaction typing and sequencing. RESULTS In the majority of samples, the number of T cells that were CCR5+ and CXCR3+ was higher in SF than in PB, and this difference was significant. One child was homozygous for the null A32 CCR5 allele; 4 others had lower expression of CXCR3 in SF than in blood. All samples showed strongly Th1-type cytokine production by synovial T cells compared with that by PB T cells. Both features were also markedly polarized within the synovial CD45RO+ subset compared with PB CD45RO+ T cells. CONCLUSION The high expression of CCR5 and CXCR3 and high interferon-gamma:interleukin-4 ratios suggest a type 1 phenotype of SF T cells in JIA. The difference between CD45RO+ T cells from SF and from PB suggests that specific activation events have occurred in synovial T cells. We suggest that the highly activated, Th1-type phenotype of T cells within the chronically inflamed joints of children with JIA may reflect specific recruitment events that contribute to the polarization of these cells.

Autoantibodies to a 140‐kd protein in juvenile dermatomyositis are associated with calcinosis

Objective The identification of novel autoantibodies in juvenile dermatomyositis (DM) may have etiologic and clinical implications. The aim of this study was to describe autoantibodies to a 140-kd protein in children recruited to the Juvenile DM National Registry and Repository for UK and Ireland. Methods Clinical data and sera were collected from children with juvenile myositis. Sera that recognized a 140-kd protein by immunoprecipitation were identified. The identity of the p140 autoantigen was investigated by immunoprecipitation/immunodepletion, using commercial monoclonal antibodies to NXP-2, reference anti-p140, and anti-p155/140, the other autoantibody recently described in juvenile DM. DNA samples from 100 Caucasian children with myositis were genotyped for HLA class II haplotype associations and compared with those from 864 randomly selected UK Caucasian control subjects. Results Sera from 37 (23%) of 162 patients with juvenile myositis were positive for anti-p140 autoantibodies, which were detected exclusively in patients with juvenile DM and not in patients with juvenile DM–overlap syndrome or control subjects. No anti-p140 antibody–positive patients were positive for other recognized autoantibodies. Immunodepletion suggested that the identity of p140 was consistent with NXP-2 (the previously identified MJ autoantigen). In children with anti-p140 antibodies, the association with calcinosis was significant compared with the rest of the cohort (corrected P < 0.005, odds ratio 7.0, 95% confidence interval 3.0–16.1). The clinical features of patients with anti-p140 autoantibodies were different from those of children with anti-p155/140 autoantibodies. The presence of HLA–DRB1*08 was a possible risk factor for anti-p140 autoantibody positivity. Conclusion This study has established that anti-p140 autoantibodies represent a major autoantibody subset in juvenile DM. This specificity may identify a further immunogenetic and clinical phenotype within the juvenile myositis spectrum that includes an association with calcinosis.

Th17 and regulatory T cells: rebalancing pro- and anti-inflammatory forces in autoimmune arthritis

Inflammatory T cells are thought to be central to the pathology of autoimmune arthritis. Th17 cells, CD4 T cells that secrete the pro-inflammatory cytokine IL-17 play a critical role in murine models of arthritis. Recent evidence from human studies suggests that Th17 cells may be important players in several autoimmune diseases, including seronegative arthritis in adults, childhood arthritis [juvenile idiopathic arthritis (JIA)]. It was surprising to find that the development of Th17 cells is closely related to that of an immunoregulatory subset called regulatory T cells (Tregs). Tregs are important in the maintenance of immune homeostasis. Defects in Treg function or reduced numbers have been documented in several human autoimmune diseases, including RA and JIA. Conditions that typically favour the development of Tregs and promote tolerance can be subverted by inflammatory signals towards supporting the generation of Th17 cells. In animal models, the enhancement of Th17 cell differentiation is at the expense of Tregs, and these combined changes trigger autoimmunity. Several mechanisms have come to light that control this reciprocal relationship between Tregs and Th17 cells, including the action of pro-inflammatory cytokines such as IL-1beta. Anti-rheumatic biologic therapies may offer a means of restoring the Th17/Treg balance in favour of Tregs and thereby re-establishing immune tolerance.

Clonal Expansions in Acute EBV Infection Are Detectable in the CD8 and not the CD4 Subset and Persist with a Variable CD45 Phenotype

We have applied a sensitive global analysis of TCR heterogeneity to compare clonal dynamics of CD4+ and CD8+ T cells in acute infectious mononucleosis. Using this approach, we are able to identify a broad representation of the total virus-specific population without the bias of in vitro culture and then to track their phenotype and fate by their unique molecular footprint. We demonstrate a large number of Ag-driven clones using different TCRs in the acute phase, all CD8+. The diverse large clones generated in the CD8 subset in response to this virus contrast with the complete lack of detectable clonal expansion in the CD4 compartment. Many of the same clones remain detectable in directly ex vivo CD8+ T cells for at least a year after resolution of infectious mononucleosis, although the clone size is reduced. Thus, memory CD8 cells following EBV infection persist at relatively high circulating frequency and represent a subset of the large range of clonotypes comprising the acute effectors. Separation of samples into CD45RA (naive) and CD45RO (memory) fractions shows the accumulation of identical CDR3 region defined clonotypes in both CD45RO and CD45RA fractions and sequencing confirms that dominant long-lived monoclonal expansions can reside in the CD45RA pool.

High expression of the ectonucleotidase CD39 on T cells from the inflamed site identifies two distinct populations, one regulatory and one memory T cell population.

The ectonucleotidase CD39 has recently been described as being highly expressed on regulatory Foxp3+ CD4 T cells. Through hydrolysis of proinflammatory extracellular ATP, CD39 activity represents a newly described mechanism of regulatory T cell action. We report a novel population of human CD4 T cells that express CD39 yet are Foxp3 negative. These cells produce the proinflammatory cytokines IFN-γ and IL-17 and fail to suppress proliferation; however, they still have high ATP hydrolysis activity. In the inflammatory site in human juvenile idiopathic arthritis, the CD39+Foxp3− population is greatly increased compared with peripheral blood of patients or healthy controls. We also show that cells expressing the AMPase CD73 are less frequent in the joint than in blood. To our knowledge, this is the first study to describe and characterize CD39 function on CD4 T cells from the target site in a human autoinflammatory condition. Our data suggest that in human CD4+ T cells from the inflamed site, CD39 can be highly expressed on two populations, one regulatory and the other of a memory phenotype.