Immune regulation in health and Juvenile Idiopathic Arthritis (JIA): molecular mechanisms and regulatory T cells

Immune regulation is essential to fighting pathogens and tolerance to self and non-harmful (food and commensal) antigens. The immune system has several mechanisms in place to keep the immune system in balance. In this study some of these mechanisms are more closely investigated in health and disease. In autoimmune Juvenile Idiopathic Arthritis (JIA) immune regulation fails at the site of disease, with large pro-inflammatory cell infiltrates and inflammation. In the first part of this thesis potential mechanisms in regulating the severity of JIA are examined through analysis of previously acquired gene expression array data from oligoarticular JIA synovial fluid cells at the time of diagnosis, stratified by disease severity outcome after one year. Expression of three genes (SMAD3, ERRFI1 and VIPR1) was tested using RT-PCR. Particular focus was put on vasoactive intestinal peptide receptor type one (VIPR1), as it was expressed at lower levels and with an apparent variant at the inflamed site of JIA. Regulatory T cells (Treg) are one essential part of immune regulation. Recent studies have suggested some pro-inflammatory potential by Treg producing cytokines including IL-17 and IFN-γ. In this thesis Treg cytokine production was linked to CD161 expression. CD161+ Treg were characterized in health, throughout development and in JIA. CD161+ Treg showed a memory effector T cell like phenotype, including expression of transcription factors (Tbet, RORCv2). However CD161+ Treg were also potent suppressors in vitro and showed a predominantly demethylated Treg specific demethylated region (TSDR). CD161+ Treg appeared pathogen specific and behaved differently to TCR stimulation as CD161- Treg. Furthermore CD161+ Treg were highly enriched in the synovial fluid of JIA and correlated with disease. This thesis suggests that immune regulation is influenced by the inflammatory environment and has more facets to it, with cytokine-producing Treg playing an important part in health and disease

T regulatory cells in childhood arthritis - novel insights.

In recent years, there have been many new developments in the field of regulatory T cells (Treg), challenging the consensus on their behaviour, classification and role(s) in disease. The role Treg might play in autoimmune disease appears to be more complex than previously thought. Here, we discuss the current knowledge of regulatory T cells through animal and human research and illustrate the recent developments in childhood autoimmune arthritis (juvenile idiopathic arthritis (JIA)). Furthermore, this review summarises our understanding of the fields and assesses current and future implications for Treg in the treatment of JIA

Targeting Tregs in Juvenile Idiopathic Arthritis and Juvenile Dermatomyositis-Insights From Other Diseases

Regulatory T cells (Tregs) are believed to be dysfunctional in autoimmunity. Juvenile idiopathic arthritis (JIA) and juvenile dermatomyositis (JDM) result from a loss of normal immune regulation in specific tissues such as joints or muscle and skin, respectively. Here, we discuss recent findings in regard to Treg biology in oligo-/polyarticular JIA and JDM, as well as what we can learn about Treg-related disease mechanism, treatment and biomarkers in JIA/JDM from studies of other diseases. We explore the potential use of Treg immunoregulatory markers and gene signatures as biomarkers for disease course and/or treatment success. Further, we discuss how Tregs are affected by several treatment strategies already employed in the therapy of JIA and JDM and by alternative immunotherapies such as anti-cytokine or co-receptor targeting. Finally, we review recent successes in using Tregs as a treatment target with low-dose IL-2 or cellular immunotherapy. Thus, this mini review will highlight our current understanding and identify open questions in regard to Treg biology, and how recent findings may advance biomarkers and new therapies for JIA and JDM

The role of FOXP3 in autoimmunity

FOXP3 controls the development and function of T regulatory cells (Tregs). Autoimmunity is linked to changes in FOXP3 activity that can occur at multiple levels and lead to Treg dysfunction. For example, changes in IL-2 signaling, FOXP3 transcription and/or post-translational modifications can all contribute to loss of self-tolerance. As additional pathways of FOXP3 regulation are elucidated, new therapeutic approaches to increase Treg activity either by cell therapy or pharmacological intervention are being tested. Early success from pioneering studies of Treg-based therapy in transplantation has promoted the undertaking of similar studies in autoimmunity, with emerging evidence for the effectiveness of these approaches, particularly in the context of type 1 diabetes

Optimized CRISPR-mediated gene knockin reveals FOXP3-independent maintenance of human Treg identity

Regulatory T cell (Treg) therapy is a promising curative approach for a variety of immune-mediated conditions. CRISPR-based genome editing allows precise insertion of transgenes through homology-directed repair, but its use in human Tregs has been limited. We report an optimized protocol for CRISPR-mediated gene knockin in human Tregs with high-yield expansion. To establish a benchmark of human Treg dysfunction, we target the master transcription factor FOXP3 in naive and memory Tregs. Although FOXP3-ablated Tregs upregulate cytokine expression, effects on suppressive capacity in vitro manifest slowly and primarily in memory Tregs. Moreover, FOXP3-ablated Tregs retain their characteristic protein, transcriptional, and DNA methylation profile. Instead, FOXP3 maintains DNA methylation at regions enriched for AP-1 binding sites. Thus, although FOXP3 is important for human Treg development, it has a limited role in maintaining mature Treg identity. Optimized gene knockin with human Tregs will enable mechanistic studies and the development of tailored, next-generation Treg cell therapies

T cell GM-CSF expression in juvenile arthritis is contingent upon Th17 plasticity.

Objectives Granulocyte monocyte colony stimulating factor (GM-CSF) is a potent inflammatory mediator responsible for recruitment and activation of innate immune cells. Recent murine data have identified Th17 cells as a key source of GM-CSF, and suggest that T cell derived GM-CSF is instrumental in the induction of autoimmune disease. We analysed the expression of T cell derived GM-CSF in the joints of patients with Juvenile idiopathic arthritis (JIA) and investigated the development links between Th17 and GM-CSF+ T helper cells. Methods 24 patients with JIA were analysed for expression of GM-CSF and the Th17 marker CD161 in synovial and peripheral blood compartments using flow cytometry and RT-PCR. A cytokine capture assay was used to purify Th17 cells and test the plasticity of cytokine production in response to IL-12 and IL-23. Results The frequency of GM-CSF producing T helper cells were significantly enriched in JIA synovial fluid mononuclear cells (SFMC) compared to PBMC (24.1% vs 2.9% of CD4+ T cells) and closely correlated with ESR levels (r(2) =0.91, p=<0.001). Synovial GM-CSF+ T cells were predominantly CD161 positive and co-expressed interferon gamma (IFNγ) but not IL-17. Culture of Th17 cells in the presence of IL-12 led to rapid upregulation of GM-CSF and IFNγ, recapitulating the phenotype of GM-CSF expressing cells within the joint. Conclusions Our results identify a novel outcome of Th17 plasticity in humans that may account for the enrichment of GM-CSF expressing T cells found within the JIA joint. The association of GM-CSF expression with systemic inflammation highlights the potential role for Th17 related cytokines in the pathology of JIA. © 2014 American College of Rheumatology

CD86 Is a Selective CD28 Ligand Supporting FoxP3+ Regulatory T Cell Homeostasis in the Presence of High Levels of CTLA-4

CD80 and CD86 are expressed on antigen presenting cells and are required to engage their shared receptor, CD28, for the costimulation of CD4 T cells. It is unclear why two stimulatory ligands with overlapping roles have evolved. CD80 and CD86 also bind the regulatory molecule CTLA-4. We explored the role of CD80 and CD86 in the homeostasis and proliferation of CD4+FoxP3+ regulatory T cells (Treg), which constitutively express high levels of CTLA-4 yet are critically dependent upon CD28 signals. We observed that CD86 was the dominant ligand for Treg proliferation, survival, and maintenance of a regulatory phenotype, with higher expression of CTLA-4, ICOS, and OX40. We also explored whether CD80-CD28 interactions were specifically compromised by CTLA-4 and found that antibody blockade, clinical deficiency of CTLA-4 and CRISPR-Cas9 deletion of CTLA-4 all improved Treg survival following CD80 stimulation. Taken together, our data suggest that CD86 is the dominant costimulatory ligand for Treg homeostasis, despite its lower affinity for CD28, because CD80-CD28 interactions are selectively impaired by the high levels of CTLA-4. These data suggest a cell intrinsic role for CTLA-4 in regulating CD28 costimulation by direct competition for CD80, and indicate that that CD80 and CD86 have discrete roles in CD28 costimulation of CD4 T cells in the presence of high levels of CTLA-4

Immunological characteristics and T cell receptor clonal diversity in children with systemic juvenile idiopathic arthritis undergoing T cell depleted autologous stem cell transplantation.

Children with systemic Juvenile Idiopathic Arthritis (sJIA), the most severe subtype of JIA, are at risk of from destructive polyarthritis and growth failure, and corticosteroids as part of conventional treatment can result in osteoporosis and growth delay. In children where there is failure or toxicity from drug therapies, disease has been successfully controlled by T cell depleted autologous stem cell transplantation (ASCT). At present, the immunological basis underlying remission post ASCT is unknown. Immune reconstitution of T, B, NK, NK-T cell and monocytes, in parallel with T cell receptor diversity by analysis of beta variable region (TCRVb) complementarity determining region-3 (CDR3) using spectratyping and sequencing, were studied in 5 children with sJIA before and after ASCT. At time of follow up (mean 11.5 years), 4 patients remain in complete remission, while 1 child had relapsed within 1 month of transplant. The CD8(+) TCRVb repertoire was highly oligoclonal early in immune reconstitution and re-emergence of pre-transplant TCRVb CDR3 dominant peaks was observed post transplant in certain TCRVb families. Further, re-emergence of pre-ASCT clonal sequences in addition to new sequences was identified post transplant. These results suggest that a chimeric TCR repertoire, comprising T cell clones developed before and after transplant, can be associated with clinical remission from severe arthritis. This article is protected by copyright. All rights reserved