Ellen J. Wehrens

Functional human regulatory T cells fail to control autoimmune inflammation due to PKB/c-akt hyperactivation in effector cells

During the last decade research has focused on the application of FOXP3(+) regulatory T cells (Tregs) in the treatment of autoimmune disease. However, thorough functional characterization of these cells in patients with chronic autoimmune disease, especially at the site of inflammation, is still missing. Here we studied Treg function in patients with juvenile idiopathic arthritis (JIA) and observed that Tregs from the peripheral blood as well as the inflamed joints are fully functional. Nevertheless, Treg-mediated suppression of cell proliferation and cytokine production by effector cells from the site of inflammation was severely impaired, because of resistance to suppression. This resistance to suppression was not caused by a memory phenotype of effector T cells or activation status of antigen presenting cells. Instead, activation of protein kinase B (PKB)/c-akt was enhanced in inflammatory effector cells, at least partially in response to TNFα and IL-6, and inhibition of this kinase restored responsiveness to suppression. We are the first to show that PKB/c-akt hyperactivation causes resistance of effector cells to suppression in human autoimmune disease. Furthermore, these findings suggest that for a Treg enhancing strategy to be successful in the treatment of autoimmune inflammation, resistance because of PKB/c-akt hyperactivation should be targeted as well.

T cells out of control—impaired immune regulation in the inflamed joint

Since the discovery of FOXP3+ regulatory T (TREG) cells over 15 years ago, intensive research has focused on their presence, phenotype and function in autoimmune disease. Whether deficiencies in TREG cells underlie autoimmune pathology and whether, or how, therapeutic approaches based on these cells might be successful is still the subject of debate. The potential role of TREG-cell extrinsic factors, such as proinflammatory cytokines and resistance of effector T cells to suppression, as the cause of regulatory defects in chronic autoimmune inflammation is an intensive area of research. It is now clear that, at the site of inflammation, antigen presenting cells (APCs) and proinflammatory cytokines drive effector T cell skewing and plasticity, and that these T cells can become unresponsive to regulation. In addition, expansion and function of TREG cells is affected by the inflammatory environment; indeed, new data suggest that, in certain conditions, TREG cells promote inflammation. This Review summarizes the latest findings on changes in effector T cell homeostasis in autoimmune disease and focuses on how mechanisms that normally regulate these cells are affected in the inflamed joints of patients with arthritis. These findings have important clinical implications and will affect the development of new therapeutic strategies for autoimmune arthritis.

CD4+CD25+ T cells regulate the intensity of hypersensitivity responses to peanut, but are not decisive in the induction of oral sensitization

Background Naturally occurring CD4+CD25+ regulatory T cells (Tregs) play a critical role in the maintenance of self‐tolerance and it has been suggested that these Tregs may also be involved in preventing allergic disease.

The CD28/CTLA-4-B7 signaling pathway is involved in both allergic sensitization and tolerance induction to orally administered peanut proteins

Dendritic cells are believed to play an essential role in regulating the balance between immunogenic and tolerogenic responses to mucosal Ags by controlling T cell differentiation and activation via costimulatory and coinhibitory signals. The CD28/CTLA-4-CD80/CD86 signaling pathway appears to be one of the most important regulators of T cell responses but its exact role in responses to orally administered proteins remains to be elucidated. In the present study, the involvement of the CD28/CTLA-4-CD80/CD86 costimulatory pathway in the induction of allergic sensitization and oral tolerance to peanut proteins was investigated. In both an established C3H/HeOuJ mouse model of peanut hypersensitivity and an oral tolerance model to peanut, CD28/CTLA-4-CD80/CD86 interactions were blocked using the fusion protein CTLA-4Ig. To examine the relative contribution of CD80- and CD86-mediated costimulation in these models, anti-CD80 and anti-CD86 blocking Abs were used. In the hypersensitivity model, CTLA-4Ig treatment prevented the development of peanut extract-induced cytokine responses, peanut extract-specific IgG1, IgG2a, and IgE production and peanut extract-induced challenge responses. Blocking of CD80 reduced, whereas anti-CD86 treatment completely inhibited, the induction of peanut extract-specific IgE. Normal tolerance induction to peanut extract was found following CTLA-4Ig, anti-CD86, or anti-CD80 plus anti-CD86 treatment, whereas blockade of CD80 impaired the induction of oral tolerance. We show that CD28/CTLA-4-CD80/CD86 signaling is essential for the development of allergic responses to peanut and that CD86 interaction is most important in inducing peanut extract-specific IgE responses. Additionally, our data suggest that CD80 but not CD86 interaction with CTLA-4 is crucial for the induction of low dose tolerance to peanut.

Autologous stem cell transplantation aids autoimmune patients by functional renewal and TCR diversification of regulatory T cells

Autologous hematopoietic stem cell transplantation (HSCT) is increasingly considered for patients with severe autoimmune diseases whose prognosis is poor with standard treatments. Regulatory T cells (Tregs) are thought to be important for disease remission after HSCT. However, eliciting the role of donor and host Tregs in autologous HSCT is not possible in humans due to the autologous nature of the intervention. Therefore, we investigated their role during immune reconstitution and re-establishment of immune tolerance and their therapeutic potential following congenic bone marrow transplantation (BMT) in a proteoglycan-induced arthritis (PGIA) mouse model. In addition, we determined Treg T-cell receptor (TCR) CDR3 diversity before and after HSCT in patients with juvenile idiopathic arthritis and juvenile dermatomyositis. In the PGIA BMT model, after an initial predominance of host Tregs, graft-derived Tregs started dominating and displayed a more stable phenotype with better suppressive capacity. Patient samples revealed a striking lack of diversity of the Treg repertoire before HSCT. This ameliorated after HSCT, confirming reset of the Treg compartment following HSCT. In the mouse model, a therapeutic approach was initiated by infusing extra Foxp3(GFP+) Tregs during BMT. Infusion of Foxp3(GFP+) Tregs did not elicit additional clinical improvement but conversely delayed reconstitution of the graft-derived T-cell compartment. These data indicate that HSCT-mediated amelioration of autoimmune disease involves renewal of the Treg pool. In addition, infusion of extra Tregs during BMT results in a delayed reconstitution of T-cell compartments. Therefore, Treg therapy may hamper development of long-term tolerance and should be approached with caution in the clinical autologous setting.

Human regulatory T cell suppressive function is independent of apoptosis induction in activated effector T cells.

Background CD4+CD25+FOXP3+ Regulatory T cells (Treg) play a central role in the immune balance to prevent autoimmune disease. One outstanding question is how Tregs suppress effector immune responses in human. Experiments in mice demonstrated that Treg restrict effector T cell (Teff) responses by deprivation of the growth factor IL-2 through Treg consumption, resulting in apoptosis of Teff. Principal Findings In this study we investigated the relevance of Teff apoptosis induction to human Treg function. To this end, we studied naturally occurring Treg (nTreg) from peripheral blood of healthy donors, and, to investigate Treg function in inflammation in vivo, Treg from synovial fluid of Juvenile Idiopathic Arthritis (JIA) patients (SF-Treg). Both nTreg and SF-Treg suppress Teff proliferation and cytokine production efficiently as predicted. However, in contrast with murine Treg, neither nTreg nor SF-Treg induce apoptosis in Teff. Furthermore, exogenously supplied IL-2 and IL-7 reverse suppression, but do not influence apoptosis of Teff. Significance Our functional data here support that Treg are excellent clinical targets to counteract autoimmune diseases. For optimal functional outcome in human clinical trials, future work should focus on the ability of Treg to suppress proliferation and cytokine production of Teff, rather than induction of Teff apoptosis.

Brief Report: Anti–Tumor Necrosis Factor α Targets Protein Kinase B/c‐Akt–Induced Resistance of Effector Cells to Suppression in Juvenile Idiopathic Arthritis

OBJECTIVE To determine whether therapeutic strategies that block interleukin-6 (IL-6) or tumor necrosis factor α (TNFα) can improve the responsiveness of Teff cells to suppression in patients with juvenile idiopathic arthritis (JIA). METHODS Synovial fluid mononuclear cells (SFMCs) from the inflamed joints of patients with JIA were cultured in the presence of etanercept or anti-IL-6 in vitro, and protein kinase B (PKB)/c-Akt activation and responsiveness to suppression were measured. In addition, the in vivo effects of TNFα blockade were investigated using peripheral blood mononuclear cells obtained from patients before and after the start of etanercept therapy. RESULTS In vitro treatment of SFMCs with anti-IL-6 led to improved Treg cell-mediated suppression of cell proliferation in some but not all patients. Blocking TNFα with etanercept, however, clearly enhanced suppression, especially that of CD8+ T cells. In the presence of etanercept, PKB/c-Akt activation of Teff cells was reduced, and cells became more susceptible to transforming growth factor β-mediated suppression, indicating that anti-TNFα directly targets resistant Teff cells. CONCLUSION This study is the first to show that anti-TNFα targets the resistance of Teff cells to suppression, resulting in improved regulation of inflammatory effector cells.

Editorial: Quality or quantity? Unraveling the role of Treg cells in rheumatoid arthritis

Approximately 25 years ago, the introduction of methotrexate revolutionized the treatment of rheumatoid arthritis (RA) and juvenile idiopathic arthritis (JIA). In the late 1990s, this was followed by a second revolution: the introduction of biologic agents. Since the introduction of these treatment approaches, progress has not stopped as new targets for biologic therapies emerge and second-generation biologic agents are being produced. Nonetheless, this development represents progress mostly in terms of diversification and optimization rather than a new revolution. The next challenge in the treatment of RA and JIA is to reestablish self-tolerance with the goal of achieving long-term medication-free remission; in other words, a cure (1). The discovery of Treg cells, first in animal models and later in humans, offered a completely new perspective regarding the establishment and maintenance of self-tolerance (2). Treg cells regulate immune responses through their ability to suppress other immune cells, such as Teff cells, natural killer cells, and antigenpresenting cells (APCs). In humans, Treg cells mostly reside within the population of CD4 CD25 CD127 T cells. They are characterized by expression of the lineage-specific transcription factor FoxP3. In various experimental models, Treg cells indeed make the difference between health and autoimmunity and were shown to be crucial for the maintenance of self-tolerance and immune homeostasis. The subsequent discovery that a gene mutation in FoxP3 leads to uncontrolled inflammation and autoimmunity in children with immune dysregulation, polyendocrinopathy, enteropathy, or X-linked syndrome raised expectations that in human autoimmune diseases, chronic inflammation may be caused by a deficiency in the Treg cell population. However, closer inspection revealed that the answer to the question of the role of Treg cells in humans was more complex. Data on peripheral FoxP3 Treg cell numbers and function in human autoimmune diseases are contradictory and remain subject to debate (3). To complicate this point further, it has become clear that FoxP3expressing T cells are not a homogenous population (Table 1). First, FoxP3 is not a definitive marker for human Treg cells: conventional T cells can temporarily up-regulate FoxP3 expression upon activation without displaying a suppressive function (4), which hampers identification and isolation of human Treg cells. Moreover, it has been demonstrated that FoxP3-positive Treg cells may have a certain degree of plasticity (5) or instability (6,7), resulting in differentiation into Teff-like subtypes with a reduced or complete loss of suppressive function, especially under inflammatory conditions. To add to this conundrum, new data have emerged indicating that Treg cells are not one of a kind: various subtypes can be determined, including specific populations that share Th cell characteristics but retain their suppressive capacities (8). Various groups of investigators have reported an increased number of Treg cells in the synovial fluid of inflamed joints in patients with RA or JIA (3,9–11). This inevitably leads to the question of why these Treg cells in the synovium are not capable of controlling inflammation. Is the proinflammatory environment simply too strong for Treg cells to overcome, or are Treg cells in inflamed synovial fluid not true Treg cells but effector cells in disguise that have temporarily up-regulated FoxP3? In this issue of Arthritis & Rheumatism, Walter et al (12) present important new data that shed light on this puzzle. In elegantly performed in vitro studies, Walter and colleagues show convincingly that activated Dr. Prakken and Ms Wehren’s work was supported by the Dutch Arthritis Foundation. Dr. van Wijk is recipient of a Veni grant from the Dutch Organization for Scientific Research. Berent Prakken, MD, PhD, Femke van Wijk, PhD: University Medical Centre Utrecht, Utrecht, The Netherlands, and Eureka Institute for Translational Medicine, Siracusa, Italy; Ellen Wehrens, MSc: University Medical Centre Utrecht, Utrecht, The Netherlands. Address correspondence to Berent Prakken, MD, PhD, Center for Molecular and Cellular Intervention, Department of Pediatrics, Child Health Program, University Medical Centre Utrecht, PO Box 85090, 3508 AB Utrecht, The Netherlands. E-mail: bprakken@ umcutrecht.nl. Submitted for publication November 23, 2012; accepted in revised form December 11, 2012.

Self‐Sustained Resistance to Suppression of CD8+ Teff Cells at the Site of Autoimmune Inflammation Can Be Reversed by Tumor Necrosis Factor and Interferon‐γ Blockade

Resistance of Teff cells to Treg cell–mediated suppression contributes to the breakdown of peripheral tolerance in the inflamed joints of patients with juvenile idiopathic arthritis (JIA). However, unanswered questions are whether this resistant phenotype is self‐sustained and whether CD8+ and CD4+ Teff cells share the same mechanism of resistance to suppression. We undertook this study to investigate intrinsic resistance of CD8+ Teff cells to suppression and to determine how this can be targeted therapeutically.

PReS-FINAL-1019: Inflammatory monocytes induce resistance of effector t cells to suppression

Ever since their discovery research has focused on whether deficiencies in FOXP3+ regulatory T cells (Treg) underlie human autoimmune pathology. Very recently however, the topic of Treg extrinsic factors as the cause of regulatory defects in chronic autoimmune inflammation has become more prominent in the discussion. It has become clear that resistance of effector cells (Teff) to suppression contributes to disturbed immune regulation in autoimmune inflammation, especially at the site of inflammation. Therefore, targeting this unresponsiveness to suppression could be a promising treatment option for patients with autoimmune disease. It remains unknown how resistance of T cells to suppression is induced.