Amy E. Anderson

Differential regulation of naïve and memory CD4+ T cells by alternatively activated dendritic cells

Promising immunotherapeutic tools for T cell‐mediated pathologies are alternatively activated dendritic cells (aaDC), which exert their effect through the regulation and tolerization of T cells. As naïve and memory T cells have different susceptibilities to tolerogenic signals, it is important to understand the modulatory effects of aaDC on these T cell subsets. We have examined regulation of naïve and memory CD4+ T cells by human aaDC generated with dexamethasone, the active form of vitamin D3, 1α,25‐dihydroxyvitamin D3, and LPS. Although aaDC induced low, primary, allogeneic responses by naïve and memory T cells, aaDC regulated the differentiation of these T cell subsets in a distinct manner. Naïve T cells primed by aaDC retained a strong, proliferative capacity upon restimulation but were skewed toward a low IFN‐γ/high IL‐10 cytokine profile. In contrast, memory T cells primed by aaDC became hyporesponsive in terms of proliferation and cytokine production. Induction of anergy in memory T cells by aaDC was not a result of the presence of CD25hi regulatory T cells and could be partially reversed by IL‐2. Both T cell subsets acquired regulatory activity and inhibited primary CD4 and CD8 responses. Addition of exogenous IL‐12p70 during T cell priming by aaDC prevented anergy induction in memory T cells and cytokine polarization in naïve T cells, indicating that the lack of IL‐12p70 is a key feature of aaDC. Our finding that aaDC differentially regulate naïve and memory T cells is important for understanding and maximizing the therapeutic potential of aaDC.

LPS activation is required for migratory activity and antigen presentation by tolerogenic dendritic cells

Autoimmune pathologies are caused by a breakdown in self‐tolerance. Tolerogenic dendritic cells (tolDC) are a promising immunotherapeutic tool for restoring self‐tolerance in an antigen‐specific manner. Studies about tolDC have focused largely on generating stable maturation‐resistant DC, but few have fully addressed questions about the antigen‐presenting and migratory capacities of these cells, prerequisites for successful immunotherapy. Here, we investigated whether human tolDC, generated with dexamethasone and the active form of vitamin D3, maintained their tolerogenic function upon activation with LPS (LPS‐tolDC), while acquiring the ability to present exogenous autoantigen and to migrate in response to the CCR7 ligand CCL19. LPS activation led to important changes in the tolDC phenotype and function. LPS‐tolDC, but not tolDC, expressed the chemokine receptor CCR7 and migrated in response to CCL19. Furthermore, LPS‐tolDC were superior to tolDC in their ability to present type II collagen, a candidate autoantigen in rheumatoid arthritis. tolDC and LPS‐tolDC had low stimulatory capacity for allogeneic, naïve T cells and skewed T cell polarization toward an anti‐inflammatory phenotype, although LPS‐tolDC induced significantly higher levels of IL‐10 production by T cells. Our finding that LPS activation is essential for inducing migratory and antigen‐presenting activity in tolDC is important for optimizing their therapeutic potential.

Low-strength T-cell activation promotes Th17 responses

We show that the strength of T-cell stimulation determines the capability of human CD4(+) T cells to become interleukin-17 (IL-17) producers. CD4(+) T cells received either high- (THi) or low (TLo)-strength stimulation via anti-CD3/CD28 beads or dendritic cells pulsed with superantigen in the presence of pro-Th17 cytokines IL-1β, transforming growth factor β, and IL-23. We found that TLo, but not THi, stimulation profoundly promoted Th17 responses by enhancing both the relative proportion and total number of Th17 cells. Titration of anti-CD3 revealed that low TCR signaling promoted Th17 cells, but only in the presence of anti-CD28. Impaired IL-17 production in THi cells could not be explained by high levels of Foxp3 or transforming growth factor β-latency-associated peptide expressed by THi cells. Nuclear factor of activated T cells was translocated to the nucleus in both THi and TLo cells, but only bound to the proximal region of the IL-17 promoter in TLo cells. The addition of a Ca(2+) ionophore under TLo conditions reversed the pro-Th17 effect, suggesting that high Ca(2+) signaling impairs Th17 development. Although our data do not distinguish between priming of naive T cells versus expansion/differentiation of memory T cells, our results clearly establish an important role for the strength of T-cell activation in regulating Th17 responses.

Raised interleukin-17 is immunolocalised to neutrophils in cystic fibrosis lung disease

Interleukin (IL)-17 is pivotal in orchestrating the activity of neutrophils. Neutrophilic inflammation is the dominant pathology in cystic fibrosis (CF) lung disease. We investigated IL-17 protein expression in the lower airway in CF, its cellular immunolocalisation and the effects of IL-17 on CF primary bronchial epithelial cells. Immunohistochemistry was performed on explanted CF lungs and compared with the non-suppurative condition pulmonary hypertension (PH). Airway lavages and epithelial cultures were generated from explanted CF lungs. Immunoreactivity for IL-17 was significantly increased in the lower airway epithelium in CF (median 14.1%) compared with PH (2.95%, p = 0.0001). The number of cells staining positive for IL-17 in the lower airway mucosa was also increased (64 cells·mm−1 compared with 9 cells·mm−1 basement membrane, p = 0.0005) and included both neutrophils in addition to mononuclear cells. IL-17 was detectable in airway lavages from explanted CF lungs. Treatment of epithelial cultures with IL-17 increased production of IL-8, IL-6 and granulocyte macrophage colony-stimulating factor. In conclusion, immunoreactive IL-17 is raised in the lower airway of people with CF and localises to both neutrophils and mononuclear cells. IL-17 increases production of pro-neutrophilic mediators by CF epithelial cells, suggesting potential for a positive feedback element in airway inflammation.

Regulatory T cells and autoimmunity.

Purpose of reviewThe identification of regulatory T cells (Tregs) as regulators of immunological self-tolerance has stimulated tremendous interest in the field. Over the past 12 months, new studies have added greatly to our understanding of the role of Tregs in autoimmune disease, details of which are presented here. Recent findingsIn this review, the mechanism of action of Tregs, their antigen specificity and their frequency and function in different autoimmune diseases is explored. Currently available data on the role of transforming growth factor-β, the reciprocal relationship between Tregs and Th17 cells, Treg markers, and current therapeutic approaches are evaluated. Other regulatory cells, which have been recently identified to play a significant role in autoimmunity, are described. SummaryIncreasing insights into understanding the complex mechanisms of action of Tregs have already led to exciting therapeutic advances. This review provides an in-depth analysis of recent advances in the field of Tregs in autoimmunity. It highlights targets for future immunomodulatory therapy that may treat and potentially cure autoimmune disease, and it identifies areas for future research.

Autologous tolerogenic dendritic cells for rheumatoid and inflammatory arthritis

Objectives To assess the safety of intra-articular (IA) autologous tolerogenic dendritic cells (tolDC) in patients with inflammatory arthritis and an inflamed knee; to assess the feasibility and acceptability of the approach and to assess potential effects on local and systemic disease activities. Methods An unblinded, randomised, controlled, dose escalation Phase I trial. TolDC were differentiated from CD14+ monocytes and loaded with autologous synovial fluid as a source of autoantigens. Cohorts of three participants received 1×106, 3×106 or 10×106 tolDC arthroscopically following saline irrigation of an inflamed (target) knee. Control participants received saline irrigation only. Primary outcome was flare of disease in the target knee within 5 days of treatment. Feasibility was assessed by successful tolDC manufacture and acceptability via patient questionnaire. Potential effects on disease activity were assessed by arthroscopic synovitis score, disease activity score (DAS)28 and Health Assessment Questionnaire (HAQ). Immunomodulatory effects were sought in peripheral blood. Results There were no target knee flares within 5 days of treatment. At day 14, arthroscopic synovitis was present in all participants except for one who received 10×106 tolDC; a further participant in this cohort declined day 14 arthroscopy because symptoms had remitted; both remained stable throughout 91 days of observation. There were no trends in DAS28 or HAQ score or consistent immunomodulatory effects in peripheral blood. 9 of 10 manufactured products met quality control release criteria; acceptability of the protocol by participants was high. Conclusion IA tolDC therapy appears safe, feasible and acceptable. Knee symptoms stabilised in two patients who received 10×106 tolDC but no systemic clinical or immunomodulatory effects were detectable. Trial registration number NCT01352858.

Treg cells in rheumatoid arthritis: an update.

Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease arising from a breakdown in immunological self-tolerance, which leads to aberrant immune responses to autoantigens. Regulatory CD4+ T-cells (Tregs) underpin one of the key mechanisms of self-tolerance and are a major focus of study in RA and other autoimmune diseases. In order to design new and improved therapies to reinstate self-tolerance, and perhaps cure disease, we need to understand the complex mechanism of action of Tregs. This review addresses recent findings in the field of Tregs in RA, with particular focus on identification of potential defects in Treg-mediated self-tolerance mechanisms present in RA patients, as well as how Tregs interact with other cells in the inflamed joints. As antigen-specific Tregs are a potential route for the reinstatement of immune tolerance, we also discuss new strategies currently being investigated which expand or induce de novo generation of Tregs.

Being sorry is not enough: the sorry state of the evidence base for improving the health of indigenous populations.

h o e ndigenous people worldwide experience worse health outcomes than other population groups onmeasures such as life expectancy, death rates, and years of life ost (i.e., premature deaths). The historical underpinings of this trend can be seen in the devastating effects of olonization on health outcomes for indigenous people, ighlighting the urgency needed in providing effective ealth interventions. Differences in health policy and in ocial, economic, cultural, geographic, and institutional actors also contribute to disparities in health outcomes. o improve the health of indigenous populations, multile strategies are needed to deal with the complexities f health inequity, taking into account all of these eterminants. Awareness is increasing of the need for an evidenceased rather than an opinion-based approach to improvng the health of indigenous populations. As the Cohrane Collaboration states: “Evidence-based health care s the conscientious use of current best evidence . . .” nvolving “information from relevant, valid research . . . .” nless an evidence-based approach is taken, there is jusifıable concern that ineffective strategies will be impleented, resulting in little improvement in health utcomes.

Online and offline recruitment of young women for a longitudinal health survey: Findings from the australian longitudinal study on women's health 1989-95 cohort

Background In 2012, we set out to recruit a cohort of at least 10,000 women aged 18-23 from across Australia. With recent research demonstrating the inadequacy of traditional approaches to recruiting women in this age group, we elected to conduct open recruiting. Objective Our aim was to report on the overall success of open recruiting and to evaluate the relative success of a variety of recruitment methods in terms of numbers and demographics. Methods We used referrals, Facebook, formal advertising, and incentives in order to recruit the cohort. Results In all, 17,069 women were recruited for the longitudinal online survey, from 54,685 initiated surveys. Of these women, most (69.94%, n=11,799) who joined the longitudinal cohort were recruited via Facebook, 12.72% (n=2145) via the fashion promotion, 7.02% (n=1184) by referral, 4.9% (n=831) via other Web activities, and 5.4% (n=910) via traditional media. Conclusions Facebook was by far the most successful strategy, enrolling a cohort of women with a similar profile to the population of Australian women in terms of age, area of residence, and relationship status. Women recruited via fashion promotion were the least representative. All strategies underrepresented less educated women—a finding that is consistent with more traditional means of recruiting. In conclusion, flexibility in recruitment design, embracing new and traditional media, adopting a dynamic responsive approach, and monitoring the results of recruiting in terms of sample composition and number recruited led to the successful establishment of a new cohort.

IL-6-driven STAT signalling in circulating CD4+ lymphocytes is a marker for early anticitrullinated peptide antibody-negative rheumatoid arthritis

Objectives A previously identified signal transduction and activator of transcription-3 (STAT3) target-enriched gene signature in circulating CD4+ T cells of patients with early rheumatoid arthritis (RA) was prominent in autoantibody-negative individuals. Here, interleukin (IL)-6-mediated STAT signalling was investigated in circulating lymphocytes of an independent early arthritis patient cohort, seeking further insight into RA pathogenesis and biomarkers of potential clinical utility. Methods Constitutive and IL-6-induced expression of phosphorylated STAT1 (pSTAT1) and pSTAT3 was determined in T and B cells using Phosflow cytometric analysis in patients with RA and controls. Contemporaneous levels of serum cytokines were measured by immunoassay. Induced gene expression was measured in cultured CD4+T cells by quantitative real-time PCR. Results Among circulating lymphocytes of 187 patients with early arthritis, constitutive pSTAT3 correlated with serum IL-6 levels maximally in CD4+ T cells. Increased constitutive pSTAT3, but not pSTAT1, was observed in circulating CD4+ T cells of patients with early anticitrullinated peptide autoantibody (ACPA)-negative RA compared with disease controls, and these levels decreased alongside markers of disease activity with IL-6R-targeted treatment. Among patients presenting with seronegative undifferentiated arthritis (UA) the ratio of constitutive pSTAT3:pSTAT1 in CD4+ T cells contributed substantially to an algorithm for predicting progression to classifiable RA during a median of 20 months follow-up (area under receiver operator characteristic curve=0.84; p<0.001). Conclusions Our findings support a particular role for IL-6-driven CD4+ T cell activation via STAT3 during the induction of RA, particularly as a feature of ACPA-negative disease. CD4+ T cell pSTAT measurements show promise as biomarkers of UA–RA progression and now require independent validation.