Theo van den Broek

Epitope-specific immunotherapy of rheumatoid arthritis: clinical responsiveness occurs with immune deviation and relies on the expression of a cluster of molecules associated with T cell tolerance in a double-blind, placebo-controlled, pilot phase II trial.

OBJECTIVE Induction of immune tolerance to maintain clinical control with a minimal drug regimen is a current research focus in rheumatoid arthritis (RA). Accordingly, we are developing a tolerization approach to dnaJP1, a peptide part of a pathogenic mechanism that contributes to autoimmune inflammation in RA. We undertook this study to test 2 hypotheses: 1) that mucosal induction of immune tolerance to dnaJP1 would lead to a qualitative change from a proinflammatory phenotype to a more tolerogenic functional phenotype, and 2) that immune deviation of responses to an inflammatory epitope might translate into clinical improvement. METHODS One hundred sixty patients with active RA and with immunologic reactivity to dnaJP1 were enrolled in a pilot phase II trial. They received oral doses of 25 mg of dnaJP1 or placebo daily for 6 months. RESULTS The dnaJP1 peptide was safe and well-tolerated. In response to treatment with dnaJP1, there was a significant reduction in the percentage of T cells producing tumor necrosis factor alpha and a corresponding trend toward an increased percentage of T cells producing interleukin-10. Coexpression of a cluster of molecules (programmed death 1 and its ligands) associated with T cell regulation was also found to be a prerequisite for successful tolerization in clinical responders. Analysis of the primary efficacy end point (meeting the American College of Rheumatology 20% improvement criteria at least once on day 112, 140, or 168) showed a difference between treatment groups that became significant in post hoc analysis using generalized estimating equations. Differences in clinical responses were also found between treatment groups on day 140 and at followup. Post hoc analysis showed that the combination of dnaJP1 and hydroxychloroquine (HCQ) was superior to the combination of HCQ and placebo. CONCLUSION Tolerization to dnaJP1 leads to immune deviation and a trend toward clinical efficacy. Susceptibility to treatment relies on the coexpression of molecules that can down-regulate adaptive immunity.

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.

Neonatal thymectomy reveals differentiation and plasticity within human naive T cells

The generation of naive T cells is dependent on thymic output, but in adults, the naive T cell pool is primarily maintained by peripheral proliferation. Naive T cells have long been regarded as relatively quiescent cells; however, it was recently shown that IL-8 production is a signatory effector function of naive T cells, at least in newborns. How this functional signature relates to naive T cell dynamics and aging is unknown. Using a cohort of children and adolescents who underwent neonatal thymectomy, we demonstrate that the naive CD4+ T cell compartment in healthy humans is functionally heterogeneous and that this functional diversity is lost after neonatal thymectomy. Thymic tissue regeneration later in life resulted in functional restoration of the naive T cell compartment, implicating the thymus as having functional regenerative capacity. Together, these data shed further light on functional differentiation within the naive T cell compartment and the importance of the thymus in human naive T cell homeostasis and premature aging. In addition, these results affect and alter our current understanding on the identification of truly naive T cells and recent thymic emigrants.

STAT3 regulates monocyte TNF-alpha production in systemic inflammation caused by cardiac surgery with cardiopulmonary bypass.

Background Cardiopulmonary bypass (CPB) surgery initiates a controlled systemic inflammatory response characterized by a cytokine storm, monocytosis and transient monocyte activation. However, the responsiveness of monocytes to Toll-like receptor (TLR)-mediated activation decreases throughout the postoperative course. The purpose of this study was to identify the major signaling pathway involved in plasma-mediated inhibition of LPS-induced tumor necrosis factor (TNF)-α production by monocytes. Methodology/Principal Findings Pediatric patients that underwent CPB-assisted surgical correction of simple congenital heart defects were enrolled (n = 38). Peripheral blood mononuclear cells (PBMC) and plasma samples were isolated at consecutive time points. Patient plasma samples were added back to monocytes obtained pre-operatively for ex vivo LPS stimulations and TNF-α and IL-6 production was measured by flow cytometry. LPS-induced p38 mitogen-activated protein kinase (MAPK) and nuclear factor (NF)-κB activation by patient plasma was assessed by Western blotting. A cell-permeable peptide inhibitor was used to block STAT3 signaling. We found that plasma samples obtained 4 h after surgery, regardless of pre-operative dexamethasone treatment, potently inhibited LPS-induced TNF-α but not IL-6 synthesis by monocytes. This was not associated with attenuation of p38 MAPK activation or IκB-α degradation. However, abrogation of the IL-10/STAT3 pathway restored LPS-induced TNF-α production in the presence of suppressive patient plasma. Conclusions/Significance Our findings suggest that STAT3 signaling plays a crucial role in the downregulation of TNF-α synthesis by human monocytes in the course of systemic inflammation in vivo. Thus, STAT3 might be a potential molecular target for pharmacological intervention in clinical syndromes characterized by systemic inflammation.

Heat shock protein-derived T-cell epitopes contribute to autoimmune inflammation in pediatric Crohn's disease.

Pediatric Crohns disease is a chronic auto inflammatory bowel disorder affecting children under the age of 17 years. A putative etiopathogenesis of Crohns disease (CD) is associated with disregulation of immune response to antigens commonly present in the gut microenvironment. Heat shock proteins (HSP) have been identified as ubiquitous antigens with the ability to modulate inflammatory responses associated with several autoimmune diseases. The present study tested the contribution of immune responses to HSP in the amplification of autoimmune inflammation in chronically inflamed mucosa of pediatric CD patients. Colonic biopsies obtained from normal and CD mucosa were stimulated with pairs of Pan HLA-DR binder HSP60-derived peptides (human/bacterial homologues). The modulation of RNA and protein levels of induced proinflammatory cytokines were measured. We identified two epitopes capable of sustaining proinflammatory responses, specifically TNF〈 and IFN© induction, in the inflamed intestinal mucosa in CD patients. The responses correlated positively with clinical and histological measurements of disease activity, thus suggesting a contribution of immune responses to HSP in pediatric CD site-specific mucosal inflammation.

Cytokine profiling at disease onset: support for classification of young antinuclear antibody-positive patients as a separate category of juvenile idiopathic arthritis

The current classification for juvenile idiopathic arthritis (JIA) separates the disease entity JIA into seven disease categories based on clinical and laboratory features1 It is postulated that the combination of early onset of disease and antinuclear antibody (ANA) positivity better classifies a homogeneous subset of patients than the current International League of Associations for Rheumatology (ILAR) categories, displaying asymmetric arthritis, female predominance and an increased risk of developing iridocyclitis.2–5 The classification by the number of joints involved might not be an adequate criterion for identifying homogeneous disease entities. We sought out to determine if this homogenous group of young ANA-positive patients could be distinguished from other JIA patients based on their soluble inflammatory profile. These profiles could aid in the differentiation of patient groups by identifying similar underlying inflammatory processes. This study prospectively followed 40 patients with JIA at onset of disease at their first visit to the paediatric rheumatology clinic for oligoarthritis (n=24) and rheumatoid factor-negative polyarthritis (n=16). Ethics approval was given by the regional review boards. Informed consent was obtained from all patients. The occurrence of iridocylitis was determined by reviewing total clinical history; the time of follow-up did not differ between the groups analysed (table 1). Apart from nonsteroidal anti-inflammatory drugs (NSAID) therapy, …

Differential homeostatic dynamics of human regulatory T-cell subsets following neonatal thymectomy.

Disclosure of potential conflict of interest: R. P. Schleimer has consultant arrangementswith Interesect ENT, GlaxoSmithKline, Allakos, and Aurasense. L. C. Grammer has received grants and travel support for meetings or other purposes from the National Institutes of Health (NIH); has received grants from the Bazley Foundation; has consultant arrangements with Atellas Pharmaceuticals; is employed by Northwestern University and the Northwestern Medical Faculty Foundation; has grants/grants pending with the NIH, the Food Allergy Network, and SC has received payment for lectures, including service on speakers’ bureaus, from theAmericanAcademy ofAllergy, Asthma & Immunology and Mount Sinai; and receives royalties from Lippincott, UpToDate, BMJ, and Elsevier. B. K. Tan has received grants from the NIH and the Triological Society/American College of Surgeons; has consultant arrangements with Acclarent, Inc, and has received travel/accommodations/meeting expenses from the Foundation for Innovation, Education, and Research in Otorhinolaryngology. T. R. Torgerson has consultant arrangements with Baxter Biosciences and BDBiosciences; has grants/grants pending with Baxter Biosciences and CSL Behring; has received payment for lectures, including service on speakers’ bureaus, from Baxter Biosciences; receives royalties from New England Biolabs; and receives payment for the development of educational presentations from Baxter Biosciences. K. E. Harris has received grants from the NIH/ National Heart, Lung and Blood Institute (grant no. R37L068546 and grant no. R01HL078860), the NIH/National Institute for Allergy and Infectious Disease (grant no. T32A1083216), and the Ernest S. Bazley Trust. A. T. Peters has received payment for lectures, including service on speakers’ bureaus, from Baxter. A. Kato has received a grant from the NIH (grant no. R21HL113913). The rest of the authors declare that they have no relevant conflicts of interest.

A sensitive protocol for FOXP3 epigenetic analysis in scarce human samples

CD4+CD25high regulatory T (Treg) cells are key players in the maintenance of peripheral immune tolerance [1]. Stable expression of the FOXP3 transcription factor is essential for Treg cells’ ability to suppress the immune responses of conventional T (Tconv) cells [2]. FOXP3 stability in murine Treg cells has been linked to FOXP3 locus demethylation at the CNS2 [3, 4], also called the Treg-specific demethylated region (TSDR) [5]. In contrast to FOXP3 expression [6, 7], complete demethylation of the TSDR [3], in addition to other genomic regions [8], is a true hallmark of human and mouse Treg cells, and its role in the early Treg lineage specification is currently under intense scrutiny in the mouse [3, 8, 9]. Unfortunately, the investigation of TSDR demethylation to define whether FOXP3+ T cells are bona fide Treg cells in translational research settings has so far been inaccessible due to limitations in clinical sample amount. Indeed, methods currently available to inspect the TSDR at single-CpG resolution [5] lack sensitivity due to bottlenecks at the amplification stage (Fig. 1A). Other methods originally aimed at counting Treg cells in unsegregated populations [10] can be adapted to study the regulatory lineage commitment of sorted cells. However, because these methods separately amplify methylated and unmethylated TSDRs, the sample amount requirement is a function of the degree of TSDR methylation, growing exponentially at the two ends of the range, where one of the two species becomes limiting. In this work, we report an inexpensive, single-CpG resolution, PCR-based protocol with very low requirements on sample amount and robust to aldehyde-based fixation, features making it of immediate relevance for sample-limited research settings. Our optimized protocol enhances the sensitivity of existing techniques [5] while retaining specificity. We introduced nested PCR, touchdown preamplification, primer tailing, and a two-step sequencing cycle (detailed protocol available as Supporting Information). The nested PCR targets 15 commonly investigated CpGs sites [5, 10], thereby allowing for direct comparison of results with previous literature. However, we found that measurements of the first CpG dinucleotide are typically noisier due to proximity to the sequencing primer, and we recommend excluding this first CpG dinucleotide, unless several technical replicates are performed. The touchdown preamplification step was introduced not to compromise specificity with the enhanced sensitivity. Moreover, we added 5′ tails to the inner primers in order to (i) increase their length, which allows raising the annealing temperature, thereby minimizing spurious amplification; (ii) introduce C and G nucleotides, which are rarer in amplicons from bisulphiteconverted templates, thereby increasing DNA complexity and, consequently, the specificity of primer annealing; (iii) extend the amplicon at the 5′ end, which allows to shift the sequencing primer upstream, thereby improving base resolution at the 5′ end; and (iv) allow the sequencing primer to anneal only to the inner-PCR product. Finally, the two-step sequencing cycle yields a better signal balance when compared with the standard three-step denaturation/annealing/extension cycle. Electropherograms were analyzed with ESME to quantitatively determine the methylated/unmethylated CpG ratio [11]. We favored Sanger sequencing for its widespread availability, but pyrosequencing is also an attractive option. Our method was able to successfully generate TSDR amplicons from much lower amounts of template than singleround PCR techniques (Fig. 1A). The TSDR methylation profiles of CD25low/− Tconv cells and CD25highCD127low/− Treg cells generated by our protocol are in line with published data [5, 8–10], demonstrating its accuracy (i.e. proximity to the true value) in measuring highly methylated (Tconv cells), highly demethylated (male Treg cells), and partially demethylated (female Treg cells, due to FOXP3 methylation on the inactive X chromosome) samples (Fig. 1B). Accuracy was linearly preserved across the whole range of TSDR methylation, indicating no amplification bias of either methylated or unmethylated TSDR templates (Fig. 1C). In addition, to carefully characterize the lower limit of detection, we calculated the percentage error (a normalized measure of the difference between observed and expected value, i.e. an estimate of accuracy) and the coefficient of variation (a normalized measure of data dispersion around the mean, i.e. an estimate of precision) of technical replicates across decreasing amounts of template gDNA. We were able to amplify down to 1.25 ng bisulphiteconverted gDNA with 100% success rates

Brief Report: Autologous Stem Cell Transplantation Restores Immune Tolerance in Experimental Arthritis by Renewal and Modulation of the Teff Cell Compartment

Autologous stem cell transplantation (ASCT) induces long‐term drug‐free disease remission in patients with juvenile idiopathic arthritis. This study was undertaken to further unravel the immunologic mechanisms underlying ASCT by using a mouse model of proteoglycan‐induced arthritis (PGIA).

Human neonatal thymectomy induces altered B-cell responses and auto-reactivity

An association between T‐cell lymphopenia and autoimmunity has long been proposed, but it remains to be elucidated whether T‐cell lymphopenia affects B‐cell responses to autoantigens. Human neonatal thymectomy (Tx) results in a decrease in T‐cell numbers and we used this model to study the development of autoreactivity. Two cohorts of neonatally thymectomized individuals were examined, a cohort of young (1–5 years post‐Tx, n = 10–27) and older children (>10 years, n = 26), and compared to healthy age‐matched controls. T‐cell and B‐cell subsets were assessed and autoantibody profiling performed. Early post‐Tx, a decrease in T‐cell numbers (2.75 × 109/L vs. 0.71 × 109/L) and an increased proportion of memory T cells (19.72 vs. 57.43%) were observed. The presence of autoantibodies was correlated with an increased proportion of memory T cells in thymectomized children. No differences were seen in percentages of different B‐cell subsets between the groups. The autoantigen microarray showed a skewed autoantibody response after Tx. In the cohort of older individuals, autoantibodies were present in 62% of the thymectomized children, while they were found in only 33% of the healthy controls. Overall, our data suggest that neonatal Tx skews the autoantibody profile. Preferential expansion and preservation of Treg (regulatory T) cell stability and function, may contribute to preventing autoimmune disease development after Tx.