words):Objective: Autoimmune responses to DNA topoisomerase-I (TOP1) are found in a subset of patients with scleroderma at high risk for interstitial lung disease (ILD) and mortality. Anti-TOP1 antibodies (ATA) are associated with specific HLA-DRB1 alleles, and the frequency of HLA-DR-restricted TOP1-specific CD4+ T cells is associated with the presence, severity, and progression of ILD.Although this strongly implicates the presentation of TOP1 peptides by HLA-DR in scleroderma pathogenesis, the processing and presentation of TOP1 has not been studied. Methods:We developed a natural antigen processing assay (NAPA) to identify putative CD4+ T cell epitopes of TOP1 presented by monocyte-derived dendritic cells (mo-DCs) from six ATA-positive patients with scleroderma. Mo-DCs were pulsed with TOP1 protein, HLA-DR:peptide complexes were isolated, and eluted peptides were analyzed by mass spectrometry. We then examined the ability of these naturally presented peptides to induce CD4+ T cell activation in 11 ATA-positive and 11 ATA-negative scleroderma patients. Results:We found that a common set of 10 TOP1 epitopes was presented by mo-DCs from scleroderma patients with diverse HLA-DR variants. Sequence analysis revealed shared peptidebinding motifs within the HLA-DRβ chains of ATA-positive patients and a subset of TOP1 epitopes with distinct sets of anchor residues capable of binding to multiple different HLA-DR variants. The NAPA-derived epitopes elicited robust CD4+ T cell responses in 73% (8/11) ATA-positive patients, and the number of epitopes recognized correlated with ILD severity (p=0.025). Conclusion:These findings mechanistically implicate presentation of a convergent set of TOP1 epitopes in the development of scleroderma.

BackgroundThis work seeks to develop a methodology for identifying reliable biomarkers of disease activity, progression and outcome through the identification of significant associations between high-throughput flow cytometry (FC) data and interstitial lung disease (ILD) - a systemic sclerosis (SSc, or scleroderma) clinical phenotype which is the leading cause of morbidity and mortality in SSc. A specific aim of the work involves developing a clinically useful screening tool that could yield accurate assessments of disease state such as the risk or presence of SSc-ILD, the activity of lung involvement and the likelihood to respond to therapeutic intervention. Ultimately this instrument could facilitate a refined stratification of SSc patients into clinically relevant subsets at the time of diagnosis and subsequently during the course of the disease and thus help in preventing bad outcomes from disease progression or unnecessary treatment side effects.The methods utilized in the work involve: (1) clinical and peripheral blood flow cytometry data (Immune Response In Scleroderma, IRIS) from consented patients followed at the Johns Hopkins Scleroderma Center. (2) machine learning (Conditional Random Forests - CRF) coupled with Gene Set Enrichment Analysis (GSEA) to identify subsets of FC variables that are highly effective in classifying ILD patients; and (3) stochastic simulation to design, train and validate ILD risk screening tools.ResultsOur hybrid analysis approach (CRF-GSEA) proved successful in predicting SSc patient ILD status with a high degree of success (>82 % correct classification in validation; 79 patients in the training data set, 40 patients in the validation data set).ConclusionsIRIS flow cytometry data provides useful information in assessing the ILD status of SSc patients. Our new approach combining Conditional Random Forests and Gene Set Enrichment Analysis was successful in identifying a subset of flow cytometry variables to create a screening tool that proved effective in correctly identifying ILD patients in the training and validation data sets. From a somewhat broader perspective, the identification of subsets of flow cytometry variables that exhibit coordinated movement (i.e., multi-variable up or down regulation) may lead to insights into possible effector pathways and thereby improve the state of knowledge of systemic sclerosis pathogenesis.Electronic supplementary materialThe online version of this article (doi:10.1186/s12859-015-0722-x) contains supplementary material, which is available to authorized users.

One Sentence Summary: Use of a novel natural antigen processing assay reveals a mechanism for the presentation of shared CD4+ T cell epitopes of topoisomerase-I in immunogenetically diverse patients with scleroderma. Abstract:Disease-associated HLA-DRB1 alleles are thought to confer risk of developing autoimmunity by favoring the presentation of select autoantigenic epitopes. However, identification of these epitopes and the principles governing their presentation has been hindered by the imprecision of currently available methods, which cannot fully recapitulate the complexity of human pathophysiology. We present a natural antigen processing assay (NAPA), which overcomes these limitations by studying the presentation of autoantigenic CD4+ T cell epitopes by monocytederived dendritic cells (mo-DCs) from patients. We applied this strategy to study the processing and presentation of topoisomerase-1 (TOP1), a prevalent autoantigen in scleroderma that is associated with lung fibrosis and high mortality. We found that a common set of 10 epitopes was presented by mo-DCs from patients with diverse HLA-DR variants, including those not previously associated with the disease. Sequence analysis revealed a shared peptide-binding motif within the HLA-DR peptide binding grooves of patients who developed anti-TOP1 autoantibodies. In addition, a subset of naturally presented TOP1 peptides were characterized by immunological promiscuity, as they could bind to diverse HLA-DR peptide binding grooves. NAPA epitopes were immunorelevant: they could stimulate autoreactive CD4+ T cells in patients, and the number of epitopes recognized correlated with lung disease severity. These findings mechanistically implicate presentation of a convergent set of TOP1 epitopes in the development of scleroderma lung disease. Precise identification of autoantigenic epitopes is key to understanding the primordial mechanisms for the loss of tolerance, studying disease-propagating autoreactive T cells, and developing antigen-specific immunotherapy.

Background Increasing scientific evidence implicates a pathogenic role of Th17 cells and associated cytokines in rheumatoid arthritis (RA). Vitamin D may have suppressive effect on Th17 cells. In vitro studies show that vitamin D suppresses IL-17 production in stimulated PMBCs1 in patients with early RA. In a recent study2, serum 25(OH)D levels were inversely associated with IL-17 levels in RA patients. However, the effect of vitamin D repletion on IL-17 levels in RA remains untested. This is of clinical importance as correction of vitamin D deficiency may reduce IL-17 related inflammation in RA. Objectives Our goal was to compare serum IL-17 levels in vitamin D sufficient vs vitamin D deficient RA patients and to investigate the effect of vitamin D repletion on IL 17 levels in vitamin D deficient RA subjects. Methods Sera from RA patients (1987 ARA criteria) enrolled in a randomized controlled trial were used. 25(OH)D levels were measured using Diasorin radioimmunoassay. Vitamin D deficiency was classified as 25(OH)D level <30ng/ml. Vitamin D deficient patients were randomly assigned to either vitamin D therapy [50,000 IU ergocalciferol/week for 8 or 16 weeks until repletion [25(OH)D≥30 ng/ml] was achieved OR placebo for 16 weeks. Sera were collected and stored at -70 degrees every study visit. IL-17 was measured in both vitamin D sufficient (n=56) and deficient (n=83) patients at baseline. IL-17 was also measured in patients who completed vitamin D therapy and achieved repletion. We compared pre- and post-treatment IL-17 levels in this group. A commercially available Ruthenium based electro-chemoluminescence platform was used to measure IL-17 (lowest quantification level =0.2 pg/ml). Unpaired and paired students t-test and regression analyses were used. Results Participants (n=139) were mostly female (80%), white (79.1%) with a mean (SD) age of 53.4 (12.3) yrs, body mass index of 30.5 (6.9) m/kg2 and disease duration of 9.9 (9) yrs. Mean 25(OH)D levels were 28.3 (11.1)ng/mL (range 6.3 – 72.8 ng/ml). 83 participants (60%) patients had 25(OH)D levels <30 ng/ml and entered the RCT. 68 participants completed vitamin D repletion therapy. Mean increase in vitamin D in this group was 17.3 (12.9) ng/ml. In a cross-sectional analysis, no significant difference was noted in IL-17 levels at baseline between vitamin D sufficient and deficient groups (mean IL 17 0.71±1.27 pg/ml vs 0. 62±0.65 pg/ml respectively, p value=0.62). Using linear regression, baseline vitamin D sufficiency/deficiency was a non-significant predictor of IL -17 levels (beta= -0.04, p=0.622). After vitamin D repletion therapy, no differences were noted in IL-17 levels before and after repletion (IL-17 at baseline vs repletion, 0.61 (0.61) pg/ml vs 0.68 (0.93) pg/ml respectively, p=0.60). Conclusions These data suggest that IL-17 levels do not differ between vitamin D deficient and sufficient RA patients. Moreover, vitamin D repletion does not impact IL-17 levels. This is the first study to investigate the effect of vitamin D repletion therapy o...