A discrete cluster of urinary biomarkers discriminates between active systemic lupus erythematosus patients with and without glomerulonephritis.

BackgroundManagement of lupus nephritis (LN) would be greatly aided by the discovery of biomarkers that accurately reflect changes in disease activity. Here, we used a proteomics approach to identify potential urinary biomarkers associated with LN.MethodsUrine was obtained from 60 LN patients with paired renal biopsies, 25 active non-LN SLE patients, and 24 healthy controls. Using Luminex, 128 analytes were quantified and normalized to urinary creatinine levels. Data were analyzed by linear modeling and non-parametric statistics, with corrections for multiple comparisons. A second cohort of 33 active LN, 16 active non-LN, and 30 remission LN SLE patients was used to validate the results.ResultsForty-four analytes were identified that were significantly increased in active LN as compared to active non-LN. This included a number of unique proteins (e.g., TIMP-1, PAI-1, PF4, vWF, and IL-15) as well as known candidate LN biomarkers (e.g., adiponectin, sVCAM-1, and IL-6), that differed markedly (>4-fold) between active LN and non-LN, all of which were confirmed in the validation cohort and normalized in remission LN patients. These proteins demonstrated an enhanced ability to discriminate between active LN and non-LN patients over several previously reported biomarkers. Ten proteins were found to significantly correlate with the activity score on renal biopsy, eight of which strongly discriminated between active proliferative and non-proliferative/chronic renal lesions.ConclusionsA number of promising urinary biomarkers that correlate with the presence of active renal disease and/or renal biopsy changes were identified and appear to outperform many of the existing proposed biomarkers

Validation of CyTOF Against Flow Cytometry for Immunological Studies and Monitoring of Human Cancer Clinical Trials

Flow cytometry is a widely applied approach for exploratory immune profiling and biomarker discovery in cancer and other diseases. However, flow cytometry is limited by the number of parameters that can be simultaneously analyzed, severely restricting its utility. Recently, the advent of mass cytometry (CyTOF) has enabled high dimensional and unbiased examination of the immune system, allowing simultaneous interrogation of a large number of parameters. This is important for deep interrogation of immune responses and particularly when sample sizes are limited (such as in tumors). Our goal was to compare the accuracy and reproducibility of CyTOF against flow cytometry as a reliable analytic tool for human PBMC and tumor tissues for cancer clinical trials. We developed a 40+ parameter CyTOF panel and demonstrate that compared to flow cytometry, CyTOF yields analogous quantification of cell lineages in conjunction with markers of cell differentiation, function, activation, and exhaustion for use with fresh and viably frozen PBMC or tumor tissues. Further, we provide a protocol that enables reliable quantification by CyTOF down to low numbers of input human cells, an approach that is particularly important when cell numbers are limiting. Thus, we validate CyTOF as an accurate approach to perform high dimensional analysis in human tumor tissue and to utilize low cell numbers for subsequent immunologic studies and cancer clinical trials

Presence of an interferon signature in individuals who are anti-nuclear antibody positive lacking a systemic autoimmune rheumatic disease diagnosis

Abstract Background Elevated levels of type I interferons (IFNs) are a characteristic feature of the systemic autoimmune rheumatic diseases (SARDs) and are thought to play an important pathogenic role. However, it is unknown whether these elevations are seen in anti-nuclear antibody–positive (ANA+) individuals who lack sufficient criteria for a SARD diagnosis. We examined IFN-induced gene expression in asymptomatic ANA+ individuals and patients with undifferentiated connective tissue disease (UCTD) to address this question. Methods Healthy ANA− control subjects and ANA+ titre (≥1:160 by immunofluorescence) participants meeting no criteria, meeting at least one criterion (UCTD) or meeting SARD classification criteria were recruited. Whole peripheral blood IFN-induced and BAFF gene expression were quantified using NanoString technology. The normalized levels of five IFN-induced genes were summed to produce an IFN5 score. Results The mean IFN5 scores were increased in all ANA+ participant subsets as compared with healthy control subjects. We found that 36.8% of asymptomatic ANA+ and 50% of UCTD participants had IFN5 scores >2 SD above the mean for healthy control subjects. In all ANA+ subsets, the IFN5 score correlated with the presence of anti-Ro/La antibodies. In the asymptomatic ANA+ subset, this score also correlated with the ANA titre, whereas in the other ANA+ subsets, it correlated with the number of different ANA specificities. Development of new SARD criteria was seen in individuals with normal and high IFN5 scores. Conclusions An IFN signature is seen in a significant proportion of ANA+ individuals and appears to be associated with ANA titre and type of autoantibodies, rather than with the presence or development of clinical SARD symptoms

The baseline interferon signature predicts disease severity over the subsequent 5 years in systemic lupus erythematosus

Abstract Objectives Type I interferons (IFNs) play an important role in the pathophysiology of systemic lupus erythematosus (SLE). While cross-sectional data suggest an association between IFN-induced gene expression and SLE disease activity, interest in this as a biomarker of flare has been tempered by a lack of fluctuation with disease activity in the majority of patients. This led us to question whether IFN-induced gene expression might instead be a biomarker of overall disease severity, with patients with high levels spending more time in an active disease state. Methods Levels of five interferon-responsive genes were measured in the whole peripheral blood at baseline visit for 137 SLE patients subsequently followed for 5 years. Log transformed values were summed to yield a composite IFN5 score, and the correlation with various disease outcomes examined. Receiver operator characteristic analyses were performed for outcomes of interest. Kaplan-Meier curves were generated to compare the proportion of flare-free patients with high and low IFN5 scores over time. Results The baseline IFN5 score was positively correlated with the adjusted mean SLE disease activity index-2000, number of flares, adjusted mean prednisone dose, and number of new immunosuppressive medications over the subsequent 5 years. Optimal cut-offs for the IFN5 score were determined using Youden’s index and predicted more severe outcomes with 57–67% accuracy. A high baseline IFN5 level was associated with a significantly increased risk of subsequent flare. Conclusions Measurement of the type I IFN signature is a useful tool for predicting the subsequent disease activity course

T Cell and Dendritic Cell Abnormalities Synergize to Expand Pro-Inflammatory T Cell Subsets Leading to Fatal Autoimmunity in B6.NZBc1 Lupus-Prone Mice

<div><p>We have previously shown that B6 congenic mice with a New Zealand Black chromosome 1 (c1) 96-100 cM interval produce anti-nuclear Abs and that at least two additional genetic loci are required to convert this subclinical disease to fatal glomerulonephritis in mice with a c1 70-100 cM interval (c1(70-100)). Here we show that the number of T follicular helper and IL-21-, IFN-γ-, and IL-17-secreting CD4⁺ T cells parallels disease severity and the number of susceptibility loci in these mice. Immunization of pre-autoimmune mice with OVA recapitulated these differences. Differentiation of naïve T cells <i>in-vitro</i> under polarizing conditions and <i>in-vivo</i> following adoptive transfer of OVA-specific TCR transgenic cells into c1(70-100) or B6 recipient mice, revealed T cell functional defects leading to increased differentiation of IFN-γ- and IL-17-producing cells in the 96-100 cM and 88-96 cM intervals, respectively. However, in<i>-vivo</i> enhanced differentiation of pro-inflammatory T cell subsets was predominantly restricted to c1(70-100) recipient mice, which demonstrated altered dendritic cell function, with increased production of IL-6 and IL-12. The data provide support for the role of pro-inflammatory T cells in the conversion of subclinical disease to fatal autoimmunity and highlight the importance of synergistic interactions between individual susceptibility loci in this process.</p> </div

Increased differentiation of naïve CD4⁺ T cells from c1 congenic mice to Th17 and Th1 cells <i>in-vitro</i>.

<p>Naïve T cells from 8-wk-old mice were stimulated under Th0, Th1, Th2, Th17, and IL-21-producing polarizing conditions and cytokine production quantified 5 days later by flow cytometry (see Methods). (A) Representative contour plots gated on CD3⁺CD4⁺ T cells from B6 and c1(70-100) mice. For each polarizing condition, plots for relevant cytokine production under Th0 conditions (-) and polarizing conditions (+) are shown. The quadrants used to define positively and negatively staining cells are indicated. (B) Scatterplots showing the percentage of T cells that are IL-21-producing (Tfh), Th17, Th1 and Th2 cells, under relevant polarizing conditions. Horizontal lines indicate the mean for each population examined. Significance levels were determined by one-way ANOVA with Dunns’ post-test. The <i>p</i> values for significant differences between B6 and congenic mouse strains are shown with *p<0.05, **p<0.01, ***p<0.001. Bars with <i>p</i> values above denote significant differences between congenic strains.</p

Altered production of IL-6 and/or IL-12 by myeloid DC from c1(88-100) and c1(70-100) mice following stimulation with TLR ligands.

<p>BMDC from 8-12 week-old mice were expanded with FLT3L and then cultured in the presence or absence of Imiquimod R837, Poly(I:C), CpG 2216, or LPS for 18h with GolgiStop (for IL-12) or GolgiPlug (for IL-6) being added for the last 6 h. The cells were then stained as outlined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075166#pone-0075166-g005" target="_blank">Figure 5</a> and the Methods. (A) Left panel shows representative dot plots indicating the regions used to gate B220 ⁺ CD11b^- pDC (top left box) and B220^-CD11b⁺ mDC (bottom right box) within the CD11c⁺ population. Shown to the right are representative histogram plots of IL-6 and IL-12 for B6 (solid grey) and c1(70-100) mice (black line) in unstimulated (Media) and stimulated (Poly(I:C) for IL-6 or CpG 2216 for IL-12) conditions. (B) Scatterplots showing the MFI for IL-6 and IL-12 expression on mDC. (C) IFN-α and IL-23 levels in the culture supernatants of BMDC as measured by ELISA. (D) MFI for TNF-α expression in mDC. Horizontal lines indicate the mean. Significance levels were determined by one-way ANOVA with Dunns’ post-test. The <i>p</i> values for significant differences between B6 and congenic mouse strains are shown with *p<0.05, **p<0.01. Bars with <i>p</i> values above denote significant differences between congenic strains.</p

Myeloid DC from c1(88-100) and c1(70-100) mice demonstrate altered function and an enhanced ability to induce differentiation of Th1 cells.

<p>BMDC from 8–12 wk-old mice were expanded with FLT3L for 7 days and then co-cultured with OVA peptide and purified naïve CD4⁺ T cells from OT-II TCR Tg mice. On day 4, the cells were re-stimulated with PMA and ionomycin for 4 h in the presence of GolgiStop or GolgiPlug, and analyzed by flow cytometry for cell surface DC (CD11c, CD11b, B220, MHC-II, B7.2) or T cell (CD3, CD4) markers and intracellular cytokine levels. (A) Scatterplots showing the percentage of IL-21-, IL-17- and IFN-γ-producing T cells. Results are clustered in groups based on the strain of T cells (top of the figure) with the DC strain shown at the bottom of the figure. Scatterplots showing the percentage of CD11c⁺CD11b⁺B220⁻ mDC (B) and CD11c⁺CD11b^-B220⁺ pDC (C) expressing elevated levels of MHCII and B7.2, or IL-6 and IL-12. Results with the different strains of T cells have been pooled as no differences were noted between strains. Horizontal lines indicate the mean. Significance levels were determined by one-way ANOVA with Dunns’ post-test. The <i>p</i> values for significant differences between B6 and congenic mouse strains are shown with *p<0.05, **p<0.01, ***p<0.001. Bars with <i>p</i> values above denote significant differences between congenic strains.</p