Flow Cytometry Core Facility Papers

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Cloud-based highly parallel execution of t-SNE and SPADE with metaclustering for analysis and visualization of large single-cell datasets
(2017-06) Ciccolella, Chris; Belkina, Anna
The use of machine learning techniques, in particular unsupervised clustering and dimensionality reduction algorithms, is quickly becoming a standard workflow for identifying and visualizing biological populations from within high-dimensional data. These methods allow researchers to approach data analysis without the bias and subjectivity that has traditionally been standard in the field. Algorithms have context-dependent strengths and weaknesses. Across algorithms, an inability to scale computation to large datasets is a common theme. Most algorithms are designed and distributed to run on individual computers where memory and CPU are quickly exhausted by large datasets. Even when high-performance compute resources are available, algorithms often don't scale to large datasets as a fundamental property of their design. If they do, it might result in an untenable increase in runtime or diminished quality of results. t-SNE and SPADE are two well-published algorithms that suffer problems as discussed above after datasets exceed a number of observations on the order of 1 million. This study introduces an alternative approach to the use of SPADE and t- SNE whereby a dataset is divided and distributed across numerous compute nodes in the cloud to process independently in parallel. The results of each computation are then combined in a metaclustering step for final visualization and analysis. The improvement in execution speed as a function of degree of parallelization is established. The method is validated against a non-parallel analysis of the same dataset to establish concordance of identified populations. The workflow is executed on Cytobank for portability to other researchers.
Reagent-driven reconfiguration/optimization of a 16-parameter BD FACSARIA II SORP to allow accurate detection of violet- and UV-excited Sirigen dyes
(2016-06) Belkina, Anna; Snyder-Cappione, Jennifer
Background. Modern flow cytometers can detect emission from a variety of commercially available fluorescent reagents. However, accurate detection of novel dyes is often difficult due to the lack of readily available quality assurance tools, for instrument manufacturer QC and optimization protocols often become available secondary to new fluorescent reagents. Aims. For standard QC and instrument calibration of the FACSARIA, BD provides a standard 'CS&T' method that includes three-peak beads and a dedicated software module. While this method allows tracking instrument state over time, it does not accurately access PMT performance on a significant part of the spectrum for violet-or UV-excited dyes. In order to ensure accurate detection of reagents within all 16 channels of the Boston University Flow Core 16-color, 4-laser BD FACSARIA SORP, we created and performed an novel optimization process that allows simultaneous accommodation of as many as nine polymeric Sirigen dyes, including those emitting in both long-wavelength violet-and UV-laser excited channels. Methods and Results. Firstly, the electronic noise of all PMTs was assessed and information was collected on rSD of non-stained cells within 100-800V range. The derived basal PMT values then provided a starting point for voltage optimization of instrument-specific panels. These values differed greatly from CS&T-deduced PMT voltages for abovementioned channels, for some the CS&T calculation of optimal PMT voltage was not possible due to a poor resolution of CS&T peaks at certain wavelengths. Several PMTs were identified with sub-par performance and were consequently replaced. Our testing of multiple commercial compensation beads found that the majority demonstrated prohibitively high backgrounds; the eBioscience UltraComp beads performed best and were therefore our reagent of choice. For instrument performance tracking, we also compared multi-peak beads from several manufacturers and found Spherotech Ultra Rainbow beads to be the sole bead type with satisfactory resolution of all peaks on long-wavelength UV channels. Finally, we developed an ergonomic protocol for facility users that includes an experiment template and electronic tables for data processing. With that protocol, a user can: (1) finely tune PMT voltages to accommodate a specific panel, (2) determine antibody concentrations for compensation control preparations, and (3) associate these optimized settings with multi-peak bead target values. Such preliminary setup allows quick panel-specific instrument calibration for each experimental run. This approach was successfully applied to several 16-color panels used in our Core facility and resulted in vastly improved reproducibility of acquired data over months of use. Conclusions. Synchronizing cutting-edge reagent technologies with existing instrument QC and maintenance methodology requires development of mix-and-match solutions not necessarily provided by the instrument manufacturer. Creating a user-friendly, accurate QC and calibration protocol that accommodates novel reagents allows dramatic expansion of our userbase's experimental capabilities.
14-color flow cytometry to determine the contribution of mitochondrial mass to differences in glycolytic capacity in human immune cell subsets
(2017-06) Nicholas, Dequina A.; Belkina, Anna; Proctor, Elizabeth A.; Raval, Forum; Apovian, Caroline M.; Lauffenburger, Douglas A.; Nikolajczyk, Barbara S.; Lp, Blanche
Mitochondrial metabolism controls immune cell function, but comprehensive tools to assess human primary immune cell metabolic capacity remain rudimentary. We previously demonstrated that CD19+ B cells rely more heavily on anaerobic glycolysis (i.e. are more glycolytic) than CD4+ T cells. Furthermore, both PBMCs and CD4+ T cells from subjects with type 2 diabetes (T2D) are more glycolytic than their counterparts from BMI-matched non-T2D controls. The contribution of mitochondrial mass, an indicator of non-glycolytic metabolism, to the various metabolic phenotypes is untested. To assess the contribution of immune cell subset identity and mitochondrial mass to the enhanced glycolytic capacity of resting B cells and PBMCs from T2D subjects, we designed a 13-color panel based on standard immune cell subset markers and chemokine receptors, and included MitoTracker Green FM (MTG), which quantitatively indicates mitochondrial mass. We used this novel panel to phenotype 63 total samples from BMI-matched subjects in three groups: non-T2D, pre-T2D, and fulminant T2D, as defined by American Diabetes Association guidelines. The panel was built in several iterations to accommodate spillover of MTG fluorescence into neighboring channels and includes, besides MTG and live-dead discriminator, the following surface markers: CD4, CD8, CD19, CD45RA, CD25, CD127, CD14, CCR4, CCR5, CCR6, CXCR3, and CD161. The PBMC samples were run on a 4-laser BD FACSARIA II SORP with pre-established panel-specific PMT voltages tracked using 6-peak Ultrarainbow beads. To normalize MTG fluorescence intensity and thus minimize batch effects, each of 5 total batches included a reference donor PBMC sample that was frozen in multiple aliquots from one blood draw. Using this approach, we quantified the percentages of immune cell populations (CD19+ B cells, CD8+ naïve and memory/effector T cells, and CD4+ cells including Tregs and populations enriched in Th1, Th2 and Th17) along with the relative mitochondrial mass in each subset. We found that CD19+ B cells in PBMCs from both ND and T2D subjects had significantly less mitochondrial mass than CD4+ cells, supporting the demonstration that B cells are more glycolytic than CD4+ T cells. Of all the CD4+ T cell subsets, Th17 cells consistently had the lowest mitochondrial mass, consistent with the interpretation that Th17s are more dependent on glycolysis than previously appreciated. Our results validate the utility of our 13-color panel to simultaneously quantify relative mitochondrial mass in numerous immune cell subsets and thereby provide a new tool to explore metabolism in human primary cells.
Evaluation of ‘Super Bright’ polymer dyes in 13-16-color human immunophenotyping panels
(2017-06) Belkina, Anna; Pihl, Riley; Snyder-Cappione, Jennifer
Sirigen Group Limited developed unique polymer 'Brilliant' dyes that have become a staple of modern multicolor panel design. Polymer-based conjugates are often 4-10 times brighter than conventional fluorochromes with similar excitation/emission parameters. A new group of polymer fluorochromes, the 'Super Bright' dyes, was recently launched by eBioscience. The performance of these new dyes in large polychromatic panels is unclear to date. Therefore, we tested several preparations of the Super Bright dyes (such as Super Bright 436 and Super Bright 600) in two polychromatic fluorescent panels (one 13-and one 16-color). Specifically, we evaluated the spillover spread matrices of both panels to evaluate the compatibility of Super Bright dyes with other fluorochromes in a setup with tight placement of fluorochrome emissions over the spectrum. We have also matched Super Bright conjugates with comparable Brilliant Violet-labeled antibodies of same specificity in an existing 13-color panel where those conjugates are staining relatively dim targets, such as CCR6 and CD25, on resting human PBMC cells. Our results show that Super Bright dyes inflict a modest spillover spread in neighboring channels. In a 16x16 spillover spread matrix (3-UV, 5-VIOLET, 5-BLUE, 3-RED) Super Bright dyes demonstrate low to moderate spillover that is very close quantitatively to the Brilliant Violet dyes. In a 13-color human immunophenotyping panel that we previously developed to quantify T cell subsets, the " brightness " (i.e. the staining index of the Super Bright-conjugated antibodies) appears to be lower than comparable Brilliant Violet dyes when titrated, although stained populations in a full panel are still well separated. As the use of up to nine Brilliant polymer dyes simultaneously in large panels is not uncommon, we also tested the performance of Super Bright dyes in staining protocols that include Brilliant Buffer (BD Biosciences) to prevent polymer dye interactions and found them compatible. Overall, we found Super Bright dyes to perform well in large polychromatic panels. This expansion of commercially available conjugated antibody repertoire with the addition of Super Brights is timely and will greatly facilitate the success of larger (13+ color) fluorescent panel design.
Automated analysis of 16-color polychromatic flow cytometry data maps immune cell populations and reveals a distinct inhibitory receptor signature in systemic sclerosis
(2015-06) Belkina, Anna; Fleury, Michelle; Vazques Mateo, Christina; Raval, Forum; Lafyatis, Robert; Dooms, Hans; Snyder-Cappione, Jennifer
Background. The phenotypic profiles of both peripheral blood and tissue-resident immune cells have been linked to the health status of individuals with infectious and autoimmune diseases, as well as cancer. In light of the promising clinical trial results of agents that block the Inhibitory Receptor (IR) Programmed Death 1 (PD-1) axis, novel flow cytometric panels that simultaneously measure multiple IRs on several immune cell subsets could provide the distinct IR signatures to target in combinational therapies for many disease states. Also, due to the paucity of human samples, larger (14+ color) ‘1-tube’ panels for immune cell characterization ex vivo are of a high value in translational studies. Development of fluorescent-based panels offer several advantages as compared with analogous mass cytometric methods, including the ability to sort multiple populations of interest from the sample for further study. However, automated platforms of multi-dimensional single cell analysis that allow objective and comprehensive population characterization are severely underutilized on data generated from large polychromatic panels. Methods. A 16-color flow cytometry (FCM) panel was developed and optimized for the simultaneous characterization and purification of multiple human immune cell populations on a 4- laser BD FACSARIA II cell sorter. FCM data of samples obtained from healthy subjects and individuals with systemic sclerosis (SSc) were loaded into Cytobank cloud, then compensated and analyzed with SPADE clustering algorithm. The viSNE algorithm was also employed to compress the data into a 2D map of phenotypic space that was subsequently clustered using SPADE. For comparison, the FCM data were also analyzed manually using FlowJo software. Results. Our novel 16-color panel recognizes CD3, CD4, CD8, CD45RO, CD25, CD127, CD16, CD56, γδTCR, vα24, PD-1, LAG-3, CTLA-4, and TIM-3; it also contains a CD1d-tetramer and a live-dead dye (with CD19 and CD14 included as a combined dump channel). This panel allows combinational IR signatures to be determined from CD4+ T, CD8+ T, Natural Killer (NK), invariant Natural Killer (iNKT), and gamma delta (γδ) immune cell subsets within one sample. We have successfully identified all subsets of interest using automatic SPADE and viSNE algorithms integrated into Cytobank services, and demonstrated a distinctive phenotype of IR distribution on healthy versus systemic sclerosis subject groups. Conclusions. Methods of automatic analysis that were originally developed for processing multi-dimensional mass cytometry can be applied to polychromatic FCM datasets and provide robust results, including subset identification and distinct IR signatures in healthy compared to diseased subject groups.
viSNE fine-tuning enables better resolution of cell populations
(2017-06-09) Belkina, Anna; Ciccolella, Chris; Snyder-Cappione, Jennifer
t-Distributed Stochastic Neighbor Embedding (t-SNE or viSNE) is a dimensionality reduction algorithm that allows visualization of complex high-dimensional cytometry data as a two-dimensional distribution or " map ". These maps can be interrogated by human-guided or automated techniques to categorize single cell data into relevant biological populations and otherwise visualize important differences between samples. The method has been extensively adopted and reported in the literature to be superior to traditional biaxial gating. The analyst must carefully choose the parameters of a t-SNE computation, as incorrectly chosen parameters might create artifacts that make the resulting map difficult or impossible to interpret. The correct choice of algorithm parameters is complicated by a lack of agreed-upon quantitative framework for assessing the quality of algorithm results. Gauging result quality currently relies on subjective visual evaluation by an experienced t-SNE user. To overcome these limitations, we used Cytobank viSNE engine for all t-SNE analyses and employed 18-parameter flow cytometry data as well as 32-parameter mass cytometry data of varying numbers of events to optimize t-SNE parameters such as total number of iterations and perplexity. We also investigated the utility of Kullback-Liebler (KL) divergence as a metric for map quality as well as SPADE clustering as an indirect measure of multidimensional data integrity when flattened into t-SNE coordinates. We have established the imperative requirement for the number of t-SNE analysis optimization steps ('iteration number') to be scaled with the total number of data points (events) in the set, suggesting that a number of existing software solutions produce unclear t-SNE maps of flow and mass cytometry data due to built-in user control restrictions. We also evaluated lower-level parameters within the t-SNE code that control the 'early exaggeration' stage initially introduced into t-SNE algorithm for better map optimization. These parameters are not available as part of the standard algorithm interface, but we found that they can be tuned to produce high quality results in shorter periods of time, avoiding unnecessary increases of both analysis duration and computation cost. Therefore, our approach allows to fine-tune the t-SNE analysis to ensure both optimal resolution of t-SNE low-dimensional maps and better faithfulness of their presentation of high-parameter cytometry data.
Epithelial cell–derived secreted and transmembrane 1a signals to activated neutrophils during pneumococcal pneumonia
(American journal of respiratory cell and molecular biology, 2016-04-11) Kamata, Hirofumi; Yamamoto, Kazuko; Wasserman, Gregory A.; Zabinski, Mary C.; Yuen, Constance K.; Lung, Wing Yi; Gower, Adam C.; Belkina, Anna; Ramirez, Maria I.; Deng, Jane C.; Quinton, Lee J.; Jones, Matthew R.; Mizgerd, Joseph P.
Airway epithelial cell responses are critical to the outcome of lung infection. In this study, we aimed to identify unique contributions of epithelial cells during lung infection. To differentiate genes induced selectively in epithelial cells during pneumonia, we compared genome-wide expression profiles from three sorted cell populations: epithelial cells from uninfected mouse lungs, epithelial cells from mouse lungs with pneumococcal pneumonia, and nonepithelial cells from those same infected lungs. Of 1,166 transcripts that were more abundant in epithelial cells from infected lungs compared with nonepithelial cells from the same lungs or from epithelial cells of uninfected lungs, 32 genes were identified as highly expressed secreted products. Especially strong signals included two related secreted and transmembrane (Sectm) 1 genes, Sectm1a and Sectm1b. Refinement of sorting strategies suggested that both Sectm1 products were induced predominantly in conducting airway epithelial cells. Sectm1 was induced during the early stages of pneumococcal pneumonia, and mutation of NF-kB RelA in epithelial cells did not diminish its expression. Instead, type I IFN signaling was necessary and sufficient for Sectm1 induction in lung epithelial cells, mediated by signal transducer and activator of transcription 1. For target cells, Sectm1a bound to myeloid cells preferentially, in particular Ly6GbrightCD11bbright neutrophils in the infected lung. In contrast, Sectm1a did not bind to neutrophils from uninfected lungs. Sectm1a increased expression of the neutrophil-attracting chemokine CXCL2 by neutrophils from the infected lung. We propose that Sectm1a is an epithelial product that sustains a positive feedback loop amplifying neutrophilic inflammation during pneumococcal pneumonia.
Role of Fas and Treg cells in fracture healing as characterized in the Fas‐deficient (lpr) mouse model of lupus
(Journal of Bone and Mineral Research, 2014-05-19) Al‐Sebaei, Maisa O.; Daukss, Dana M.; Belkina, Anna; Kakar, Sanjeev; Wigner, Nathan A.; Cusher, Daniel; Graves, Dana; Einhorn, Thomas; Morgan, Elise; Gerstenfeld, Louis C.
Previous studies showed that loss of tumor necrosis factora (TNFa) signaling delayed fracture healing by delaying chondrocyte apoptosis and cartilage resorption. Mechanistic studies showed that TNFa induced Fas expression within chondrocytes; however, the degree to which chondrocyte apoptosis ismediated by TNFa alone or dependent on the induction of Fas is unclear. This questionwas addressed by assessing fracture healing in Fas‐deficient B6.MRL/Faslpr/Jmice. Loss of Fas delayed cartilage resorption but also lowered bone fraction in the calluses. The reduced bone fraction was related to elevated rates of coupled bone turnover in the B6.MRL/Faslpr/J calluses, as evidenced by higher osteoclast numbers and increased osteogenesis. Analysis of the apoptoticmarker caspase 3 showed fewer positive chondrocytes and osteoclasts in calluses of B6.MRL/Faslpr/J mice. To determine if an active autoimmune state contributed to increased bone turnover, the levels of activated T cells and Treg cellswere assessed. B6.MRL/Faslpr/J mice had elevated Treg cells in both spleens and bones of B6.MRL/Faslpr/J but decreased percentage of activated T cells in bone tissues. Fracture led to 30% to 60% systemic increase in Treg cells in bothwild‐type and B6.MRL/Faslpr/J bone tissues during the period of cartilage formation and resorption but either decreased (wild type) or left unchanged (B6.MRL/Faslpr/J) the numbers of activated T cells in bone. These results show that an active autoimmune state is inhibited during the period of cartilage resorption and suggest that iTreg cells play a functional role in this process. These data show that loss of Fas activity specifically in chondrocytes prolonged the life span of chondrocytes and that Fas synergized with TNFa signaling tomediate chondrocyte apoptosis. Conversely, loss of Fas systemically led to increased osteoclast numbers during later periods of fracture healing and increased osteogenesis. These findings suggest that retention of viable chondrocytes locally inhibits osteoclast activity or matrix proteolysis during cartilage resorption.
Inhibition of Ubc13-mediated ubiquitination by GPS2 regulates multiple stages of B cell development
(The Journal of Biological Chemistry, 2016-12-30) Lentucci, Claudia; Belkina, Anna; Cederquist, Carly Theresa; Chan, Michelle; Johnson, Holly E.; Prasad, Sherry; Lopacinski, Amanda; Nikolajczyk, Barbara S.; Monti, Stefano; Snyder-Cappione, Jennifer; Tanasa, Bogdan; Cardamone, M. Dafne; Perissi, Valentina
Non-proteolytic ubiquitin signaling mediated by Lys63 ubiquitin chains plays a critical role in multiple pathways that are key to the development and activation of immune cells. Our previous work indicates that GPS2 (G-protein Pathway Suppressor 2) is a multifunctional protein regulating TNF signaling and lipid metabolism in the adipose tissue through modulation of Lys63 ubiquitination events. However, the full extent of GPS2-mediated regulation of ubiquitination and the underlying molecular mechanisms are unknown. Here, we report that GPS2 is required for restricting the activation of TLR and BCR signaling pathways and the AKT/FOXO1 pathway in immune cells based on direct inhibition of Ubc13 enzymatic activity. Relevance of this regulatory strategy is confirmed in vivo by B cell-targeted deletion of GPS2, resulting in developmental defects at multiple stages of B cell differentiation. Together, these findings reveal that GPS2 genomic and non-genomic functions are critical for the development and cellular homeostasis of B cells.
Abstract 803: Targeting β-catenin/CBP signaling in OSCC
(AACR Publications, 2017-07) Alamoud, Khalid Abdulrahman M.; Sadykov, Khikmet; Kartha, Vinay; Monti, Stefano; Belkina, Anna; Snyder-Cappione, Jennifer; Pai, Sara; Kukuruzinska, Maria
OBJECTIVES: Oral squamous cell carcinoma (OSCC) is an aggressive malignancy characterized by molecular heterogeneity and locoregional spread associated with high morbidity. Aggressive cancers are thought to arise from populations of cancer initiating cells (CICs) that exhibit the properties of stem cells and drive tumor development, recurrence and resistance to therapy. The transcriptional regulator, β-catenin, has been implicated in OSCC CICs. Nuclear β-catenin has been shown to recruit the chromatin remodeling CREB binding protein (CBP) to drive expression of proliferation and survival genes, as well as genes that maintain stem-like phenotypes. We hypothesized that targeting β-catenin-CBP interaction will inhibit CICs in oral tumors and restore an epithelial phenotype. METHODS: To test tumor aggressive potential of OSCC CICs, we used zebrafish as a model system. We isolated CD44+CD24hiCD29hi cells fom aggressive HSC-3 OSCC cells by FACS and assayed their ability to drive tumor growth and metastases in zebrafish compared to unsorted and CD44+CD24lowCD29low cells. In addition, we examined the role of the β-catenin/CBP axis in the aggressive phenotype of these cells. We also assessed whether the β-catenin/CBP axis affected CICs in tumors from immune competent HPV+ mice. RESULTS: Zebrafish injected with subpopulation of cells co-expressing CD44+CD24hiCD2hi primitive cell surface markers drove rapid tumor growth and metastases, followed by unsorted and sorted CD44+CD24lowCD29low. Treatment of CD44+CD24hiCD29hi cells with a small molecule inhibitor of the β-catenin-CBP interaction, ICG-001, interfered with tumor growth and metastases in zebrafish. Further, ICG-001 inhibited tumor growth in immunocompetent HPV+ murine model. On a cellular level, ICG-001 promoted membrane localization of β-catenin, enhanced E-cadherin adhesion and restored epithelial phenotype. Significantly, ICG-001 gene signatures tracked with reduced overall patient survival in the cancer genome atlas, TCGA. Conclusion: Our studies indicate that the β-catenin/CBP axis promotes OSCC CICs and that ICG-001 may be an effective therapeutic agent for this malignancy.
Targeting tumor multicellular aggregation through IGPR-1 inhibits colon cancer growth and improves chemotherapy
(Nature Publishing Group, 2017-09-18) Woolf, N.; Pearson, B.E.; Bondzie, Philip Apraku; Meyer, R.D.; Lavaei, M.; Belkina, Anna; Rahimi, N.
Adhesion to extracellular matrix (ECM) is crucially important for survival of normal epithelial cells as detachment from ECM triggers specific apoptosis known as anoikis. As tumor cells lose the requirement for anchorage to ECM, they rely on cell-cell adhesion 'multicellular aggregation' for survival. Multicellular aggregation of tumor cells also significantly determines the sensitivity of tumor cells to the cytotoxic effects of chemotherapeutics. In this report, we demonstrate that expression of immunoglobulin containing and proline-rich receptor-1 (IGPR-1) is upregulated in human primary colon cancer. Our study demonstrates that IGPR-1 promotes tumor multicellular aggregation, and interfering with its adhesive function inhibits multicellular aggregation and, increases cell death. IGPR-1 supports colon carcinoma tumor xenograft growth in mouse, and inhibiting its activity by shRNA or blocking antibody inhibits tumor growth. More importantly, IGPR-1 regulates sensitivity of tumor cells to the chemotherapeutic agent, doxorubicin/adriamycin by a mechanism that involves doxorubicin-induced AKT activation and phosphorylation of IGPR-1 at Ser220. Our findings offer novel insight into IGPR-1's role in colorectal tumor growth, tumor chemosensitivity, and as a possible novel anti-cancer target.
Increased frequency and compromised function of T regulatory cells in systemic sclerosis (SSc) Is related to a diminished CD69 and TGFβ expression
(Public Library of Science, 2009-6-22) Radstake, Timothy R. D. J.; Van Bon, Lenny; Broen, Jasper; Wenink, Mark; Santegoets, Kim; Deng, Yanhui; Hussaini, Anila; Simms, Robert; Cruikshank, William W.; Lafyatis, Robert
BACKGROUND. Regulatory T cells (Tregs) are essential in the control of tolerance. Evidence implicates Tregs in human autoimmune conditions. Here we investigated their role in systemic sclerosis (SSc). METHODS/PRINCIPAL FINDINGS. Patients were subdivided as having limited cutaneous SSc (lcSSc, n=20) or diffuse cutaneous SSc (dcSSc, n=48). Further subdivision was made between early dcSSc (n=24) and late dcSSc (n=24) based upon the duration of disease. 26 controls were studied for comparison. CD3+ cells were isolated using FACS and subsequently studied for the expression of CD4, CD8, CD25, FoxP3, CD127, CD62L, GITR, CD69 using flow cytometry. T cell suppression assays were performed using sorted CD4CD25highCD127- and CD4CD25lowCD127high and CD3+ cells. Suppressive function was correlated with CD69 surface expression and TGFβ secretion/expression. The frequency of CD4+CD25+ and CD25highFoxP3highCD127neg T cells was highly increased in all SSc subgroups. Although the expression of CD25 and GITR was comparable between groups, expression of CD62L and CD69 was dramatically lower in SSc patients, which correlated with a diminished suppressive function. Co-incubation of Tregs from healthy donors with plasma from SSc patients fully abrogated suppressive activity. Activation of Tregs from healthy donors or SSc patients with PHA significantly up regulated CD69 expression that could be inhibited by SSc plasma. CONCLUSIONS/SIGNIFICANCE. These results indicate that soluble factors in SSc plasma inhibit Treg function specifically that is associated with altered Treg CD69 and TGFβ expression. These data suggest that a defective Treg function may underlie the immune dysfunction in systemic sclerosis.
The pronounced Th17 profile in systemic sclerosis (SSc) together with intracellular expression of TGFβ and IFNϒ distinguishes SSc phenotypes
(Public Library of Science, 2009-6-17) Radstake, Timothy R. D. J.; Van Bon, Lenny; Broen, Jasper; Hussiani, Anila; Hesselstrand, Roger; Wuttge, Dirk M.; Deng, Yanhui; Simms, Robbert; Lubberts, Erik; Lafyatis, Robert
BACKGROUND. Systemic sclerosis (SSc) is an autoimmune disease where controversy on Th1/Th2 balance dominates. We investigated whether the recently discovered Th17 pattern was present in SSc. METHODOLOGY AND PRINCIPAL FINDINGS. Patients were subdivided as having limited cutaneous SSc (lcSSc, n=12) or diffuse cutaneous SSc (dcSSc, n=24). A further arbitrary subdivision was made between early dcSSc (n=11) and late dcSSc (n=13) based upon the duration of disease. As a comparator group 14 healthy controls were studied. CD3+ cells were isolated using FACS and subsequently studied for the expression of CD4, CD8, CD25, CD45Ro, CD45Ra, IL-23, GITR, CD69 and intracellular expression of IL-17, TGFβ and IFNϒ using flow cytometry. Levels of IL-17, IL-6, IL-1a and IL-23 were measured using Bioplex assays. SSc patients had more and more activated CD4+ cells. In addition, CD4, CD45Ro and CD45Ra cells from all SSc patients highly expressed the IL23R, which was associated with a higher IL-17 expression as well. In contrast, IFNϒ and TGFβ were selectively up regulated in SSc subsets. In line with these observation, circulating levels of IL-17 inducing cytokines IL-6, IL-23 and IL-1a were increased in all or subsets of SSc patients. CONCLUSION AND SIGNIFICANCE. The combination of IL-17, IFNϒ and TGFβ levels in CD45Ro and CD45Ra cells from SSc patients is useful to distinguish between lSSc, ldSSc or edSSc. Blocking Th17 inducing cytokines such as IL-6 and IL-23 may provide a useful tool to intervene in the progression of SSc.

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