Automated analysis of 16-color polychromatic flow cytometry data maps immune cell populations and reveals a distinct inhibitory receptor signature in systemic sclerosis

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

A Role of Myocardin Related Transcription Factor-A (MRTF-A) in Scleroderma Related Fibrosis.

In scleroderma (systemic sclerosis, SSc), persistent activation of myofibroblast leads to severe skin and organ fibrosis resistant to therapy. Increased mechanical stiffness in the involved fibrotic tissues is a hallmark clinical feature and a cause of disabling symptoms. Myocardin Related Transcription Factor-A (MRTF-A) is a transcriptional co-activator that is sequestered in the cytoplasm and translocates to the nucleus under mechanical stress or growth factor stimulation. Our objective was to determine if MRTF-A is activated in the disease microenvironment to produce more extracellular matrix in progressive SSc. Immunohistochemistry studies demonstrate that nuclear translocation of MRTF-A in scleroderma tissues occurs in keratinocytes, endothelial cells, infiltrating inflammatory cells, and dermal fibroblasts, consistent with enhanced signaling in multiple cell lineages exposed to the stiff extracellular matrix. Inhibition of MRTF-A nuclear translocation or knockdown of MRTF-A synthesis abolishes the SSc myofibroblast enhanced basal contractility and synthesis of type I collagen and inhibits the matricellular profibrotic protein, connective tissue growth factor (CCN2/CTGF). In MRTF-A null mice, basal skin and lung stiffness was abnormally reduced and associated with altered fibrillar collagen. MRTF-A has a role in SSc fibrosis acting as a central regulator linking mechanical cues to adverse remodeling of the extracellular matrix

Low field vortex matter in YBCO: an atomic beam magnetic resonance study

We report measurements of the low field structure of the magnetic vortex lattice in an untwinned YBCO single-crystal platelet. Measurements were carried out using a novel atomic beam magnetic resonance (ABMR) technique. For a 10.7 G field applied parallel to the c-axis of the sample, we find a triangular lattice with orientational order extending across the entire sample. We find the triangular lattice to be weakly distorted by the a-b anisotropy of the material and measure a distortion factor, f = 1.16. Model-experiment comparisons determine a penetration depth, lambda_ab = 140 (+-20) nm. The paper includes the first detailed description of the ABMR technique. We discuss both technical details of the experiment and the modeling used to interpret the measurements.Comment: 44 pages, 13 figures, submitted to Phys. Rev. B Revision includes Postscript wrapped figures + minor typo

The HLA-B*35 allele modulates ER stress, inflammation and proliferation in PBMCs from Limited Cutaneous Systemic Sclerosis patients

Introduction\ud HLA-B*35 is associated with increased risk of developing pulmonary hypertension in SSc patients. We previously reported that HLA-B*35 induces endothelial cell dysfunction via activation of ER stress/UPR and upregulation of the inflammatory response. Because PBMCs from lcSSc-PAH patients are also characterized by activation of ER stress/UPR and inflammation, the goal of this study was to assess whether the presence of HLA-B*35 contributes to those characteristics.\ud \ud Methods\ud PBMCs were purified from healthy controls (n = 49 HC) and lcSSc patients, (n = 44 with PAH, n = 53 without PAH). PBMCs from each group were stratified for the presence of HLA-B*35. Global changes in gene expression in response to HLA-B*35, HLA-B*8 or empty lentivirus were investigated by microarray analysis in HC PBMCs. Total RNA was extracted and qPCR was performed to measure gene expression.\ud \ud Results\ud ER stress markers, in particular the chaperones BiP and DNAJB1 were significantly elevated in PBMC samples carrying the HLA-B*35 allele. IL-6 expression was also significantly increased in HLA-B*35 lcSSc PBMCs and positively correlated with ER stress markers. Likewise, HMGB1 was increased in HLA-B*35-positive lcSSc PBMCs. Global gene expression analysis was used to further probe the role of HLA-B*35. Among genes downregulated by HLA-B*35 lentivirus were genes related to complement (C1QB, C1QC), cell cycle (CDNK1A) and apoptosis (Bax, Gadd45). Interestingly, complement genes (C1QC and C1QB) showed elevated expression in lcSSc without PAH, but were expressed at the low levels in lcSSc-PAH. The presence of HLA-B*35 correlated with the decreased expression of the complement genes. Furthermore, HLA-B*35 correlated with decreased expression of cyclin inhibitors (p21, p57) and pro-apoptotic genes (Bax, Gadd45) in lcSSc B35 subjects. FYN, a tyrosine kinase involved in proliferation of immune cells, was among the genes that were positively regulated by HLA-B*35. HLA-B*35 correlated with increased levels of FYN in lcSSc PBMCs.\ud \ud Conclusions\ud Our study demonstrates that HLA-B*35 contributes to the dysregulated expression of selected ER stress, inflammation and proliferation related genes in lcSSc patient PBMCs, as well as healthy individuals, thus supporting a pathogenic role of HLA-B*35 in the development of PAH in SSc patients