Reagent-driven reconfiguration/optimization of a 16-parameter BD FACSARIA II SORP to allow accurate detection of violet- and UV-excited Sirigen dyes

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

viSNE fine-tuning enables better resolution of cell populations

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

Evaluation of ‘Super Bright’ polymer dyes in 13-16-color human immunophenotyping panels

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

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 Comprehensive Ex Vivo Functional Analysis of Human NKT Cells Reveals Production of MIP1-α and MIP1-β, a Lack of IL-17, and a Th1-Bias in Males

NKT cells contribute to the modulation of immune responses and are believed to be important in the pathogenesis of autoimmune and infectious diseases, as well as cancer. Variations in the composite NKT cytokine response may determine individual disease susceptibility or severity. Due to low frequencies in peripheral blood, knowledge of the breadth of ex vivo human NKT cell functions has been limited. To bridge this gap, we studied highly purified NKT cells from PBMC of healthy donors and assessed the production of 27 effector functions using sensitive Elispot and multiplex bead assays. We found the ex vivo human NKT cell response is predominantly comprised of the chemokines MIP1-α, and MIP1-β as well as the Th1 cytokines IFN-γ and TNF-α. Although lower in magnitude, there was also significant production of IL-2, IL-4, and perforin after mitogen stimulation. Surprisingly, little/no IL-5, IL-6, IL-10, or IL-13 was detected, and no subjects' NKT cells produced IL-17. Comparison of the NKT functional profiles between age-matched male and female subjects revealed similar IL-4 responses, but higher frequencies of cells producing IFN-γ and MIP1-α, from males. There were no gender differences in the circulating NKT subset distribution. These findings implicate chemokines as a major mechanism by which NKT cells control responses in humans. In addition, the panoply of Th2 and Th17 cytokine secretion by NKT cells from healthy donors may not be as pronounced as previously believed. NKT cells may therefore contribute to the gender bias found in many diseases

Abstract 803: Targeting β-catenin/CBP signaling in OSCC

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.Support: Evans Center for Interdisciplinary Biomedical Research ARC funding AU 5303015 8000000

Multivariate Computational Analysis of Gamma Delta T Cell Inhibitory Receptor Signatures Reveals the Divergence of Healthy and ART-Suppressed HIV+ Aging

Even with effective viral control, HIV-infected individuals are at a higher risk for morbidities associated with older age than the general population, and these serious non-AIDS events (SNAEs) track with plasma inflammatory and coagulation markers. The cell subsets driving inflammation in aviremic HIV infection are not yet elucidated. Also, whether ART-suppressed HIV infection causes premature induction of the inflammatory events found in uninfected elderly or if a novel inflammatory network ensues when HIV and older age co-exist is unclear. In this study we measured combinational expression of five inhibitory receptors (IRs) on seven immune cell subsets and 16 plasma markers from peripheral blood mononuclear cells (PBMC) and plasma samples, respectively, from a HIV and Aging cohort comprised of ART-suppressed HIV-infected and uninfected controls stratified by age (≤35 or ≥50 years old). For data analysis, multiple multivariate computational algorithms [cluster identification, characterization, and regression (CITRUS), partial least squares regression (PLSR), and partial least squares-discriminant analysis (PLS-DA)] were used to determine if immune parameter disparities can distinguish the subject groups and to investigate if there is a cross-impact of aviremic HIV and age on immune signatures. IR expression on gamma delta (γδ) T cells exclusively separated HIV+ subjects from controls in CITRUS analyses and secretion of inflammatory cytokines and cytotoxic mediators from γδ T cells tracked with TIGIT expression among HIV+ subjects. Also, plasma markers predicted the percentages of TIGIT+ γδ T cells in subjects with and without HIV in PSLR models, and a PLS-DA model of γδ T cell IR signatures and plasma markers significantly stratified all four of the subject groups (uninfected younger, uninfected older, HIV+ younger, and HIV+ older). These data implicate γδ T cells as an inflammatory driver in ART-suppressed HIV infection and provide evidence of distinct “inflamm-aging” processes with and without ART-suppressed HIV infection

Lower numbers of natural killer T cells in HIV-1 and Mycobacterium leprae co-infected patients

Natural killer T (NKT) cells are a heterogeneous population of lymphocytes that recognize antigens presented by CD1d and have attracted attention because of their potential role linking innate and adaptive immune responses. Peripheral NKT cells display a memory-activated phenotype and can rapidly secrete large amounts of pro-inflammatory cytokines upon antigenic activation. In this study, we evaluated NKT cells in the context of patients co-infected with HIV-1 and Mycobacterium leprae. The volunteers were enrolled into four groups: 22 healthy controls, 23 HIV-1-infected patients, 20 patients with leprosy and 17 patients with leprosy and HIV-1-infection. Flow cytometry and ELISPOT assays were performed on peripheral blood mononuclear cells. We demonstrated that patients co-infected with HIV-1 and M.leprae have significantly lower NKT cell frequencies [median 0.022%, interquartile range (IQR): 0.0070.051] in the peripheral blood when compared with healthy subjects (median 0.077%, IQR: 0.0320.405, P < 0.01) or HIV-1 mono-infected patients (median 0.072%, IQR: 0.0300.160, P < 0.05). Also, more NKT cells from co-infected patients secreted interferon-? after stimulation with DimerX, when compared with leprosy mono-infected patients (P = 0.05). These results suggest that NKT cells are decreased in frequency in HIV-1 and M.leprae co-infected patients compared with HIV-1 mono-infected patients alone, but are at a more activated state. Innate immunity in human subjects is strongly influenced by their spectrum of chronic infections, and in HIV-1-infected subjects, a concurrent mycobacterial infection probably hyper-activates and lowers circulating NKT cell numbers.National Institutes of Health [R01-AI52731, AI060379]Fogarty International Center [D43 TW00003]Fundacao de Amparo a Pesquisa do Estado de Sao Paulo [04/15856-9/Kallas, 2010/05845-0/Kallas]Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq)Brazilian Ministry of Science and Technology [484230/2011-5]New York Community Trus

Metformin Enhances Autophagy and Normalizes Mitochondrial Function to Alleviate Aging-Associated Inflammation

Age is a non-modifiable risk factor for the inflammation that underlies age-associated diseases; thus, anti-inflammaging drugs hold promise for increasing health span. Cytokine profiling and bioinformatic analyses showed that Th17 cytokine production differentiates CD4+ T cells from lean, normoglycemic older and younger subjects, and mimics a diabetes-associated Th17 profile. T cells from older compared to younger subjects also had defects in autophagy and mitochondrial bioenergetics that associate with redox imbalance. Metformin ameliorated the Th17 inflammaging profile by increasing autophagy and improving mitochondrial bioenergetics. By contrast, autophagy-targeting siRNA disrupted redox balance in T cells from young subjects and activated the Th17 profile by activating the Th17 master regulator, STAT3, which in turn bound IL-17A and F promoters. Mitophagy-targeting siRNA failed to activate the Th17 profile. We conclude that metformin improves autophagy and mitochondrial function largely in parallel to ameliorate a newly defined inflammaging profile that echoes inflammation in diabetes

Skewed Distribution of Circulating Activated Natural Killer T (NKT) Cells in Patients with Common Variable Immunodeficiency Disorders (CVID)

Common variable immunodeficiency disorder (CVID) is the commonest cause of primary antibody failure in adults and children, and characterized clinically by recurrent bacterial infections and autoimmune manifestations. Several innate immune defects have been described in CVID, but no study has yet investigated the frequency, phenotype or function of the key regulatory cell population, natural killer T (NKT) cells. We measured the frequencies and subsets of NKT cells in patients with CVID and compared these to healthy controls. Our results show a skewing of NKT cell subsets, with CD4+ NKT cells at higher frequencies, and CD8+ NKT cells at lower frequencies. However, these cells were highly activated and expression CD161. The NKT cells had a higher expression of CCR5 and concomitantly expression of CCR5+CD69+CXCR6 suggesting a compensation of the remaining population of NKT cells for rapid effector action