Nadia Roan, PhD; Assistant Professor of Urology, Dept. of Urology, UC San Francisco
“Use of CyTOF to characterize HIV entry, replication, and remodeling in tissue CD4+ T cells”
The primary targets of HIV are CD4+ T cells, but different subsets of CD4+ T cells are differentially susceptible to infection by HIV in a manner influenced by both cell-extrinsic and cell-intrinsic factors. To characterize intrinsic cellular susceptibility to HIV, we phenotyped infected tonsillar T cells by single-cell mass cytometry (CyTOF) and created comprehensive maps to identify which subsets of CD4+ T cells support HIV fusion and productive infection. By comparing HIV-fused and HIV-infected cells through dimensionality reduction, clustering, and nearest-neighbor statistical approaches to account for viral perturbations, we identified a subset of memory CD4+ T cells that support HIV entry but not viral gene expression. These cells express high levels of CD127, the IL-7 receptor, and are believed to be long-lived lymphocytes. In HIV-infected patients, CD127-expressing cells preferentially localize to extrafollicular lymphoid regions with limited viral replication. The statistical approaches used for these in vitro assays are currently being applied to characterize by CyTOF the properties of productively- and latently-infected cells in HIV-infected individuals, and to identify signatures of other immune cells that associate with disease outcome. In conclusion, CyTOF-based phenotyping combined with analytical approaches to distinguish between selective infection and receptor modulation by viruses can be used as a discovery tool.
Dr. Nadia Roan received her BA in Molecular and Cellular Biology from UC Berkeley, and then completed her PhD in Immunology and Microbiology at Harvard Medical School, where in the lab of Dr. Michael Starnbach she established a system to track and characterize mucosal T cell responses to the bacterium Chlamydia trachomatis. For her postdoctoral work, she joined the lab of Dr. Warner Greene at the J. David Gladstone Institutes, where she characterized the properties of mucosal T cells and other mucosal factors on HIV infection and pathogenesis. Since starting her own lab in the Dept. of Urology at UCSF, she has focused on understanding the extrinsic and cell-intrinsic factors that affect T cell function, particularly in the context of HIV infection and reproductive health. Recently, her lab has used mass cytometry (CyTOF) and high-dimensional single-cell analysis tools to characterize the features of T cells that are targeted for HIV entry, and quantitatively characterized remodeling of T cells upon HIV productive infection. CyTOF is also being used in the Roan lab to characterize the phenotypes and effector functions of various human tissue-resident immune subsets, and to identify novel biomarkers of various human diseases. Dr. Roan has extensive experience in the fields of mucosal immunology, infectious diseases, and reproductive biology, and has published 35 manuscripts in these research areas.
Joe Trotter, Principal Scientist, BD Fellow BD Biosciences
“Setting up and Sorting: Making it all work”
The setting up of an electrostatic cell sorter to classify and separate a wide variety of particles and cell types presents a host of challenges that range from sample preparation nuances to instrument configuration choices and sample classification strategies. This presentation will attempt to shed some light on some of the problems, both simple and complex, that can result in poor results in viability, purity or yield – by pointing them out and providing some suggestions that might benefit certain troublesome or difficult cell sorting experiments.
Joe Trotter, Principal Scientist, BD Fellow, is a key flow cytometry expert of BD Biosciences. He was honored as a 2010 Wesley J. Howe Award of BD Fellow not only for his outstanding contributions to the flow cytometry business of BD Biosciences, but for his extensive contributions over 30 years to the field of flow cytometry. Joe has been contributing to the development of flow cytometry beginning with collaborations in the 1970’s, while in the laboratories of Robert Holley and Renato Dulbecco at the Salk Institute, with the early Los Alamos National Laboratory flow cytometry engineering efforts which helped to establish flow as a key tool in biology and medicine. While at Salk, to address user needs he built several early high-performance cytometers, including a multi-laser/multi-parameter cell sorter in the mid 1980’s with customized software developed at Los Alamos that could be configured with either a cuvette based or a jet-in-air flow cell design. He served as Director of Flow Cytometry at the Salk Institute for well over a decade before taking the role of Flow Cytometry Core Lab Director at The Scripps Research Institute in the mid 1990’s. Prior to joining BD Biosciences in 2000, Joe’s interest in flow cytometry software and data visualization motivated him to develop the flow cytometry data analysis WinMDI, which for many years was downloaded and used by thousands of investigators worldwide to analyze and publish their data.
Since joining BD Biosciences, Joe has continued to be a key contributor to the evolution of flow cytometry. His insights and understanding of customer needs was key to the development of the successful BD™ LSR-II product line. Joe was responsible for the system’s design and implementation as well as a critical contributor to the team’s success. He worked with Bob Hoffman and others within BD to develop and implement instrument calibration and standardization, which has set the industry standard and resulted in BD’s recognition as a leader in the field. He led the development of BD FACS™ Sortware, the client side application for the BD Influx™ and FACSJazz™ cell sorting platforms.
Joe’s understanding of flow cytometry and his reputation with cytometerists worldwide has provided him multiple opportunities to demonstrate his expertise. He is a regular contributor and teacher at domestic and international flow cytometry courses. He has published key papers on flow cytometry and frequently is invited to speak and teach at cell analysis conferences and cytometry workshops. He is currently in the role of solution architect and working on next generation systems within the Advanced Technology Group at BD Biosciences.
Anna C. Belkina, MD/PhD, Associate Director, Flow cytometry Core Facility, Department of Pathology at Boston University School of Medicine;
“Revealing disease phenotypes with computational analysis of high-dimensional flow cytometry data”
Advances in both cytometer capabilities and breadth of reagent availability have led to the expansion of individual flow cytometry panels; however, despite this newer ease in generation of high-parameter flow datasets, the proper extraction of results from larger panels is currently bottlenecked due the limitations of available analysis tools that cannot properly analyze such large datasets. Computational analysis is imperative for the proper investigation of high-dimensional flow and mass cytometry datasets. In this seminar, I will discuss several recent developments from the BUSM Flow Core and other sites that enable more efficient and comprehensive computational analysis of large flow cytometry datasets. Specifically, I will present flow and mass cytometry human immunophenotyping data analyzed using our own adaptation of a popular t-SNE algorithm that can accommodate large datasets typical for flow cytometry but beyond reach for the traditional t-SNE implementations. To demonstrate the potency of various newer and adapted computational approaches, I will show an assembly of methods we used to characterize the inhibitory receptor (IR) landscape of various immune subsets in HIV individuals and to propose specific IR phenotypes to be investigated as potential biomarker readouts in HIV and aging.
Anna C. Belkina is the Associate Director of the Flow Cytometry Core Facility and an Instructor (appointment pending final confirmation) in the Department of Pathology at Boston University School of Medicine. She received her M.D. from Russian State Medical University in Moscow and her Ph.D degree from Boston University School of Medicine investigating the epigenetic regulation of inflammatory responses driven by bromodomain proteins. Anna’s research is focused on the intersection of immunology and computational biology, for her current research efforts include investigating the immune landscape of chronic inflammatory diseases and developing computational techniques to assess high-parameter single cell cytometry data. Anna is an active member of ISAC (International Society for the Advancement of Cytometry) and has been named 2015-2019 ISAC SRL Emerging Leader.
Ilhem Messaoudi, PhD, Associate Professor, Department of Molecular Biology and Biochemistry , Ayala School of Biological Sciences, University of California Irvine;
“Mechanisms of Ebola Virus Pathogenesis”
Zaire Ebolavirus (ZEBOV) continues to pose a significant threat to human health as highlighted by the recent epidemic that originated in West Africa and the ongoing outbreak in the Democratic Republic of the Congo. Although the ZEBOV variant responsible for this epidemic (Makona) shares significant genetic similarity with previously identified variants (Kikwit and Mayinga), the pathogenesis caused by the new variant is not fully understood. To address this question, we measured changes
in immune cell frequencies, plasma levels of immune mediators, and differentially expressed genes (DEGs) within whole blood (WB) and peripheral blood mononuclear cells (PBMC). Our combined approach revealed a link between: 1) increased interferon-stimulated gene expression, IFNα levels, and activated plasmacytoid dendritic cells; 2) higher proinflammatory gene expression, cytokine and chemokine levels, and non-classical monocytes; 3) gene signature of leukocyte activation and increased granulocytes; and 4) decreased expression of lymphocyte related genes and lymphopenia. In addition, we carried out a longitudinal analysis of transcriptional changes in purified monocytes, T-cells, and B-cells isolated from cynomolgus macaques following infection with ZEBOV-Makona. Our data reveal monocytes as one of the major immune cell subsets that supports ZEBOV replication in vivo. Moreover, we detected a marked increase in the transcription of genes involved in inflammation, coagulation, and vascular disease within monocytes, suggesting that monocytes contribute to EVD manifestations. Further, genes important for antigen presentation and regulation of immunity were downregulated, potentially subverting development of adaptive immunity. Indeed, lymphocytes, which do not support ZEBOV replication, showed transcriptional changes limited to a small number of interferon-stimulated genes (ISGs) and a failure to upregulate genes associated with an antiviral effector immune response. Collectively, these data suggest that ZEBOV-infected monocytes play a significant role in ZEBOV-Makona pathogenesis.
Dr. Ilhem Messaoudi is an associate professor in the Department of Molecular Biology and Biochemistry within the Ayala School of Biological Sciences, University of California Irvine. She received her B.Sc. in Biochemistry from Lafayette College and PhD in immunology from The Weill Graduate School of Medical Sciences of Cornell University and Memorial Sloan Kettering Cancer Center. She completed her post-doctoral training at at the Vaccine and Gene Therapy Institute, Oregon Health and Science University, where she investigated causes and consequences of immune senescence and the impact of caloric restriction on delaying immune senescence. Dr. Messaoudi became an assistant professor at OHSU, then moved to University of California-Riverside before joining UCI in January 2017. Research in Dr. Messaoudi’s laboratory is focused on understanding how immunity is dysregulated by age, nutrition and viral infections.