JUN 03, 2020 12:00 PM PDT

Single cell multiomic analysis of T-cell exhaustion

Sponsored by: BD
C.E. Credits: P.A.C.E. CE Florida CE
Speaker
  • Director, Precision Immunology Laboratory, Perlmutter Cancer Center, NYU Langone Health
    Biography
      Pratip Chattopadhyay, PhD is an Associate Professor (Pathology) and founding Director of NYU Langone Health's Precision Immunology Laboratory (PIL). His laboratory performs independent research in tumor immunology and provides cutting-edge immune monitoring services for a wide variety of biomedical disciplines (cancer, infectious disease, rheumatology). He uses high parameter cytometry technologies, including 30-parameter flow cytometry and combined protein/mRNA analysis by RNA sequencing (molecular cytometry), to reveal biomarkers that predict patient outcomes, better understand disease pathogenesis, and inform rational design of combination drug therapies. Dr. Chattopadhyay has a history of innovative work in the cytometry and immunoassay space. In 2005, as a post-doctoral fellow in Mario Roederer's laboratory (VRC/NIH), he developed a novel assay for enumeration and isolation of antigen-specific CD4+ T-cells, based on CD154 expression (Nature Medicine). In 2006, he reported the first 18-color flow cytometry experiments, which were used to comprehensively characterize antigen-specific T-cells against multiple antigenic epitopes in the same tube (Nature Medicine); this study was the first use of quantum dots for flow cytometry. This work increased the multiplexing capability of advanced flow cytometers, culminating in the development of an advanced 30-parameter flow cytometry system (co-led with the Roederer Lab and BD Biosciences). He also contributed to the development of CITE-seq (Nature Methods 2017). Finally, Dr. Chattopadhyay played a key role in the development of a number of bioinformatics tools for analyzing high parameter data, including SPICE (Roederer), FlowType and R-chyoptimyx (Aghaheepour and Brinkman), and CytoBrute, and a unique tool for building high parameter flow cytometry panels, known as Color Wheel. Dr. Chattopadhyay is a proud graduate of the Johns Hopkins Bloomberg School of Public Health (Ph.D.) and the University of Virginia (B.A.).

    Abstract

    A key step in the clinical production of CAR T cells is the expansion of engineered T cells. To generate enough cells for viable adoptive cell therapy, cells must be robustly stimulated, which raises the risk of inducing T-cell exhaustion and reducing therapeutic efficacy. We sought to answer fundamental questions about the impact of in vitro manipulation on T-cell identity to by performing single-cell multiomic analysis using BD® AbSeq and BD Rhapsody™ Single-Cell Analysis system to simultaneously measure expression of 38 proteins and 399 genes in human T cells expanded in vitro. Comprehensive immunophenotypic and transcriptomic analysis at day 0 enabled a refined characterization of T-cell maturational states and the identification of a donor-specific subset of terminally differentiated T cells that would have been otherwise overlooked using canonical cell classification schema. As expected, T-cell activation induced downregulation of naïve-associated markers and upregulation of effector molecules, proliferation regulators, co-inhibitory and co-stimulatory receptors. Our deep kinetic analysis further revealed clusters of proteins and genes identifying unique states of activation defined by markers temporarily expressed upon 3 days of stimulation, markers constitutively expressed throughout chronic activation, and markers uniquely up-regulated upon 14 days of stimulation. These data indicate heterogeneity and plasticity of chronically stimulated T cells in response to different kinetics of activation. We demonstrate the power of a single-cell multiomic approach to comprehensively characterize T cells and to precisely monitor changes in differentiation, activation and exhaustion signatures in response to different activation protocols. For Research Use Only. Not for use in diagnostic or therapeutic procedures. BD, the BD Logo, and Rhapsody are trademarks of Becton, Dickinson and Company or its affiliates. © 2020 BD. All rights reserved.

    Learning Objectives:

    1. The complexity of T-cell exhaustion and utility of multiomic approaches in studying complex biological processes such as T-cell exhaustion

    2. Use of unsupervised data analysis approaches in analyzing heterogeneous cell population

    3. Comparing and contrasting data from traditional flow cytometry and BD® AbSeq


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