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How Flow Cytometry can be used in Vaccine Research

How Flow Cytometry can be used in Vaccine Research. (Buffalo Presentation, 6/11/2012) http:// www.bioontology.org/wiki/index.php/Immunology_Ontologies_and_Their_Applications_in_Processing_Clinical_Data Yongqun “Oliver” He University of Michigan Medical School Ann Arbor, MI 48109. Outline.

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How Flow Cytometry can be used in Vaccine Research

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  1. How Flow Cytometry can be used in Vaccine Research (Buffalo Presentation, 6/11/2012) http://www.bioontology.org/wiki/index.php/Immunology_Ontologies_and_Their_Applications_in_Processing_Clinical_Data Yongqun “Oliver” HeUniversity of Michigan Medical SchoolAnn Arbor, MI 48109

  2. Outline  • The applications of flow cytometry in vaccine research • Introduction of flow cytometry and vaccine research • Flow cytometry uses in vaccine research • Ontology representation of flow cytometry in vaccine research • Possible representation of flow cytometry in OBI/VO for vaccine research • Scientific questions to address • Challenges

  3. What is Flow Cytometry • A technological process that allows for individual measurements of cell fluorescence and light scattering. • Performed at rates of thousands to >10,000 of cells per second. • Can be used to individually sort or separate subpopulations of cells. • Flow cytometry integrates electronics, fluidics, computer, optics, software, and laser technologies in a single platform.

  4. Why vaccine research needs flow cytometry? • Vaccine studies are both clinical and basic • Understanding how individuals control disease is crucial to vaccine development • Study behavior of immune cells to discern protective versus harmful reactions. • Vaccines attempt to elicit beneficial responses. • Critical to quantify and characterize immune cells • Fluorescence-activated cell sorting (FACS) is well suited since it simultaneously examines multiple features of immune cells.

  5. Bolton and Roederer, 2009. PMID: 19485757

  6. Flow Cytometry Analysis of Vaccines Bolton and Roederer, 2009. PMID: 19485757

  7. Pathogenesis Differences of Mechanisms Immune Defense

  8. Humoral vs. Cellular Immune Responses Humoral: B-cells interact with virus and then differentiate into antibody-secreting plasma cells. CMI: starts with antigen presentation via MHC I and II molecules by dendritic cells, which then leads to activation, proliferation and differentiation of antigen-specific CD4+ or CD8+ T cells. These cells gain effector cell function to help or directly release cytokines, or mediate cytotoxicity following antigen recognition. Ref: http://www.influenzareport.com/ir/pathogen.htm

  9. Pathways of Antigen Presentation: Exogenous (MHCII) and Endogenous (MHCI)

  10. Use case 1: Analysis of Vaccine Antigen-specific T Cell-Mediated Immunity to Bovine Respiratory Disease Viruses using Flow CytometryCells to measure: T Helper cells (CD4+)Cytotoxic T cells (CD8+)Gamma Delta (γδ) T cells (CD25) Refs. Platt, R., W. Burdett, and J. A. Roth. 2006. Induction of antigen specific T cell subset activation to bovine respiratory disease viruses by a modified-live virus vaccine. Am J Vet Res, 67:1179-1184, 2006. James A. Roth presentation: http://www.ars.usda.gov/SP2UserFiles/Place/36253000/BVD2005/12_DenverBVDmtg2006.pdf

  11. T-c T-c Various Cytokines Changes of cell markers after T Cell Activation Unstimulated cells Activated cells Antigen stimulation Regular cell surface markers CD25 (IL-2R, IL-2 receptor alpha chain) Ref: James A. Roth presentation: http://www.ars.usda.gov/SP2UserFiles/Place/36253000/BVD2005/12_DenverBVDmtg2006.pdf

  12.  T CD25 IL-4 CD4 CD8 T T IFN Multi-protein Flow Cytometry General Strategy: Measures the expression of CD25, intracellular IFN-and IL-4 in T cell subsets. After incubation of PBMCs with antigens for days, cells are strains for cell surface markers and intracellular cytokines and analyzed by flow cytometry. Ref: James A. Roth presentation: http://www.ars.usda.gov/SP2UserFiles/Place/36253000/BVD2005/12_DenverBVDmtg2006.pdf

  13. Use case 2: YF17D as Yellow fever vaccine induces integrated multilineage and polyfunctional immune responses • YF17D: yellow fever (YF) vaccine 17D • Functional genomics and polychromatic flow cytometry to define the signature of the immune response to YF17D in a cohort of 40 volunteers followed for up to 1 year after vaccination. Reference: GaucherD, et al. Yellow fever vaccine induces integrated multilineage and polyfunctional immune responses. J Exp Med. 2008 Dec 22;205(13):3119-31. PMID: 19047440.

  14. DCs pulsed with YF17D virus (live or UV-inactivated) primed a strong antigen-specific response of naive T cells with up to 4% cells expressing CD154 and IFN-g in response to live or UV-inactivated YF17D. Ref: Gaucher D, et al. J Exp Med. 2008. 205(13):3119-31. PMID: 19047440

  15. Use case 3:Brucella cattle vaccine RB51 induces caspase-2-mediated programmed cell death of macrophages and dendritic cells (DCs) (my own lab research) • RB51, but not its parent virulent strain S2308, induces caspase-2-mediated macrophage cell death • RB51 and S2308 both induce cell death in DCs. Reference: Chen F, He Y. Caspase-2 mediated apoptotic and necrotic murine macrophage cell death induced by rough Brucella abortus. PLoS One. 2009 Aug 28;4(8):e6830

  16. Brucella-induced macrophage cell death: Apoptosis, necrosis, or pyroptosis? So, it’s a novel cell death pathway!! Proposed name: Caspase-2-mediated pyroptosis? • Question: Can caspase-2 mediates antigen presentation? Bergsbaken et al. Nat Rev Microbiol. 2009;7(2):99-109.

  17. Use case 3: RB51 also induces caspase-2-mediated programmed cell death of dendritic cells (my own lab research) • RB51, but not its parent virulent strain 2308, induces caspase-2-mediated DC activation • Flow cytometry markers: CD40, CD80, CD86, MHC class I and II.

  18. Outline  • The applications of flow cytometry in vaccine research • Introduction of flow cytometry and vaccine research • Flow cytometry uses in vaccine research • Ontology representation of flow cytometry in vaccine research • Possible representation of flow cytometry in OBI/VO for vaccine research • Scientific questions to address • Challenges

  19. OBI/PRO/CL/VO Representation of Vaccine Flow Cytometry Data • Ontology uses for vaccine data standardization: • OBI for medical investigations • PRO for proteins; • CL for cell types, CLO for cell lines • VO for vaccines • Melanie will present an overview of the representation of flow cytometry assays in OBI • VO can be used in combination with OBI to represent how flow cytometry can be used in vaccine research.

  20. Capturing Vaccine Flow Cytometry-derived Knowledge in VO • Interesting stories for VO representation: • What’s unique in vaccine-stimulated cell molecular markers • Why are they unique and critical? • Pathways of the induction of cell types with the markers. • Collaboration among ontology teams is key to make the story successful

  21. Challenges • There has no centralized flow cytometry data repository like GEO and ArrayExpress for microarray data • How to use ontologies to analyze vaccine flow cytometry data needs demonstrations. • Why adding ontologies benefit?

  22. References Bolton DL, Roederer M. Flow cytometry and the future of vaccine development. Expert Rev Vaccines. 2009 Jun;8(6):779-89. Review. PMID: 19485757 Diane L. Bolton and Mario Roederer. Flow cytometry and the future of vaccine development. Expert Rev Vaccines. 2009. 8(6), 779-789. PMID: 19485757. Gaucher D, et al. Yellow fever vaccine induces integrated multilineage and polyfunctional immune responses. J Exp Med. 2008 Dec 22;205(13):3119-31. PMID: 19047440.

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