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 SchoolAnn Arbor, MI 48109

OutlineI. The applications of flow cytometry in vaccine

researchi. Introduction of flow cytometry and vaccine research

ii. Flow cytometry uses in vaccine research

II. Ontology representation of flow cytometry in vaccine research

i. Possible representation of flow cytometry in OBI/VO for vaccine research

ii. Scientific questions to address

III. Challenges

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.

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.

Bolton and Roederer, 2009. PMID: 19485757

Flow Cytometry Analysis of Vaccines

Bolton and Roederer, 2009. PMID: 19485757

PathogenesisAdherence to mucosa Mucosal antibody (IgA)Parasites IgE antibody

Exotoxin and Endotoxin Neutralizing antibodyViremia Neutralizing antibodySepticemia Opsonizing antibodyIntracytoplasmic growthVirus and bacterial replication

Cytotoxic T cells (CTL) Type-1 and -2 Interferons

Intracellular growth Th1 cytokinesInfect epithelial cells Gamma delta T cells

Immune Defense

Differences of Mechanisms

Humoral vs. Cellular Immune Responses

Ref: http://www.influenzareport.com/ir/pathogen.htm

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.

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

Use case 1: Analysis of Vaccine Antigen-specific T Cell-Mediated Immunity to Bovine Respiratory

Disease Viruses using Flow Cytometry

Cells 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

Changes of cell markers after T Cell Activation

Antigen

stimulation

Unstimulated cells Activated cells

T-c T-c

CD25 (IL-2R, IL-2 receptor alpha chain)

Regular cell surface markers Various Cytokines

Ref: James A. Roth presentation: http://www.ars.usda.gov/SP2UserFiles/Place/36253000/BVD2005/12_DenverBVDmtg2006.pdf

Multi-protein Flow Cytometry

After incubation of PBMCs with antigens for days, cells are strains for cell surface markers and intracellular cytokines and analyzed by flow cytometry.

General Strategy: Measures the expression of CD25, intracellular IFN- and IL-4 in T cell subsets.

CD4

T

T

TIFN

IL-4

CD8

CD25

Ref: James A. Roth presentation: http://www.ars.usda.gov/SP2UserFiles/Place/36253000/BVD2005/12_DenverBVDmtg2006.pdf

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: 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.

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

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

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

• RB51 and S2308 both induce cell death in DCs.

Brucella-induced macrophage cell death: Apoptosis, necrosis, or pyroptosis?

Bergsbaken et al. Nat Rev Microbiol. 2009;7(2):99-109.

Apoptosis Pyroptosis Rough Brucella-induced M death

Inflammatory - + +

Caspase-1 ? + -

Caspase-2 + ? +

So, it’s a novel cell death pathway!!

Proposed name: Caspase-2-mediated pyroptosis?

• Question: Can caspase-2 mediates antigen presentation?

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.

OutlineI. The applications of flow cytometry in vaccine

researchi. Introduction of flow cytometry and vaccine research

ii. Flow cytometry uses in vaccine research

II. Ontology representation of flow cytometry in vaccine research

i. Possible representation of flow cytometry in OBI/VO for vaccine research

ii. Scientific questions to address

III. Challenges

OBI/PRO/CL/VO Representation of Vaccine Flow Cytometry Data

• Ontology uses for vaccine data standardization:o OBI for medical investigationso PRO for proteins; o CL for cell types, CLO for cell lineso 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.

Capturing Vaccine Flow Cytometry-derived Knowledge in VO

• Interesting stories for VO representation:o What’s unique in vaccine-stimulated cell

molecular markerso Why are they unique and critical?o Pathways of the induction of cell types with

the markers.

• Collaboration among ontology teams is key to make the story successful

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?

References1) Bolton DL, Roederer M. Flow cytometry and the

future of vaccine development. Expert Rev Vaccines. 2009 Jun;8(6):779-89. Review. PMID: 19485757

2) Diane L. Bolton and Mario Roederer. Flow cytometry and the future of vaccine development. Expert Rev Vaccines. 2009. 8(6), 779-789. PMID: 19485757.

3) 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.