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
 I.
The applications of flow cytometry in vaccine
research
i.
Introduction of flow cytometry and vaccine research
ii.
Flow cytometry uses in vaccine research
II.
III.
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
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
Differences of Mechanisms
Pathogenesis
Immune Defense
Adherence to mucosa
Mucosal antibody (IgA)
Parasites
IgE antibody
Exotoxin and Endotoxin
Neutralizing antibody
Viremia
Neutralizing antibody
Septicemia
Opsonizing antibody
Intracytoplasmic growth
Virus and bacterial replication
Cytotoxic T cells (CTL)
Type-1 and -2 Interferons
Intracellular growth
Th1 cytokines
Infect epithelial cells
Gamma delta T cells
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
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_Denver
BVDmtg2006.pdf
Changes of cell markers
after T Cell Activation
Unstimulated cells
T-c
Activated cells
Antigen
T-c
stimulation
Regular cell surface markers
Various Cytokines
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
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
CD25
T
IL-4
CD4
CD8
T
IFN
T
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 UVinactivated) 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
• 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
Brucella-induced macrophage cell death:
Apoptosis, necrosis, or pyroptosis?
Apoptosis
Pyroptosis
Rough Brucellainduced M death
Inflammatory
-
+
+
Caspase-1
?
+
-
Caspase-2
+
?
+
So, it’s a novel cell death pathway!!
Proposed name:
Caspase-2-mediated pyroptosis?
Bergsbaken et al. Nat Rev
Microbiol. 2009;7(2):99-109.
• Question: Can caspase-2 mediates
antigen presentation?
Use case 3: RB51 also induces caspase2-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.
Outline
 I.
The applications of flow cytometry in vaccine
research
i.
Introduction of flow cytometry and vaccine research
ii.
Flow cytometry uses in vaccine research
II.
III.
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
Challenges
OBI/PRO/CL/VO Representation of
Vaccine Flow Cytometry Data
•
Ontology uses for vaccine data standardization:
o OBI for medical investigations
o PRO for proteins;
o CL for cell types, CLO for cell lines
o 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 markers
o 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?
References
1) 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):311931. PMID: 19047440.