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Cytomics in Action: Cytokine
Network Cytometry
Jonni S. Moore, Ph.D.
Director, Clinical and Research Flow
Cytometry and PathBioResource
Associate Professor of Pathology & Laboratory
Medicine
University of Pennsylvania School of Medicine
What is Cytomics ?
Functional relationships between the cell
(Cytome) and the metabolic pathways
(Proteome) resulting from genetic control
mechanisms (Genome). It is the study of the
role of the cell within the context of genomics
and proteomics…centered on the concept that
the cell is the final arbiter in the production of
metabolic products.
Cytomics links Molecular and Cellular
Biology Using the Technologies of
Cytometry
• Microscopy: transmission, fluorescence imaging, digital
imaging, confocal , multiphoton, laser dissection
• Computational resources for 3D imaging
• Bioengineering resources for cellular manipulation
• Multiparameter analytical flow cytometry and sorting
of single cells, colonies, particles or bead
• Bioinformatics
Cytomics Combines Structural and
Functional Cellular Measurements
• Cytometric determination of structural components
has been in use for years (DNA content, antigen
content, natural pigments etc); cannot follow rapid
changes
• Functional changes are early indicators of cell growth,
death and differentiation (intracellular pH, energy
production, phosphorylation events etc). Recognition
of functional change can be
PREDICTIVE!
Predictive Medicine by Cytomics
Evidenced Based Medicine
at the Cellular Level
• Multiparametric cytometric determination of functions
or constituents in disease associated cytomes
• Analysis of all measured numeric parameters for all
cell populations
• Data pattern classification against future disease course
(learning set)
• Classification of a test set of patient data
• Prospective classification of data from new patients
during clinical evaluation phase.
Advantages of Cytomics Approach to
Predictive Medicine
• Independent of exact knowledge of the ultimate
molecular causes of disease
• All measurements can be expressed as numeric data
and subjected to bioinformatics analyses
• Predictive molecular phenotypic patterns can be
hypothesis generating (data mining); complications
may be predictable for individual patients
• Provides information on disease associated cytomes as
well as disease involved cytomes
• Dynamic; capable of rapid re-classification
• High information content due to heterogeneity
Cytokine Networks in Clinical PracticeA Model for Evidenced-based Medicine
• Use of cytokine profiles for diagnosis and
prognosis
• Measurement of cytokine network components
as therapeutic monitors
• Cytokine therapy
– Anti-cytokine antibodies
– Cytokine binding proteins
– Receptor antagonists
– Gene therapy
Cytokines Regulate Cell Fate
Apoptosis
Cytokine producing cell
Proliferation
Target cell
Autocrine loop
Common Features of Cytokines
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Produced transiently
Bind to high affinity receptors
Redundancy
Pleiotropic effects
Function in networks
Cytokine Network Cytometry
Target Cell Receptor
( WHERE)
Cytokine Secretion
( WHAT )
Intracellular Cytokine Cell Function
( HOW )
( WHO )
Determine the Cytokine of Interest
THE WHAT
• Bioassay
– Cell lines for detection of functional
cytokines
– Time consuming; non-specific
• Immunoassay
– Radioimmunoassays, solid-phase ELISA
– FlowELISA (suspension arrays)
• CAN BE MULTIPLEXED!!
CAVEAT FOR MEASUREMENT OF SOLUBLE
CYTOKINES IN BIOLOGIC FLUIDS
Cytokines are generally used in the immediate
vicinity of their production and externally
administered cytokines may have a very short halflife or be immediately bound, thus a negative result
in the measurement of soluble cytokines in biologic
fluids may be of limited significance without other
network measurements.
Identify the Cytokine-Producing Cell
THE WHO
• Molecular detection of cytokine mRNA
– ? Relationship to actual protein production
• Flow cytometric detection of intracellular
cytokine protein
– Provides a frequency measurement for
determination of cytokine bias
– Requires careful attention to assay performance and
interpretation
Step 1: In Vitro Stimulation…or
Not!
• Customize to biologic question
• Specific (antigen) or non-specific
(PMA,PHA, anti-CD3, other cytokines)
• Spontaneous-difficult
• Combination protocols (antigen +
mitogen)
• Timing is everything!
Critical Concerns for the Detection
of Intracellular Cytokines
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•
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•
•
permeablization
fixation
selection and titration of probes
stability of target antigens
appropriate selection of controls
Controls for Intracellular Cytokine
Detection
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Permeablization: anti-actin or vimentin
Activation control- ex. CD69
Isotype control-BE CAREFUL!!
Ligand blocking control
Antibody blocking control
Compensation-on unstimulated cells
Permeablization Control with
Anti-Beta Actin
Not Permed
Adequately Permed
The Devil is in the Details!!
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Paraformadehyde must be fresh (same day)
Always use a permeablization control
Extended wash procedure required
Optimum time for one cytokine may not be the
same as another
• Do not use frozen cells*
• Analyze as soon as possible after staining
Receptors Target the Cytokine Effects
THE WHERE
• Immunofluorescent detection using antireceptor antibodies or directly labeled
cytokine
• Requires selection of antibodies NOT
directed to cytokine binding site
• May need to use MFI measurements
• Use multiparameter flow cytometry to
determine cell lineage of potential targets
Biologic Effects of Cytokines
THE HOW
• Multiplex functional assays with Who, What
and Where for complete network evaluation
• Typical functional assays
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Proliferation
Apoptosis
Cell cycle
Secondary cytokines
Activation markers
Adhesion molecules
Cytokine Network Cytometry
Target Cell Receptor
( WHERE)
Cytokine Secretion
( WHAT )
Intracellular Cytokine Cell Function
( HOW )
( WHO )
Cytokine Network Cytometry
Interferon-gamma in CLL
Zaki et al. Leuk Res 2000 Jul;24(7):611-621
Cytokine Network Cytometry: Practical
Considerations
• Requires thorough understanding of
cytokine biology
• Rigorous quality control
– Reference standards
– Instrument performance
– Precise reagent titration
Cytokine Network Cytometry:
Limitations
• Complexity of cytokine networks
• Normal ranges difficult to determine
• Cytokine profiles differ from one biologic
site to another
• Cytokine binding proteins can affect
results
• Redundancy of cytokine networks
The Future of Flow Cytometric
Evaluation of Cytokine Networks is
“BRIGHT” and “COLORFUL” ! It’s
time to bask in the glow of a
multiparameter world!