Cytomics in Action: Cytokine Network Cytometry

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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

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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
proteomicscentered on the concept that the cell
is the final arbiter in the production of
metabolic products.
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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

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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!

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Predictive Medicine by CytomicsEvidenced 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.

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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

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Cytokine Networks in Clinical Practice- A 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

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Cytokines Regulate Cell Fate
Apoptosis
Proliferation
Cytokine producing cell
Target cell
Autocrine loop
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Common Features of Cytokines

• Produced transiently
• Bind to high affinity receptors
• Redundancy
• Pleiotropic effects
• Function in networks

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Cytokine Network Cytometry
Target Cell Receptor ( WHERE)
Cytokine Secretion ( WHAT )
Cell Function ( HOW )
Intracellular Cytokine ( WHO )
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Determine the Cytokine of InterestTHE WHAT

• Bioassay
• Cell lines for detection of functional cytokines
• Time consuming non-specific
• Immunoassay
• Radioimmunoassays, solid-phase ELISA
• FlowELISA (suspension arrays)
• CAN BE MULTIPLEXED!!

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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
half-life 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.
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Identify the Cytokine-Producing CellTHE 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

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Step 1 In Vitro Stimulationor 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!

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Critical Concerns for the Detection of
Intracellular Cytokines

• permeablization
• fixation
• selection and titration of probes
• stability of target antigens
• appropriate selection of controls

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Controls for Intracellular Cytokine Detection

• Permeablization anti-actin or vimentin
• Activation control- ex. CD69
• Isotype control-BE CAREFUL!!
• Ligand blocking control
• Antibody blocking control
• Compensation-on unstimulated cells

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Permeablization Control with Anti-Beta Actin
Not Permed
Adequately Permed
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The Devil is in the Details!!

• 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

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Receptors Target the Cytokine EffectsTHE WHERE

• Immunofluorescent detection using anti-receptor
  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

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Biologic Effects of CytokinesTHE HOW

• Multiplex functional assays with Who, What and
  Where for complete network evaluation
• Typical functional assays
• Proliferation
• Apoptosis
• Cell cycle
• Secondary cytokines
• Activation markers
• Adhesion molecules

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Cytokine Network Cytometry
Target Cell Receptor ( WHERE)
Cytokine Secretion ( WHAT )
Cell Function ( HOW )
Intracellular Cytokine ( WHO )
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Cytokine Network Cytometry Interferon-gamma in CLL
Zaki et al. Leuk Res 2000 Jul24(7)611-621
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Cytokine Network Cytometry Practical
Considerations

• Requires thorough understanding of cytokine
  biology
• Rigorous quality control
• Reference standards
• Instrument performance
• Precise reagent titration

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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

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The Future of Flow Cytometric Evaluation of
Cytokine Networks is BRIGHT and COLORFUL !
Its time to bask in the glow of a multiparameter
world!