Cytomics in Action: Cytokine Network Cytometry

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1 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

2 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.

3 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

4 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!

5 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.

6 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

7 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

8 Cytokines Regulate Cell Fate Apoptosis Cytokine producing cell Proliferation Autocrine loop Target cell

9 Common Features of Cytokines Produced transiently Bind to high affinity receptors Redundancy Pleiotropic effects Function in networks

10 Cytokine Network Cytometry Target Cell Receptor ( WHERE) Intracellular Cytokine ( WHO ) Cytokine Secretion ( WHAT ) Cell Function ( HOW )

11 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!!

12 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.

13 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

14 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!

15 Critical Concerns for the Detection of Intracellular Cytokines permeablization fixation selection and titration of probes stability of target antigens appropriate selection of controls

16 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

17 Permeablization Control with Anti-Beta Actin Not Permed Adequately Permed

18 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

19 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

20 Biologic Effects of Cytokines THE 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

21 Cytokine Network Cytometry Target Cell Receptor ( WHERE) Intracellular Cytokine ( WHO ) Cytokine Secretion ( WHAT ) Cell Function ( HOW )

22 Cytokine Network Cytometry Interferon-gamma in CLL Zaki et al. Leuk Res 2000 Jul;24(7):

23 Cytokine Network Cytometry: Practical Considerations Requires thorough understanding of cytokine biology Rigorous quality control Reference standards Instrument performance Precise reagent titration

24 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

25 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!