in Action: Cytokine Network Cytometry

Jonni S. Moore, Ph.D. Director, Clinical and Research 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 () resulting from genetic control mechanisms (Genome). It is the study of the role of the cell within the context of and …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 • 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! 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 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 Cytokines Regulate Cell Fate Apoptosis

Cytokine producing cell Proliferation

Target cell Autocrine loop Common Features of Cytokines

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

Target Cell Receptor Cytokine Secretion ( WHERE) ( WHAT ) Intracellular Cytokine Cell Function ( WHO ) ( HOW ) 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 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. 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

• permeablization • fixation • selection and titration of probes • stability of target antigens • appropriate selection of controls 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 Permeablization Control with Anti-Beta Actin

Not Permed Adequately Permed 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 Receptors Target the Cytokine Effects THE 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 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 Cytokine Network Cytometry

Target Cell Receptor Cytokine Secretion ( WHERE) ( WHAT ) Intracellular Cytokine Cell Function ( WHO ) ( HOW ) 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!