8th SEMINAR SEPARATION AND MEASUREMENT OF THE ACTIVITY OF IMMUNECOMPETENT CELLS

CELL SEPARATION Physical isolation of the cells of interest from a heterogeneous population Differences in the physical, biological or immunological properties of the cells are utilized to separate the cells. (Differences in cell surface receptor expression is often available – there is a possibility to further investigate the separated living cells).  physical – density, size  cell biological – adherence, phagocytosis, sensitivity to the medium  immunological – antigen differences (surface marker) Consideration taken to: purity, recovery, yield and viability of the cells

TWO SEPARATION STRATEGIES Positive separation Labeling and separation of the cells of interest e.g. labeling a cell surface molecule by a fluorescent antibody. The cells become affected both by the separation environment and the antibodies bound to the receptors. The purity of the separation is generally high. Negative separation Labeled the unwanted cells (depletion) The cells become affected only by the separation environment, hence this is the preferred strategy in functional examinations.

peripheral blood (or buffy coat) pipetting cells on ficoll centrifugation separated cells plasma ficoll Red blood cells mononuclear cells (PBMC) Neutrophil granulocytes pipettig the „ring” containing the mononuclear cells to a new tube to get rid of Ficoll FICOLL-PAQUE DENSITY BASED CELL SEPARATION

(Nature Protocols (from Google pictures)

Magnetic-Activated Cell Sorting (MACS) paramagnetic bead antigen specific antibody SEPARATION METHODS BASED ON THE IMMUNOLOGICAL PROPERTIES OF THE CELLS

MAGNET column depleting or selecting unlabeled cells (negative separation)

Fluorescence-Activated Cell Sorting (FACS) SEPARATION METHODS BASED ON THE IMMUNOLOGICAL PROPERTIES OF THE CELLS Example: NKT cell separation (CD3/CD56) NK cells NKT cells lymphocytes blood sample T cells

The fluid stream break up into droplets by the vibration of the flow cell. breakoff point

Laser vibration (nozzle orifice of the flow cell) If the wanted cell reaches the breakoff point, the stream become charged for the short time of drop formation, so the formed drop become charged charged deflection plate collection tube waste

MEASURING THE ACTIVITY OF IMMUNECOMPETENT CELLS PHAGOCYTIC CELLS – PHAGOCYTOSIS ASSAY Using killed pathogens (bacteria: E. coli, S. aureus; yeast: S. cerevisiae) labeled with different fluorophores Phagocytosis can be detected by fluorescent microscopy or by flow cytometry

The activation of lymphocytes by a specific antigen is hardly detectable (low numbers of the antigen specific cells) The activation of lymphocytes by a polyclonal activator can help investigate abnormal lymphocyte functions MEASURING LYMPHOCYTE ACTIVITY For detection of immunodeficiencies affecting T and/or B cell functions

POLYCLONAL ACTIVATION OF B AND T CELLS  Lectins (like concavalin A and PHA) act through crosslinking receptors  Intracellular signaling cascade activators (PMA – PKC activator, Ionomycin – increased intracellular Ca 2+ levels)  Specific antibodies (anti-IgM, anti-CD3, anti-TCR)

POLYCLONAL B CELL ACTIVATORS POLYCLONAL T CELL ACTIVATORS Pokeweed mitogen (PWM) Staphylococcus protein A superantigen (SpA) Epstein Bar Virus (EBV) (transforming) Anti-IgM antibody Phytohaemagglutinin (PHA) Concanavalin A (ConA) Anti-CD3, Anti-TCR antibodies Canavalia ensiformis Phytolacca americana Phaseolus vulgaris

Receptor crosslinking (immediate) phosphorylation steps (seconds-minutes) -Western blot i.c. Ca 2+ increase -flow cytometry -fluorescent microscopy Gene activation -qRT-PCR  mRNA -Western blot  protein Cytokine synthesis Cytokine secretion -i.c. cytometry - ELISA - ELISPOT Antigen receptors (TCR, BCR), cytokine receptors, etc. Viability/apoptosis-dies specific to dead cells Cell division - 3 H-thymidine -CFSE -MTT Lymphocyte activation The examination often requires specific Ag-Ab reactions

Fluorescence proportional with Intracellular Ca 2+ level time basic signal activation of cells Fluo-3 or Indo-1 MEASUREMENT OF CA 2+ SIGNAL BY FLOW CYTOMETRY An increase in cytoplasmic Ca 2+ levels can be detected by fluorescent indicator dyes /Fluo-3 or Indo-1/

INTRACELLULAR CYTOKINE DETECTION BY IMMUNOFLUORESCENCE cytokine specific antibodies with fluorescent labeling cytokines  the cell membrane should be permeabilized (detergent)  but first the cells should be fixed to avoid decomposition (using e.g. aldehyde fixation)  optionally the cells can also be labeled by cell type (CD marker) specific antibodies

Activation of cells can be monitored by the detection of mRNA transcription of the activated genes e.g. activation of cytokine genes QUANTITATIVE (REAL-TIME) PCR (qPCR/qRT-PCR)  cells  RNA isolation  RNA  reverse transcription (RT-PCR)  cDNA  cDNA  polymerase chain reaction (PCR)  determination of quantity (investigation of gene activation on protein level  WB) INVESTIGATION OF GENE ACTIVATION the more mRNA the sample contains, the less time (cycles) it will take to reach the threshold

ELISPOT Enzyme Linked Immuno-Spot  Similar principles as in ELISA  Determination of the number of cells that produce Ig, cytokines, chemokines, granzymes and other soluble effector molecules  Sensitive. Allows the determination of 1 activated cell among 300,000 others. (Can reveal activated effector cells not only after polyclonal but after antigen specific activation).

- coating with antigen specific capture antibodies - blocking - administration of the cells - administration of biotin conjugated antigen-specific secondary antibody - avidin-enzyme conjugate - administration of the insoluble chromogenic substrate (AEC 3-amino-9-ethylcarbazol ) (activation, incubation) - washing A spot showing the place of the cytokine producing cell Upper view of a well on an ELISPOT plate with the generated spots ELISPOT Enzyme Linked Immuno-Spot

Spot number and size determination is valuated slowly and manually by microscopy or using “ELISPOT plate reader” which is fast and standardizable

VIABILITY ASSAYS MTT (Dimethyl thiazolyl diphenyl tetrazolium salt) Colorimetric test for measuring viability (apoptotic cells). NADPH-dependent cellular oxyreductase enzymes that reduce MTT dye to an insoluble purple color (formazan). PI (propidium iodide) A fluorescent molecule intercalating with nucleic acids for measuring cell viability by flow cytometry. It is impermeable to viable cells. 7-AAD (7-aminoactinomycin) A fluorescent chemical intercalating with dsDNA. Won’t pass intact cells so is used for cell viability by flow cytometry.

PROLIFERATION ASSAYS 3 H-labeled thymidine- measures the increasing DNA content by β decomposition, and does not answer the numbers of cell division, and the dividing cell number. Bromodeoxyuridin (BrdU) A Thymidine-analogue can be administered to experimental animals, or cell cultures, and the proliferating cells can be detected by labelling with BrdU specific antibody (microscopy, FACS). CFSE (Carboxyfluorescein succinimidyl ester) A fluorescent dye easily penetrating cells binding intracellular amine structures for long periods. Studies of cell divisions, prolifearation, migration and positioning.

CFSE TRACKING THE CELL DIVISIONS  „Cell tracer” dye enters the cell, and becomes trapped there  The apolar CFSE can bind covalently to the cellular proteins  Progressively halved within daughter cells  Used in vitro and in vivo to monitor lymphocyte proliferation CFSE-labeled cells that were not treated with polyclonal activator (control)