Methods for analysis of cell
mediated immunity
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Methods
• 1.Flow cytometry
• 2.Functional tests of lymphocytes
• 3.Functional tests of phagocyting cells
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What is flow cytometry/ FACS ?
• Flow ~ cells in motion,
Cyto ~ cell, Metry ~ measure
• FACS (fluorescent analysed cell sorting)
• measuring various properties of cells or particles
(e.g. synthetic beads with surface bound antibody to
detect cytokines) while in a fluid
stream (biological, chemical,
physical)
(pH, membrane potential, size,
granularity, viability etc.)
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Flow cytometry
• Measurement of several parameters at the same time:
- number of cells
- size (FSC)
- granularity (SSC) of cells
- fluorescent signal (FL)
(2 or
multiple depending on number of lasers)
• staining of cells with mononuclear antibodies against:
cell surface molecules
cytoplasmatic molecules
nuclear molecules
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Flow cytometry
• Material: whole blood, bioptic samples of bone
marrow, separated cell subpopulations,
or other cell suspensions obtained by tissue
desintegration
• Immunofluorescent staining of cells:
Direct or indirect
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Cell staining in flow cytometry
Direct staining
excitation
(laser)
Indirect staining
emission
fluorescence
emission
fluorescence
Fluorochrome-labelled
secundar Ab/streptavidin
Fluorochromelabelled mAb
purified/
Cell surface marker
biotinylated mAb
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List of common fluorochromes used
in flow cytometry
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Principle of flow cytometry
• One-by-one stream of cells moves rapidly through
the flow cytometer
• Cells pass through a focused beam of light from a
laser
– Photons scattered in all directions
• Photodetectors capture scattered light and generate
digital signals to define cellular characteristics
– Size, internal complexity, antigen makeup
• Information stored and analyzed by computer
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Optical system in
flow cytometer
Detectors of
fluorescence
APC
PE
PE-Cy7
FITC
FSC
LASER
SSC
stained cells
PC analysis
Page 9
Typical Dot plot of cell subpopulations in
blood samples and double staining
Dot plot graphs
1 dot/event
= 1 cell
Lympho gate
Single parameter histogram
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Phenotypisation of lymphocytes
• CD3+ T lymphocytes (50-75%)
• CD3+/CD4+ Th lymphocytes (52-69%)
• CD3+/CD8+ cytotoxic T lymphocytes (27-46%)
• CD3+/CD16+/CD56+ NKT lymphocytes ( only 0.2%)
• CD16+/CD56+ NK cells (4-18%)
• CD19+ B lymphocytes (7-18%)
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Application of cytometric analysis
• phenotypisation of cells (diagnostic of primar
immunodeficiency, autoimmune diseases, leukemie and
lymphoms, etc.)
• functional tests of leukocytes and thrombocytes
(proliferating activity – measurement of DNA content)
• Evaluation of spermatogenesis, detection of viruses,
bacteries and parasites, analysis of chromosomes,
assessment of enzymatic activity, measurement of
intracellular calcium
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Functional tests of lymphocytes
• Proliferation
• Expression of activated markers
• Cytotoxicity
• Cytokine secretion
• Production of antibodies
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Isolation of lymphocytes from blood
• gradient centrifugation (cell separation
according to differences in their density)
Diluted
plasma
Lymphocytes
Separative
solution
Erythrocytes
Granulocytes
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Proliferation of lymphocytes
• Important for the process of
immune reactions
• Diagnostics of innate
immunodeficiency
• Activation of T cell receptor
(TCR) → activation of
intracellular signal cascade
→ signal transduction into
nucleus → transcription of
genes for proliferation
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Blastic transformation test
• Ability of T lymphocytes to response on polyclonal
stimuli
3H-
tymidin test
• Cultivation of isolated lymphocytes in medium with
stimulators (3-7 days/37°C)  incubation with
radioactive stained tymidin (3H)  incorporation of 3Htymidin into DNA of proliferative lymphocytes 
detection of -radiation (-counter)
• more  proliferation, more  measuring radioactivity
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Activation of lymphocytes
Assessment of expression of
specific activated markers
Early activated markers
(CD69)
Late activated markers
(CD25, CD71 )
→ measured by flow cytometry
CD69
CD4
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Cytometric DNA analysis
• DNA ploidity
• Analysis of cell cycle
•
Count of
cells
DNA fluorochrome binding
(Propidiumiodid, akridin
orange)
• Intensity of fluorescence
directly proportional to DNA
content in cell
Intensity of fluorescence
(DNA content)
• G2/M phase- proliferative
phase,i.e. % of prolife.cells
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Cytotoxicity
→ Ability of T cytotoxic lymphocytes and NK cells to kill transformed/
tumor or virus infected cells
Different mechanisms:
Fas-FasL
Perforines, granzines
TNF- TNFR
Page 19
Cytotoxic tests
• test based on release of radioactive labelled
chrom (51Cr) from tumor cells
• Vital staining of tumor cells  microscopic or
cytometric analysis
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Performing of test with
51Cr
isoloted lymphocytes
+
incubation
(37°C/3,5h)
Tumor cells
incubated with
51Cr
51Cr
Calculation of % cytotoxic activity
100x (activity of sample-SPON)/(MAX-SPON)
Detection of radiation in
supernatant
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Measurement of cytokine secretion
• ELISA
• intracelullar assessement by flow cytometry
• ELISPOT
 detection and quantification of T lymphocytes
reacting on antigen stimulus by secretion of specific
cytokines
 Number of spots in well = number of
cells secreting cytokines
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ELISPOT
primar Ab against cytokine
cytokine
T ly
secundar Ab labelled
by biotin
streptavidin-enzym
substrate
Page 23
Application of functional tests of
lymphocytes
• Diagnostic of SCID
• Diagnostic and prognostic of malignant tumours
• Monitoring of cellular immunity (secundar
immunodeficiency, sepsi, post-operative state)
• Monitoring of development of graft after transplantation
of bone marrow
• Testing of new drugs (pharmacology, anti-cancer
immunotherapy)
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Functional tests of phagocytic cells
• Phagocytosis
• Testing of oxidative burst
• Determination of adhesive molecules expression
• Testing of chemotaxis
• Bactericidal test
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Phagocytosis
• Phagocytic cells: Neutrophils, monocytes/macrophages
• Target for phagocytosis: bacteria, cellular debris
• Phases of phagocytosis:
Diapedesis- chemotaxis- recognition- ingestion- killing and
destruction of target particles- antigen presentation
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Examination of ingestion phase
(engulfment of microorganism)
• substrate  Saccharomyces cerevisiae, Candida
albicans, polymer particules
• Suspension of particules or yeasts + blood (incubation
37°C/1h)  making of blood smear  fixation and
staining  reading in light microscope
• Positive cells- 3 and more engulfed
particles
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Examination of ingestion phase
(Engulfment of microorganism)
• Phagocyting activity-% phagocytes with absorbed
particles from all phagocyting cells
• Another kind of examination:
flow cytometry- fluorescent labelled particles
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Testing of oxidative burst
• Examination of phagocyte`s ability to build 02
radicals (activation of NADPH oxidase)
Measurement by flow cytometry
• DHR-123 test: Full blood + phorbol esters +
dihydrorhodamin  rhodamin (effect of 02 radicales
 measurement of fluorescenct intensity
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Testing of oxidative burst
• NBT (nitroblue-tetrazolium chlorid) and INT (iod-nitroblue
tetrazolium chlorid) tests
• Ability to reduce colourless tetrazolium salts
(result of 02 radicals)
• Full blood + amyloid grains + colourless liquid of
NBT or INT  colourful formazan (02 radicals)
 microscopic or spectrophotometric analysis
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Bactericidal test
• Assessment of phase of killing the engulfed particle
• substrate  Staphylococus aureus, Candida albicans
• Incubation of blood together with microorganism
(37°C/1hod)
Analysis:
• microscopic (vital staining with trypan blue)
• cytometric (vital staining with PI, Hoechst)
• Inoculation on plates (counting of colonies)
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Activation of basophils by alergens
• Anafylactic degranulation of basophils – fast morphological changes,
exocytosis of IC granules and release of modified mediators
• „Piecemeal“ degranulation – slow morphological changes without
degranulation
• CD63, CD203c, CRTH2-FITC /CD203c-PE/CD3-PC7
• CD69, Cd107a, CD123, CD 164, basogranulin and etc.
• Detection of mediators and enzymes
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Baso study in insect allergy
• Diagnostic
– Examination of double positivity (CCD)
• Biomarker of anaphylaxis
– ↑ expression of CD69 –exposition in vitro and in
vivo
• Monitoring of immunotherapy
– Change in reactivity
– Prediction of undesirable effects
• Control of effect – exposure test, persistence of
treatment effectivity
• Research of alergens
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Application of functional tests of
phagocytic cells
• Diagnostic of primar immunodeficiency
(LAD-1, LAD-2, chronic granulomatosis)
• Testing of new drugs
• Anticancer immunotherapy (test of DC maturity)
Page 34