Methods for testing cell-mediated immunity

Inrtoduction: Cell-mediated immunity

• White blood cells – leukocytes • Nonspecific immunity:

» Neutrophils » Eosinophils » Basophils » Macrophages » NK cells

• Specific immunity» T cells» B cells

How to work with a blood sample?

• Anticoagulant → anticoagulated blood 

• Transport to the laboratory within 2h

• Storing at 4 °C → Slowdown "aging" 

• Funcinal tests – intact cells• Gentle fast work → vitality 

→

What can we observe? (1/2)

• Features• Biological

» Size» Granularity» Viability

• Chemical» pH

• Physical» Membrane potential

• Representation (numbers) of individual population = Phenotyping

What can we observe? (2/2)

• Individual cell activities• Directly – detection of

function itself

• Indirectly – expression of specific molecules eg:» Activation markers

       – expression of specific products eg:

» Cytokines» Reactive oxygen intermediates – Oxidative burst» Enzymes» DNA

Flow cytometry • Simultaneous measurement of cell

(particle) features by its passing through the laser ray

• Physical features• Chemical features• Biological features

• Separate measurements of each cell (particle)• Not an average values of measured suspension

↓

Qualitative and quantitative analysis of up to

10,000 cells (particles)/s

Light refraction (FCS x SSC)

• Refraction of light passing through a suspension (cell) stream – monitoring the direction of the refraction angle 

• Direct scattering (Forward Scatter – FSC)» Refraction of 2°-13° – proportional to cell size 

• Side scattering (SSC)» Scattering of 90° – indicating

cell structure (granularity)

Analysis of labeled cells (1/3)

• Using fluid system • Cells carryed by sheath fluid • Under pressure drawn into a special space • Encountering laser beam and cell

Analysis of labeled cells (2/3)

• Optical system• Excitation part

» Laser» The system of lenses and prisms – directing a

light beam

• Collecting part» Optical mirror + filters – detection of light

quanta with specific wavelenghth  

Analysis of labeled cells (3/3)• Elektronic system

• Converting of optical signals to electronic • Digitization (for computer analysis) → data file 

Graphic display of measured values (1/2)

• Single parameter histogram • X-axis – signal strength • Y-axis – number of measured cells

Graphic display of measured values (2/2)

• Two-parameter histogram • X-axis – first signal intensity • Y-axis – second signal intensity• Number of measured cells

» Dot plot – density of points» Contour plot – density corresponds

to a bars suggesting contours

Tests of cell-medieted immunity

Detection of cells, structures, molecules by using antibodies

→ →

Cell phenotyping

• Using an antigen-antibody reaction

• Visualization – fluorochrome

Characterization of cells by using specific membrane or cytoplasmic markers

Phenotyping of leukocytes by CD attributes 

CD features = systematic designation of most of leukocyte surface molecules

Human natural killer cells: a unique innate immunoregulatory role for theCD56bright subsetMegan A. Cooper,  Todd A. Fehniger, Sarah C. Turner, Kenneth S. Chen, BobakA.Ghaheri,  Tariq Ghayur, William E. Carson, and  Michael A. Caligiuri

Feature Cell population

CD45 leukocytes

CD3 lymphocytes

CD3/CD4 Th (helper) cells

CD3/CD8 Tc (cytotoxic) cells

CD19 B cells

CD16/CD56 Natural Killers

CD203 Basophiles

Graphical representation

Fig.1 Th cell phenotyping Fig.2 Tc cell phenotyping

Fig.3 B cell phenotyping Fig.4 NK cell phenotyping

Testing of leukocytes functions – Basophil activation (1/2)

• Determination of allergic reaction degree

• Basophils identification – CD203c

• Activated basophils identification – CD63 • The resting basophils – CD63 a component

of granule containing histamine membrane • Basophil activation → fusion of granules with

cytoplasmic membrane = exposure of CD63 molecule on cell surfaces

Procedure of basophil activation test

• Incubation of the blood sample with IL-3 • Promoting of basophil stimulation – eg increased

histamine release • The possibility of CD63 expression increase

→ increased sensitivity of the test

• Incubation of the blood sample with an allergen

• Basophil activation

• Staining of molecules:• CD203c• CD63

Testing of leukocytes functions (2/2)

• Cytotoxicity test of T cells• Cytotoxicity = ability of T cells and NK cells to

kill tumor and infected cells » Target cells contain for example 51Cr» Killing =  51Cr release» Measurement of supernatant radioactivity 

The importance of lympohcyte population testing 

• Monitoring of cell-mediated immunity • Secondary immunodeficiency • Injuries

• Sepsis

• Postoperative states

• Diagnostics and prognostication of malignant tumors • Diagnostics of SCID and hypogammaglobulinaemia

• Monitoring a graft development after bone marrow

transplantation • Testing new drugs (pharmacology, cancer

immunotherapy)

Phagocytosis

• Phagocytosis phases: 

• Diapedesis

• Chemotaxis

• Ingestion

• Killing and destruction of phagocytosed particles

An absorption of particles by the cell

Testing phagocytic cell functions• Expression of adhesion molecules

• Adherence to the surface • Antigen – antibody reaction

» Eg. FACS

• Chemotaxis

• Ingestion• Incubation of whole blood with polymer particles or

bacterias• Phagocytosis of bacteria or particles • Evaluated by light microscopy

chemotaxin (fMLP)

suspension of tested cells

medium

Chemotaxis

migration natural migration

Testing phagocytic cell functions

• Respiratory burst = the ability to generate reactive oxygen radicals

• Oxygen radicals – reaction with differnt substances → detection of colored substances

• FACS analysis – dihydrorhodamin → rhodamin• Microscopic, spectrophotometric analysis –

colorless tetrazolium salt (př.NBT) → collored formazan

Bactericidal test

• Testing all stages of phagocytosis• Incubation of whole blood (containing opsonins)

with bacterias (C. albicans, S. aureus, E. coli)

↓↓• Evaluation of an ability to kill microorganisms:

» Inoculation of microorganisms on soil – counting colonies» Microscopically – vital staining (trypan blue = dead

microorganisms)

The importance of phagocytosis testing

FUNCTION TEST MALFUNCTION

ADHESION Expresion of CD11/ CD18 LAD1 syndrom

CHEMOTAXIS Under-agarose migration

assay

Disorders are relatively rare;transient in infections, burns, traumas

INGESTION Opsonization and engulf-ment of particles(methacrylate particles,microorganisms)

Disorder affects opsonization

RESPIRATORYBURST

NBT Defective in chronic granulomatousdisease (CGD);transient disorder of infections,traumas, malnutritions

Fluorescence

Chemoluminiscence

THE WHOLE PROCESSPHAGOCYTOSIS INCLUDING MICRO-ORGANISMS DEATH

Baktericidal test Warnning of one phagocytosis stagefailure 

(Litzman a spol., 2007)

Thank you for your attention