1. Page 1 Methods for analysis of cell mediated immunity

2. Page 2 Methods 1.Flow cytometry 2.Functional tests of lymphocytes 3.Functional tests of phagocyting cells

3. Page 3 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.)

4. Page 4 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

5. Page 5 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 Flow cytometry

6. Page 6 Cell staining in flow cytometry excitation (laser) emission fluorescence Cell surface marker Fluorochrome- labelled mAb Direct staining Indirect staining purified/ biotinylated mAb Fluorochrome-labelled secundar Ab/streptavidin

7. Page 7 List of common fluorochromes used in flow cytometry

8. Page 8 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

9. Page 9 LASER stained cells Dete c tors of fluorescence APC Optical system in flow cytometer PE FITC PE-Cy7 SSC FSC PC analysis

10. Page 10 Typical Dot plot of cell subpopulations in blood samples and double staining Lympho gate Dot plot graphs 1 dot/event = 1 cell Single parameter histogram

11. Page 11 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%)

12. Page 12 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

13. Page 13 Functional tests of lymphocytes Proliferation Expression of activated markers Cytotoxicity Cytokine secretion Production of antibodies

14. Page 14 Isolation of lymphocytes from blood gradient centrifugation (cell separation according to differences in their density) Diluted plasma Separative solution Lymphocytes Erythrocytes Granulocytes

15. Page 15 Important for the process of immune reactions Diagnostic s of innate immunodeficiency Activation of T cell receptor (TCR) → activation of intracellular signal cascade → signal transduction into nucleus → transcription of genes for proliferation Proliferation of lymphocytes

16. Page 16 Blastic transformation test Ability of T lymphocytes to response on polyclonal stimuli 3 H- tymidin test Cultivation of isolated lymphocytes in medium with stimulators (3-7 days/37°C)  incubation with radioactive stained tymidin ( 3 H)  incorporation of 3 H- tymidin into DNA of proliferative lymphocytes  detection of  -radiation (  -counter) more  proliferation, more  measuring radioactivity

17. Page 17 Activation of lymphocytes CD69 CD4 Assessment of expression of specific activated markers Early activated markers (CD69) Late activated markers (CD25, CD71 ) → measured by flow cytometry

18. Page 18 Intensity of fluorescence (DNA content) DNA ploidity Analysis of cell cycle DNA fluorochrome binding (Propidiumiodid, akridin orange) Intensity of fluorescence directly proportional to DNA content in cell G2/M phase- proliferative phase,i.e. % of prolife.cells Cytometric DNA analysis Count of cells

19. Page 19 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

20. Page 20 Cytotoxic tests test based on release of radioactive labelled chrom ( 51 Cr) from tumor cells Vital staining of tumor cells  microscopic or cytometric analysis

21. Page 21 Performing of test with 51 Cr + isoloted lymphocytes Tumor cells incubated with 51 Cr incubation (37°C/3,5h) Detection of  - radiation in supernatant 51 Cr Calculation of % cytotoxic activity 100x (activity of sample-SPON)/(MAX-SPON)

22. Page 22 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

23. Page 23 ELISPOT primar Ab against cytokine T ly cytokine secundar Ab labelled by biotin streptavidin-enzym substrate

24. Page 24 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)

25. Page 25 Functional tests of phagocytic cells Phagocytosis Testing of oxidative burst Determination of adhesive molecules expression Testing of chemotaxis Bactericidal test

26. Page 26 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

27. Page 27 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

28. Page 28 Phagocyting activity-% phagocytes with absorbed particles from all phagocyting cells Another kind of examination: flow cytometry- fluorescent labelled particles Examination of ingestion phase (Engulfment of microorganism)

29. Page 29 Testing of oxidative burst Examination of phagocyte`s ability to build 0 2 radicals (activation of NADPH oxidase) Measurement by flow cytometry DHR-123 test: Full blood + phorbol esters + dihydrorhodamin  rhodamin (effect of 0 2 radicales  measurement of fluorescenct intensity

30. Page 30 NBT (nitroblue-tetrazolium chlorid) and INT (iod-nitroblue tetrazolium chlorid) tests Ability to reduce colourless tetrazolium salts (result of 0 2 radicals) Full blood + amyloid grains + colourless liquid of NBT or INT  colourful formazan (0 2 radicals)  microscopic or spectrophotometric analysis Testing of oxidative burst

31. Page 31 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)

32. Page 32 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

33. Page 33 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

34. Page 34 Diagnostic of primar immunodeficiency (LAD-1, LAD-2, chronic granulomatosis) Testing of new drugs Anticancer immunotherapy (test of DC maturity) Application of functional tests of phagocytic cells