Methods for analysis of cell mediated immunity

1. Methods for analysis of cell mediated immunity

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

3. 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.) What is flow cytometry/ FACS ?

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. 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

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

7. List of common fluorochromes used in flow cytometry

8. 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 Principle of flow cytometry

9. Detectors of fluorescence Optical system in flow cytometer APC PE PE-Cy7 FITC FSC LASER SSC stainedcells PC analysis

10. 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

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. Application of cytometric analysis • phenotypisation of cells (diagnostic of primar immunodeficiency, autoimmune diseases, leukemie and lymphoms, etc.) • functional testsof 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 intracellularcalcium

13. Functional tests oflymphocytes • Proliferation • Expression of activated markers • Cytotoxicity • Cytokine secretion • Production of antibodies

14. gradient centrifugation (cell separationaccording todifferencesin theirdensity) Isolation of lymphocytes from blood Diluted plasma Lymphocytes Separative solution Erythrocytes Granulocytes

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

16. Blastic transformation test • Ability of T lymphocytes to response on polyclonal stimuli 3H- tymidin test • Cultivation of isolated lymphocytesin mediumwith stimulators (3-7 days/37°C)  incubationwith radioactive stained tymidin (3H)  incorporation of3H- tymidin into DNA of proliferative lymphocytes detection of -radiation (-counter) • more  proliferation, more  measuring radioactivity

17. 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

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

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. test based onrelease of radioactive labelled chrom (51Cr) fromtumorcells Vitalstaining oftumor cells microscopicor cytometricanalysis Cytotoxic tests

21. Performing of test with51Cr isoloted lymphocytes incubation (37°C/3,5h) + 51Cr Tumorcells incubated with51Cr Detection of-radiation in supernatant Calculation of % cytotoxic activity 100x (activityof sample-SPON)/(MAX-SPON)

22. ELISA intracelullarassessementby flow cytometry ELISPOT  detection and quantification of T lymphocytes reactingon antigen stimulusby secretion of specific cytokines Number of spotsin well = number of cells secreting cytokines Measurement of cytokine secretion

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

24. Diagnostic of SCID Diagnostic and prognosticof malignanttumours Monitoring ofcellular immunity (secundar immunodeficiency, sepsi, post-operative state) Monitoring ofdevelopment ofgraftafter transplantation of bone marrow Testing of new drugs (pharmacology, anti-cancerimmunotherapy) Applicationof functional tests of lymphocytes

25. Functional tests of phagocytic cells • Phagocytosis • Testing of oxidativeburst • Determination of adhesive molecules expression • Testing of chemotaxis • Bactericidal test

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. Examination of ingestion phase (engulfment of microorganism) • substrate Saccharomyces cerevisiae, Candida albicans, polymer particules • Suspension of particulesoryeasts + blood (incubation 37°C/1h)  making of blood smear  fixation andstaining  reading in light microscope • Positive cells- 3 and more engulfed particles

28. Examination of ingestion phase (Engulfment of microorganism) • Phagocyting activity-% phagocyteswithabsorbedparticlesfrom allphagocytingcells • Another kind of examination: flow cytometry- fluorescent labelled particles

29. 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

30. 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 + amyloidgrains + colourless liquid of NBT or INT  colourful formazan (02 radicals)  microscopic or spectrophotometric analysis

31. Assessment of phaseof killing the engulfed particle substrate Staphylococus aureus, Candida albicans Incubation of bloodtogether with microorganism (37°C/1hod) Analysis: microscopic (vitalstaining with trypan blue) cytometric (vitalstaining with PI, Hoechst) Inoculation on plates (counting ofcolonies) Bactericidal test

32. 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 Activation of basophils by alergens

33. 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 Baso study in insect allergy

34. Applicationof 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)