Methods for analysis of cell mediated immunity Page 1 Methods • 1.Flow cytometry • 2.Functional tests of lymphocytes • 3.Functional tests of phagocyting cells Page 2 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.) Page 3 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 Page 4 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 Page 5 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 Page 6 List of common fluorochromes used in flow cytometry Page 7 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 Page 8 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 Page 10 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%) Page 11 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 Page 12 Functional tests of lymphocytes • Proliferation • Expression of activated markers • Cytotoxicity • Cytokine secretion • Production of antibodies Page 13 Isolation of lymphocytes from blood • gradient centrifugation (cell separation according to differences in their density) Diluted plasma Lymphocytes Separative solution Erythrocytes Granulocytes Page 14 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 Page 15 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 Page 16 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 Page 17 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 Page 18 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 Page 20 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 Page 21 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 Page 22 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) Page 24 Functional tests of phagocytic cells • Phagocytosis • Testing of oxidative burst • Determination of adhesive molecules expression • Testing of chemotaxis • Bactericidal test Page 25 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 Page 26 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 Page 27 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 Page 28 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 Page 29 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 Page 30 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) Page 31 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 Page 32 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 Page 33 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