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• Preparative and analytical methods based on antibody-antigen interactions • Functional investigations of the cells of the immune system (1st part) The characterisation and separation of the cells of the immune system Flow cytometry (FACS) The sensitivity of immunoassays Many cell types of the immune system are indistinguishabe by simple morphology (e.g. lymphocyte subtypes) The most important cell surface molecules (”antigens”) of the lymphocyte subtypes Cell surface molecules – as markers – can be used for identifying cell types The ligands of the cell surface receptors would be used to label the cells Problems: • affinity • large scale manufacturing CD40 CD40L CD40 CD40L Cell surface antigen specific antibodies can be easily produced CD40 anti-CD40 The affinity/avidity of the antibodies can be appropriate for the purpose To recognise the antigens on the cells, the label should be also detectable The antibodies should be also “visualised” possibilities: • isotopes • enzyme (e.g. colour change catalysed by the enzyme) • fluorescence • microparticles (electron microscopy – “immunogold”) • …etc…etc… • … Amino acid side chains of the proteins can be simply coupled to labels with reactive chemical groups 1. e.g. to amines on lysine side chains: isothyocyanate group (…ITC) succinimidyl ester (…SE) group: Amino acid side chains of the proteins can be simply coupled to labels with reactive chemical groups 1. e.g. to thiols on cysteine side chains: with haloalkalnes (alkyl halides): with maleimide groups: Fluorescent labels/dyes used in flow cytometry or microscopy The ideal fluorescent labels: • photostable slow fading • narrow excitation and emission spectra multiple dyes can be used together Fluorescent spectra internet tools: http://www.bdbiosciences.com/us/s/spectrumviewer http://www.biolegend.com/spectraanalyzer https://www.thermofisher.com/hu/en/home/life-science/cell-analysis/labelingchemistry/fluorescence-spectraviewer.html https://www.chroma.com/spectra-viewer Fluorescent dyes Fluorescein excitation emission FITC (fluorescein isothiocyanate) fluorescein-5-isothiocyanate 2-(6-Hydroxy-3-oxo-3H-xanthen-9-yl)-5isothiocyanatobenzoic acid Lots of isomers exist with slightly different fluorescent spectra! Fluorescent dyes Phycoerythrin, PE (protein) 3Z-phycoerythrobilin (PEB), the chromophore group of the phycoerythrin protein Phycoerythrin is an accessory pigment in different algae. Helps the function of the main chlorophyll pigments responsible for photosynthesis. FLOW CYTOMETRY An immunofluorescent method that mutually complements the fluorescent microscopy Large part of the method is using monoclonal antibodies Detection and analysis of different cells or particles travelling at high velocity in flow Detects fluorescence intensity and scattered light of the labeled cells Can give indirect morfological data (size, granularity) Statistical method: Can investigate enormous number of cells in a short period of time ADVANTAGES OF THE FLOW CYTOMETRY IN IMMUNOLOGY Most cells in the immune system can be found in free or loosely adherent form. They can be easily dispersed and labeled by fluorescent antigen specific antibodies, and then they can be examined cell by cell in suspension The cells’ light scatter and immunofluorescent properties can be analyzed statistically (e.g. percentages of different cell populations) Rare cell populations can be identified and examined (e.g. antigen specific lymphocytes) The method provide qualitative and quantitative data – it can detect the presence of different antigens in the cell, and the expression levels of these antigens. Changes in the expression of certain molecules can be followed after different treatment of the specimen. (e.g. cell activation, disease progression) CHARACTERIZATION OF IMMUNE CELLS USING CELL SURFACE MARKERS Cell types, differentiation stages can be identified using a combination of cell surface markers. Used in diagnostics: - ratio of different cell types - altered expression of cell surface markers Examples: - Inflammatory processes – increased neutrophil numbers - HIV progression – decrease of CD4+ T cell count CD4+ : CD8+ = ~1.6 Normal CD4+ T cell count = 600 – 1400/l AIDS = CD4+ T cell count <200/l, altered CD4+ : CD8+ ratio - increase of CD5+ B cells – typical for some B cell leukemias Some typical markers of the main immune cell types CD antigen cell types function ligand CD3 T cells T cell antigen receptor signalling chain complex CD4 helper T cells, plasmacytoid dendritic cells (pDC), monocytes co-receptor of the T cell antigen receptor, (also HIV entry receptor) MHC II, (HIV) CD5 T cells, (B cell sub-population: B1) cell adhesion, signalling (costimulation) CD72 CD8 cytotoxic T cells, (NK, some T co-receptor of the T cell antigen receptor MHC I cells) CD14 Monocytes, macrophages, some granulocytes part of the LPS signalling receptor complex LPS, LBP CD19 B cells part of the B cell Ag receptor co-receptor complex C3d, C3b (CD19/CR2(CD21)/CD81) CD28 T cells co-stimulation (B7-1, B7-2) CD80, CD86 CD34 hematopoietic progenitor cells, endothelial cells cell adhesion, … CD62L (L-selectin) CD56 NK cells, (some T and B cell sub- homoadhesion (N-CAM isophorm) population) CD80, CD86 (B7-1, -2) professional APC: DC, B, monocytes, macrophages co-stimulation, cell adhesion CD28, CD152 Some picture from the flow cytometers’ history: From the first cell-counting machines to the high speed cell sorters ©J.Paul Robinson Early cell counter. Katherine Williams and C.S. Sanders (Atomic Energy Research Establishment) 1948 - Unclassified in 1956. (Photo taken in Science Museum, London UK) (cytometers from the 2000s) benchtop flow cytometer (BD FACSCalibur™) sorter flow cytometer (BD FACSDiVa™) benchtop sorter flow cytometers from the recent ages BD FACS Aria™ Beckman Coulter MoFlo™ XDP FACS (acronym) • original meaning: Fluorescence Activated Cell Sorting • nowadays generally: flow cytometry Basic principles of the flow cytometry The cells could be rapidly and easily investigated in laminar flow a: The erythrocytes are arranged in the laminar flow stream and travel in elongated manner by the hydrodynamic forces b: In turbulence the cells are disoriented, scattered and they are travelling with different velocity in the flow Image fromV. Kachel, et al. Hydrodynamic focusing in the laminar flow The ink is arranged to a thin stream in the laminar flow There is no mixing between the ink and the sheath fluid The original position of the ink have only slight influence on the position of the thin stream in the laminar flow V. Kachel, H. Fellner-Feldegg & E. Menke The hydrodynamic system of the flow cytometers sheath fluid reservoir Flow cell injector +++ +++ +++ sheath fluid sample 6-10m/s velocity Fluorescent signals Focused laser beam Measured parameters: Light scattering (FSC, SSC) Any particle can scatter the light FALS Sensor FSC Laser (forward angle light scatter or forward scatter) The FSC has some correlation with the particle size 90 (side) scatter SSC (side scatter) Smaller structures, organeles inside the cells effectively scatter the blue light in this way Measured parameters: Fluorescence Laser FSC High sensitivity photon detectors (photomultipliers) (photoelectron multiplier tubes – PMT) detection of the fluorescence: fluorescent dyes (and autofluorescence by the presence of pyridines and flavins) The scheme of a flow cytometer detectors dicroic mirrors and filters PMT 4 sample PMT 3 flow cell PMT 2 FSC detector PMT 1 Laser(s) IMMUNOPHENOTYPING BY FACS Example: Measurement of CD4+ (helper) and CD8+ (cytotoxic) T cell ratio (e.g. monitoring AIDS progression) Labeling: FITC labeled anti-CD4 antibody(α-CD4-FITC) PE labeled anti-CD8 antibody (α-CD8-PE) Th NK Tc Lymphocytes in the peripheral blood sample Fluorescent microscopy B high velocity flow stream detecting CD4-FITC labeled (TH) cell (in cuvette or stream in air) detector signal processing unit CD8 PE screen increasing light intensity a dot representing a CD4+ CD8- cell CD4 FITC microscopy: detecting the PE labeled cell (CD8-PE) CD8 PE detector signal processing unit increasing light intensity CD4 FITC detecting the unlabeled cell (e.g. B cell) by autofluorescence CD8 PE detector Signal processing unit increasing light intensity microscopy: dim (autofluorescent) cell CD4 FITC CD8 PE 18% 44% animation! 0% quadrant statistics CD4 38% FITC GRAPHICAL REPRESENTATIONS 1. dot-plot contourplot densityplot GRAPHICAL REPRESENTATIONS 2. Histogramm Numeral intensity values (MFI): ~7 ~ 1300 homogenous cell population is normally distributed (Gaussian) Different cell types - characteristic light scattering granulocytes side light scattering (SSC) (e.g. granulated) monocytes lymphocytes forward light scattering (FSC) („size”) EXAMINATION OF PERIPHERAL BLOOD BY HAEMATOLOGY AUTOMATS Measured parameters: peroxydase staining (the presence of myeloperoxydase, x – axis) light scatter (high on large granular cells, y – axis) 1 (Noise) 2 Nucleated Red Blood Cells 3 Platelet Clumps 4 Lymphocytes and Basophils 5 Large Unstained Cells (LUC) 6 Monocytes 7 Neutrophils 8 Eosinophils The major cell types can be easily identified by the help of only two parameters! The "gating" helps to identify the properties of the cell populations lymphocytes granulocytes all cells granulocyte gate monocytes monocyte gate limfocyte gate Are they really CD4+ or it is autofluorescence? Autofluorescence or real expression? In the case of the lymphocytes the CD4 staining is obvious: There were CD4+ (positive) and CD4- (negative) populations CD4- CD4+ But there are more difficult cases: e.g. CD1a, MHC-like molecule expression on dendritic cells Where are the CD1a positive cells? Comparison with an unstained sample ? (or with an “isotype control” stained sample) human CD1a specific mouse IgG1 atibody “control” mouse IgG1 antibody Where to draw the border? ? It is rewarding to draw the specidic fluorescence together with the autofluorescence: IgG1 isotype CD1a control antibody A control sample stained with isotype control antibody No staining can be seen within the area where the specific staining will be expected The histogram “hides” this cells autofluorescence autofloureszcencia The histogram representation could be misleading Cell Biology / Immunology The cellular functions can be also examined by flow cytometry with fluorescent indicators, the so called fluorescent “probes”: • • • • • • DNA content intracellular ion concentrations redox state pH organelle number …etc… (They will be discussed on the next lecture also) (Cell biology methods) Investigation of the cell function The intensity of the cell metabolism can be characterised by the numbers of the mitochondria. Functional mitochondrion specific "probes" (e.g. Mitotracker dyes) can be used for this purpose, which can make distinction between living and dead cells also. The nucleic-acid content of the cells can be measured by fluorescent dyes that bind stoechiometrically to the nucleic-acids. Such kind of double stranded nucleic-acid (DNA, dsRNA) incorporating stains are: •propidium jodid •ethidium bromid Propidium NH2 Ethidium NH2 NH2 MitoTracker Red CH N+ 2 C2H2)3 N+ NH2 N+ 5 CH3 C2H5 C2H5 The size of the cycling cells are increased – called blast transformation Cell-cycle Possibilities of the examination Stimuli (e.g. antigen) resting lymphocyte (G0) effector cell - transcription (RT-PCR) - protein synthesis memory cell (Immunoassay) changes in the RNA- and protein synthesis, in the cell membrane and in the transports cell division change in the number of the cells DNA-synthesis (MTT, CFSE) DNA quantification (fluorescent DNS intercalating agents, 3H-thymidine) The cell cycle can be examined by fluorescent dye G2 G0 M that intercalates stoechiometrically into the double stranded DNA (e.g. propidium iodide, PI) DNA analysis G1 cell number s G0G1 G2 M s 0 200 400 600 800 1000 4N 2N DNA content Distribution of a normal cycling cell-population by DNA content (flow cytometry) DNA Analysis A typical DNA Histogram sub G0/G1 cells 0 200 400 2N 600 800 4N PI Fluorescence 1000 Cell cycle specific protein detection can be used for the examination of dividing cells: Ki-67, PCNA, modified cyclins – can be shown by antibodies Apoptosis detection: •DNA loss during apoptosis sub G0/G1 cells •Inter nucleosomal cleavages result free DNS ends which can be enzymatically (Tdt) labeled by BrDU or dUTP normal cycling cells •Apoptotic changes in the plasmamembrane (extracellular appearance of the intracellular phosphatidylserine) can be detected by fluorescent dye conjugated Annexin V protein Chromosome analysis and separation Chromosomes from cell cultures can be investigated (and separated) by flow cytometry. Two fluorescent dyes are used: Hoechst 33258 binds the AT-rich regions, and the chromomycin A3 binds the GC-rich regions in the DNA. Because of the different AT/GC ratios of the chromosomes, they can be discriminated Separated chromosomes, chromosome pools are useful in the genomics Hoechst 33258 Normal human Human X hamster Normal hamster Normal mouse Chromomycin A3 J.W. Gray & L.S. Cram Kinetic measurements by flow cytometry (the time can be also measured as a parameter) In homogenous cell population the consecutive measurement of some property in different cells is represent the change of that property: measurement in one cell measurement in homogenous cell population The intracellular Ca2+ signal can be measured with fluorescent Ca2+ indicators: e.g. Fluo-3 or Indo-1 Single cell microscopic kinetic measurement: Ca2+ cell signalling Intracellular Ca2+ signal in one cell: B cell Antigen presenting B cell activates a T cell which is preloaded with a Ca2+ indicator dye T cell e.g. Fluo-3 or Indo-1 Fluorescence intensity representing the intracellular Ca2+ concentration Intracellular Ca2+ signal measurement by flow cytometry activation of the cells time base signal (the time can be also measured as a parameter) Intracellular Ca2+ signal measurement by flow cytometry Ca2+ signalling in Influenza virus hemagglutinin protein derived peptide specific T cell hybridome induced by the peptide pre-loaded antigen presenting cells activated T cells (no measurement during the cell activation: the APCs and the T cells are centrifuged to close together) non-activated T cells Imaging cytometry (More detailed morphologic information – sophisticated picture analysis softwares) CIRCULARITY: cell differentiation cell division (blasts) migrating cells source: www.amnis.com Investigation of the phagocytosis phagocytosis of fluorescent latex particles by dendritic cells – conventional flow cytometry Measurements of the “difficult” small cell populations can be achieved by multicolour cytometry e.g. identification and investigation of different monocyte cell subpopulations in the mouse peripheral blood used antibodies: CD45+ cells: leukocyte marker for excluding the erythrocytes Ly6C+ cells: myeloid cell marker (monocytes, MF, neutrophyls) Ly6G- cells: Ly6G is a good neutrophil marker in the mouse CD115+ cells: M-CSF receptor mainly on macrophages F4/80+ cells: different macrophages an monocytes can express it MHCII+ cells: a good marker for antigen presenting cells 4-8 colour can be relatively easily measured by the recent cytometers (You should use “fluorescence compensation controls” to consider the spectral overlaps of the different fluorescent dyes) Using more and more colours increase the hardness/trickiness of the experiments Spectral cytometry The detailed measurements of the fluorescent spectra helps to identify different overlapping colours in the multicolour experiments (including mathematical methods) by David Buschke, Other possibility for “multicolour” cytometry Using different type, pin sharp “spectral” labels: heavy metal–conjugated antibodies (mainly lantanoids) In mass spectroscopy this labels are very easily discriminated from each other MASS CYTOMETRY Cell separation Physical isolation of the cells of interest from a heterogeneous population Differences in the physical , biological or immunological properties of the cells are utilized to separate the cells physical – density, size cell biological – adherence, phagocytosis, sensitivity to the medium immunological – antigen differences (surface!) Differences in cell surface receptor expression are convenient possibilities for the separation of different cell types Characteristics of the separation: • purity • recovery, yield, loss • viability of the cells Separation Base strategies: positive separation – labeling and separation of the cells of interest e.g. Labeling of a cell surface molecule (receptor!) by a fluorescent antibody. The cells become affected both by the separation environment and the antibodies bound to the receptors. The purity of the separation is generally high. negative separation – get rid of the labeled unwanted cells (depletion) The cells become affected only by the separation environment This is the preferred strategy in the functional examinations. The two procedure can be used in combinations Peripheral blood is an easily accessible source for the separation of human immune cells and for plasma components Separation of the plasma from the cellular components: Separation by filtration (simple membrane or holofiber „membrane”) Pore diameter for plasma separation: 0.2 to 0.6μm The different density parts of the anticoagulated blood is separated to three parts in undisturbed tube: • bottom: sedimented red blood cells • top: cell free plasma • the intermediate layer is called „buffy coat” contains the leukocytes, platelets The process can be accelerated by centrifugation (clinical procedures) Apheresis (ancient greek ἀφαίρεσις) -“to take away” Separating one particular component of the blood and returning the remainder to the circulation • Donor apheresis • Therapeutic apheresis Donor apheresis: • Plasmapheresis – processed (e.g. IVIG) or fresh frozen plasma for immunodefficient patients or in acute infections as passive immunization • Plateletapheresis (trombocytapheresis) – concentrated platelets for inherited or induced thrombocytopenia (infections, chemotherapy, irradiation) or in the case of thrombocyte disfunction • Red blood cells (erythrocytapheresis) - for patients with anemia (inherited or internal/external blood loss by surgery or trauma) • Leukapheresis – buffy coat, mainly for autotransplantation • isolating and protecting the leukocytes before chemotherapy • monocyte separation for dendritic cell therapy • leukapheresis after the mobilization of bone marrow stem cells – for stem cell therapy or bone marrow transplantation (autologous, allogeneic) (clinical procedures) Therapeutic apheresis: • Removing abnormal/disfunctional components of the blood (humoral or cellular) • Replacing a component with a healthy donor apheresis product • Blood component alteration (ex vivo therapy) • Leukapheresis – Extreme high leukocyte number can lead to hemostatic disorders in leukemia (accompanying shortness of breath, vision changes). Inflammatory cell number can be decreased in autoimmune chronic inflammatory diseases (ulcerative colitis, rheumatoid arthritis) • LDL apheresis – Removal of low density lipoprotein from the plasma in patients with familial hypercholesterolemia (adsorption with ApoB affinity column or precipitation with acetate) • Thrombocytapheresis – In essential trombocythemia/e.trombocytosis (rare disease) the disfunctional very high thrombocyte number (with thrombosis and bleedings) can be lowered rapidly in the rare cases of life threatening emergency situations • Erythrocytapheresis - Removing abnormal red blood cells in patients experiencing sickle cell crisis (in sickle-cell anemia) • Plasmaexchange – Removing/replacement the plasma with autoimmune antibodies in various autoimmune diseases (combined with immunosuppression) (eg. Myasthenia gravis, Guillain-Barré syndrome, lupus, Goodpasture syndrome, Antiphospholipid antibody syndrome, Behcet syndrome, etc….) • Immunoadsorption with protein A/G – removal of allo- and autoantibodies (in autoimmune diseases, transplant rejection, hemophilia) by directing plasma through protein A or G-agarose columns Continuous Flow Apheresis Systems Continuous Flow Centrifugation (CFC) Cross section representation of an old fashioned washable apheresis centrifuge bowl Thrombocytes and thrombocyte free plasma can be separated by appropriate CFC methods The blood or the “buffy coat” cells are too “contaminated” with erythrocytes for some purposes Ficoll-Paque (1.077g/ml) Ficoll helps to separate the near similar density cells (from Google pictures) (Nature Protocols http://www.nature.com/nprot/journal/v3/n6/images/nprot.2008.69-F1.jpg) Ficoll-Paque - density based cell separation peripheral blood centrifugation pipetting the cell layer into a new tube plasma mononuclear cells (PBMC) ficoll Ficoll can be filled under the blood with a thin pipette or the blood can be layered on the Ficoll neutrophil granulocytes RBCs, dead cells separated peripheral blood mononuclear cells Granulocytes are also missing from the Ficoll separated blood samples Separation with the lysis of the erythrocytes Density based cell separation (Ficoll) missing granulocytes Percoll – best way to separate neutrophil granulocytes Physical chemical characterization of Percoll. I-III. Laurent, T.C. et al. Colloid Interface Sci. 76, 124–145 (1980). stepwise or continuous pecoll gradient Romanian J. Biophys., Vol. 14, Nos. 1–4, P. 53–58, Bucharest, 2004 Rosette formation Red blood cells can be coupled to other cells with the help of antibodies (a special type of agglutination) The unwanted cells closed into erythrocyte rosettes can be depleted by Ficoll separation: Red blood cell rosettes can be used for cell separation (depletion) negative separation Separating or depleting adherent cells (both for negative and positive separation) Simple, cheap, but only for adherent cells. The purity is not too high Antibody mediated separation methods Complement mediated lysis cell or cell type specific antibodies complement factors LYSIS only for negative separation (depletion) (Erythrocytes can be selectively lysed in a slightly hypotonic ammonium chloride buffer) Antibody mediated separation methods Antibody ”panning” (both for negative and positive separation) cell specific antibodies on the surface magnetic cell separation Simple magnetic separation of macrophages Phagocytic cells engulf small iron particles. A strong magnet can be used to separate these cells from the others. Immuno magnetic cell separation – “MACS” (1st step) cell specific antibody paramagnetic bead MACS (2nd step) Magnetic cell separation - MACS (3rd step) separation of the labelled cells (positive separation) MAGNET MAGET separation column lots of similarities with the procedures of the affinity column depletion or separation of the non labelled cells (negative separation) Magnetic separation columns CliniMACS – closed system magnetic cell separation CliniMACS Plus CliniMACS® Prodigy The paramagnetic particles are very small and possibly don’t influence the cells’ functions CD8+ T cells „DETACHaBEAD” Polyclonal antibodies against the Fab part of the "paramagnetic" antibodies. Possibly involving anti-idiotype antibodies. They can help to detach the "paramagnetic" antibodies from the cell surface. (competition between the cell surface antigen and the anti-idiotype antibodies) FACS (Fluorescence Activated Cell Sorting) any detected cell population can be separated • light scattering • fluorescence PMT 4 sample PMT 3 PMT 2 Flow cell PMT 1 Laser CD antigen cell types function ligand CD3 T cells T cell antigen receptor signalling chain complex CD4 helper T cells, plasmacytoid dendritic cells (pDC), monocytes co-receptor of the T cell antigen receptor, (also HIV entry receptor) MHC II, (HIV) CD5 T cells, (B cell sub-population: B1) cell adhesion, signalling (costimulation) CD72 CD8 cytotoxic T cells, (NK, some T co-receptor of the T cell antigen receptor MHC I cells) CD14 Monocytes, macrophages, some granulocytes part of the LPS signalling receptor complex LPS, LBP CD19 B cells part of the B cell Ag receptor co-receptor complex C3d, C3b (CD19/CR2(CD21)/CD81) CD28 T cells co-stimulation (B7-1, B7-2) CD80, CD86 CD34 hematopoietic progenitor cells, endothelial cells cell adhesion, … CD62L (L-selectin) CD56 NK cells, (some T and B cell sub- homoadhesion (N-CAM isophorm) population) CD80, CD86 (B7-1, -2) professional APC: DC, B, monocytes, macrophages co-stimulation, cell adhesion CD28, CD152 examples: •B1 cell separation: CD19+ & CD5+ •NKT cell separation: CD3+ & CD56+ NKT cells NK cells lymphocytes Multiple step magnetic separations are needed to achieve similar result: Can you figure it out? FACS: The constant vibration of the flow cell produces drops from the flow stream in a stable position breakoff point vibration + + + + + + + + + Laser electrically charged deflection plates + + + + When the targeted cell reach the breakoff point, a short charge is applied on the stream, and the detaching drop get charged. + + + + + - electrically charged deflection plates --- collecting tube collecting tube The control panels of a FACS sorter