LAB 1, Immunology. Laboratory manual Immunology and Infection Biology Biomedicine course Autumn 2007

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1 LAB 1, Immunology Laboratory manual Immunology and Infection Biology Biomedicine course Autumn 2007 QUANTIFICATION OF CYTOTOXIC T LYMPHOCYTE ACTIVITY AND DETECTION OF FAS/TRAILR INDUCED APOPTOSIS RESPONSIBLE: Michael Uhlin, Helen Karlsson, Mantas Okas and Bruno Vanherberghen 1. INTRODUCTION AND AIM: T cells use their T cell receptors (TCR) to recognise antigen presenting cells (APC) and infected cells. The TCR bind to antigenic peptides in combination with Major Histocompatibility Complex (MHC) molecules. This recognition is very specific and a given TCR will only recognise one type of MHC molecule in combination with one specific peptide bound in the peptide-binding grove of the MHC molecule. There are two types of MHC molecules, class I and class II. The MHC molecules are extremely polymorphic, ie only rarely do two persons express the same MHC molecules. T cell receptors on CD4+ T cells bind to MHC class II molecules with the help of the coreceptor CD4, while CD8+ T cells bind MHC class I. Upon recognition of a MHC/peptide complex on the surface of an APC, the naive T cells will proliferate (clonal expansion) and differentiate into effector T cells. The effector T cell may either produce cytokines (helper T cells, CD4 + ) or kill the recognised cell (cytotoxic T lymphocytes (CTL), CD8 + ). AIMS: Lab A) We will explore the mechanism of T-cell mediated cytotoxicity and test the concept of MHC restriction, the central paradigm of adaptive immune response. The activity of cytotoxic CD8 + T cells will be measured by the release of radioactive Cr from killed target cells. Lab B) We will investigate the effect of co stimulation. We will work with a T cell line which has been activated and grown in vitro. The way we will detect stimulation of the T cell is by investigating how many cells produce interferon γ (IFNγ) upon triggering. We will stain for intracellular IFNγ and analyse by FACS (fluorescence activated cell scanner). 1

2 GENERAL SCHEME: The class will be divided into two groups. Each group will be divided into subgroups (approx. 3-4 students per subgroup). DAY 1: WHOLE DAY GROUP 1: Cytotoxicity assay (lab A). GROUP 2: IFN gamma lab (lab B). Morning session: Theoretical and practical introduction. Plan your experiment. Identify cells to use, materials and reagents. Plan your assay in the 96 well plates. Set up your experiment (either for cytotoxicity assay or for IFN gamma assay) Preparation of cell lines, counting of the cells, set up cultures in micro wells (96-well plates). Dilution of reagents to proper concentrations. Afternoon session: GROUP 1: 4h incubation followed by harvesting and counting of cytotoxicity assay in gamma counter machine. GROUP 2: 2h incubation followed by CD3 and IFN gamma staining and reading of cytokine assay in FACS. DAY 2: WHOLE DAY GROUP 1: IFN gamma assay (Lab B). GROUP 2: Cytotoxicity assay (Lab A). Scheme as day 1. DAY 3: 4 hours in the afternoon GROUP 1: Analysis of FACS results, preparation of lab report GROUP 2: Analysis of Cr release assay, preparation of lab report 2. PRESENTATION OF RESULTS: Lab report: Each subgroup will prepare their results for presentation and discussion during a short oral presentation on Thursday 6 th of September. The presentation should contain a basic introduction to the method, how the method was performed, the results and discussion of its usefulness. Around 5-7 minutes. Which group to present will be decided by lottery. Also write down possible mistakes and interpret your data. You shall prepare diagrams presenting your results using e.g. Excel. These diagrams plus lab report should be handed in latest 6 th of September at the oral presentation. 2

3 A) Cr RELEASE CYTOTOXICITY TEST: 1. ASSAY PRINCIPLE: CD8 + cytotoxic T cells kill target cells upon recognition of complexes composed of antigenic peptides and MHC class I molecules on the target cell surface. CD8 + T cells are important in the host response to tumors and viruses (and some intracellular bacteria). Cytotoxic CD8 + effector T cells can be generated from naive T lymphocytes following specific stimulation by antigens presented by stimulator cells. The activity of these effector cells can be measured in a Chromium release assay by detecting radioactive chromium isotope released from the cytoplasm of labeled target cells. In chromium release assays the target cells are labeled with radioactive chromium ( 51 Cr) in the form of Na 2 51 CrO 4. The labeled target cells are mixed with activated CD8 + T cells and coincubated. If the target cells are lysed, the chromium is released into the supernatant. Released chromium is in a reduced form and can not re-enter other cells. Thus, the amount of released chromium (measured as radioactivity in the supernatant) is a good measure of the cytolytic activity of the effector cells. We will test the specific killing mediated by an EBV-specific cytotoxic T cell clone (effector). The T cell clone has expanded from a single T cell and all cells thus express identical TcRs. The target cells have to express the proper combination of MHC and antigenic peptide to get killed. 2. MATERIALS: Effector T cells: A human CD8 + T cell clone specific for the MHC molecule HLA-A11 in conjunction with a peptide derived from the Epstein-Barr Virus (EBV). Target cells: C1R cells are a B-lymphoblastoid cell-line (BLCL) normally negative for expression of HLA-A and B alleles. The cell lines used in this lab are transfected with HLA- A11 or with an irrelevant HLA molecule. The cells will be incubated with or without the correct peptide and labeled with radioactive 51 Cr (see Appendix C for safety rules). The following target cells are provided: 1. C1R-HLA A11 2. C1R-HLA A11 + peptide 3. C1R-irrelevant HLA 4. C1R-irrelevant HLA + peptide Complete cell culture media. RPMI 1640, 10% FCS (Appendix A) Trypan blue stain and Bürker chamber (see Appendix B) for counting cells. 96 well plate (V-bottom) 3. EXPERIMENTAL PROCEDURE: Plan your Assay and make calculations You will have one type of effector T cell and four types of target cells (see above). Effector and target cells should be mixed at 4 different effector:target (E:T) ratios (3:1, 1:1, 0.33:1, 0.11:1). Each E:T ratio should be tested in triplicate. 3

4 The number of target cells per well should always be 5000, and you should add 100µl of effector cells and 100µl of target cell to each experimental well. In addition, for each target cell (1-4), three wells for spontaneous Cr-release and three wells for maximum Cr-release must be prepared: Spontaneous release: 100 µl of target cells µl of medium (no effector cells!!!). Maximum release: 100 µl of target cells µl of lysis buffer (2% Tween in H 2 O) Plan your assay in the 96 well plate, draw a map below and label the plate with a marker. For the target cells, calculate: What concentration of target cells should you have? What is the total volume of each target cell type needed? (add at least 0.5 ml extra for losses of liquid in tubes, pipettes etc) What volume should you take out from the tube of cells provided? For the effector cells: You will make a dilution series of the effector cells in the plate so that the volume of the effector cells is 100 µl in each well after dilution. The highest E:T ratio is 3:1 What concentration of effector cells should you have for the highest E:T ratio? The following E:T ratios are 1:1; 0.33:1 and 0.11:1. Calculate how to make the dilution series to get the wanted E:T ratios. Check with the amanuens before you start setting up the experiment! Preparing the effector cells Check the cultured cells in flasks in the inverted microscope; check for bacterial/fungal contamination (a cloud of tiny particles). How do the cells look? Healthy, activated T cells are often large and somewhat irregularly shaped. Resuspend the cells and move to a 15 ml falcon tube. Centrifuge at 1500 rpm, 10 min. Pour off the medium and resuspend cells in 2 ml fresh 4

5 medium. Take out an aliquot for counting (dilute 20µl resuspended cells in 20µl Trypan blue; see appendix B) and adjust the volume to get the concentration calculated above. Preparing the target cells: You will be provided with prepared target cells that have already been labelled with Cr, washed and counted, as described below. Place 1 x 10 6 cells from each target in a 5 ml falcon tube. Spin down the cells, discard the supernatant and take away the last drop by placing the tube up side down on a Kleenex tissue. Resuspend the pellet, add 10 µl of Na 2 52 CrO 4 (high activity), add peptide to bind to MHC class I and mix. Incubate 45 min at 37 o C. Wash the cells three times with PBS. The labeling and washing will be done in the isotope lab. Resuspend the Cr-labeled and peptide-coated cells in 1 ml medium and count. The labeled, washed and counted target cells will be provided by the amanuens. Always work with gloves and on bench-paper when handling radioactive isotopes! All Cr-contaminated plastic and liquid waste should be collected in labeled containers! Refer to Appendix C for Safety Rules! Keep the labeled cells on ice until needed. The amanuens will tell you the stock concentration of the target cells. From your estimate of number of target cells needed, calculate the corresponding volume, take out cells and adjust to the wanted concentration with medium. Setting up the assay Set up your assay according to the plan you have made. Start with adding media and lysis buffer in the wells for spontaneous and maximum release, respectively. Then add effector T cells in the other wells and do the dilution series. Finish with adding the target cells. Check by eye that you have the same volume in all wells (200µl). Incubate plates in 37 o C, 5% CO 2 for 4 hours. After 4 h, spin plates for 2 min, 1500rpm. Set up small precipitin tubes in a clean 96-well plate (in the same positions as your assay wells). Carefully harvest 100µl supernatant and transfer to the precipitin tubes. Make sure not to let the pipette tips touch the cell pellet or stir the cells up. NB! For the three maximum release wells, resuspend cells before harvesting! Label the plate with your group number Give it together with a map of the setup to the amanuens The amanuens will measure radioactivity counts in gamma counter machine and bring back results the next day. 5

6 Analysis of results from cytotoxicity assay (Day 2) The amount of released radioactivity in the experiment wells (containing effectors and target cells), and the control wells (spontaneous and maximum) is used to calculate the % specific lysis. This value corresponds to the number of target cells killed by the effector T cells and is calculated according to the formula below: (experimental release spontaneous release) % specific lysis = (maximum release spontaneous release) 100 The gamma counter makes this calculation for you and provides the % specific lysis values. You should however check that the spontaneous release is not too high compared to the maximum release (up to 25 % is OK). High spontaneous release may be a sign of that the target cells were in bad condition before the experiment. You should also check the triplicates: if one of the three values differs a lot from the other two, the outlier can be deleted and a new mean value calculated. Compare this new value to the old one. 6

7 Lab B: The importance of costimulation during T cell activation: Intracellular cytokine staining of a human T cell line. T cells that have not yet been activated in secondary lymphoid tissue are called naïve T cells. These naïve T cells continuously circulate from blood, into lymph nodes and exit the lymphoid tissues via lymphatic vessels to again drain into the blood. While in the lymph node, naïve T cells interact with antigen presenting cells (APC), such as dendritic cells (DC), searching for the MHC-peptide complex for which they have a specific T cell receptor. If such a fit is encountered, the naïve T cell is activated and will start proliferating causing a so called clonal expansion of this particular T cell. However, a naïve T cell needs not only the MHC peptide complex to be activated, but also the second signal in the form of costimulation. Some APCs, ex DC and B cells, express a high concentration of costimulatory molecules on their cell surface, ex CD80 and CD86. CD80 and CD86 bind to CD28 on the naïve T cell and thus potentiates the signal received through the T cell receptor, activating the T cell. The activated T cell, or all the thousands of cells springing from this particular T cell, will then express cell surface molecules enabling them to extravasate into inflamed tissue and respond to antigens at the site of infection. Activated T cells are not dependent on costimulation, and can thus be activated by cells presenting the correct peptide but lacking costimulatory molecules. In this lab, we will investigate the effect of costimulation. We will work with a T cell line which has been activated and grown in vitro. Since the T cell line which we will use has been activated, it is not naïve, and thus has no absolute need of costimulation. We will stimulate the T cell line with antibodies specific for one of the chains of the T cell receptor, CD3, and combine this stimulation with antibodies to CD28, the costimulatory molecule. Each group in the class will receive a different ratio of stimulation via CD3 and CD28, with the aim to investigate whether we can detect a situation where a weak signal through CD3 is potentiated by CD28 stimulation even in this preactivated T cell line. The way we will detect stimulation of the T cell is by investigating how many cells produce interferon γ (IFNγ). We will stain for intracellular IFNγ and analyse by FACS (fluorescence activated cell scanner). Before lunch (text in italics is intended as explanation for the procedures) You will receive a tube of T cells, total volume is 1 ml. Please determine the concentration of this cell suspension (refer to appendix B) and concentrate the cells to 2 million cells/ml by spinning the cells down 5 min 1500 rpm and after that adding medium to the correct concentration. You will also receive a 96 well flat bottomed plate, where marked wells have been incubated with antibodies since yesterday. This will cause the antibodies to stick to the plastic surface. Plastic bound antibodies have a higher stimulation capacity compared to antibodies in suspension since they are able to crosslink the cell surface receptor, bringing them close together, and thus increase the intracellular signal. Four wells have been preincubated with antibody: a. anti-cd3 in different concentration for each lab group (5ug/ml less than 0,1ug/ml). Remember which concentration your group has received! b. anti-cd28, 10ug/ml c. anti-cd3+anti-cd28 d. no antibody 7

8 Wash away excess antibody by pipetting 200ul PBS into each used well. Flip off the liquid and repeat this washing four times. Resuspend your cells by pipetting up and down a few times. Pipett 200ul of cells into each well (giving a total of cells/ well). You will receive a tube of GolgiStop. This chemical inhibits the Golgi complex inside of the cell from secreting the cytokines produced by the cell. This is necessary to be able to increase the intracellular concentration of IFNγ in order to detect it. Add 50 ul of GolgiStop to each well. Cover the plate and put it in the incubator at 37 C for at least 2 hours. After lunch To minimize cell loss during washing procedures, please transfer your cells to a 96 well v-bottomed plate by suspending the cells (pipett up and down at least 5 times before transferring cells to v-bottom shaped plate, avoid bubbles). You will stain the cells with fluorescently labeled antibodies to the T cell receptor (CD3 on the cell surface) and intracellularly for cytokine (IFNγ). NB! Fluroescently labeled antibodies and cells should not be exposed to strong light for long periods or the fluorescence will fade away. Surface stain: spin plate 3 min 1500 rpm, discard supernatant by flipping the plate forcefully once (!!) over the yellow waste bin (this procedure is termed a wash ). Add to each well 50ul PBS containing CD3-PerCP. Suspend by pipetting up and down. Cover the plate and incubate 10 min in fridge (+4 C). Add 200ul PBS, resuspend and spin down 3 min 1500 rpm. Flip away supernatant. Add 100 ul fixation buffer, resuspend the cells, cover the plate and incubate 10 min in fridge. The fixation causes the antibody to bind covalently to the cells, and also ensures that the cell is kept intact even when permeabilising the cell using a detergentcontaining buffer (see c). Add 100ul PBS, resuspend and spin down. Flip away supernatant. Add 200ul permeabilisation buffer, which partly disintegrates the cell membrane allowing us to stain intracellular proteins. Resuspend this and spin down again and flip away supernatant. Add 50ul of the IFNg-PE solution to each well, suspend the cells, cover the plate and incubate 30 min in fridge (4 C). Wash twice with 200ul permeabilisaton buffer and once with 200 ul PBS (Every wash: add solution, spin down and flip away supernatant). Resuspend in 200ul PBS + 50ul fixation buffer. Analysis Transfer the contents of each well into small precipitin tubes for FACS analysis. A PhD student will help you calculate the percentage of IFNγ producing T cells, and also a relative measurement of how much each cell produces (median fluorescence intensity). 8

9 Appendix A, Media and solutions Phosphate Buffered Saline (PBS) 1.9 mm NaH2PO4 8.1 mm Na2HPO4 154 mm Na Cl Adjust to ph 7.4 Complete RPMI Medium RPMI U/ml Penicillin 100 mg/ml Streptomycin 50 mm 2-Mercaptoethanol 1% Nonessential aminoacids 1 mm Sodium pyruvate 10% Foetal Bovine Serum (FBS) 9

10 Appendix B, Counting cells Two dyes are commonly used to determine the concentration of cells in a solution. Türk stain is incorporated the nuclei of all cells (living as well as dead) while Trypan blue stains the cytoplasm of dead cells. In addition, Türk lyses erythrocytes. Hence, Türk is commonly used to distinguish cell types with different morphology of the nucleus, while Trypan blue is used for counting of live cells and determining the degree of cell death. We will use Trypan blue during the course. Counting Cells Take out 20 µl of the cell suspension and mix it with 20 µl stain in an eppendorf tube. Fill the Bürker chamber under the cover glass with the cell stain mix. Count live cells in three A squares and calculate the mean of the number of cells in one A square. The volume of one A square is ml. To calculate the number of cells per ml: Number of cells in one A square dilution / ml = number of cells per ml. 10

11 Appendix C, Work with isotopes If you have any doubts consult the amanuens!!! In course lab#1, radioactive chromium, 51 Cr, is used. 51 Cr is a gamma-emitting isotope. Gamma irradiation has high energy and can travel through most material, with the exception of lead. The 51 Cr-labelled cells have been washed extensively and therefore have a low radioactive activity. Therefore, you are allowed to work in a normal laboratory and the amount of irradiation that you are exposed to is minimal and is drastically reduced with distance from the source. To ensure safe usage of this gamma-emitting isotope the following rules must be followed: 1. Use gloves and lab coat 2. Cover your lab bench with bench paper 3. Discard all contaminated waste in labelled containers 4. When centrifuging tubes or plates, always use lids 11

12 Appendix D, Flow cytometry Cells labelled with fluorochromes can be analysed by a FACS machine (fluorescence activated cell sorting). The cells pass a beam of light from an argon laser. The light has a wavelength of 488nm. If a fluorochrome that can be excited by light with a wavelength of around 488nm is attached to the cell, the light emitted can be analysed. The FACS machines that we use in the course (FACScan) are equipped with three detectors that can detect light with different wavelengths. The detectors are called FL1 (detection range 530±15nm), FL2 (585±44nm) and FL3 (>600nm). In addition, each cell is also defined by the scattering of light as the cell passes through the laser beam. This spread of light is analysed by forward scatter, indicating the size of the cell, and by side scatter, relating to the granularity of the cell. Hence, it is possible to analyse three colours (ex antibodies conjugated to three different fluorochromes and binding to three different proteins on the cell) in addition to size and granularity of each cell in the test tube. As the emission spectra of many fluorochromes leak into several detectors, it is often necessary to compensate the machine. Compensation is an electronic way of erasing light from ex the fluorochrome phycoerythrin (PE) from the FL3 detector. PE emits light with a wavelength of ca nm and its main peak of emission is in the range of the FL2 detector (585±44nm), while a small amount of light will leak into the FL3 detector (>600nm) 12