Institut für Medizinische Immunologie, Berliner Hochschulmedizin Charite, Charité Campus Mitte, Berlin, Germany

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1 Flow Cytometric Methods Journal of Biological Regulators and Homeostatic Agents A protocol for combining proliferation, tetramer staining and intracellular cytokine detection for the flow-cytometric analysis of antigen specific T-cells L. TESFA, H.-D. VOLK, F. KERN Institut für Medizinische Immunologie, Berliner Hochschulmedizin Charite, Charité Campus Mitte, Berlin, Germany ABSTRACT: Flow-cytometry can be used in different ways in order to analyze or enumerate antigen specific T-cells. The three basic principles are direct staining of the T-cell receptor using so called tetramer reagents, staining intracellular cytokines following antigen-specific ex vivo T-cell activation or staining with dyes that are incorporated (increase in staining) or distributed between daughter cells (decrease in staining) upon proliferation in response to a specific antigen challenge. Each system has its advantages and disadvantages. Here we demonstrate that tetramer staining, cytokine flow cytometry and staining with CFDA-SE can be combined permitting the analysis of proliferation and cytokine production with a subset of T-cells specific for a single peptide antigen. (J Biol Regul Homeost Agents 2003; 17: ) KEY WORDS: Antigen-specific T-cells, Cytokine flow cytometry, CFDA-SE, Tetramers Received: January 10, 2003 Revised: May 23, 2003 Accepted: October 11, 2003 INTRODUCTION Antigen specific T-cells can be detected by flowcytometry in different ways. First, direct staining of the T-cell receptor (TCR) by tetrameric or dimeric reagents combining several recombinant MHC-molecules with bound peptides (1). Second, by stimulating T-cells (usually in PBMC or whole blood) with specific antigens such as viral or bacterial lysates (for example CMV-lysate or PPD), recombinant proteins or synthetic peptides (2, 3). This is followed by flow-cytometric detection of intracellular cytokines (most frequently IFN-γ). Third, by stimulating cells in the same way as above, however, detection not of intracellular cytokines (which requires fixation and permeabilization) but secreted cytokines that are bound to the cell surface via a catch antibody which in turn is attached to CD45 (4). Fourth, by the detection of antigen induced proliferation using for example staining with BrDU (DNA-staining) or CFDA-SE (protein staining) (5). TCR staining, cytokine production and proliferation are all signs of antigen specificity, have, however, slightly different implications. While TCR staining with MHC/peptide-tetramers is probably the most precise way of determining the frequency of T-cells with a given antigen-specificity it says little about the functionality of the detected T-cells. Detection of intracellular cytokines used on its own, on the other hand, limits analysis to functional cells, or, more precisely to cells that are able to produce the one or several detected cytokines. Proliferation implies that cells were efficiently activated by the antigens added, however, it is difficult to determine frequencies with reasonable precision. Since all mentioned methods focus on different aspects of the same cells, it may be interesting to combine them in order to analyze how proliferation and cytokine production are interrelated within a subset of antigen specific T-cells and to look at the overall proportion of antigen-specific cells that are activated or proliferate. This interest prompted us to develop the following experimental setup and staining protocol using tetramer staining, peptide stimulation and intracellular cytokine induction as well as CFDA-SE based measurement of proliferation at the same time. To measure proliferation by flow-cytometry, we favor the use of Caroboxyfluroscein diacetate succinimidyl ester (CFDA-SE)-staining, because it is simple, reliable, and gives high resolution. Other methods such as BrDU incorporation are more difficult to handle (6). The staining intensity of (this is produced following enzymatic degradation of CFDA-SE within the cell) is lost in regular increments as the intracellular protein content is passed on to the next generation by dividing cells (each generation of proliferated cells is distinguishable). CFDA-SE is non-toxic, fluoresceinrelated, and spontaneously penetrates cell membranes thanks to two acetate side chains. Inside cells, the acetate groups are removed by esterases turning it into Carboxyfluorescein. The latter leaves the cells at a much slower rate, permitting coupling to lysine side chains and other available amines of cellular proteins X/ $15.00 Wichtig Editore, 2003

2 Tesfa et al PBMC-Preparation CFDA-SE staining 144 hours Stimulated Un tetramer no tetramer tetramer no tetramer 6 hours Fig. 1 - Procedure overview. The diagram shows the two phases of the experiment, i.e. primary stimulation (144 hours) and restimulation (6 hours). All tubes should be run in duplicate if possible. also reacts with molecules that are rapidly degraded or transported out of the cell, and thus, a lot of the CFDA-SE that is initially taken up by cells and transformed into is lost within 24 h of labeling. What is left is bound to more stable components and may be used for tracking cell proliferation (5). The following protocol describes the combination of i) tetramer staining, ii) cytokine production and iii) proliferation of antigen-specific T lymphocytes. Lab equipment MATERIALS NEEDED Dispenser pipettes Standard incubator (37 C, humidified Co 2 atmosphere) Standard centrifuge 4-colour flow-cytometer (e.g. FACScalibur) Racks for 50ml tubes (Nalgene, USA) Racks for 2054 tubes (BD) Plastic materials 50 ml conical polypropylene tubes (BD, Heidelberg, Germany) 5 ml round bottom polyethylene 2054 tubes (BD) Reagents PBS (Gibco, Paisley,UK) RPMI-1640 medium (Biochrom, Berlin, Germany) Fetal calf serum (FCS, Biochrom) Penicillin / Streptomycin (Biochrom) Carboxyfluorescein diacetate succinimidyl ester (CFDA-SE; Molecular Probes, Leiden, Netherlands) Recombinant human IL-2 (Biosource) Brefeldin A (BFA; Sigma, Steinheim, Germany) EDTA (Sigma) Bovine albumin serum (BSA; Serva, Heidelberg, Germany) Sodium azide (NaN 3 ) (Serva) FACS lysing solution (BD, San Jose, USA) Permeabilizing solution 2 (BD) EXPERIMENTAL PROCEDURE Figure 1 gives an overview of the procedure. Cells Citrated blood from a donor with a relevant HLA type is used to isolate peripheral blood mononuclear cells using the standard Ficoll-Paque centrifugation. Cells are washed twice with phosphate-buffered saline (PBS) and resuspended in RPMI-1640 containing 2mmol/L L-glutamine, 10% (v/v) fetal calf serum (FCS, Biochrom, Germany), and 100 IU penicillin/streptomycin (Biochrom) ( supplemented media ). CFDA-SE staining of PBMC Cells are resuspended at a concentration of 10 7 /ml in PBS in 50 ml conical polypropylene tubes (Falcon) and an equal volume of 5 µm CFDA-SE (Molecular probes, Leiden, Netherlands) solution in PBS is added and mixed for 3 min at room temperature. A volume of PBS containing 10% FCS (v/v) equal to the total volume is added to wash out the unbound CFDA- SE or its deacetylated form,. Proliferation assay CFDA-SE-labelled cells are resuspended at a concentration of 1x10 6 cells/ml in supplemented media. Note: In establishing this protocol, we used FCS as a supplement in our media. However, AB serum may be preferable for longer culture periods, because it tends to cause less background proliferation especially in CD4 T-cells. 1 ml of the cell suspension is aliquotted into each 5 ml round bottom polyethylene tube (Falcon 2054). The relevant peptide for stimulation is added (e.g. NLVPMVATV). We usually add the peptide in a volume of 4 µl of 100% DMSO. Our peptide working solution concentration is 0.25 mg/ml. 367

3 A protocol for combining proliferation Note: The peptide should have a final concentration of about 1 µg/ml. If peptides are dissolved in 100% DMSO, care must be taken that the final DMSOconcentration does not exceed 1%. Un samples should be run as control. Tubes are incubated in an upright position in a standard incubator (37 C, humidified 5% CO 2 atmosphere). After 48h 25 IU/ml of recombinant IL-2 is added. Note: This amount of IL-2 was sufficient for stimulation with the particular peptides used at the given concentration. Depending on the number of peptide reactive cells and the intensity of the proliferative response, more IL-2 may be needed. At 144 h, cells are spun down (430 g, 8 min, 4 C) and the supernatant is decanted carefully. Note: Depending on the antigen used and your interests the best time to stop the assay may differ. Finally, cells are resuspended in 100 µl culture media (s.a.) for subsequent tetramer staining. Tetramer staining Tetramer staining is performed prior to restimulation. Previous experiments indicated that tetramer staining was unsatisfactory after peptide stimulation, probably due to TCR downregulation. Resuspended cells are stained with the relevant tetramer (we used an HLA-A * 0201/NLVPMVATV- Tetramer) at an end concentration of 10 µg/ml for 15 min in a standard incubator. 300 µl of supplemented media is added (total volume 400 µl). Re-stimulation and intracellular cytokine detection The procedure is continued by adding peptide for re-stimulation, adding Brefeldin A to inhibit cytokine secretion, and stopping the experiment after a total of 6 hours. For each tube to be 4 µl of peptide stock (0.25 mg/ml in 100% DMSO) is added to 96 µl of supplemented media (it is wise to prepare a little bit more than actually needed). 100 µl of this peptide solution is added to each tube. At 2 hours 500 ml of supplemented media containing Brefeldin A (BFA, Sigma) at a concentration of 20 µg/ml is added for the remaining 4 hours. The final concentration of BFA in the assay is 10 µg/ml, the final concentration of peptide 1 µg/ml. After additional 4 hours incubation period 2 ml of ice-cold PBS are added to stop the reaction and the cells are centrifuged (430 g, 8 min, 4 C). 3 ml of PBS containing 2 mm EDTA (Sigma) are added to remove adherent cells. Tubes are incubated for 10 min at 37 C water bath. Cells are then spun down (430 g, 8 min, 4 C) and the supernatant is decanted. 3ml of PBS containing 0.5% (w/v) bovine serum albumin (BSA) and 0.1% (w/v) sodium azide (washing buffer). Cells are then spun down again (430 g, 8 min, 4 C) and the supernatant is decanted. The procedure is then completed by fixing, permeabilizing and staining the cells. This protocol was established using the following BD Biosciences reagents: Lysing solution (BD, San Jose, USA) for fixation and BD Perm 2 solution for permeabilization. Note: If fixation sensitive epitopes (e.g. CD45RA or RO, CD57, TCRα/β, and others) are stained, staining should be done before fixation. Please refer to existing protocols. 1 ml BD lysing solution is supplemented to each tube. Tubes are then incubated for 10 min at room temperature in the dark. 3 ml of washing buffer is added and tubes are centrifuged (430 g, 8 min, 4 C). Pellets are stained in remaining fluid. 1 ml of permeabilizing solution is added to each tube. Tubes are incubated for 10 min at room temperature in the dark. After a final addition of 3 ml of washing buffer and cells are centrifuged (430 g, 8 min, 4 C). Cells are now ready for staining. Antibody staining Staining was done in a total staining volume of 100 µl with Peridinin chlorophyll protein (PerCP)- conjugated anti-cd3 or anti-cd8 (BD), Allophyocyanin (APC)-conjugated anti-ifn-γ or anti-tnf-α.we have not tested other fixation or permeabilization systems, for example based on saponin in combination with these or other antibodies. These may work as well as the system we have used. Data collection and analysis Flow cytometric analysis was done using FACSCalibur flow cytometer (BD). For each analysis 250,000 events were collected from each sample using CellQuest Software. Data analysis was carried out using either CellQuest or PAINT-A-GATE plus software (BD). Figure 2 shows a typical evaluation. First, intact lymphocytes were gated based on scatterlight distribution in an SSC vs. FSC dot-plot. Next, CD3 or CD8 positive events (depending on the staining) were gated with an additional region (polygon) in an SSC vs. FL-3 dot-plot. A third (rectangular) region was placed around Tetramerpositive events in a (FL-1) vs. Tetramer (FL-2) 368

4 Tesfa et al A: IFN- γ B: TNF- α restimulation no restimulation restimulation no restimulation A2 TET A2 TET IFN γ TNF α 4 4 Fig. 2 - Discrimination of tetramer positive events and cytokine positive events in a population of T-cells proliferated in response to peptide stimulation. Following 144 hours of ex vivo stimulation with NLVPMVATV (HCMV pp65) cells were stained with the HLA-A * 0201/NLVPMVATV tetramer and re (left diagrams in A and B) or not re (right diagrams in A and B) for 6 hours with the same peptide (Brefeldin A was added after 2 hours). Tetramer positive events/cytokine positive can be readily distinguished by setting the threshold on the bulk of non-proliferated/non-cytokine producing cells. The upper panels show Tetramer staining, the lower panels cytokine staining. A shows intracellular INF-γ; B shows intracellular TNF-α. Tetramer positive events are highlighted black in all diagrams. Dotted lines in the lower left diagram indicate approximate boundaries of different generations of dividing cells. Each generation is at the same distance from the previous generation on a log. scale. Axes show log. Fluorescence intensity. dot-plot. Tetramer positive events were highlighted black and shown in a (FL-1) vs. IFN-γ (FL-4) or TNF-α (FL-4) dot-plot. This way the proportion of Tetramer-positive events within the CD3 or CD8 T-cell population could be determined as well as the proportion of IFN-γ or TNF-α positive events within the Tetramer-positive population, and, if required within each distinguishable subset of proliferated cells. Controls Ideally, controls should be available for every part of the procedure. While tubes may control for the effect of the stimulation, good staining controls are more complicated. Isotype antibodies are expensive but usually not very helpful, neither for surface nor for intracellular staining (7). If surface markers are used that require control staining, we recommend that staining be controlled by using identical samples stained with the identical antibodies except the one of interest. This is sometimes referred to as FMO controls (fluorescence minus one) (7). With regard to the tetramer staining we found in several initial experiments, that tetramer positive cells could be readily distinguished by setting the positive/negative gate according to the nonproliferated (i.e. CFDA-SE bright) population. Using a non-tetramer stained control for gating produced the same results. Generally with a good stain, population gating is possible, and the control is unhelpful. In some experiments this control was, therefore, omitted for lack of sufficient amounts of cells. We recommend that it be included whenever possible. Since dead cells do not produce cytokines, and, in our example almost all tetramer positive cells produced cytokines, the risk of staining dead cells was very low. In principle however, live/dead staining would be of advantage. When cells are fixed and permeabilized, on the other hand, dead-cell exclusion is difficult because reagents such as propidium-iodide can permeate in and out of the cells freely, which makes the results unreliable. 369

5 A protocol for combining proliferation DISCUSSION This protocol has a range of potential applications. Studying the sequence of cytokine production of proliferating cells following antigen challenge and correlating this to the phenotype would be an idea that comes to mind first. This application would be even more interesting if additional fluorescence channels were available to perform a detailed analysis of the phenotypes. With the new generation of flowcytometers, this is a realistic option. The study of cytokine memory is another possible application, especially if cells are sorted following staining. This was previously done without tetramers (8). The addition of tetramers would allow for example sorting antigen specific cells with much higher specificity than if proliferation in response to an antigen was used as an indicator of antigen specificity on its own. The combination of these technologies in principle allows not only to identify the total number of cells exhibiting a T-cell receptor recognizing a given peptide antigen (tetramer), but also to work out the proportion of cells that are able to produce cytokines (intracellular cytokine staining) and proliferate (CFDA-SE). This protocol was established last but not least to demonstrate the feasibility of combining the advantages of the three most prominent methods used for the detection of antigen specific T cells to date. With a modern instrument, additional fluorescence channels will allow us to introduce additional functional or phenotype markers. Ideally, live/dead discrimination should be included, T-cells should be defined using CD3, CD4 and CD8 in combination, etc. The advantage over more conventional assays clearly lies in the simultaneous visualization of quite distinct properties of identifiable single cells (phenotype, antigen specificity, cytokine production and proliferative capacity). Reprint requests to: Lidia Tesfa Institut für Medizinische Immunologie Berliner Hochschulmedizin Charite Charité Campus Mitte Schumannstrasse, Berlin, Germany lidia.tesfa@charite.de REFERENCES 1. Altman JD, Moss PAH, Goulder PJR, et al. Phenotypic analysis of antigen-specific T lymphocytes [published erratum appears in Science 1998; 280: 1821]. Science 1996; 274: Waldrop SL, Pitcher CJ, Peterson DM, Maino VC, Picker LJ. Determination of antigen-specific memory/ effector CD4+ T cell frequencies by flow cytometry: evidence for a novel, antigen-specific homeostatic mechanism in HIV-associated immunodeficiency. J Clin Invest 1997; 99: Kern F, Surel IP, Brock C, et al. T-cell epitope mapping by flow cytometry. Nat Med 1998; 4: Brosterhus H, Brings S, Leyendeckers H, et al. Enrichment and detection of live antigen-specific CD4(+) and CD8(+) T cells based on cytokine secretion. Eur J Immunol 1999; 29: Lyons AB. Analysing cell division in vivo and in vitro using flow cytometric measurement of dye dilution. J Immunol Methods 2000; 243: Bohmer RM, Ellwart J. Cell cycle analysis by combining the 5-bromodeoxyuridine/33258 Hoechst technique with DNA-specific ethidium bromide staining. Cytometry 1981; 2: De Rosa SC, Brenchley JM, Roederer M. Beyond six colors: A new era in flow cytometry. Nat Med 2003; 9: Richter A, Lohning M, Radbruch A. Instruction for cytokine expression in T helper lymphocytes in relation to proliferation and cell cycle progression. J Exp Med 1999; 190: