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Immunology and Cell Biology (1999) 77, 509–515 Special Feature Divided we stand: Tracking cell proliferation with carboxyfluorescein diacetate succinimidyl ester A B RU C E LYO N S Discipline of Pathology, The University of Tasmania, Hobart, Tasmania, Australia Summary Most techniques for assessing cell division can either detect limited numbers of cell divisions (bromodeoxyuridine incorporation) or only quantify overall proliferation (tritiated thymidine incorporation). In the majority of cases, viable cells of known division history cannot subsequently be obtained for functional studies. The cells of the immune system undergo marked proliferation and differentiation during the course of an immune response. The relative lack of an organized structure of the lymphohaemopoietic system, in contrast with other organ systems, makes lineage interrelationships difficult to study. Coupled with the remarkable degree of mobility engendered by recirculation, the differentiation occurring along with cell division in the immune system has not been readily accessible for investigation. The present article reviews the development of a cell division analysis procedure based on the quantitative serial halving of the membrane permeant, stably incorporating fluorescent dye carboxyfluorescein diacetate succinimidyl ester (CFSE or CFDA, SE). The technique can be used both in vitro and in vivo, allowing eight to 10 successive divisions to be resolved by flow cytometry. Furthermore, viable cells from defined generation numbers can be sorted by flow cytometry for functional analysis. Key words: carboxyfluorescein diacetate succinimidyl ester, dye dilution, lymphocyte division. Introduction The technique for analysing cell division using serial dilution of the fluorescein-based dye carboxyfluorescein diacetate succinimidyl ester (CFSE), described in 1994,1 developed from our attempts to achieve long-term tracking of intravenously transferred lymphocytes. Previously, workers in the Parish laboratory at the John Curtin School of Medical Research had successfully tracked lymphocytes in vivo for 8 weeks using CFSE,2 a membrane-permeant dye that covalently attaches to free amines of cytoplasmic proteins. However, one of our aims was to combine fluorescein-based CFSE staining with an antibody against fluorescein, thereby allowing the position of CFSE-labelled lymphocytes to be determined many weeks after transfer. During the process of investigating the longevity of CFSE-labelled lymphocytes after transfer using flow cytometry, I noted a small but significant number of events that had one-half, one-quarter and one-eighth of the fluorescence of the majority of events, as shown in Fig. 1. Simultaneous staining with a B cell-specific antibody revealed the events of decreased fluorescence intensity to be confined to the B cell compartment.1,3 Such a pattern could most adequately be explained by cell division. Correspondence: A Bruce Lyons, Discipline of Pathology, The University of Tasmania, GPO Box 252-29, Hobart, Tas. 7001, Australia. Email: <[email protected]> Received 11 August 1999; accepted 11 August 1999. Demonstration that cell division is responsible for CFSE serial dilution To formally demonstrate that the dilution of the fluorescence was due to cell division, CFSE staining was used in conjunction with the well-established Hoechst quenching method. Bromodeoxyuridine (BrdU) is incorporated into the DNA of dividing cells, which allows the identification of up to two rounds of cell division because BrdU-substituted DNA quenches the fluorescence of the DNA-binding dye Hoechst 33342. Figure 2 shows that, as predicted, cells with half and one-quarter the fluorescence intensity of CFSE fell within the first and second cell divisions identified by the Hoechst quenching technique. Long-term survival and division tracking of transferred lymphocytes using CSFE Due to the exquisite sensitivity of modern flow cytometers, CFSE-labelled murine lymphocytes transferred intravenously to syngeneic recipients can be tracked in vivo for at least 6 months. After transfer, spleen cell suspensions were stained with anti-CD45R-phycoerythrin (PE) to identify B cells. Figure 3a–c shows contour plots of CD45R expression against CFSE fluorescence of transferred cells, demonstrating division within the B cell compartment. Note that a small amount of T cell division is apparent from 12 weeks post transfer. Figure 3d–f shows the fluorescence intensity of CFSE-labelled lymphocytes compared with the autofluorescence levels of the recipient’s own cells. Even at 24 weeks post injection, 510 AB Lyons Figure 1 A proportion of carboxyfluorescein diacetate succidimidyl ester (CFSE)-labelled lymphocytes intravenously transferred to 4-week-old mice undergo serial halving of CFSE label. The original observation. Syngeneic splenic lymphocytes (3 × 107) labelled with CFSE were injected intravenously into unmanipulated BALB/c recipients. Ten days after injection, spleen cell suspensions were prepared and 50 000 events collected by flow cytometry. Dot plot of forward scatter against CFSE fluorescence. (b) Histogram of CFSE fluorescence. Note peaks evenly spaced on a logarithmic scale, suggestive of cell division. FL1, green fluorescence, logarithmic scale. resolution of at least two division cycles is possible (Fig. 3f), demonstrating the ability of this technique to allow the analysis of long-term survival and proliferative behaviour of transferred lymphocytes. Studies into the longevity and proliferation behaviour of T lymphocytes are beginning to provide insight into the still controversial areas of immunological memory and immune class regulation.4–7 Independent assessment of cell division within subpopulations of lymphocytes is possible using CSFE One of the major advantages of the CFSE division technique is that dividing cells can be identified in complex mixtures by immunophenotyping with appropriately conjugated monoclonal antibodies. Because CFSE acquires identical spectral characteristics as fluorescein when carboxyl groups are cleaved by cytoplasmic esterases, it is efficiently excited by the 488 nm argon laser standard in most flow cytometers, allowing concurrent use of fluorochromes including phycoerythrin (PE) and tandem dyes such as PE/Texas Red and PE/Cy5 or peridinin chlorophyll-alpha protein (PerCP). Even more complex studies can be performed using multilaser instruments, allowing further fluoro-chromes such as allophycocyanin (APC) to be used. Figure 4 shows the response of murine splenic lymphocytes to different combinations of anti-CD3 and bacterial lipopolysaccharide, demonstrating the power of this technique used in vitro. Table 1, derived from the data generated from Fig. 4f, demonstrates the fidelity of partitioning of CFSE fluorescence over a number of cell divisions. This approach has also been used to probe for differentiation changes in homogenous populations of cells given appropriate stimulus, such as B lymphocyte differentiation and isotype switching,8–11 T lymphocyte differentiation,4,5,12 differentiation of haemopoietic precursors13 and NK cells14 and in evaluation of clinically relevant antigen-specific responses of human peripheral T cells15 (see also further reviews in this issue). Carboxyfluorescein diacetate succinimidyl ester can be used to label populations of lymphocytes at different fluorescence intensities, allowing them to be independently tracked in vivo A major feature of CFSE staining is the ability to manipulate the final fluorescence intensity of a stained population of cells. As the fluorescence intensity obtained is directly proportional to the CFSE concentration during the staining process, as well as the duration of staining, it is possible to differentially label two or more cell populations. Figure 5 illustrates an experiment where murine lymphocytes were ‘fully’ labelled with CFSE, or labelled using one-quarter or one-sixteenth of the CFSE concentration during staining. Equal numbers of fully and one-quarter or one-sixteenth labelled cells were mixed and cells injected into recipient mice. After 4 days, spleens were removed and suspensions made. After staining with a PE-conjugated B cell-specific antibody (CD45R), events above a threshold of CFSE fluorescence were collected by flow cytometry. Figure 5 (lower panels) shows that the two populations can be independently Analysis of division by dilution of CFSE 511 Figure 2 Cell cycle analysis of a proliferating carboxyfluorescein diacetate succidimidyl ester (CFSE)-stained population of lymphocytes, coupled with bromodeoxyuridine (BrdU) quenching of Hoechst 33342 fluorescence, demonstrates that sequential halving of CFSE fluorescence is due to cell division. (a) Fluorescence histograms of CFSE fluorescence. A, unstained control anti-CD3/LPS-stimulated (proliferating) population; B, CFSE-stained anti-CD3/LPS-stimulated (proliferating) population; C, CFSE-stained unstimulated (nonproliferating) population. Note region markers over histogram B delineating undivided (M1), first division (M2) and second division (M3). (b) Bivariate contour plot showing 7-AAD and Hoechst 33342 staining of cells in histogram B of (a). Vertical axis shows 7-AAD binding, which distinguishes the G0/G1, S and G2/M phases of the cell cycle as shown. As a result of culture in the presence of bromodeoxyuridine (BrdU) these cells show typical quenching of Hoechst 33342 (horizontal axis), allowing two rounds of cell division to be visualized. The regions shown delineate undivided (R1), first division (R2) and second division (R3). (c) Bivariate contour plot of cells under region marker M1 of cells in histogram B (a). Note that the majority of these events fall within region 1, corresponding to undivided cells. (d) Bivariate contour plot of cells under region marker M2 of cells in histogram B of (a). Note that the majority of these events fall within region 2, corresponding to the first generation. (e) Bivariate contour plot of cells under region marker M3 of cells in histogram B of (a). Note that the majority of these events fall within region 3, corresponding to the second generation. (Reprinted from Journal of Immunological Methods, volume 171, AB Lyons and CR Parish, Determination of lymphocyte division by flow cytometry, pages 131–7, Copyright 1994, with permission from Elsevier Science.) 512 AB Lyons Figure 3 Murine lymphocytes labelled with carboxyfluorescein diacetate succidimidyl ester (CFSE) can be tracked in vivo for at least 24 weeks. Murine splenocytes (5 × 107 per mL) were incubated at 37°C for 30 min at a concentration of 10 µmol/L CFSE, washed and 3 × 107 cells transferred to unmanipulated syngeneic BALB/c recipients via tail vein injection. At various intervals up to 24 weeks, recipients were killed and splenocyte suspensions prepared. Suspensions were stained with CD45R-phycoerythrin (PE) to allow B cells to be identified. (a–c) Contour plots of CFSE fluorescence (gated above autofluorescence of unstained control splenocytes) against CD45R-PE fluorescence of samples taken 1, 12 and 24 weeks post-transfer, respectively, demonstrating division within the B cell compartments. (d–f) Histograms derived from the same data of CFSE fluorescence of transferred (j) versus unstained splenocytes (—), demonstrating that up to two division cycles can still be resolved after 24 weeks. FL1, green fluorescence, logarithmic scale. tracked in vivo. Antibody staining allows even more complex analyses to be achieved, as shown in Fig. 5 (upper panels) where four cell subpopulations are tracked. This approach has been successfully used to determine the behaviour of transgenic B cells specific for hen egg lysozyme (HEL) after transfer into different antigenic environments, using quarter CFSE labelled non-transgenic B cells as an injection control, allowing accurate measurement of cell survival and migration.16 It is also possible to analyse cell division under these conditions, provided antibodies are available to distinguish between the different transferred populations.16 some in vivo experiments, especially those using transgenic T and B cells, the majority of transferred cells may undergo division. Therefore, it is important to be able to determine the fluorescence intensity of non-divided cells to enable kinetic analyses to be performed. Figure 6 shows data from an experiment where labelled splenic lymphocytes are both transferred to syngeneic recipients, and set up in culture under different conditions. Over a typical time course of 3 days, the peak of undivided cells has the same fluorescence intensity in vitro or in vivo, allowing combined in vivo/ in vitro experiments to be performed. Labelled lymphocytes maintain identical fluorescence characteristics in vitro and in vivo Discussion As seen in Fig. 3a, CFSE fluorescence intensity in nondivided cells declines slowly over time due to catabolism. In Since its introduction in 1994, the CFSE dye dilution technique has become the method of choice for investigating cell division-linked differentiation of lymphocytes and Analysis of division by dilution of CFSE 513 Figure 4 In vitro culture of splenic lymphocytes stained with carboxyfluorescein diacetate succidimidyl ester (CFSE) allows differential B and T cell division in response to polyclonal stimuli to be analysed. (a) Bivariate dot plot of cells from a control unstimulated culture. Vertical axis shows anti-CD45R-phycoerythrin (PE) staining, horizontal axis shows CFSE staining. (b) Bivariate dot plot of cells from an LPS-stimulated culture shows division occurring in a subpopulation of anti-CD45R staining B lymphocytes. (c) Bivariate dot plot of cells from an anti-CD3 stimulated culture shows division occurring in a subpopulation of non-antiCD45R staining T lymphocytes, as well as in a minority of B lymphocytes. (d) Bivariate dot plot of cells from a culture stimulated with both LPS and anti-CD3 demonstrates pronounced cell division of B and T lymphocytes. (e) Fluorescence histogram of gated B lymphocytes (R1) from (d) demonstrates sequential half loss of CFSE fluorescence. (f) Fluorescence histogram of gated T lymphocytes (R2) from (d) demonstrates sequential half loss of CFSE fluorescence. Analysis of the fluorescence intensity of peaks under markers was used to generate Table 1. (Reprinted from the Journal of Immunological Methods, volume 171, AB Lyons and CR Parish, Determination of lymphocyte division by flow cytometry, pages 131–7, Copyright 1994, with permission from Elsevier Science.) Table 1 Peak M1 M2 M3 M4 M5 M6 The CFSE fluorescence intensity of proliferating T cells closely follows the predicted sequential halving due to cell division Mean fluorescence intensity 1775 869 432 221 114 61 Fraction of undivided cell fluorescence Actual Predicted 1.0 0.49 0.243 0.125 0.064 0.034 1.0 0.5 0.25 0.125 0.0625 0.03125 Data is a comparison between mean fluorescence intensities of peaks in Fig. 4f. (Reprinted from the Journal of Immunological Methods, volume 171, AB Lyons and CR Parish, Determination of lymphocyte division by flow cytometry, pages 131–7, Copyright 1994, with permission from Elsevier Science). CFSE, carboxyfluorescein diacetate succinimidyl ester. 514 AB Lyons Figure 5 Differential labelling with carboxyfluorescein diacetate succidimidyl ester (CFSE) allows two populations of lymphocytes to be tracked simultaneously in vivo. Murine splenocytes were stained with CFSE at a final concentration of 5 µmol/L (fully labelled), 1.25 µmol/L (one-quarter labelled) and 0.3125 µmol/L (one-sixteenth labelled) for 10 min at 37°C. After washing away excess stain, equal quantities of fully labelled and one-quarter or one-sixteenth labelled cells were mixed and 3 × 107 cells injected intravenously into syngeneic BALB/c recipients. After 4 days, spleen suspensions were prepared and stained with a phycoerythrin (PE)-conjugated anti-CD45R B cell specific antibody and CFSE-labelled events collected for analysis. The upper pair of dot plots represent the coinjected fully and one-quarter labelled cells (a) and the fully and one-sixteenth labelled cells (b) stained with anti-CD45R-PE. Note that due to the intensity of CFSE fluorescence at 4 days, adequate compensation between the PE and CFSE collecting channels is not possible. However, by day 6 the fluorescence has dropped sufficiently to allow complete compensation (not shown). The lower panels are histograms of CFSE fluorescence derived from the upper panels, demonstrating the accuracy of resolution of the coinjected populations. FL1, green fluorescence, logarithmic scale. haemopoietic cells and for the investigation of kinetics of proliferation during immune responses (reviewed by Lyons and Doherty17 and Lyons et al.18). Carboxyfluorescein diacetate succinimidyl ester is equally partitioned between daughter cells with remarkable fidelity. With a uniformly sized starting population, such as resting B or T lymphocytes, more than eight sequential cell divisions can be visualized without the need for computer-based deconvolution.8 In addition to being able to explore cell surface molecule changes linked to cell division, the technique has also been combined with techniques measuring production of bioactive materials such as cytokines,4,5 as well as with uptake of BrdU to allow rates of division to be determined.9 A major advantage of the technique is the ease in which viable cells of defined generation number can be obtained by flow cytometric cell sorting for functional investigations. An everexpanding group of different cell types have proved amenable to study by this technique, including T and B lymphocytes, NK cells and haemopoietic precursors. The ability to use the technique for both in vivo and in vitro studies has greatly enhanced our understanding of the immune system at the individual cell level. Analysis of division by dilution of CFSE 515 Figure 6 Lymphocytes labelled with carboxyfluorescein diacetate succidimidyl ester (CFSE) can be simultaneously tracked in vivo and in vitro, because undivided fluorescence intensity remains constant. Splenic lymphocytes were labelled with CFSE and either injected via the lateral tail vein into syngeneic hosts or cultured in vitro with maximally stimulating amounts of anti-CD3 plus anti-CD28. At intervals of 24 (a), 48 (b) and 72 h (c), spleen cell suspensions from recipients were prepared and CFSE fluorescence of cultured (j) and in vivo samples (—) analysed by flow cytometry. The markers identify the undivided cell peaks and the means differ by less than 5%. Thus, decay of undivided cell fluorescence remains constant in vivo and in vitro, allowing direct comparison of cultured and transferred cell division behaviour. FITC LOG, green fluorescence, logarithmic scale. References 1 Lyons AB, Parish CR. Determination of lymphocyte division by flow cytometry. J. Immunol. Meth. 1994; 171: 131–7. 2 Weston SA, Parish CR. New fluorescent dyes for lymphocyte migration studies. Analysis by flow cytometry and fluorescence microscopy. J. Immunol. Meth. 1990; 133: 87–97. 3 Lyons AB. Pertussis toxin pretreatment alters the in vivo division behaviour and survival of B lymphocytes, after intravenous transfer. Immunol. Cell Biol. 1997; 75: 7–12. 4 Gett AV, Hodgkin PD. Cell division regulates the T cell cytokine repertoire, revealing a mechanism underlying immune class regulation. Proc. Natl Acad. Sci. USA 1998; 95: 9488–93. 5 Bird JJ, Brown DR, Mullen AC et al. Helper T cell differentiation is controlled by the cell cycle. Immunity 1998; 9: 229–37. 6 Kurts C, Carbone FR, Barnden M et al. CD4+ T cell help impairs CD8+ T cell deletion induced by cross-presentation of self-antigens and favors autoimmunity. J. Exp. Med. 1997; 186: 2057–62. 7 Lee WT, Pelletier WJ. Visualizing memory phenotype development after in vitro stimulation of CD4+ T cells. Cell. Immunol. 1998; 188: 1–11. 8 Hodgkin PD, Lee JH, Lyons AB. B cell differentiation and isotype switching is related to division cycle number. J. Exp. Med. 1996; 184: 277–81. 9 Hasbold J, Lyons AB, Kehry MR, Hodgkin PD. Cell division number regulates IgG1 and IgE switching of B cells following CD40L and IL-4 stimulation. Eur. J. Immunol. 1998; 28: 1040–51. 10 Kindler V, Zubler RH. Memory, but not naive, peripheral blood B lymphocytes differentiate into Ig-secreting cells after CD40 ligation and costimulation with IL-4 and the differentiation factors IL-2, IL-10, and IL-3. J. Immunol. 1997; 159: 2085–90. 11 Hodgkin PD, Bartell G, Mamchak A, Doherty KV, Lyons AB, Hasbold J. The importance of efficacy and partial agonism in evaluating models of B lymphocyte activation. Int. Rev. Immunol. 1997; 15: 101–27. 12 Wells AD, Gudmundsdottir H, Turka LA. Following the fate of individual T cells throughout activation and clonal expansion. Signals from T cell receptor and CD28 differentially regulate the induction and duration of a proliferative response. J. Clin. Invest. 1997; 100: 3173–318. 13 Nordon RE, Ginsberg SS, Eaves CJ. High resolution cell division tracking demonstrates the Flt3 ligand dependence of human marrow CD34+CD38- cell production in vitro. Br. J. Haematol. 1997; 98: 528–39. 14 Warren HS, Kinnear BF. Quantitative analysis of the effect of CD16 ligation on human NK cell proliferation. J. Immunol. 1999; 162: 735–42. 15 Angulo R, Fulcher DA. Measurement of Candida-specific blastogenesis: Comparison of carboxyfluorescein succinimidyl ester labelling of T cells, thymidine incorporation, and CD69 expression. Cytometry 1998; 34: 143–51. 16 Fulcher DA, Lyons AB, Korn S et al. The fate of self-reactive Bcells depends primarily on the degree of antigen receptor engagement and availability of T-cell help. J. Exp. Med. 1996; 183: 2313–28. 17 Lyons AB, Doherty KV. Flow cytometric analysis of cell division by dye dilution. In: Robinson JP, Darzynkiewicz Z, Dean P et al. (eds). Current Protocols in Cytometry. New York: John Wiley and Sons, 1998; Unit 9.11. 18 Lyons AB, Hasbold J, Hodgkin PD. Flow cytometric analysis of cell division history using dilution of CFSE, a stably integrated fluorescent probe. In: Darzynkiewicz Z, Crissman HA, Robinson JP (eds). Methods in Cell Biology; Cytometry. San Diego: Academic Press, 1999; in press.