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Immunology and Cell Biology (1999) 77, 516–522 Special Feature Quantitative analysis of lymphocyte differentiation and proliferation in vitro using carboxyfluorescein diacetate succinimidyl ester J H A S B O L D, AV G E T T, J S RU S H , E D E E N I C K , D AV E RY, J J U N a n d P D H O D G K I N Immune Regulation Group, Medical Foundation, University of Sydney, The Centenary Institute of Cancer Medicine and Cell Biology, Sydney, New South Wales, Australia Abstract Mature T and B lymphocytes respond to receptor-delivered signals received during and following activation. These signals regulate the rates of cell death, growth, differentiation and migration that ultimately establish the behaviour patterns collectively referred to as immune regulation. We have been pursuing the philosophy that in vitro systems of lymphocyte stimulation, when analysed quantitatively, help reveal the logical attributes of lymphocyte behaviour. The development of carboxyfluorescein diacetate succinimidyl ester (CFSE) to track division has enabled the variable of division number to be incorporated into these quantitative analyses. Our studies with CFSE have established a fundamental link between differentiation and division number. Isotype switching, expression of T cell cytokines, surface receptor alterations and changes to intracellular signalling components all display independent patterns of change with division number. The stochastic aspects of these changes and the ability of external signals to independently regulate them argue for a probabilistic modelling framework for describing and understanding immune regulation. Key words: carboxyfluorescein diacetate succinimidyl ester, cell division, flow cytometry, immune regulation, lymphocyte differentiation. Introduction The discovery by Lyons and Parish that cells labelled with the dye (5- and 6-) carboxyfluorescein diacetate succinimidyl ester (CFSE) accurately apportion fluorescence between daughter cells upon mitosis made it possible for the division history of a cell population to be monitored by flow cytometry.1 Importantly, cellular differentiation turned out to be unaffected by the dye, because labelled lymphocytes were shown to isotype switch, develop cytokine secretion potential and alter their cell surface phenotype as normal.2–4 Thus, it became possible to explore how lymphocytes integrate simultaneous proliferation- and differentiation-inducing signals and compute a net outcome. Methods Methods used in our laboratory have been discussed in some detail previously;5 however, a brief summary of important practical information follows. Labelling method Typically we use purified naïve lymphocytes for differentiation and proliferation studies. For B cells, percoll is used to separate high density cells,6 whereas T cells with the desired phenotype are sorted from pooled lymph nodes.4 Naïve cells are excellent subjects for Correspondence: Philip D Hodgkin, Centenary Institute of Cancer Medicine and Cell Biology, Locked Bag 6, Newtown, NSW 2042, Australia. Email: <[email protected]> Received 2 September 1999; accepted 2 September 1999. CFSE labelling, because they are relatively homogeneous in size and the level of labelling with the dye is approximately proportional to cell volume. The CFSE method is less well suited to labelling more heterogeneous populations, such as dividing cell lines. A stock solution of CFSE should be prepared by dissolving CFSE (Molecular Probes, Eugene, OR, USA) in DMSO at a concentration of 5 mmol/L. This stock can be stored frozen in small aliquots to avoid excessive freeze and thaw cycles. To label cells with CFSE, they are suspended at 107 cells/mL in PBS with 0.1% BSA (PBS/BSA). Sufficient CFSE stock solution is added directly to make a final concentration of 10 mmol/L. The suspension should be vortexed immediately following CFSE addition, to ensure rapid dispersal, and then left to incubate for 10 min in a 37°C water bath. After labelling, a 10× volume of PBS/0.1% BSA is added and cells are washed twice in culture medium. Labelled cells can then be placed directly in culture under conditions where they are stimulated to divide. After harvesting they are run on the flow cytometer under standard conditions for monitoring fluorescein. Label concentration The fluorescence intensity of labelled cells is directly proportional to the concentration of CFSE.5 However, at high concentrations the CFSE load becomes toxic to cells (giving very bright, but very dead cells). It is advisable to titrate the CFSE concentration and therefore labelling intensity for each new cell type because the optimal conditions will vary. Curiously, T cells take up twice as much dye as B cells and human cells are more brightly stained than mouse lymphocytes. Controls for flow cytometry When CFSE-labelled lymphocytes are simply left in culture at 37°C, the intensity of fluorescence decays rapidly over the first 48 h.5 Lymphoctye differentiation analysis with CFSE 517 Figure 1 Monitoring lymphocyte responses in vitro using carboxyfluorescein diacetate succinimidyl ester (CFSE). (a) Basic features of a CFSE tracking experiment for cell division using B cell stimulation by CD40L for 4 days. The dashed line at the right illustrates the CFSE intensity of B cells cultured unstimulated with IL-4 alone for the same time period. These cells serve as the control to locate the undivided cell position. The solid line shows the dividing B cell population revealing the typical asynchronous profile. Progressive divisions are apparent by the accurate two-fold dilutions of the fluorescence intensity, which on the log scale appear as even spacings. The dashed line to the left illustrates the position of an equivalent population that was not labelled with CFSE, allowing the position of the autoflourescent intensity to be determined. (b) The same cells presented as a forward versus side scatter plot. The live cell gate set is illustrated, as is the position of CaliBRITE beads used to determine the total number of cells in culture. (c) Use of computer fitting to determine the best series of Gaussian curves to accommodate the experimental data. The proportion of cells in each division can be determined by the relative area under each normal curve which, when combined with the ratio of total live cells compared to the beads, can be used to determine the absolute number of cells in each division in the culture (d). (e) Use of simultaneous surface staining with a different fluorochrome is illustrated by IgG1 expression. Clearly, the most divided cells are more likely to express IgG1. This is further illustrated by ‘slicing’ the data and calculating the proportion of cells in each division that express IgG1 (f). In addition to cell surface labelling, CFSE-stained cells can be fixed and permeabilized for concurrent monitoring of intracellular components. This is illustrated in (g), which shows labelling of bromodeoxyuridine (BrdU) incorporation following a 3 h pulse. (h) STAT-1 expression diminishes progressively with each division. 518 J Hasbold et al. Presumably this is due to catabolism of CFSE-bound proteins in the cell. This loss can be as high as 10-fold and occurs in the absence of cell division. After 48 h cells will continue to lose CFSE, although at a much reduced rate. For this reason it is always important to know where the undivided peak would be when analysing a proliferating group of cells. Fortunately, this is easily determined by controls, because the rate of loss of fluorescence in unstimulated cells is identical to that observed for stimulated and activated cells.5 Therefore, the CFSE intensity of unstimulated cells can be used to determine the position of the undivided peak in stimulated cultures where no undivided cells are apparent (Fig. 1a). Another useful and important control is to set up cells activated under the same conditions but not labelled with CFSE. This autofluorescence control is useful for determining the limits of resolution of division tracking (Fig. 1a). Monitoring the total cell number in culture If it is important to measure the total number of cells in each division, a known number of beads can be added prior to harvesting cells from culture wells. We typically add 5000 CaliBRITE beads (Becton-Dickinson, San Jose, CA, USA) to a 1 mL culture. These beads are easily distinguished from cells by the scatter profile following acquisition on the flow cytometer (Fig. 1b). Because the number of beads per well is known, a simple ratio determination can be used to estimate final cell numbers accurately. For example, if 5000 beads were added to the culture well and, following analysis by flow cytometry, the ratio of beads to live cells was 2:1 then there were 2500 cells in the culture. This number can be combined with the determination of the proportion of cells in each division to calculate the total cell number in each division (see later). How many divisions can be tracked? The number of divisions that can be followed after CFSE labelling is limited by the autofluorescent level of unlabelled cells. Thus, as the cells divide and dilute CFSE accurately in two, the fluorescence approaches the level of unlabelled cells. Typically, the intensity is plotted on a log scale and therefore the geometric two-fold reductions will appear as even spacings, as shown in Fig. 1a and by Lyons and Parish1 and Hodgkin et al.2 As the cells approach the autofluorescent level, the peaks appear to get closer together. For most applications, seven to eight divisions is the practical limit for accurate resolution. The expected position of each peak for any division (Di, where i is the division number) can be accurately calculated using the formula:5 Di = ((D0 – A)/2i) + A [1] where D0 is the position of the undivided peak and A is the average (geometric) autofluorescence intensity. Computer-based fitting We have used PROFIT (Quantumsoft, Zurich, Switzerland), or EXCEL (Microsoft, Redmond, WA, USA) to fit a series of Gaussian curves to CFSE data using the above formula to set the position of each division peak. Typically, the results are consistent with the expected features of sequential log-normal Gaussian distributions (Fig. 1c). By fitting a series of curves, the area under each peak can be expressed as the proportion of live cells in each division, which, by reference to bead numbers, can then be used to calculate the total final cell number per division (Fig. 1d). Examples of using this method for monitoring the impact of cytokines on the rates of growth and death of dividing cells are given in Hasbold et al.7 Analysing differentiation The phenotype or behaviour of cells from different divisions is often markedly different. We have used a number of methods to explore this phenomenon. The expression of a cell surface marker in comparison with CFSE intensity can be monitored by flow cytometry (Fig. 1e). Although bright, CFSE-labelled cells can be simultaneously examined with any conventional combination of fluorochromes (except fluorescein isothiocyanate (FITC)), including phycoerythrin (PE), tricolour, PerCP, Texas red and allophycocyanin (APC). Immediately after labelling at standard CFSE concentrations, lymphocytes are too bright to compensate for the use of PE. Therefore, a lower concentration of CFSE can be used, although this will limit the number of divisions that can be monitored at later times. Alternatively, the early phenotype of very bright CFSE-labelled cells can be determined using a two-laser system employing APC as a second colour. To monitor differentiation, a two-dimensional plot of CFSE versus the second fluorochrome is drawn as shown in Fig. 1e, using CELLQUEST software (Becton Dickinson). The position of each division peak is determined and gates set denoting the interval along the CFSE axis maximally occupied by each division. By appropriate gating, the proportion of cells of interest in each division can then be determined. This process we refer to as ‘division slicing’.3 By this method the proportion of positive (or negative) cells per division can be calculated and plotted, as in Fig. 1f, for expression of IgG1 by B cells cultured with IL-4. This information conforms to a divisionbased map as discussed further below. Because harvested CFSE labelled cells are viable, they can be sorted and their behaviour monitored further in vitro. This method has proved useful for monitoring the changing patterns of cytokine production with cell division.4 It is also possible to extract mRNA or DNA for PCR8 or even protein for western blotting from cells in different divisions. Intracellular staining Cell fixation and permeabilization conditions can be used that preserve CFSE profiles and allow simultaneous intracellular labelling with antibodies. Cells harvested from culture are washed and resuspended in 4% formaldehyde or 2% paraformaldehyde (in PBS) for 20 min at room temperature. Fixed cells are then rinsed with PBS and stored in PBS/0.1% BSA at 4°C, protected from light until staining. To stain intracellular components, fixed cells must be permeabilized. We have used two methods successfully: 1. Saponin at 0.5% with 0.1% BSA in PBS. For this method, all antibody labelling and cell washing is performed in this solution. All steps can be carried out at room temperature. This is the method of choice for intracellular cytokine staining.4 For analysis by flow cytometry, cells can be resuspended in PBS/0.1% BSA. 2. Tween 20.9 Cells are fixed for 10 min at room temperature before suspension volume is increased four-fold by addition of PBS and Tween 20 to a final concentration of Tween 20 of 0.1%. These cells are left overnight at room temperature. The next day cells are washed and resuspended in PBS/0.1% BSA and left on ice for 30 min. These cells can now be stained with antibodies according to standard methods. This permeabilization method is optimal for staining bromodeoxyuridine (BrdU)-labelled cells,3,7 as illustrated in Fig. 1g, or intracellular signalling molecules such as STAT-1 (Fig. 1h). Lymphoctye differentiation analysis with CFSE 519 Differentiation and cell division Isotype switching is linked to cell division B lymphocytes can be activated by T-dependent (TD) or T-independent (TI) protocols in vitro by the use of CD40L or LPS, respectively. B lymphocytes initially express IgM and IgD as antigen receptors, but may switch to another isotype during an immune response. The nature of this isotype switch is affected by the activating stimulus and by the presence of cytokines.10 When B cells are stimulated with the TD stimulus CD40 L, CFSE labelling reveals that they proliferate asynchronously but do not switch from IgM expression over the first 7–8 division cycles.3 When IL-4 was included in culture the CD40 L stimulated B cells switched to IgG1 and IgE in a division related manner, with approximately 50% of the cells switching to IgG1 between divisions 3–8, whereas the switch to IgE occurred at the later divisions.3 The appearance of switched cells only at later divisions suggested isotype switching was either under the regulation of a form of counting mechanism or that a subpopulation of switched cells or cells destined to switch divided faster, allowing these cells to dominate at the later divisions. This latter possibility was ruled out by studying the kinetics of appearance of switched cells. B cells stimulated by CD40L divide asynchronously and enter division at widely disparate times.3 If cells entering division at different times had different switching potential, then the ratio of switched cells within each division would be different when monitored at different times. Remarkably, however, the proportion of switched cells in each division remains constant, regardless of when the cultures are harvested.3 Furthermore, the switched and unswitched cells at later divisions are dividing at the same rate,3 a result inconsistent with the hypothesis that the switched cells are dominating the cultures due to a faster division rate. To explain these results we have adopted a probabilistic view of switching and differentiation.3,6 By this view, cells vary in sensitivity to CD40L and therefore the time of entry to division; however, that variation bears no relationship to the probability of a cell switching, which is dictated by division number and not time spent in any division. Simultaneously, each cell also varies in the division number at which it will switch under the influence of IL-4. Thus, there are two independent probabilities encapsulated in each B lymphocyte that direct its differentiation after stimulation.6 Further analysis has revealed that the switch to all isotypes displays a relation with division that is independent of time. Stimulation by LPS alone is sufficient to induce cells to switch to IgG3, whereas the addition of transforming growth factor (TGF)-β induces the same cultures to additionally switch to IgG2b.11 B cells stimulated by the TD stimulus CD40L will switch to IgG2a when IFN-γ is added,7 to IgA if TGF-β is present11 and as already noted, to IgG1 and IgE if IL-4 is included.2,3 T cell differentiation to cytokine secretion is also division related Altering the B cell isotype is an example of class switching by the immune system. The equivalent process for T cells is the development of different cytokine repertoires. The T cell can become a Th1- or Th2-like cell after culture under appro- Figure 2 The source of asynchrony in the standard model of cell division. Under a given set of stimulation conditions, individual cells in a lymphoctye population will exhibit a variable time to division that conforms to a probability distribution (top panel). At the time of harvest, cells that fall to the left of the harvest time within the time division distribution will have entered division whereas those to the right make up the undivided population. The dividing cells are moving through at a similar average division time (b) such that the number of cells in their first, second and later divisions can be determined (lower panel). CFSE, carboxyfluorescein diacetate succinimidyl ester. priate conditions.12,13 Typically, culture in IL-4 generates cells capable of secreting a range of cytokines, including IL-4 itself, IL-10 and IL-5. In contrast, culturing the same cells with IL-12 leads to a Th1 phenotype, typified by the secretion of IFN-γ.14 To test whether induction of T cell differentiation is also division linked and time independent, purified naïve CD4 T cells (CD44 lo, CD62Lhi and CD25–ve) were incubated with immobilized anti-CD3 in the presence of IL-4, the same cytokine that induced division-linked differentiation in B cells. To examine cytokine secretion by cells in different divisions, cells were sorted from overlapping divisions taken from cultures at different times. The results of these experiments showed clearly that the secretion of IL-4 increased with division.4 Furthermore, the production of IL-4 strongly correlated with division number and not time after stimulation.4 The pattern of other cytokines also varied, with IL-2 increasing then decreasing and IL-10, IL-5 and IFN-γ appearing at late divisions. Again, as for B cells, the relationship between division and cytokine is highly influenced by the presence of exogenous cytokines. The addition of IL-12 induces the appearance of IFN-γ secreting cells at early divisions, whereas IL-4 generates IL-4 secreting cells.4,15 520 J Hasbold et al. Figure 3 Progressive cell division in the standard model. (a) Effect of different harvest times on the standard model presented in Fig. 2. The time of harvest is shown by the dashed arrow. The corresponding carboxyfluorescein diacetate succinimidyl ester (CFSE) profiles are presented at the right. (b) Stimulation dose also alters the time to first division profiles as illustrated progressively for weak (upper panels) to strong (lower panels) stimulation. The effect on the CFSE profiles is similar to the time course in (a) and the CFSE patterns could arise from either scenario. Cell surface marker expression alters with division Asynchronous division Lymphocyte differentiation is also associated with alteration to cell surface molecules that subsequently influence the cell’s sensitivity to the environment and alter its migratory behaviour. We have noted that many of these cell surface changes proceed in a division-related manner, with each marker displaying an independent and distinct relationship with division.2 Therefore, division number represents a useful reference framework for describing differentiation. By plotting the probability of a change in expression versus division number, a differentiation ‘map’ can be generated.2–4,11 For many division-linked changes so far monitored, the corresponding maps are approximated by a normal distribution upon the division scale.11 Curiously, these maps need not involve all the cells in the dividing population.2–4 We were interested in the underlying cause of division asynchrony in lymphocyte culture. It was possible the cells were dividing at different rates, with those having undertaken the most divisions being the fastest dividing cells. Alternatively, cells could be dividing at the same rate and vary in the time they enter into the first division round. To distinguish between these possibilities, we pulsed cells with BrdU prior to harvesting and then simultaneously monitored BrdU incorporation and CFSE intensity (as illustrated in Fig. 1g). Because BrdU is incorporated into DNA, a brief pulse can indicate the number of cells in S phase of the cell cycle. Our analysis has revealed a range of outcomes. We refer to the most common outcome as the standard model of proliferation. T cells stimulated by anti-CD3 or B cells stimulated by Lymphoctye differentiation analysis with CFSE 521 Figure 4 Evidence for independence of time to division rate and probability of undergoing differentiation. (a) The progeny of cells from different parts of the time to first division plot reach each subsequent division at different times. Irrespective, the proportion that change phenotype (for example, switch isotype) is not altered. Therefore, the probability of switching is an independent property of the time to division. The cumulative expression of the marker with division can be converted to a ‘map’ by plotting the change per division as illustrated by the arrows. Maps often conform to a normal distribution. (b) The average time to division plot is also altered by stimulation strength; however, again the differentiation map followed is related to division number and is independent of proliferation kinetics. Figure 5 Independent regulation of proliferation and differentiation by cytokines. Extrinsic signals can alter both proliferation and differentiation. These processes may be linked, as shown in (a), where increasing amounts of IL-4 reduce the time to division of CD40Lstimulated B cells and reduce the divisions at which switching to IgG1 takes place. In contrast, IL-12 acting on T lymphocytes does not alter the proliferation rate but profoundly alters the divisions at which acquisition of IFN-γ secreting ability occurs. CD40 L and IL-4 conform to this standard. By this model, the cells in division 1 or greater take up BrdU at the same rate and are dividing evenly. Therefore, most of the asynchrony in division number derives from variation in the time of entry into the first division cycle. Underlying this pattern must be a distribution presenting the proportion of cells entering division at each time after stimulation. We have measured this distribution and found it to be approximately Gaussian, consistent with the difference in times to first division between cells being stochastic rather than being due to different cell populations. Figures 2 and 3 illustrate how this standard model of growth gives rise to the characteristic asynchronous CFSE profiles. In other examples of proliferation, the rate of BrdU incorporation does change and increase with progressive division number. This can be observed with CD40L stimulation of B cells without IL-4, or B cells stimulated with anti-Ig reagents (J Hasbold et al., unpubl. obs., 1999). Independence of cellular parameters Together, these studies precipitate a novel understanding of lymphocytes that requires the adoption of a probabilistic view of cell behaviour. This view is illustrated in Figs 4,5. It is convenient to imagine each lymphocyte as comprised of a series of machines that control the rates of growth and 522 J Hasbold et al. differentiation. Furthermore, these machines are capable of being controlled independently. The independence of proliferation and differentiation is illustrated in Fig. 4a. That is, a cell population will vary in the time taken to divide due to variations in the construction of the cell. This can be presented as a variable time to first division plot, as shown (Fig. 4a). At the same time, cells will possess an innate differentiation program for linking division number and some differentiation change; however, not all cells are constructed identically and therefore the division at which the change will occur is spread around a mean division number. Because the time of division program and the differentiation program are not related in the cell, the differentiation map of change per division is not altered by the timing of entry into the first cycle. A variation of this behaviour is illustrated in Fig. 4b. Here the stimulation dose is varied, which has the effect of changing the average time to first division. Despite this change, the division-linked differentiation pattern is unaffected. This is the case for altering CD40L dose during B cell switching induced by IL-4 to IgG1.3 Further evidence that the time to division and the differentiation program are capable of independent regulation is revealed by studying the effect of cytokines. Some cytokines can hasten or lessen the time to first division and also alter the division-linked differentiation response. Interleukin-4 has this effect on both T and B cells (Fig. 5a). In contrast, IL-12 does not alter the timing of division, but will alter the divisions at which T cells acquire IFN-γ secreting ability (Fig. 5b and AV Gett and PD Hodgkin, unpubl. data, 1999). Conclusions The discovery that lymphocyte differentiation displays a predictable stochastic relationship with division number, which is altered by cytokines and independent of the time spent in traversing a division, provides a simplifying paradigm for further analyses. The discovery indicates that there are only three components required to predict the biological properties of a population of lymphocytes after activation. These are: 1. The distribution of cells in each division cycle. This is complex, but can be determined empirically with CFSE. 2. Maps of division linked changes. Such maps can be prepared experimentally and include information on the effect of engaging receptors from the cell surface, including cytokines. 3. The rules of integration for different signals. For instance, how does the combination of IFN-γ and IL-4 affect the separate differentiation probabilities of each? This must also be determined experimentally. Because division number provides a constant reference point, development of differentiation maps is cumulative and can incorporate an inventory of components at the cell surface and inside the cell, as well as describing the functional properties of cells on restimulation. It is anticipated that development of these models will facilitate further dissection of the lymphocyte response by helping us to look inward to determine the mechanism of how these predictable relationships are controlled by intracellular processes. Furthermore, they will enable us to look outward from the cell to develop models that predict how the programmed response of lymphocytes will dictate their behaviour during ongoing immune reactions in vivo. Acknowlegements This work was supported by the Medical Foundation of the University of Sydney and the National Health and Medical Research Council. References 1 Lyons AB, Parish CR. Determination of lymphocyte division by flow cytometry. J. Immunol. Meth. 1994; 171: 131–7. 2 Hodgkin PD, Lee J-H, Lyons AB. B cell differentiation and isotype switching is related to division cycle number. J. Exp. Med. 1996; 184: 277–81. 3 Hasbold J, Lyons AB, Kehry MR, Hodgkin PD. Cell division number regulates IgG1 and IgE switching of B cells following stimulation by CD40 ligand and IL-4. Eur. J. Immunol. 1998; 28: 1040–51. 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 Lyons AB, Hasbold F, Hodgkin PD. Flow cytometric analysis of cell division history using dilution of CFSE, a stably integrated fluorescent probe. Meth. Cell Biol. 1999; in press. 6 Hodgkin PD, Kehry MR. Methods for polyclonal activation to proliferation and Ig secretion in vitro. In: Herzenberg LA, Weir DM, Herzenberg LA, Blackwell C (eds). Weir’s handbook of experimental immunology, vol. 3. Oxford: Blackwell Science, 1996. 7 Hasbold J, Hong JS-Y, Kehry MR, Hodgkin PD. Integrating signals from IFN-γ and IL-4 by B cells: Positive and negative effects on CD40 ligand-induced proliferation, survival and division-linked isotype switching to IgG1, IgE, and IgG2a. J. Immunol. 1999; in press. 8 McCall MN, Hodgkin PD. Switch recombination and germ-line transcription are division-regulated events in B lymphocytes. Biochim. Biophys. Acta 1999; 1447: 43–50. 9 Tough DF, Sprent J. Turnover of naive- and memory-phenotype T cells. J. Exp. Med. 1994; 179: 1127–35. 10 Snapper CM, Mond JJ. Towards a comprehensive view of immunoglobulin class switching. Immunol. Today 1993; 14: 15–17. 11 Deenick EK, Hasbold J, Hodgkin PD. Switching to IgG3, IgG2b and IgA is division-linked and independent revealing a stochastic framework for describing differentiation. J. Immunol. 1999; in press. 12 Mosmann TR, Coffman RL. TH1 and TH2 cells: Different patterns of lymphokine secretion lead to different functional properties. Annu. Rev. Immunol. 1989; 7: 145–73. 13 Kelso A. Cytokines: Principles and prospects. Immunol. Cell Biol. 1998; 76: 300–17. 14 O’Garra A, Murphy K. Role of cytokines in determining T-lymphocyte function. Curr. Opin. Immunol. 1994; 6: 458–66. 15 Bird JJ, Brown DR, Mullen AC et al. Helper T cell differentiation is controlled by the cell cycle. Immunity 1998; 9: 229–37.