A central role for IL-2 in fate determination of mature T cells—I: role in determining the Th1/Th2 profile in primary T cell cultures

Kaliannan Raju, Brian A. Rabinovich, Laszlo G. Radvanyi,1, David Spaner,2 and Richard G. Miller

Departments of Medical Biophysics and Immunology, University of Toronto and Ontario Cancer Institute, 610 University Avenue, Toronto M5G 2M9, Canada

Correspondence to: R. G. Miller


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
IL-2 signaling appears to play a significant role in enabling the synthesis of Th2 cytokines in an in vitrosystem for studying primary T cell responses. When T cells from C57BL/6J or BALB/c strains of mice were activated in vitroand re-stimulated through their TCR complex 48 h later, CD4+ T cells producing the Th2 cytokines IL-4 and IL-10 were found only when IL-2 was present. IL-2 also enhanced IFN-{gamma} synthesis in C57BL/6J cells but not in BALB/c cells. By up-regulating production of anti-inflammatory Th2 cytokines during a primary response, IL-2 may play a critical role in limiting Th1-mediated responses.

Keywords: autoimmunity, IFN-{gamma}, IL-2, IL-4, IL-5, IL-10, Th1, Th2, tumor necrosis factor-{gamma}


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Differentiation of naive T cells upon stimulation through their TCR into either IFN-{gamma}-producing Th1 or IL-4- and IL-10-producing Th2 helper cells has been the subject of intensive study over the last several years (for a recent review, see 1). Previous studies have indicated that IL-12 induces Th1 cell development (1–4), while IL-4 induces differentiation of Th2-type cells (3–7). Further, Th1 and Th2 cytokines are known to inhibit the development of Th2 and Th1 effector cells respectively. In mice, induction of a Th1 or Th2 response to certain infections has been shown to be strain dependent. Thus, infection with Leishmania major results in a Th1 response in resistant C57BL/6 (B6), while susceptible BALB/c mice mount a Th2 response (8,9). The reason for this strain difference is not understood.

IL-2 is a potent immunoregulatory cytokine that plays a central role in a number of T cell functions. Although IL-2 has traditionally been grouped with the Th1 cytokines, it is now thought that IL-2 is not a true Th1 cytokine since naive CD4+ T cells are known to produce it (10). There is scattered evidence suggesting a role for IL-2 in the regulation of Th1 and/or Th2 cytokines. Thus, IL-2 was shown to be required for production of modest amounts of IL-4 by T cells activated in vitro in the absence of accessory cells, suggesting a possible role for IL-2 in the differentiation of Th2 effector cells (11). Using an ELISA method, Seder (12) has shown a requirement for IL-2 in priming CD4+ TCR transgenic T cells to produce IL-4 as well as IFN-{gamma}. There is also indirect evidence for IL-2-mediated enhancement of Th2 cytokine synthesis that can resolve Th1-mediated diseases (13,14). Despite the above evidence, there is not widespread agreement that IL-2 plays a critical role in Th1/Th2 determination.

In the present study we have investigated the role of IL-2 in Th1 and Th2 cytokine synthesis in short-term primary T cell cultures derived from C57BL/6J and BALB/c mice. Most previous studies in vitro on the role of IL-2 and other cytokines in Th cell differentiation have been carried out using established cell lines. In the studies reported here, we have characterized ex vivo primary T cells activated with or without IL-2 blockade for their ability to produce Th1 and Th2 cytokines on being re-stimulated through their TCR before developing their fully differentiated potential, a sequence of events that is likely to happen during an actual in vivo response. Significant numbers of CD4+ cells producing the Th2 cytokines IL-4 and IL-10 were seen only when IL-2 was present. IL-2 also enhanced IFN-{gamma} production in B6 CD4+ T cells but not in BALB/c cells.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Mice
B6 and BALB/c mice were purchased from the Jackson Laboratory (Bar Harbor, ME). Mice that carry a transgenic TCR specific for chicken ovalbumin (OVA) peptide (OVA326–336) in a SCID background were made by mating OVA transgenic mice carrying the TCR from the T cell hybridoma D0-11.10 (a gift from Dr Dennis Y. Loh, Washington University School of Medicine, St Louis) to C.B-17 SCID mice for six generations and selecting for the presence of the transgenic TCR by flow cytometry using the mAb KJ1-26 that is specific for this TCR (15). All mice were housed under specific pathogen-free conditions at the Ontario Cancer Institute, Toronto, Canada.

Reagents and antibodies
mAb to mouse CD3{varepsilon} (hybridoma clone 145-2C11) (16) was purified from hybridoma culture supernatants as described previously (17). Murine rIL-2 obtained from the culture supernatants of the mouse myeloma clone X63BCMGNeo-mIL-2 transfected with mouse IL-2 gene (a gift from Dr Hajime Karasuyama, University of Tokyo, Japan) (18) was assayed for biological activity as described before (17). Ascites fluid containing mAb to murine IL-2 (S4B6.1) was prepared from C.B-17 SCID mice as described (19). The mAb to IL-2 was purified from these ascites by using a Protein G-affinity column. For flow cytometry, the following antibodies to mouse cytokines and T cell-surface proteins as well as the isotype controls (all from PharMingen, San Diego, CA) were used: FITC-conjugated anti-tumor necrosis factor (TNF)-{alpha}, anti-IFN-{gamma}, anti-CD69, rat IgG1 and rat IgG2a; phycoerythrin-conjugated anti-IL-4, anti-IL-10, anti-CD45RB, anti-CD25, rat IgG2b and rat IgG1. All other reagents were as described before (17).

Anti-CD3-induced T cell activation and reactivation
Initial activation of T cells from the spleens of B6, BALB/c and OVA-SCID transgenic mice and re-cross-linking of activated cells were done essentially as described before (17) except that the mAb to CD3 was bound directly to the bottom of 96-well plates. For initial activation in 24-well plates, anti-CD3 was used at a concentration of 1 µg/ml. For TCR re-stimulation, flat-bottom, high-binding 96-well plates (Costar, Cambridge, MA) were coated with 100 ng of anti-CD3 in 100 µl of PBS per well at 37°C for 1 h. The wells were then washed 3 times with PBS and unbound sites were blocked with 10% FCS in PBS for 30 min at room temperature. Reagents and procedures for cell culture and viability measurement were as described (17).

Flow cytometry
Staining of cell-surface antigens with fluorescent mAb and flow cytometric analysis using a Becton Dickinson (Mountain View, CA) FACScan were done as described previously (20). Intracellular cytokine staining was done essentially as described by Ferrick et al. (21). Briefly, activated T cells were incubated with Brefeldin A (5 µg/ml) for 3 h at 37°C. The cells (2–4x105 per group) were then harvested, washed with PBS without Ca2+ and Mg2+, and incubated with antibody to Fc receptor (2.4G2) and fixation medium (solution A; Caltag, Burlingame, CA; 75 µl) in a total volume of 90 µl for 15 min at room temperature. The cells were then washed with Ca2+- and Mg2+-free PBS and incubated with different combinations of fluorescent mAb to various cytokines and 75 µl of permeabilization medium (solution B; Caltag) in a total volume of 125 µl for 30 min at room temperature. Cells were then washed and resuspended in 200 µl of Ca2+- and Mg2+-free PBS and analyzed by flow cytometry.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
In all the studies reported here we have used an in vitro system of T cell activation and reactivation previously developed in our laboratory (17). Briefly, spleen cells were first activated with soluble anti-CD3 mAb (145-2C11) in 24-well plates for 48 h. After this initial activation, viable activated T cell blasts were purified by centrifugation through a lympholyte density gradient and re-stimulated by re-cross-linking with plate-bound anti-CD3 mAb in 96-well plates or (control) grown in IL-2-containing medium (20 U/ml) without re-cross-linking for another 16 h. These activation and reactivation steps were designed to simulate the initial priming of naive T cells and their probable re-encounter with the priming stimulus before differentiating into mature effector cells.

Cytokine profile of CD4+ T cells during TCR re-stimulation in the presence and absence of IL-2 signal
We studied the production of various cytokines by activated BALB/c and B6 CD4+ T cells in the in vitro system described above using flow cytometry to measure cytoplasmic cytokine levels as described in Methods. We could not detect any of the cytokines analyzed (IL-4, IL-10, IFN-{gamma} and TNF-{alpha}) after 48 h of activation or after culturing the 48-h activated and purified T cells for another 16 h in IL-2-containing medium without further TCR stimulation (data not shown). Upon re-cross-linking, however, cytokine production was observed and preliminary experiments suggested that the presence or absence of IL-2 in the re-cross-linking cultures had a major effect on the levels of IL-4 and IL-10 produced. Table 1Go is a summary of pooled results from four repetitions of the same experiment explicitly designed to test this possibility. One sees that addition of IL-2 at the time of re-cross-linking significantly increased the production of both IL-4 and IL-10 from both B6 and BALB/c T cells. Compare `no additions' groups with `IL-2' groups. We found that even the `No additions' groups contained some IL-2 as a result of endogenous production, ~0.5 U/ml (data not shown). When this was removed by adding anti-IL-2 mAb (`IL-2 + anti-IL-2' groups), IL-4 and IL-10 production levels fell to barely significant (BALB/c) or insignificant (B6) levels in comparison with `threshold control'. Manipulation of IL-2 levels in BALB/c cultures did not have a significant effect on either TNF-{alpha} or IFN-{gamma} production. In B6 cultures, addition of IL-2 significantly augmented both TNF-{alpha} and IFN-{gamma} production, but IL-2 blockade left production levels well above background. We conclude that addition of IL-2 in the re-cross-linking cultures greatly enhances production of IL-4 and IL-10, and that production of these cytokines is zero or near zero in the complete absence of IL-2.


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Table 1. Effect of IL-2 level during re-cross-linking on the synthesis of IL-4, IFN-{gamma}, IL-10 and TNF-{alpha} in BALB/c and B6 CD4+ T cellsa
 
Anti-IL-2 mAb was used to rule out the possibility that the observed changes in cytokine profiles produced by addition of exogenous IL-2 were due to an unknown factor in the IL-2 preparation. Addition of increasing amounts of anti-IL-2 mAb to the re-cross-linked cultures at the same time IL-2 was added led to near (BALB/c, Fig. 1Go) or complete (B6, Fig. 2Go) inhibition of the synthesis of IL-4 and IL-10. With B6 cultures this inhibition of cytokine synthesis could be achieved at a much lower level of anti-IL-2 than with BALB/c. A non-specific antibody with the same isotype as anti-IL-2 did not have any effect even at the highest concentration tested (Figs 1F and 2FGoGo). There was no significant variation in the levels of activation markers CD44, CD45RB and CD69 in the different groups of samples at the time of cytokine measurements (data not shown). However, the levels of CD25, the high-affinity component of the IL-2 receptor, were slightly greater in IL-2 blocked cells.



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Fig. 1. Effect of IL-2 level on the synthesis of IL-4 and IFN-{gamma} in BALB/c CD4+ T cells. The data form part of one of the experiments summarized in Table 1Go. The figure shows the results obtained for cells with the forward and side light scatter properties of live, activated T lymphocytes that stained positive for CD4. Cells stained with flourescein-conjugated isotype control antibodies were used as negative controls. The numbers indicate the percentage of CD4+ viable T cells in each quadrant. At least 20,000 events were analyzed for each profile. Cytokine profiles shown are: (A) no added IL-2, (B) exogenous IL-2 (20 U/ml) added during re-cross-linking, and (C)–(F) exogenous IL-2 (20 U/ml) added during re-cross-linking along with 1, 10 or 20 µg/ml of mAb to IL-2 or 20 µg/ml of a non-specific mAb with the same isotype as the mAb toIL-2 respectively.

 


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Fig. 2. Effect of IL-2 level on the synthesis of IL-4 and IFN-{gamma} in B6 CD4+ T cells. See legend to Fig. 1Go for experimental details.

 
IL-2 signal is required throughout the activation and reactivation steps for efficient production of Th2 cytokines
A low level of endogenously produced IL-2 can be found in the primary cultures at 48 h. Its presence during at least the late stages of the primary cultures may have had an effect on the ability to produce various cytokines during the re-stimulation cultures. To test this, we blocked IL-2 signal in the initial spleen cultures by adding anti-IL-2 at 24 h during the first culture stage. The IL-2 blocked cultures were then re-stimulated as before in the presence or absence of exogenous IL-2 and the cytokine profiles analyzed after 16 h of reactivation. As shown in Table 2Go, IL-2 blockade in the initial cultures inhibited the production of both IL-4 and IL-10 in both BALB/c and B6 cells to levels barely different from background. This occurred whether or not IL-2 was added to the re-cross-linking cultures. IL-2 blockade in the priming culture had little to no effect on IFN-{gamma} or TNF-{alpha}, except that IFN-{gamma} production was reduced in B6 cultures although it remained at levels well above background. These results are similar to those obtained when IL-2 was blocked only during the reactivation step (Table 1Go), and suggest that exposure to IL-2 during both the priming and reactivation cultures plays a critical role in programming a T cell to make the Th2 cytokines IL-4 and IL-10. Similar results were obtained when the cytokine profiles were analyzed after 24 h of re-stimulation although the percentages of cells producing various cytokines were generally less at this point (data not shown). This suggests that IL-2 blockade during initial activation did not simply delay the production of different cytokines during re-cross-linking.


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Table 2. IL-2 signal is required throughout the primary response for efficient production of IL-4 and IL-10 in both BALB/c and B6 CD4+ T cellsa
 
The presence of anti-IL-2 mAb did not affect the activation of T cells, as suggested by the number of T cell blasts obtained after initial activation and the results of analysis of various activation markers at the time of cytokine measurements (data not shown). Only small differences were seen with some markers (e.g. CD44) being actually higher in IL-2 blocked cultures. At the concentration of anti-IL-2 used, we could not detect any free IL-2 in the IL-2 blocked cultures as shown by the inability of the medium in these wells to support the growth of CTLL-2 cells (data not shown).

IL-2 signal requirement for Th2 cytokine synthesis in monoclonal transgenic TCR-carrying CD4+ cells
The results above indicate that the presence of IL-2 enables the production of the Th2 cytokines IL-4 and IL-10 in normal B6 and BALB/c mice. As a further test of this and to see whether CD8+ cells were playing any indirect role, we used T cells from C.B-17 SCID mice that carry a transgenic TCR specific for a chicken OVA peptide (OVA326–336). Because of the SCID mutation, essentially all (>99%) of the T cells in this mouse are CD4+ and carry the transgenic TCR which is recognized specifically by the mAb KJ1-26.1. Activation and reactivation of the OVA-SCID T cells and subsequent analysis of cytokine profiles indicated that IL-2 signaling was required for enhanced Th2 cytokine production in both anti-CD3 and KJ1-26.1 stimulated OVA-SCID T cells (Table 3Go). The IFN-{gamma} levels in OVA-SCID T cells, which have primarily a BALB/c background, were not significantly affected by the IL-2 blockade.


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Table 3. Analysis of cytokine profile in OVA-transgenic SCID cells activated and reactivated with anti-CD3 or KJ1-26.1a
 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
In this paper, we describe the application of an in vitro system to investigate the differentiation of naive primary T cells into Th1 and Th2 effector cells upon TCR stimulation and the role of IL-2 in this process. The salient feature of this in vitro system is that the primary response of previously unactivated T cells is split into two stages. (i) Initial activation with soluble anti-CD3 mAb for 48 h in spleen cell cultures. (ii) Purification and re-stimulation (or re-cross-linking) of the activated T cells with plate-bound anti-CD3 mAb for a further 16 h followed by analysis of cytokine production with a sensitive flow cytometric technique by which one can observe cytokine production in individual cells. At the time of re-cross-linking, the cytokines IL-4, IL-10, IFN-{gamma} and TNF-{alpha} were not detectable in either B6 or BALB/c. After re-cross-linking of either B6 or BALB/c cells, significant numbers of Th2 cytokine producers were observed. However, this number dropped to zero or near-zero values when IL-2 was blockaded at either culture stage. IL-2 blockade during the initial activation culture remained effective in blocking IL-4 and IL-10 production even when IL-2 was added to the re-cross-linking stage (Table 2Go). Similarly, IL-2 blockade at the re-cross-linking stage was effective even when IL-2 was not blockaded during the initial activation stage (Table 1Go). Thus IL-2 must be present during both culture stages to obtain an appreciable number of IL-4- and IL-10-producing T cells.

Previous work on the role of IL-2 in promoting IL-4 synthesis has been done only at the population level (11,12). Recently, Saparov et al. (22) showed that production of IL-4 and IFN-{gamma} by OVA transgenic T cells following re-stimulation is only from cells that produced IL-2 during the initial activation. Exposure to exogenous IL-2 did not induce effector cytokine synthesis in cells that were derived from non IL-2 producers even if the high-affinity IL-2 receptor (CD25) was present on their surface. However, whether IL-2 signal was required during reactivation for the synthesis of effector cytokines by cells that initially produced IL-2 was not tested. Our results suggest that a functional IL-2 signal is required throughout the activation and reactivation steps for efficient production of these cytokines. We also suggest that IL-2 should not be grouped with either the Th1 or Th2 cytokines since it promotes synthesis of Th2 cytokines.

At sufficiently low antigen doses, one gets a Th1 response. This switches to a Th2 response when antigen dose is sufficiently increased (23,24). Thus the presence of IL-2 might be having the same effect as a higher antigen dose. However, our results from the analysis of various activation markers suggest that IL-2 does not alter the level of activation in mediating its effect on Th1/Th2 cytokine synthesis since there was no correlation between levels of activation markers and development of Th2 cytokine producers (data not shown).

In general, we observed that the production of the Th2 cytokines IL-4 and IL-10 was greater under all conditions for BALB/c T cells than B6 T cells. The opposite held for the Th1 cytokines IFN-{gamma} and TNF-{alpha}. In particular, when IL-2 was present throughout, BALB/c T cells produced a dominantly Th2 response as measured by percentages of cytokine-producing T cells. The opposite was true for B6. Thus our in vitro results mimic the response of these two mouse strains to infection with L. major. The requirements for inducing a Th2 response with the resulting susceptibility to L. major are not well understood. Conflicting results on the role of IL-4 in counteracting a Th1 response and rendering BALB/c mice susceptible to L. major have been reported (25–27). Although our experiments did not directly address these questions, the role of IL-2 in modulating Th1/Th2 differentiation may be important in determining the outcome of infection with L. major. Susceptibility to L. major in BALB/c mice has been shown to be reversed by administering anti-IL-2 (14).

The role of IL-2 in up-regulating the synthesis of IFN-{gamma} has been documented in several studies. IFN-{gamma} production by T cells activated by LCMV infection of mice that were predominantly of B6 genotype has been shown to be enhanced by IL-2 (28). Cleavage of the {alpha} subunit of the IL-2 receptor CD25 from human peripheral T cells was found to inhibit IFN-{gamma} secretion (29). Our results show that IL-2 can induce the synthesis of IL-4 and IL-10 in addition to increasing IFN-{gamma} synthesis in B6 T cells, and suggest a modulatory function for IL-2 in regulating Th1 and Th2 balance during an immune response. This function of IL-2 may depend on genetic factors since IFN-{gamma} production by BALB/c T cells was not significantly affected by IL-2. Since IL-4-producing cells are known to inhibit the differentiation of CD4+ T cells into IFN-{gamma}-producingTh1 cells, it is also possible that the larger number of IL-4 producers in BALB/c may account for this lack of IL-2 effect on IFN-{gamma} synthesis.

The shifting of T cells away from Th2 in the absence of an IL-2 signal that we have observed may have significant implications for development of autoimmune diseases. Th1 cells are generally thought to be responsible for the development of many autoimmune diseases (30–32). Since IL-4 and IL-10 have anti-inflammatory properties (33–35), one of the important functions of the IL-2 signal during T cell activation and differentiation may be to reduce the inflammatory response by enhancing the production of IL-4 and IL-10. There is direct evidence that IL-2 administration in vivo can alleviate autoimmune diseases: IL-2 treatment reduced the severity of experimental autoimmune uveoretinitis (36). Studies with IL-2 knockout mice partly support this conclusion. When kept in a conventional mouse facility, they develop ulcerative colitis (37) which appears to be Th1-mediated (38). However, these mice also have a generalized overproduction of autoantibodies (39), a condition which can be corrected by crossing them onto an IL-4 knockout mouse (40). Further, when mice carrying the OVA transgenic TCR (the same as studied here) were bred onto an IL-2 knockout background, their T cells could be activated in vitro on OVA-pulsed IL-2 knockout dendritic cells to make either IL-4 or IFN-{gamma} by including IL-4 or IL-12 in the culture (41). Although we tried, we could not test the capacity of IL-2-deficient T cells to produce Th2 cytokines in our in vitro system since cells taken from IL-2 knockout mice did not get activated properly (data not shown). Nor have we tested the effect of adding cytokines such as IL-4 or IL-12 to our IL-2-blockaded cultures and we cannot rule out that the Th2-promoting effects of IL-2 signaling are due to secondary production of other cytokines rather than a direct effect of the signal itself. The fact remains that T cells undergoing their first activation in our in vitro culture system fail to make Th2 cytokines when IL-2 is blockaded. We conclude that IL-2 has a central role in the induction of cells that produce anti-inflammatory cytokines and that this might be important for the prevention/amelioration of autoimmunity.


    Acknowledgments
 
We thank John Shannon for excellent technical assistance. This work was supported by a grant to R. G. M. from the Medical Research Council of Canada.


    Abbreviations
 
B6 C57BL/6
OVA ovalbumin
TNF tumor necrosis factor

    Notes
 
1 Present address: Immunology Department, Aventis Pasteur, Sunnybrook and Women's College Health Science Centre, Toronto M4N 3N5, Canada Back

2 Present address: Division of Cancer Biology Research, Sunnybrook and Women's College Health Science Centre, Research Building, Toronto M4N 3N5, Canada Back

Transmitting editor: J. W. Schrader

Received 25 August 2000, accepted 24 August 2001.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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