|Home | About | Journals | Submit | Contact Us | Français|
An early reaction of CD4+ T lymphocytes to antigen is the production of cytokines, notably IL-2. In order to detect cytokine dependent responses, naive antigen-specific T cells were stimulated in vivo and the presence of phosphorylated STAT5 molecules was used to identify the cell populations responding to IL-2. Within hours of T-cell priming, IL-2-dependent STAT5 phosphorylation occurred primarily in Foxp3+ regulatory T cells. In contrast, the antigen-specific T cells received STAT5 signals only after repeated antigen exposure or memory differentiation. Regulatory T cells receiving IL-2 signals proliferated and developed enhanced suppressive activity. These results indicate that one of the earliest events in a T cell response is the activation of endogenous regulatory cells, potentially to prevent autoimmunity.
Immune responses to foreign antigens have to be precisely regulated to prevent inadvertent activation of self-reactive lymphocytes or collateral damage to normal tissues. Regulatory T cells (Tregs) are one of the major mechanisms that limit responses to self and foreign antigens (1–4). The conventional view is that most Tregs are generated by self-antigen recognition in the thymus and work in peripheral tissues to prevent reactions against these self antigens (5, 6). Some Tregs may be generated in peripheral tissues as immune responses develop, to stop these responses from becoming harmful (7). Although the Treg population as a whole appears chronically activated, expressing activation markers such as CD25 and CTLA-4 (1), a fraction can be identified that is in a more proliferative state, presumably responding to tissue self-antigens (8). Tregs that are controlling T-cell responses to microbial infections may be activated by self antigens as well, released by tissue damage, or by direct recognition of microbial antigens (9). Identifying when and how Tregs are activated during the course of a physiological immune response is of great interest to better understand the way immune responses develop and are controlled. Given the well-established capacity of Tregs to limit unrelated antigen responses through bystander suppression (10), we hypothesized that communication between antigen-activated T cells and Tregs of different specificity is required to control the initiated response and limit potential damaging side effects. One interesting candidate to deliver a signal from antigen-activated T cells to Tregs is interleukin-2 (IL-2), which is produced early after T cell activation and is known to be essential for the generation and maintenance of Tregs. The functions of IL-2 during a T cell response are seemingly antagonistic, since the cytokine not only supports the development of effector and memory cells (11–13) but is also critical for the maintenance of Foxp3+ Tregs (14)(15). Since Tregs do not produce IL-2, it is likely that they receive IL-2 signals from other T cells that are responding to antigen. To address the question what the cellular targets of IL-2 are during a conventional immune response, we measured IL-2-dependent biochemical signals in different cell populations in vivo. Surprisingly, we found that Tregs, and not the actual IL-2-producing cells, are the first responders to IL-2, leading to their activation and proliferation.
BALB/c mice were purchased from Charles River Laboratories and used at 6–8 wk of age. DO.11.10 transgenic mice were a gift from K. Murphy (Washington University, St. Louis, MO), and were crossed on to Rag2−/− mice. DO.11.10 × Il-2−/− mice were generated in our laboratory by crossing DO.11.10 mice with Il-2−/− mice, all on the Rag−/− background. Foxp3-GFP mice (16) were backcrossed 10 generations to BALB/c. All mice were bred and maintained in accordance with the guidelines of the Laboratory Animal Resource Center of the University of California, San Francisco and approved Stanford Administrative Panel on Laboratory Animal Care/Institutional Animal Care and Use Committee (APLAC/IACUC) protocols.
Adoptive transfer of Il-2+/+ and Il-2−/− DO.11.10 Rag−/− T-cells was performed as previously described (11, 17). DO11 Rag−/− TCR transgenic T cells were adoptively transferred into wild-type BALB/c mice and then immunized with 100 µg OVA peptide +/− lipopolysaccharide, or with mature OVA-loaded bone-marrow derived dendritic cells (BMDCs). Serum-free medium (Cellgro, Mediatech) was used for the last 48–72h of culture, since BMDCs loaded with FCS-derived peptides were capable of stimulating a nonspecific T cell response (data not shown). In some experiments, mice were initially exposed to antigen and then re-stimulated 60 hours or 5 weeks after initial antigen priming.
Following stimulation in vivo the spleens of recipient mice were harvested as previously described (18). In brief, mice were killed at various time points by a brief (30 sec) CO2 administration followed by cervical dislocation. Spleens were excised and immediately dissociated into a 10 ml PBS solution containing 1.6% paraformaldehyde (Electron Microscopy Sciences). Following a 15 min fixation period cell suspensions were transferred through a 70-µm pore size mesh and ice-cold methanol was directly added to a final concentration of 80% methanol.
Fixed and permeabilized cells were prepared for flow cytometric analysis as previously described (19, 20). The following antibodies were used in 9-color panels to identify and analyze donor and recipient cell populations (purchased from BD unless otherwise noted): pSTAT5 (Y694, clone 47), Ki67 (B56), Foxp3 (eBioscience, NRRF-30), B220 (RA3-6B2), CD25 (PC-61), CD90.2 (Biolegend, 30-H12), CD4 (RM4-5), CD11b (M1/70), anti-KLH control Ab (X40), and anti-DO11 TCR (KJ1-26). For staining of other cell populations the following antibodies were used: CD11c (HL3), CD49b (DX5), F4/80 (eBioscience, BM8), CD44 (IM7), CD122 (TM-β1) and CD45RB (Biolegend, C363-16A). CD49b, F4/80, and CD122 staining was performed prior to methanol permeabilization. Note that all antibody concentrations were titrated for optimal staining in a 100 µl staining volume with no more than 4×106 methanol permeabilized cells per sample.
24 hrs following 100 µg OVA peptide stimulation, DO11 T cells enter the mitotic “blast phase” of activation that is characterized by increased median values of forward-scatter and side-scatter. Early experiments determined that every mouse and rabbit antibody tested for intracellular staining had increased fluorescence in T-cell blasts even when specific for an antigen not present in mammalian cells. To normalize median fluorescence between naïve and activated T cells, the following equation was used as a derived parameter in Flowjo: [(median phospho X at time Y value)/ (median SSC value at time Y/ median SSC Value of PBS control)]/10. Derived parameters are shown in Flowjo on standard “analog” style scaling from 1 to 104 units, while digital data displays negative numbers to 2 × 105 units. Because there is a 10-fold difference in the maximum values of these two scaling types, we divided the digital data by 10 to place it at a similar position on the histogram plot when displayed on an analog scaling. Normalization using control antibody specific for keyhole limpet hemocyanin (KLH) in place of SSC yielded similar results. The biexponential transformation could not be applied to derived parameters (Flowjo limitation), thus normalized data is shown with 4-log scales.
To follow the kinetics of IL-2 production by DO11 T cells primed in vivo, lymph nodes and spleen were harvested at various times after immunization and IL-2 secretion was measured during 45 min of incubation at 37 C without restimulation, using an IL-2 capture assay (Miltenyi).
Naïve CD4+ T cells and Tregs were sorted from BALB/c Foxp3-GFP reporter mice using a MoFlo high speed cell sorter (DAKO-Cytomation). Cells were lysed using a non-denaturing cell lysis buffer containing 1% Triton. Protein extracts of 5×105 cells were analysed by Western blotting for SOCS-3 presence using a polyclonal anti-SOCS-3 Ab (CST) at a 1:1000 dilution, followed by an anti-rabbit/HRP Ab (Zymed). β-actin was probed to ensure equal loading of protein.
IL-2/anti-IL-2 mAb complexes with Treg specific effects were prepared as described (21). 48 hours after intraperitoneal injection of IL-2 complexes, BALB/c splenocytes were harvested and prepared for flow cytometry. For suppression assays, BALB/c mice were injected with IL-2 complexes every other day for one week. Two days following initial IL-2 administration, CFSE-labeled DO11 T cells were transferred into IL-2-treated and untreated mice and stimulated with OVA. Spleens were harvested for analysis three days later.
Vaccinia virus (wild-type strain, Western Reserve) was obtained from ATCC. Various amounts of virus based on plaque forming unit (pfu) counts were injected via the tail vein into BALB/c mice.
To define the cells that respond to IL-2 during an immune response, we used single cell biochemical analysis of distinct lymphocyte populations (22–24). Such assays exploit phospho-specific antibodies as sensitive indicators of activated signaling pathways in complex cell populations, coupled with the visualization of phenotypic markers to allow the identification of individual cell types. IL-2 production was induced during an antigen-induced T cell reaction by transferring CD4+ T cells specific for chicken ovalbumin (OVA), from the DO11.10 T cell receptor (TCR) transgenic mouse, into normal BALB/c recipients and immunizing with OVA peptide. At various times after immunization, splenocytes were harvested, fixed, and stained with a 9-color panel of antibodies that could detect phosphorylated STAT5 (pSTAT5), the canonical IL-2-induced transcription factor, and various immune cell populations, including the transferred DO11 T cells. Although other signaling pathways (PI3K, Shc) are induced by IL-2 as well, many IL-2-induced functions in T cells are mediated by STAT5 (25) and measuring pSTAT5 is thus a powerful read-out to compare qualitative differences in IL-2 signaling between cell populations. We found that 6 hours after OVA immunization, increased STAT5 phosphorylation was totally absent in endogenous naïve T and B cells and minimally induced in a fraction of natural killer and dendritic cells. In contrast, the majority of Foxp3-expressing CD4+ T cells showed high levels of sustained STAT5 activation (Fig. 1). The induction of pSTAT5 in Foxp3+ cells was rapid and was seen with different forms of antigen administration, including OVA + the soluble adjuvant LPS and injection of mature bone marrow derived dendritic cells (BMDC) incubated with OVA (Fig. 2A and B). Although in most experiments we followed pSTAT5 induction in endogenous Foxp3+ T cells in the spleen, the same phenomenon was seen in lymph nodes (data not shown). Furthermore, the magnitude of pSTAT5 induction was proportional to the frequency of responding DO11 cells (Fig. 2C). To ask if the induction of pSTAT5 in Tregs followed the kinetics of IL-2 production by the antigen-responsive T cells, we measured IL-2 secretion by DO11 T cells in lymph nodes and spleen at various times after antigen administration. Confirming published data (26)(27), DO11 T cells started producing IL-2 as early as 2 hours after antigen administration and stopped producing the cytokine by 24 hours. Thus, emergence of pSTAT5 in Tregs correlates with the timing of IL-2 secretion (Fig. 2D).
To formally show that IL-2 produced by DO11 T cells was required to induce pSTAT5 in Tregs, we transferred either normal (Il-2+/+) or Il-2−/− DO11 T cells, immunized the recipients with OVA or control buffer, and stained for pSTAT5 in different cell populations after 9 hours. Only when the DO11 T cells were capable of producing IL-2 was pSTAT5 induced in endogenous Foxp3+ T cells (Fig. 2E). These results established that IL-2 produced during an antigen-specific T cell response acts rapidly on a large number of endogenous Foxp3+ regulatory T cells. It is noteworthy that 10–15% of Tregs are pSTAT5-positive in the absence of immunization (Fig. 1 and Fig.2). However, the reduced population of Tregs present in Il-2−/− mice (15) does not show significant pSTAT5 signaling (data not shown), supporting previous reports demonstrating that homeostasis of Tregs in the steady-state depends on low levels of IL-2 present in the environment (28, 29).
Because the best-established function of IL-2 is as an autocrine growth and differentiation factor, it is predicted to act on the antigen-responsive, IL-2-producing T cells. Surprisingly, at the times that Foxp3+ cells showed clear induction of pSTAT5, the OVA-stimulated DO11 cells remained negative (Fig. 3A). This was not attributable to a failure to express the high affinity IL-2 receptor, because after antigen exposure a large fraction of the DO11 cells expressed high levels of CD25 (comparable to the expression of CD25 on endogenous Foxp3+ cells) (Fig. 3B). Upon activation, the T cells also expressed other components of the IL-2R, the IL-2/15Rβ and common γ chains (Fig. 3C). Early induction of pSTAT5 in Tregs but not in the antigen-responding DO11 cells was also seen after immunization with antigen and complete Freund’s adjuvant (data not shown). It has been suggested that induction of an effective immune response requires repeated or prolonged exposure to antigen (30, 31). To ask if repeated stimulation with antigen was required to induce pSTAT5 in T cells, naïve DO11 T cells were primed in vivo with antigen-pulsed DCs and re-exposed to antigen either 60 hours or five weeks later, the latter reflecting a recall response of previously primed memory T cells (11). Repeated stimulation of IL-2-producing DO11 cells or DO11 memory cells induced a rapid autocrine pSTAT5 response in the antigen-responsive T cells (Fig. 3D and E). Furthermore, bystander memory T cells were also capable of inducing pSTAT5 in response to naive DO11 T-cell activation (Fig. 3F). Thus, the ability to initiate STAT5 signaling is acquired only after repeated antigenic stimulation and is preserved in memory T cells, but it is absent in naïve T cells and is an inherent feature of regulatory T cells. The failure to activate IL-2-dependent STAT5 phosphorylation in the antigen-specific T cells may be because the secreted IL-2 is rapidly consumed by Tregs or because biochemical pathways relaying IL-2 signals are actively inhibited in naïve T cells and repeated stimulation or memory cell differentiation is required to remove this inhibition and acquire responsiveness to IL-2 signals. When naïve DO11 T cells were primed in Treg-deficient hosts, they still failed to activate autocrine STAT5 signals, indicating that consumption of IL-2 by Foxp3+ Tregs (32) does not explain the absence of pSTAT5 in DO11 T cells (Figure 4A). Furthermore, administration of potent IL-2/anti-IL-2 antibody complexes (21) during priming also failed to induce pSTAT5 in DO11 cells (Fig. 4 B). These results suggest the possibility of an inhibitory mechanism in naïve T cells that is released upon repeated antigen encounter. Suppressors of cytokine signaling (SOCS) proteins inhibit STAT signaling, and expression of SOCS-3 mRNA and protein is known to decrease after activation of helper T cells (33). We confirmed that SOCS-3 mRNA expression is reduced in activated DO11 T cells (data not shown), suggesting this protein may regulate STAT5 signaling in antigen-stimulated T cells. Western blot analysis revealed diminished presence of the protein in CD4+Foxp3+ Tregs compared to naïve T cells (Fig. 4C), confirming a previous report showing translational regulation of SOCS-3 in Tregs (34). These data potentially explain the enhanced responsiveness of Tregs to IL-2 and are in agreement with other recent studies suggesting that diminished levels of SOCS-1 and -3 are required for robust STAT5 signaling in Tregs (34, 35).
The activation of STAT5 indicates that Foxp3+ cells are responding to IL-2 but it does not reveal the functional outcome of this response. To address this question, we assayed cycling and functional activity of Foxp3+ T cells exposed to IL-2. Following antigen-induced activation of DO11 T cells, the endogenous Foxp3+CD25+ cells showed increased expression of both Foxp3 and CD25 (Fig. 5A). Furthermore, 48 hours following antigen exposure, up to 50% of these cells stained positively for Ki-67, a marker of cell division, and the frequency of Foxp3+ cells almost doubled over time (Fig. 5B). To ask if exposure to IL-2 also altered the functional activity of the Foxp3+ regulatory T cells, BALB/c mice were treated with IL-2/anti-IL-2 antibody complexes. This treatment induced high levels of pSTAT5 specifically in Foxp3+ T cells, as well as expansion of these cells (Fig. 5C). In these treated mice, antigen-induced proliferation of transferred DO11 cells, as measured by CFSE dye dilution, was markedly inhibited (Fig. 5D). Thus, IL-2 acts as a signal for Foxp3+ T cells to become potent suppressors. It is noteworthy that Il-2−/− T cells show enhanced responses to antigen under certain conditions (26), presumably because they are unable to activate Tregs.
In most of the experiments described thus far, IL-2 was induced in vivo from transferred TCR transgenic cells exposed to their cognate antigen. The reasons for using this approach are that we could formally establish the physiologic requirement for IL-2 production from cells responding to the antigen (by using cells from the same TCR transgenic crossed with Il-2−/− mice), and we could readily distinguish the effects of IL-2 on the antigen-specific cells from those on endogenous (“bystander”) cells. However, a concern with studies using cells from TCR transgenic mice is that the high frequency of antigen-specific lymphocytes may not accurately reflect the normal immune repertoire. To ask if activation of endogenous Foxp3+ cells also accompanies a conventional immune response, normal mice were infected with vaccinia virus and STAT5 phosphorylation was assayed by flow cytometry. Viral infection led to a rapid dose- and time-dependent induction of pSTAT5 in Foxp3+ CD4+ T cells (Fig. 6A)(36). Mice that had been previously infected showed a more rapid response of Foxp3+ T cells upon rechallenge with the virus, presumably reflecting the accelerated kinetics of IL-2 production in memory T cells (Fig. 6B and C). The Foxp3+ T cells also stained for Ki-67, indicating commitment to cell division (Fig. 6D). Thus, in a conventional immune response to a virus, as we observed using a TCR transgenic approach, the endogenous Foxp3+ T cells are early responders to IL-2 produced by virus-specific T cells.
Our experiments looking at the single cell biochemistry of lymphocytes in an immune response in vivo have shown that endogenous Foxp3+ regulatory T cells receive signals from antigen-stimulated cells early in the response. Surprisingly, IL-2, generally believed to function as the key growth factor for antigen-reactive T cells, was identified as the “messenger” cytokine instructing Foxp3+ T cells to proliferate, increase expression of their essential transcription factor Foxp3, and enhance their suppressive activity. The autocrine action of IL-2 on the antigen-responding T cells, as measured by pSTAT5 induction, is not seen in the early phase of the response, despite high levels of CD25 expression. Since the absence of STAT5 signaling was also observed in cells stimulated in the absence of Tregs and competing endogenous T cell populations (Fig. 4A), a cell-intrinsic inhibitory mechanism likely regulates IL-2 signal transduction. SOCS proteins play important roles in innate and adaptive immune responses by regulating cytokine responses (37). SOCS-1 and SOCS-3 have been shown to inhibit STAT5 signals downstream of IL-2 (38)(39). SOCS-1-deficient T cells show enhanced responsiveness to γc cytokines and such uncontrolled cytokine responses may result in autoimmunity (37). Thus the presence of SOCS-1 and SOCS-3 in naive T cells may explain their initial inability to transduce STAT5 signals in response to IL-2. Consequently, it would be predicted that T cells signaling through STAT5, such as Tregs and memory cells, express low levels of these inhibitory molecules. In support of this hypothesis, several recent studies have demonstrated a reduced presence of SOCS proteins in Tregs and activated T cells (34)(33)(35), resulting in increased responsiveness to IL-2. Our results thus suggest that there are important biochemical differences in how Tregs respond to their environment as compared to the naïve T cells they are meant to regulate. On the other hand, memory cells responded to IL-2 in a similar manner as Tregs, phosphorylating STAT5 early after antigenic stimulation. Although the functional significance of these early STAT5 signals in memory cells is unclear at this time, some intriguing hypotheses can be made. For example, the generation of fast recall responses to antigen by memory cells may be dependent on their competence to receive early IL-2 signals. In addition, the enhanced sensitivity to cytokines of memory cells may render these cells less susceptible to regulation by Tregs, which potentially also contributes to their increased antigen reactivity.
What might be the functional consequences of the IL-2-dependent activation of endogenous regulatory T cells? It is likely that most endogenous Tregs are thymus-derived, “natural” Tregs, presumably specific for self-antigens (5, 40). It may be that early activation of these Tregs is a mechanism to minimize the risk of autoimmunity during an immune response to foreign antigens. Early activation of Tregs may also provide a constraint, or buffer, on immune responses to foreign antigens, in order to prevent pathologic side-effects of physiologic immunity. Only after this “safety net” of regulatory cells is established would the antigen-specific response develop. Although rather high precursor frequencies of TCR transgenic T cells were necessary to elicit STAT5 signaling in the majority of Tregs residing in the lymphoid organs, our experiments with vaccinia virus infection demonstrate that a broad activation of Tregs is feasible under physiological circumstances. Also, while systemic Treg activation is unlikely after initiation of an immune response by a limited number of antigen-specific T cells, it is likely that Tregs localized in the vicinity of the responding T cells will receive IL-2 signals and control the environment where the response takes place. The strong activation of the regulatory compartment after the initiation of an immune response also raises the obvious question of how protective immune responses develop in the presence of a large number of activated Tregs. One possibility is that in the event of an infection specific signals, e.g. through the Toll pathway, are overriding the suppressors creating a permissive environment for the response to develop, as has been suggested by Pasare and Medzhitov (41). In a prolonged or secondary response, effector/memory T cells may directly inhibit Treg generation (42).
Thus, IL-2 may be the prototype of a signaling molecule that serves to first establish controls on immune responses and then to promote the development of such responses. Such a scenario also suggests that limiting the transient IL-2-dependent activation of regulatory cells may serve to maximize the effectiveness of vaccines.
Supported by NIH grants RO1 AI073656 and PO1 AI35297 (AKA) and RO1 AI065824, P01 AI36535 and NHLBI contract N01-HV-28183 (GN).