Peripheral tolerance is maintained in part by thymically derived CD25+CD4+ T cells (regulatory T cells (Tregs)). Their mechanism of action has not been well characterized. Therefore, to get a better understanding of Treg action, we investigated the kinetics of murine Treg activity in vitro. Tregs were suppressive within a surprisingly narrow kinetic window: necessary and sufficient only in the first 6–10 h of culture. Visualization of this time frame, using a sensitive single-cell assay for IL-2, revealed the early elaboration of target cell IL-2 producers in the first 6 h despite the presence of CD25+CD4+ Tregs. However, after 6 h, a rapid rise in the number of IL-2 producers in the absence of Tregs was dramatically abrogated by the presence of Tregs. Importantly, the timing of suppression was dictated by the kinetics of target T cell activation suggesting that early target T cell signals may alter susceptibility to suppression. Modulating target T cell activation signals with provision of CD28, IL-2, or high Ag dose all abrogated suppression of proliferation late in culture. However, only CD28 signals enabled target T cells to resist the early Treg-induced down-regulation of IL-2. Therefore the quality of early target T cell activation signals, in particular engagement of CD28, represents an important control point in the balance between vulnerability and resistance to Treg suppression.

The CD25+CD4+ regulatory T cell (Treg)4 is emerging as a key regulator of immune responses (1). Although first described to regulate peripheral self-tolerance and to protect against autoimmunity, the CD25+ Tregs have also been implicated in suppressing a number of other immune responses including those to infectious agents, tumors, and transplants (2, 3, 4). Whether such disparate immune responses are regulated by the same Treg population or by functionally distinct subsets of suppressors is uncertain. However, given their broad suppressive action, it is unclear how the activity of Tregs is regulated to ensure that beneficial immune responses to infectious stimuli proceed unabated, whereas other responses are suppressed. The up-regulation of B7 expression (CD80 and CD86) by APCs represents a central event in the activation of naive T cells to infectious agents and may serve as a mechanism to disrupt Treg tolerance either by directly abrogating Treg activity or by rendering effector T cells unresponsive to suppression.

The mechanism of Treg activity is poorly understood. IL-2 from nonregulatory CD4+ T cells appears critical for the survival and suppressive activity of CD25+CD4+ Tregs (5, 6, 7, 8, 9) and is ironically also a target of Treg suppression (10, 11). Although IL-2 mRNA and protein production in T cells are ablated in the presence of Tregs, the kinetics and mechanism of shut-down is not well defined (12). It remains controversial whether the down-regulation of IL-2 mRNA is an active process or a passive one due to IL-2 consumption (7, 13). Interestingly, although not required for in vitro suppression, the cytokines IL-10 and TGFβ appear important mediators of Treg function in vivo (14, 15, 16). Thus Tregs may regulate immune responses in a number of different ways, and the mechanism may depend on whether Tregs are suppressing the initiation of T cell activation or down-regulating existing immunity.

The role of CD28/CTLA-4 and B7-mediated (CD80 and CD86) regulation of immune responses to both self and foreign Ags is complex (17). Addition of anti-CD28 mAb, or provision of APC expressing high levels of CD80 and CD86, to the in vitro suppression assay clearly abrogates suppression (10, 18, 19). However, it has been difficult to interpret interventions that target these costimulatory pathways in vitro and in vivo because it is unclear which T cell subset is being targeted (effector or regulator). Although CD28 expression is required for Treg homeostasis (20, 21) it appears not to be required for their effector function (22, 23). However, it is not known, in circumstances where B7 is up-regulated such as inflammatory sites, whether CD28 signals may directly antagonize Treg activity or enable effector T cells to resist suppression.

To address Treg activity in a mechanistic fashion, we developed assays to define the timing at which Tregs exert their function with respect to the down-regulation of IL-2 production. We demonstrate an early window of Treg action in the first 6–10 h of T cell activation, the kinetics of which is driven by the activation status of the target T cell population. Analysis of IL-2 production at the single-cell level reveals a distinct Treg-induced down-regulation of IL-2 after 6 h of stimulation. CD28 engagement on the responders, not the Tregs, disrupts the early IL-2 kinetics by accelerating and amplifying the IL-2 response of target T cells. In parallel, increasing CD28 signaling blocks the ability of Tregs to down-regulate early IL-2. Although CD28, IL-2, and high Ag dose all abrogate suppression of proliferation at late stages in the culture, only CD28 engagement enables target T cells to resist the early down-regulation of IL-2 by Tregs. Thus we have identified an early CD25+ T cell regulatory event that occurs before cell division and that is uniquely abrogated by CD28 engagement.

Female wild-type (WT), CD28-deficient, and DO11.10+ BALB/c mice (both Thy1.2+) were purchased from The Jackson Laboratory. BALB/c Thy1.1+ mice were obtained from Dr. J. Sprent (The Scripps Research Institute, San Diego, CA) via Dr. R. Dutton (Trudeau Institute, Saranac Lake, NY). WT15 TCR transgenic mice (Leishmania homolog of human receptor for activated C kinase (LACK) reactive) were provided by N. Killeen (University of California, San Francisco, CA) (24). All mouse maintenance and experimentation has been reviewed and approved by the University Committee for Animal Resources, University of Rochester Medical School.

Cells were cultured in RPMI 1640 (25) with 10% heat-inactivated FCS. All Abs were purchased from BD Pharmingen.

CD4+ T cells from 6- to 10-wk-old mice were enriched from spleen and lymph node by C′ lysis of CD8, MHC class II, and heat-stable Ag-bearing cells as described previously (25). CD25+ and CD25 CD4+ T cells were further isolated by MACS (Miltenyi Biotec) or by FACS (BD Biosciences; FACSVantage and Aria) where indicated. T-depleted splenocytes (APCs) were prepared by depletion of Thy1.2-bearing cells by C′ lysis and given 2500 rad irradiation. Isolation or removal of Tregs from coculture was performed by MACS-positive selection for Thy1.2-expressing cells (97 ± 1.4% effective).

T cells (107/ml) were incubated with 5 μM CFSE (Molecular Probes) for 5 min at room temperature and washed three times before culture. A total of 1 × 105 CD25CD4+ T cells were cultured in triplicate with 1 × 105 APC and 1 μg/ml anti-CD3 mAb (2C11) with/without CD25+CD4+ T cells in 96-well plates. Where indicated, 1 μg/ml anti-CD28 mAb (37.51) was added to cultures. Cells were incubated for 72 h at 37°C and proliferation was measured by FACS or [3H]thymidine incorporation (1 μCi for the last 6 h of culture). Exogenous IL-2 (50 U/ml) was added to cultures where indicated. Ag-specific assays were performed with CD25+CD4+ Tregs from DO11.10 TCR transgenic mice and CD25CD4+ responders from the LACK-reactive WT15 TCR transgenic mice (24). Both transgenes encode an IAd-restricted TCR and are >10 generations backcrossed to BALB/c. A total of 1 × 105 CD25CD4+ WT15 T cells (LACK specific) were cultured in triplicate with 1 × 105 APC, both LACK and OVA peptide, and with/without CD25+CD4+ DO11.10 T cells (OVA-specific). Ag doses were determined empirically. Peptide was kept constant for the DO11.10 Tregs (0.2 μM OVA). WT15 responders were only fully suppressed at 0.08 μM LACK peptide (low [Ag]) and fully resistant to suppression at 0.2–1 μM LACK peptide (high [Ag]).

Real-time fluorogenic 5′ nuclease (RT) PCR for IL-2 was performed, on Thy1.1+ responders purified from coculture, with primers from ABI systems using the ABI Prism 7700 Sequence BioDetector. CD3δ was used as the endogenous control and mRNA from unstimulated CD4+ T cells used as the calibrator. The cytokine capture assay was performed essentially as described (26). In vitro-activated cells were labeled with the bifunctional Ab “catch” reagent (CD45/IL-2; Miltenyi Biotec) for 5 min on ice and warmed to 37°C for 45 min to allow for IL-2 secretion. trans-capture of the cytokine was avoided by reducing the cell number used (6 × 105 T cells per time point) and by performing the secretion step in a large volume of medium (3 × 104 cells/ml) (26). Cell surface-bound IL-2 was detected using a second fluorochrome-conjugated IL-2 mAb and analyzed by FACS. T cells were gated for viability by exclusion of 7-aminoactinomycin D (7AAD) and expression of CD4.

Studies of Treg suppression have been hampered by a lack of information on exact timing of Treg activity in the 3-day in vitro coculture system (CD25 responders/targets, CD25+ Tregs, APC, and anti-CD3). Recent studies showing Tregs require IL-2 from the responders (7) and the down-regulation of glucocorticoid-induced TNFR family-related receptor ligand (27) for suppressive activity, have suggested that Treg activity is a relatively late event and may occur subsequent to, rather than during, the initiation of an immune response. We first looked at when and for how long Tregs were required in coculture to mediate suppression. Using congenic markers on the T cell responders (CD25CD4+) and regulators (CD25+CD4+), we either removed Tregs at various time points after T cell activation or added in Tregs at various time points and measured proliferation at 72 h (Fig. 1). For the Treg removal studies, supernatants from coculture were isolated and Tregs were depleted by positive magnetic bead separation (based on Thy1.2 expression) at various time points. The remaining cells (CD25 T cells and APC, routinely 98% negative for CD25+ Tregs) were resuspended in their original supernatant and returned to culture. Control cultures not containing Tregs were treated in the same way (passed over a depletion column and returned to culture in their own supernatant) and proliferated similarly to unmanipulated cultures of CD25 T cells at 72 h. Surprisingly, the presence of Tregs in the culture for as little as 2 h had a clear effect on subsequent proliferative ability of the responder cells (50% reduction in proliferation) (Fig. 1 A). Between 6 and 12 h, suppression was complete and the presence of Tregs was no longer required to maintain the suppressive phenotype; despite this being a time point at which the control responder cells were still dependent on APC signals for proliferation and when stimulatory APC function was still evident (data not shown). In agreement with published data, supernatants from the cocultures were not sufficient to mediate the decreased proliferation observed on removal of Tregs (data not shown). These results suggest that Tregs mediate their activity within the first 6–10 h of culture, before cell division, and are not subsequently required for the maintenance of the nonproliferative phenotype in the short-term, 72-h suppression assay.

FIGURE 1.

Early kinetic window of target T cell susceptibility to CD25+CD4+ T cell suppression. A, CD25CD4+Thy1.1+ T cells were stimulated with anti-CD3 mAb and APC alone (▨) or in coculture with CD25+CD4+Thy1.2+ T cells (▪) (1:1 ratio, CD25+:CD25). At indicated time points, Thy1.2+ T cells were removed from culture by MACS separation and remaining Thy1.1+ T cells (96% Thy1.2 negative) and APC were returned to culture with original supernatants for the remainder of a 72-h culture (▦). Cultures were pulsed with [3H]TdR for the last 6 h of culture, and cpm are the mean of triplicate wells. Data points are pooled (mean ± SE) from three separate experiments. B, Cultures were set up as in A. CD25+CD4+Thy1.2+ T cells were either added at the start of culture (0 h) or to established cultures of CD25CD4+Thy1.1+ cells at indicated time points (▦). The CD25+CD4+Thy1.2+ T cells were isolated from parallel anti-CD3-stimulated cocultures by MACS. Their suppressive potential was tested by addition to the start of culture of freshly isolated CD25 T cells (▪). Cultures were incubated for 72 h from time of CD25CD4+ T cell stimulation and pulsed as in A. Results represent one of three comparable experiments.

FIGURE 1.

Early kinetic window of target T cell susceptibility to CD25+CD4+ T cell suppression. A, CD25CD4+Thy1.1+ T cells were stimulated with anti-CD3 mAb and APC alone (▨) or in coculture with CD25+CD4+Thy1.2+ T cells (▪) (1:1 ratio, CD25+:CD25). At indicated time points, Thy1.2+ T cells were removed from culture by MACS separation and remaining Thy1.1+ T cells (96% Thy1.2 negative) and APC were returned to culture with original supernatants for the remainder of a 72-h culture (▦). Cultures were pulsed with [3H]TdR for the last 6 h of culture, and cpm are the mean of triplicate wells. Data points are pooled (mean ± SE) from three separate experiments. B, Cultures were set up as in A. CD25+CD4+Thy1.2+ T cells were either added at the start of culture (0 h) or to established cultures of CD25CD4+Thy1.1+ cells at indicated time points (▦). The CD25+CD4+Thy1.2+ T cells were isolated from parallel anti-CD3-stimulated cocultures by MACS. Their suppressive potential was tested by addition to the start of culture of freshly isolated CD25 T cells (▪). Cultures were incubated for 72 h from time of CD25CD4+ T cell stimulation and pulsed as in A. Results represent one of three comparable experiments.

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Conversely, responding T cells become refractory to Treg suppression by 12 h (Fig. 1,B). CD25 T cells were stimulated with APC and anti-CD3 mAb and CD25+CD4+ T cells added at the start of culture (0 h) or at various times after CD25 T cell activation. To ensure appropriate activation conditions for the Tregs, the CD25+CD4+ T cells added to the culture were isolated from parallel cocultures in which Tregs were actively exerting suppressive activity. Indeed, Tregs isolated from coculture at all time points were able to suppress freshly stimulated CD25 responders (Fig. 1 B). However, CD25+CD4+ Tregs were only able to reverse the proliferative program if added to established cultures before 12 h of culture. CFSE-dilution experiments confirmed that the majority of counts were derived from proliferating responders. However, higher proliferative counts were seen in coculture when Tregs were added at 12 and 24 h and data from CFSE experiments were consistent with the additional counts coming from proliferating Tregs (data not shown). These data define a narrow window of susceptibility to Treg suppression before cell division; the presence of Tregs being necessary and sufficient within the first 6–12 h of initial activation.

Our studies on the time at which Tregs were active in coculture suggested that much of the suppressive activity occurred before cell division. A number of recent observations suggest that IL-2 production by T cells is rapid and remarkably transient, with the decline in IL-2 production occurring before cell division (26, 28). To visualize early events in IL-2 production that might be regulated by Tregs we used congenic T cell markers and the cytokine capture assay (Miltenyi Biotec) to track the emergence and decline of IL-2 secretors within the responder population in coculture (Fig. 2). In the absence of Tregs, IL-2 secretors were detectable as early as 2 h, peaked at 12 h, and rapidly declined by 24 h (Fig. 2, A and B), similar to the transient kinetics observed upon in vivo activation (26). Cell division was not detectable by CFSE dilution at the 24-h time point (data not shown). To control for T cell density when interpreting the addition of an equal number of Tregs (1 × 105 CD25 and 1 × 105 CD25+), we analyzed the IL-2 response in the presence of twice as many responders (2 × 105 CD25 cells) and observed similar kinetics and proportion of IL-2 secretors to that of 1 × 105 CD25 T cells (Fig. 2,B). Unless otherwise stated, subsequent experiments controlled for cell density in this way. Interestingly, the early 4- to 6-h kinetics and magnitude of IL-2 production in anti-CD3 cultures were unperturbed in the presence of Tregs. However, the presence of Tregs blocked the rapid rise in the number of IL-2 producers after 6 h and led to a gradual decline in the number of IL-2 secretors (Fig. 2,B). The IL-2 secretion profile closely mirrored the pattern of IL-2 mRNA expression in the responders (Fig. 2,C) and preceded detection of IL-2 in supernatants by ELISA (Fig. 2 D). We found no evidence that this decline was due to the death of IL-2-producers in the cocultures (using 7AAD as a viability marker). Thus, as implicated in previous publications and now observed at the single-cell level, the presence of Tregs in the coculture supports the initial activation of responders (7, 29), but is quickly followed by a dramatic block in the rapid rise in IL-2.

FIGURE 2.

Single-cell kinetic analysis of CD25+CD4+ T cell suppression. A, CD25CD4+ Thy1.1+ T cells were cultured as in Fig. 1 alone or in coculture with CD25+CD4+Thy1.2+ T cells (1:1 ratio) for various time points before measurement of T cells positive for IL-2 secretion by the cytokine capture assay. FACS plots are gated on live CD4+7AAD cells. The number in the top right quadrant is the Thy1.1+ T cells positive for IL-2. The average percentage of IL-2+Thy1.1+ T cells before stimulation was 0.10 ± 0.09%. Percentage of IL-2+ Thy1.1-negative Tregs (Thy1.2+) ranged from 0.10 to 1.1%. Control plots show background staining directly ex vivo without stimulation (0 h, lower left plot) and responders alone stimulated with anti-CD3 for 12 h (peak of IL-2 response) stained for IL-2 in the absence of the bifunctional (CD45/IL-2) Ab catch reagent (no catch, lower right plot). Plots represent 1 of 10 comparable experiments. B, Cells were stimulated as in A alone (open symbols) or in coculture (closed symbols) and measured for IL-2 secretion at various time points; the percentage represents the proportion of live Thy1.1+ responders that were IL-2 positive. C, Quantitative RT-PCR on mRNA from Thy1.1+ responders isolated from cultures at the same time points as cytokine capture analysis. D, Supernatants from cells cultured as in B, were assayed for IL-2 by ELISA; detection limit for IL-2 was 0.01 ng/ml. Results in BD are from a single experiment and represent one of three similar experiments.

FIGURE 2.

Single-cell kinetic analysis of CD25+CD4+ T cell suppression. A, CD25CD4+ Thy1.1+ T cells were cultured as in Fig. 1 alone or in coculture with CD25+CD4+Thy1.2+ T cells (1:1 ratio) for various time points before measurement of T cells positive for IL-2 secretion by the cytokine capture assay. FACS plots are gated on live CD4+7AAD cells. The number in the top right quadrant is the Thy1.1+ T cells positive for IL-2. The average percentage of IL-2+Thy1.1+ T cells before stimulation was 0.10 ± 0.09%. Percentage of IL-2+ Thy1.1-negative Tregs (Thy1.2+) ranged from 0.10 to 1.1%. Control plots show background staining directly ex vivo without stimulation (0 h, lower left plot) and responders alone stimulated with anti-CD3 for 12 h (peak of IL-2 response) stained for IL-2 in the absence of the bifunctional (CD45/IL-2) Ab catch reagent (no catch, lower right plot). Plots represent 1 of 10 comparable experiments. B, Cells were stimulated as in A alone (open symbols) or in coculture (closed symbols) and measured for IL-2 secretion at various time points; the percentage represents the proportion of live Thy1.1+ responders that were IL-2 positive. C, Quantitative RT-PCR on mRNA from Thy1.1+ responders isolated from cultures at the same time points as cytokine capture analysis. D, Supernatants from cells cultured as in B, were assayed for IL-2 by ELISA; detection limit for IL-2 was 0.01 ng/ml. Results in BD are from a single experiment and represent one of three similar experiments.

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The 4- to 6-h delay in suppressive activity in the coculture could be driven by time restraints on the acquisition of Treg activity or be due to the time-dependent acquisition of a critical “target for suppression” by the responders. To determine the cell type that drives the kinetics of suppression, we preactivated CD25+CD4+ T cells in coculture conditions that support the functional activation of Tregs, isolated them from coculture at various time points, and added them to a fresh culture of CD25 responders. Surprisingly, the 4- to 6-h lag in suppressive activity was still observed even with preactivated Tregs (Fig. 3). Therefore, the kinetic restraint on suppression in the first 4–6 h appears to be dictated by the activation status of the responders and not the Tregs.

FIGURE 3.

Kinetics of suppression set by the target T cells. CD25CD4+Thy1.1+ T cells were cocultured as in Fig. 1 alone (○), with freshly isolated CD25+CD4+Thy1.2+ T cells (•) or with 6-h preactivated CD25+CD4+Thy1.2+ T cells isolated from anti-CD3-stimulated cocultures (▦) before measurement of IL-2 secretion by cytokine capture at given time points. Percentage denotes proportion of live Thy1.1+ responders that were IL-2 positive. Similar results were obtained with Tregs preactivated for 9 or 12 h. Results represent one of three comparable experiments.

FIGURE 3.

Kinetics of suppression set by the target T cells. CD25CD4+Thy1.1+ T cells were cocultured as in Fig. 1 alone (○), with freshly isolated CD25+CD4+Thy1.2+ T cells (•) or with 6-h preactivated CD25+CD4+Thy1.2+ T cells isolated from anti-CD3-stimulated cocultures (▦) before measurement of IL-2 secretion by cytokine capture at given time points. Percentage denotes proportion of live Thy1.1+ responders that were IL-2 positive. Similar results were obtained with Tregs preactivated for 9 or 12 h. Results represent one of three comparable experiments.

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We predicted that the early window of susceptibility to suppression would represent an important control point in the balance between vulnerability and resistance to suppression. CD28 signaling is a key regulator of early IL-2 production and is also known to disrupt CD25+CD4+ Treg suppression (10, 18). We used the cytokine capture assay to assess the contribution of CD28 signaling to the early activation and suppression events (Fig. 4). In the absence of Tregs, addition of CD28 mAb led to an accelerated onset of IL-2 production peaking at 6 h. In addition, there was an increase in the magnitude of the response, 40% IL-2-positive at the peak with CD3+CD28 compared with 15–20% with CD3 alone. This shift in the kinetic response correlated with a complete abrogation of CD25+CD4+ T cell-mediated early IL-2 down-regulation (Fig. 4,A). Neither preactivation of the Tregs to compensate for the earlier kinetics (Fig. 4 B), nor the presence of 2- to 3-fold more Tregs to compensate for the increase in the number of IL-2 producers with CD28 (data not shown), restored suppressive activity.

FIGURE 4.

CD28 disrupts the early window of responder T cell susceptibility to suppression. A, CD25CD4+ and CD25+CD4+ T cells were isolated from congenic WT mice and stimulated alone or in coculture for various time points before measurement of T cells positive for IL-2 secretion by cytokine capture. Percentage denotes proportion of live Thy1.1+ responders that were IL-2 positive. Data points are pooled (mean ± SE) from seven separate experiments. B, CD25+CD4+Thy1.2+ T cells were freshly isolated (▦) or preactivated as in Fig. 3 (△) and added to cultures of CD25CD4+ T cells stimulated with APC and anti-CD3 ± anti-CD28 mAb. Results represent one of three comparable experiments.

FIGURE 4.

CD28 disrupts the early window of responder T cell susceptibility to suppression. A, CD25CD4+ and CD25+CD4+ T cells were isolated from congenic WT mice and stimulated alone or in coculture for various time points before measurement of T cells positive for IL-2 secretion by cytokine capture. Percentage denotes proportion of live Thy1.1+ responders that were IL-2 positive. Data points are pooled (mean ± SE) from seven separate experiments. B, CD25+CD4+Thy1.2+ T cells were freshly isolated (▦) or preactivated as in Fig. 3 (△) and added to cultures of CD25CD4+ T cells stimulated with APC and anti-CD3 ± anti-CD28 mAb. Results represent one of three comparable experiments.

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It is not known whether the disruption of suppression with CD28 engagement directly abrogates Treg suppressive activity or rather provides signals to the responders to escape suppression. We used cells isolated from CD28-deficient or WT mice to restrict CD28 signals to either the Tregs or responders and performed standard suppression assays (Fig. 5). FACS-purified CD25+CD4+ Tregs were used in these studies as they themselves fail to proliferate in response to anti-CD3 and anti-CD28 (7, 30) (Fig. 5, “CD25+ alone” columns). As described by others, addition of anti-CD28 to WT cultures abrogated suppression (Fig. 5, left panel). Restriction of CD28 ligation to the CD25+CD4+ subset failed to abrogate their suppressive activity (Fig. 5, middle panel). In contrast, provision of additional CD28 signals restricted to the CD25 responders rendered them refractory to suppression (Fig. 5, right panel). Thus the target for CD28-mediated reversal of suppression appears to be the CD25 responder T cell. We suggest that CD28 provides a distinct signal to the target T cells that renders them resistant to early IL-2 suppression.

FIGURE 5.

CD28 engagement does not abrogate Treg activity but does enable targets to escape suppression. CD25CD4+ and CD25+CD4+ T cells were isolated from WT or CD28-deficient (CD28−/−) mice as designated and cultured alone or in coculture with APC and anti-CD3 mAb ± anti-CD28 mAb. CD28 engagement was restricted to CD25+ T cells (middle panel) or to target CD25 T cells (right panel). Cultures were pulsed with [3H]TdR for the last 6 h of a 72-h culture. Results represent one of three comparable experiments.

FIGURE 5.

CD28 engagement does not abrogate Treg activity but does enable targets to escape suppression. CD25CD4+ and CD25+CD4+ T cells were isolated from WT or CD28-deficient (CD28−/−) mice as designated and cultured alone or in coculture with APC and anti-CD3 mAb ± anti-CD28 mAb. CD28 engagement was restricted to CD25+ T cells (middle panel) or to target CD25 T cells (right panel). Cultures were pulsed with [3H]TdR for the last 6 h of a 72-h culture. Results represent one of three comparable experiments.

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In addition to CD28, a variety of stimuli that increase the potency of T cell stimulation have also been shown to abrogate Treg function including high Ag dose, TLR signals, and provision of exogenous IL-2 (10, 18, 19, 31). Although the readout of these manipulations is the reversal of suppression as measured by proliferation at 72 h, this end point may have been reached via disparate mechanisms. To begin to address potential mechanistic differences we compared the effects of CD28, exogenous IL-2, and high Ag dose on the early 6- to 12-h IL-2 regulatory events described here. Fig. 6 compares the early IL-2 down-regulation before division, using the cytokine capture assay, with 72-h read outs for proliferation using both CFSE and [3H]thymidine assays. CD28 signaling was distinct in its ability to both accelerate the kinetics of IL-2 production and to ablate the early down-regulation of IL-2 production by Tregs in both Ab and peptide-stimulated cultures (Figs. 6,A and 7). In contrast, exogenous IL-2 (a potential downstream mediator of the CD28 effects) failed to alter the kinetics or magnitude of IL-2 production and did not block the early down-regulation of IL-2 by CD25+CD4+ T cells (Fig. 6 B), in accordance with previous work (7). That the reduction in the number of IL-2 producers by Tregs occurred in the presence of excess exogenous IL-2 argues against a simple IL-2-consumption model for the regulatory effects of CD25+CD4+ T cells in vitro. In addition, the data suggest that the early abrogation of Treg suppression by CD28 cannot be explained by a simple enhancement in the available IL-2 pool.

FIGURE 6.

Distinct action of CD28 in the regulation of IL-2 suppression. Measurements for IL-2 secretion (cytokine capture) and proliferation (CFSE and [3H]TdR incorporation) were compared in cocultures of anti-CD3-stimulated CD25CD4+ and CD25+CD4+ T cells with the addition of anti-CD28 mAb (A) or exogenous IL-2 (B). C, Peptide-stimulated cultures: CD25+CD4+ T cells from DO11.10 TCR transgenic mice and CD25CD4+ T cell from LACK-reactive WT15 TCR transgenic mice were stimulated with APC and 0.2 μM OVA and LACK peptide (0.08 μM for low [Ag] and 1 μM LACK peptide for high [Ag]). For cytokine capture, cells were harvested from cultures at given time points for measurement of IL-2 secretion. Percentage denotes proportion of live CD4+ targets (Thy1.1+ cells for Ab stimulations, Vβ4-positive KJ1-26-negative cells for peptide stimulation) that were IL-2 positive. Middle panel, FACS analysis of CFSE-labeled responder cells was performed at 72 h of culture. Right panel, Cultures were pulsed with [3H]TdR for the last 6 h of a 72-h culture. Results represent one of at least three comparable experiments.

FIGURE 6.

Distinct action of CD28 in the regulation of IL-2 suppression. Measurements for IL-2 secretion (cytokine capture) and proliferation (CFSE and [3H]TdR incorporation) were compared in cocultures of anti-CD3-stimulated CD25CD4+ and CD25+CD4+ T cells with the addition of anti-CD28 mAb (A) or exogenous IL-2 (B). C, Peptide-stimulated cultures: CD25+CD4+ T cells from DO11.10 TCR transgenic mice and CD25CD4+ T cell from LACK-reactive WT15 TCR transgenic mice were stimulated with APC and 0.2 μM OVA and LACK peptide (0.08 μM for low [Ag] and 1 μM LACK peptide for high [Ag]). For cytokine capture, cells were harvested from cultures at given time points for measurement of IL-2 secretion. Percentage denotes proportion of live CD4+ targets (Thy1.1+ cells for Ab stimulations, Vβ4-positive KJ1-26-negative cells for peptide stimulation) that were IL-2 positive. Middle panel, FACS analysis of CFSE-labeled responder cells was performed at 72 h of culture. Right panel, Cultures were pulsed with [3H]TdR for the last 6 h of a 72-h culture. Results represent one of at least three comparable experiments.

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FIGURE 7.

CD28 abrogates suppression at high Ag dose. CD25+CD4+ T cells from DO11.10 TCR transgenic mice and CD25CD4+ T cell from LACK-reactive WT15 TCR transgenic mice were stimulated with APC and 0.2 μM OVA and 1 μM LACK peptide (high [Ag]) as in Fig. 6 C. Cultures were stimulated with or without the further addition of anti-CD28 mAb. Percentage denotes proportion of live CD4+ targets (Vβ4 positive, KJ1-26-negative cells) that were IL-2 positive. Results represent one of two comparable experiments.

FIGURE 7.

CD28 abrogates suppression at high Ag dose. CD25+CD4+ T cells from DO11.10 TCR transgenic mice and CD25CD4+ T cell from LACK-reactive WT15 TCR transgenic mice were stimulated with APC and 0.2 μM OVA and 1 μM LACK peptide (high [Ag]) as in Fig. 6 C. Cultures were stimulated with or without the further addition of anti-CD28 mAb. Percentage denotes proportion of live CD4+ targets (Vβ4 positive, KJ1-26-negative cells) that were IL-2 positive. Results represent one of two comparable experiments.

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It is unclear whether CD28 engagement leads to a qualitatively different T cell signal or whether it simply amplifies the TCR signal cascade (32). If CD28 does lead to quantitative differences in TCR signal then we might expect to see a similar alteration in the kinetics of IL-2 production and suppression when amplifying TCR signals alone. To enable manipulation of the target T cell signals alone and not the Tregs, we used a TCR transgenic system for in vitro suppression (CD25+ Tregs from DO11.10 and CD25 responders from LACK-reactive WT15 TCR transgenics) where increasing Ag dose enables target T cells to “escape” suppression (19) (Fig. 6,C). Analysis of the elaboration of IL-2 producers revealed a similar pattern of Treg suppression with this peptide based stimulation compared with Ab stimulation; although the overall timing of IL-2 production was delayed (peaked at 24 h). As with Ab stimulation, there was an initial increase in the number of IL-2 producers both in the presence or absence of Tregs (6–12 h) that was followed by a rapid rise in IL-2 numbers in the absence of Tregs and concomitant contraction in the presence of Tregs (Fig. 6,C, low [Ag]). Increasing Ag dose failed to accelerate the kinetics of IL-2 production (high- and low-dose Ag peaking at 24 h) but did significantly alter the magnitude of the IL-2 response over that seen from low-dose Ag (Fig. 6,C). Despite such alterations in the magnitude of the IL-2 response, CD25+CD4+ T cells were still able to mediate an early reduction (albeit incomplete) in the frequency of IL-2 producers under high Ag dose conditions (Fig. 6 C).

To directly test whether CD28 provided a distinct signal to target T cells to resist suppression, anti-CD28 was added to the high Ag dose cultures (Fig. 7). Similar to its effect on anti-CD3 cultures, addition of anti-CD28 to peptide-stimulated cultures also accelerated the timing and enhanced the number IL-2 producers (peaking at 12 h compared with 24 h with peptide alone). In addition, anti-CD28 prevented Treg down-regulation of early IL-2 production (Fig. 7). Thus CD28 does appear to provide distinct signals that disrupt Treg activity that cannot be compensated for by enhanced TCR signaling. We suggest that, unlike CD28, the apparent “abrogation” of suppression by exogenous cytokine and increased Ag dose is not mediated by qualitative changes in the target T cell’s ability to resist suppression but simply due to quantitative changes in available IL-2.

Our data provide new insight into the kinetics of CD25+CD4+ T cell mediated suppression of T cell proliferation and identify an early regulatory event before cell division. Interestingly, once suppression was established in the first 6–10 h, Tregs were no longer required to maintain the nonproliferative phenotype in short-term culture. With similar kinetics, 12-h activated responders become refractory to Treg suppression of proliferation. Thus the first 12 h of T cell activation represents an important window of susceptibility to Treg control. Highly reproducible was a surprising 50% suppression of proliferation by as little as 2 h of Treg encounter. Current literature would suggest that responder-derived IL-2 is an important mediator of Treg suppression (7, 9). The small amount of IL-2 produced at the 2-h time point (2–3% IL-2 producers by the capture assay) is not obviously consistent with such a scenario. From our analysis of both when Tregs are required and the kinetics of IL-2 suppression, we suggest that Tregs might have two separate effects on target T cells: an early curtailment of T cell activation that is IL-2 independent (2–6 h, Fig. 1,A) and a later IL-2-dependent phase (6–12 h, Fig. 2).

The use of the cytokine capture assay enabled the visualization of the early Treg “ambush”. Although the initiation of the IL-2 response proceeded unabated by Tregs, after 6 h Tregs abruptly terminated the developing IL-2 response. The kinetics of suppression was driven by the activation state of the target T cells. Therefore, we suggest that the timing of suppression is driven by the requirement for the acquisition of a suppressive target rather than the time for Tregs to acquire suppressive activity. However, the bi-phasic nature of IL-2 production and suppression in the cocultures may not be target T cell intrinsic but could also reflect cellular heterogeneity within the target T cell population. There may be a small proportion of cells that produce IL-2 early, but transiently, that are resistant to suppression. One obvious source of such early IL-2 is the small numbers of memory cells within the CD4+CD25 population (8% of the CD4+CD25 fraction). However both FACS-purified naive and memory CD4+CD25 cells show identical kinetics of early IL-2 production and suppression with the only difference being in the magnitude of the IL-2 response (2-fold more early IL-2 producers in the memory fraction) (D. K. Sojka and D. J. Fowell, unpublished observations). Therefore the biphasic nature of the kinetics cannot be explained by differences in the regulation of naive and memory T cells. The use of the IL-2 capture assay will enable future analysis of the sensitivity to early Treg-induced modulation of IL-2 production within additional subpopulations of the pool of CD25CD4+ T cell targets.

CD28, IL-2, and high Ag dose all abrogate suppression of proliferation at late stages in the culture. However only CD28 engagement enables target T cells to resist the early down-regulation of IL-2 by Tregs. These results suggest that some of the actions of Tregs can be dissociated from the routine measurement of proliferative arrest. Therefore, it is tempting to speculate that such cells that were modified early by Tregs but that escaped the proliferative block (conditions of high IL-2 or high Ag) would nevertheless be functionally altered/disabled on subsequent activation in the absence of Tregs. The data suggest that a productive immune response depends not on the ability to directly disable Tregs but on the early provision of distinct signals to the target T cells that enable escape from suppression. It is likely that the types of signals that confer resistance to suppression are tightly regulated to avoid unwanted activation of autoreactive T cells. Indeed, enhanced availability of Ag or the presence of exogenous cytokines are not sufficient to disrupt Treg control, rather a “microbial” cue appears to be required (the up-regulation of B7 being one consequence of microbial encounter).

We formally show that CD28 signals do not inhibit the intrinsic regulatory capacity of CD25+CD4+ T cells, but rather make the responding population resistant to suppression. This distinction is an important one, that CD28 engagement does not directly disable Treg activity and enables, within the same local environment, the initiation of an immune response to infectious stimuli without compromising self-tolerance. How CD28 “arms” T cells against suppression remains to be determined. Previous reports have suggested that CD28 “licenses” T cell resistance to suppression by enhancing the expression and function of GITR (27). Our kinetic analysis does not support such a mechanism for the early down-regulation of IL-2 in our system, as CD28-induced GITR expression occurs subsequent to (12–18 h; A. Hughson and D. J. Fowell, unpublished observations) the early changes in IL-2 expression (6–12 h) observed here. CD28 engagement both accelerates and amplifies the IL-2 response; therefore, abrogation of suppression might be mediated through either, or both, of these events. We think the amplification of the response is unlikely to be the primary mechanism for escaping suppression as provision of high Ag dose also enhanced the magnitude of the IL-2 response but failed to abrogate early Treg suppression. Attempts to address the accelerated kinetics induced by CD28 engagement by delaying the addition of anti-CD28 later in the culture were unsuccessful as the ability of CD28 to “costimulate” was lost if TCR and CD28 signals were separated in time (D. K. Sojka and D. J. Fowell, unpublished observations). We propose that the ability of CD28 to quickly stabilize IL-2 transcripts (33) enables target T cells to counterbalance transcriptional down-regulation by Tregs.

We thank the members of the Fowell laboratory for helpful discussion and technical assistance. Special thanks to Tim Mosmann, Jim Miller, and Ben Segal for critical review of the manuscript.

The authors have no financial conflict of interest.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

This work was supported by the Juvenile Diabetes Research Foundation, Research Grant 1-2000-609 (to D.J.F.).

4

Abbreviations used in this paper: Treg, regulatory T cell; WT, wild type; LACK, Leishmania homologue of human receptor for activated C kinase; 7AAD, 7-aminoactinomycin D.

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