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The Journal of Immunology, 2007, 179, 7924 -7931
Copyright © 2007 by The American Association of Immunologists, Inc.
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Effective Proliferation of Human Regulatory T Cells Requires a Strong Costimulatory CD28 Signal That Cannot Be Substituted by IL-21

Andreas A. Hombach2, David Kofler, Anja Hombach, Gunter Rappl and Hinrich Abken

Zentrum für Molekulare Medizin Köln and Klinik I für Innere Medizin, Labor Tumorgenetik, Universität zu Köln, Köln, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The strength of immune repression by regulatory T (Treg) cells is thought to depend on the efficiency of Treg cell activation. The stimuli and their individual strength required to activate resting human Treg cells, however, have so far not been elucidated in detail. We reveal here that induction of proliferation of human CD4+C25+ Treg cells requires an extraordinary strong CD28 costimulatory signal in addition to TCR/CD3 engagement. CD28 costimulation, noteworthy, cannot be substituted by IL-2 to induce proliferation of Treg cells, which is in contrast to CD4+CD25– T cells. IL-2, in contrast, prevents spontaneous apoptosis of Treg cells, but does not initiate their amplification. IL-2 and CD28 costimulation clearly exhibit disparate effects on Treg cells which are in contrast to those on CD4+CD25– T cells. Moreover, the prerequisites for Treg cell proliferation differ strikingly from those for effector T cells, implying a balanced orchestration in initiating and limiting a T cell immune response. In addition, data are of relevance for the design of therapeutic strategies involving IL-2 administration and CD28 costimulation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The CD4+ regulatory T (Treg)3 cells represent 1–5% of total peripheral blood CD4+ T cells and are characterized by high expression of both CD25 and FoxP3. Treg cells repress proliferation of activated T cells and are therewith key players in the control of ongoing cellular immune responses and, moreover, of T cell hemostasis (1, 2). Accordingly, the balance between self-tolerance and elimination of pathogens depends on the maintenance of regulatory T cells in sufficient numbers in the periphery and the induction of their effector functions. Complete activation of resting T cells requires, in accordance with the dual signaling concept, a costimulatory signal simultaneously with engagement of the CD3-TCR complex (3). For nearly all effector T cells, the costimulatory signal can be provided by either CD28 or alternatively by IL-2, resulting in T cell proliferation and execution of effector functions. Activation of Treg cells, however, seems to be under different control mechanisms compared with CD4+CD25– T cells since Treg cells remain in a resting state with low proliferative capacities under conditions that initiate activation of CD4+CD25– T cells (4). The conditions to activate Treg cells are so far not understood in detail. One prerequisite to induce Treg cell activation seems to be a strong TCR signal because high-affinity TCR binding, as it occurs by binding to MHC-encountered self-Ag, results in Treg cell activation (5). Accordingly, high-strength signaling via Ag-pulsed dendritic cells induced activation of CD4+CD25+ Treg cells in a transgenic mouse model (6). It remains unresolved so far whether and to which extent the CD3/TCR signal needs to be modulated by costimulatory signals to drive Treg cell activation. The situation is obviously more complex since several mouse models suggest a predominant role for IL-2 in generating and expanding Treg cells (7, 8), whereas other animal models support the impact of CD28 costimulation for Treg homeostasis and function (9, 10). For human CD4+CD25+ Treg cells, in contrast, only a few data are available and the contribution of TCR engagement and costimulatory signals to induce Treg cell activation still remains to be resolved.

In this study, we recorded in detail the requirements to activate resting human Treg cells with respect to induce cytokine secretion and proliferation. The analyses revealed that CD4+CD25+ Treg and CD4+CD25– T cells fundamentally differ in their stimulatory requirements, particularly in the strength of TCR/CD3 engagement and CD28 costimulation. An extensive CD28 costimulatory signal is required to induce Treg cell activation which cannot be substituted by IL-2 that, in contrast, prevents human Treg cells from spontaneous apoptosis in vitro. The different requirements for the activation of Treg cells and CD4+CD25– T cells imply a well-tuned balance in initiating and repressing a T cell immune reaction by the strength of CD28 costimulation. Moreover, the results are of relevance for the therapeutic application of IL-2 and CD28 costimulation as well as for the design of therapeutic strategies that aim to manipulate T effector cell functions without activating Treg cells or vice versa.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Abs and reagents

The hybridoma cell line OKT3 that produces the anti-CD3 mAb OKT3 was obtained from American Type Culture Collection (ATCC CRL 8001). The hybridoma cell line that produces the anti-CD28 mAb 15E8 (11) was a gift from R. van Lier (Academic Medical Center, Amsterdam, The Netherlands). mAbs were affinity purified from hybridoma supernatants using an agarose-immobilized goat anti-mouse IgG1 (Sigma-Aldrich)- or a Sepharose (BD Biosciences)-immobilized goat anti-mouse IgG2a Ab (Southern Biotechnology Associates). The FITC-conjugated anti-CD4 mAb and the PE- and FITC conjugated anti-CD8 mAbs, respectively, were purchased from DakoCytomation. The PE-conjugated anti-CD25 mAb was purchased from Miltenyi Biotec. The allophycocyanin-conjugated anti-human FoxP3 mAbs PCH101 and 3G3 were purchased from Natutec and Miltenyi Biotec. The neutralizing anti-human IL-2 mAb 297C16F11 was purchased from BioSource International. Isotype control Abs were purchased from BD Biosciences.

MACS

PBL from healthy donors were isolated by density centrifugation and monocytes were depleted by plastic adherence. Nonadherent lymphocytes were washed with cold PBS containing 0.5% (w/v) BSA, 1% (v/v) FCS, and 2 mM EDTA. CD4+CD25+ Treg cells and CD4+CD25– T cells were isolated by MACS using a CD25 T cell isolation kit (Miltenyi Biotec) as recommended by the manufacturer, with modifications: The amount of anti-CD25 beads was 5 µl/107 lymphocytes. The purity of isolated CD4+CD25+ Treg cells and CD4+CD25– T cells was determined by flow cytometry. Isolated T cells were washed, counted, and used for cell activation experiments. We obtained typically ~1–3 x 106 CD4+CD25+ T cells from 2 x 108 CD4+ T cells. The purity of isolated CD25+FoxP3+ T cells was typically >80%.

Immunofluorescence analysis

MACS-isolated T cells were labeled with FITC-conjugated anti-CD4 and PE-conjugated anti-CD25 mAbs. Expression of the transcription factor FoxP3 was monitored as follows: isolated resting CD4+CD25+ Treg cells and CD4+CD25– T cells were stained by the anti-CD25 PE mAb, fixed, and permeabilized using a FoxP3 labeling kit (Natutech) or FoxP3 staining buffers from Miltenyi Biotec according to the manufacturer’s recommendations. Permeabilized cells were incubated with the allophycocyanin-conjugated anti-FoxP3 mAbs PCH101 or 3G3. Labeled cells were analyzed on a FACSCanto cytofluorometer equipped with the FACSDiva research software (BD Bioscience).

Suppressor assay

To test the suppressor activity of the CD4+CD25+ T cells, freshly isolated CD4+CD25– T cells (1 x 107 cells/ml) were labeled with CFSE (1 µM; Molecular Probes) on ice for 5 min. Cells were washed once in RPMI 1640 medium/10% (v/v) FCS and incubated for 5 min on ice. Subsequently, cells were washed three times in RPMI 1640 medium/10% (v/v) FCS and rested for 30 min at 37°C, washed once again, and used for additional experiments. T cells (5 x 104/well) were stimulated with soluble OKT3 mAb (10 µg/ml) and anti-CD28 mAb 15E8 (1 µg/ml) for 6 days either alone or along with 5 x 104 (suppressor:responder ratio = 1:1) or 1 x 104 (suppressor:responder ratio = 1:5) CD4+CD25+ T cells or control cells, respectively. Cell division was monitored by flow cytometric recording of the decrease in fluorescence intensity of CFSE-labeled cells. The number of proliferating cells (percent) was calculated as follows: (1 – nonproliferating CFSE-labeled cells (percent) under stimulatory conditions/nonproliferating CFSE-labeled cells (percent) under nonstimulatory conditions) x 100.

Proliferation of CD4+CD25+ and CD4+CD25– T cells

Microtiter plates (Polysorb; Nunc) were coated overnight with anti-CD3 or anti-CD28 mAbs in different concentrations (0.01–10 µg/ml PBS). The plates were washed twice and freshly isolated human CD4+CD25+ and CD4+CD25– T cells, respectively (each 2.5 x 104 cells/well), were incubated in RPMI 1640 medium with 10% (v/v) FCS. In a second set of experiments, we incubated freshly isolated CD4+CD25+ and CD4+CD25– T cells, respectively, in the presence of soluble mAbs or plate-bound anti-CD3 mAb and soluble IL-2 (Chiron), respectively. After 6 days of culture, cells were pulsed for 6 h with BrdU and incorporated BrdU was determined by ELISA (Cell Proliferation ELISA, BrdU colorimetric; Roche) as recommended by the manufacturer. Alternatively, freshly isolated CD4+CD25+ and CD4+CD25– T cells were labeled with CFSE as described above and incubated in the presence or absence of solid-phase bound agonistic anti-CD3 and anti-CD28 mAbs (10 µg/ml) or solid-phase bound agonistic anti-CD3 mAb with IL-2 (1000 U/ml), respectively. After 6 days, cells were removed and simultaneously stained with anti-CD25 and different anti-FoxP3 mAbs as described above and cell proliferation was determined by flow cytometry.

Cytokine detection

Cytokines in the culture supernatants were recorded by ELISA using matched pairs of Abs specific for IL-2 (clones B33-2 and 5344.111), IL-10 (clones JES3-9D7 and JES3-12G8), and IFN-{gamma} (clones NIB 42 and B133.5), all purchased from BD Life Sciences. The detection limits of the assays were 15 pg/ml IL-2, 15 pg/ml IL-10, and 15 pg/ml IFN-{gamma}, respectively.

Detection of apoptotic cells

Apoptosis of CD4+CD25+ and CD4+CD25– T cells was recorded by binding of annexin V (BD Biosciences). Briefly, isolated human CD4+CD25+ and CD4+CD25– T cells were cultured in the presence or absence of added IL-2 (500 U/ml) in microtiter plates that were coated either with anti-CD3 and anti-CD28 mAbs (each 5 µg/ml) or with PBS as control. After 7 days, the cells were incubated with FITC-conjugated annexin V (BD Biosciences) and analyzed by flow cytometry as described above. Dead cells were excluded from the analysis by staining with 7-aminoactinomycin D (1 µg/ml; Sigma-Aldrich).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Human CD4+CD25+ Treg and CD4+CD25– T cells were isolated from the peripheral blood by magnetic cell sorting procedures as described in Materials and Methods. Isolated Treg cells expressed high amounts of both CD25 and FoxP3 compared with CD4+CD25– T cells (Fig. 1). To confirm the functional activity of isolated CD4+CD25+FoxP3+ Treg cells, we coincubated freshly isolated CD4+CD25+ T cells with CFSE-labeled CD4+CD25– T cells in the presence of the soluble agonistic anti-CD3 mAb OKT3 (10 µg/ml) and anti-CD28 mAb 15E8 (1 µg/ml), respectively. As demonstrated in Fig. 2, CD4+CD25+ T cells repressed proliferation of CFSE-labeled CD4+CD25– T cells, whereas CD4+CD25– T cells did not. We conclude that the population of isolated CD4+CD25+ T cells constitutes highly enriched, functionally active Treg cells.


Figure 1
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  		FIGURE 1. Isolation and characterization of human CD4+CD25+ Treg cells from the peripheral blood. CD4+CD25+ and CD4+CD25– T cells were isolated from the peripheral blood by magnetic cell sorting procedures and analyzed for CD25 and FoxP3 expression by intracellular staining using the anti-FoxP3 Ab PCH101 as described in Materials and Methods. Staining with isotype Abs served as control. The number of cells in quadrants is expressed as percentage of total cells.

 

Figure 2
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  		FIGURE 2. Isolated human CD4+CD25+ T cells exhibit repressor activities. Freshly isolated CD4+CD25– T cells were labeled with CSFE and cocultivated (5 x 104 cells/well) with either nonlabeled CD4+CD25– and CD4+CD25+ T cells (1 x 104 cells/well), respectively, or alone in the presence of the soluble anti-CD3 mAb OKT3 (10 µg/ml) and anti-CD28 mAb 15E8 (1 µg/ml), respectively. After 6 days, cells were recovered and the numbers of specific cycling CFSE-labeled cells was determined by flow cytometry and calculated as described in Materials and Methods. The assay was performed in triplicates and the mean was determined. Typical histograms are shown in A. The summary of a typical experiment of three is shown in B. w/o, Without.

 
To elucidate the requirements for induction of Treg cell proliferation in detail, we stimulated isolated CD4+CD25+ Treg and CD4+CD25– T cells via the CD3-TCR complex with and without CD28 costimulation or addition of IL-2. Cells were incubated 1) in microtiter plates with the soluble agonistic anti-CD3 mAb OKT3 and the anti-CD28 mAb 15E8, 2) in microtiter plates with the solid-phase bound anti-CD3 and anti-CD28 mAbs, and 3) on solid-phase bound anti-CD3 mAb in the presence of IL-2. The stimulatory agents were cross-titrated to determine the individual activation threshold for both CD4+CD25+ Treg and CD4+CD25– T cells. As summarized in Fig. 3, A and B, Treg cells did not proliferate in the presence of the soluble anti-CD3 plus anti-CD28 mAbs in the range of 0.01–10 µg/ml, whereas CD4+CD25– T cells did very efficiently, e.g., in the presence of 0.1 µg/ml OKT3 mAb plus 1 µg/ml 15E8 mAb. The data clearly indicate that Treg cells compared with CD4+CD25– T cells require different prerequisites to induce proliferation. Moreover, conditions under which Treg cells repress proliferation of CD4+CD25– T cells, i.e., 10 µg/ml soluble OKT3 mAb plus 1 µg/ml soluble anti-CD28 mAb 15E8, are obviously not suitable to induce proliferation of Treg cells.


Figure 3
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  		FIGURE 3. Human CD4+CD25+ and CD4+CD25– T cells require different signal strengths to induce proliferation. A and B, Freshly isolated CD4+CD25+ and CD4+CD25– T cells (2.5 x 104 cells/well each) were incubated in the presence of the cross-titrated soluble mAbs OKT3 and mAb 15E8 (0–10 µg/ml each). C and D, Microtiter plates that were coated with cross-titrated OKT3 and 15E8 mAbs (0.01–10 µg/ml each) and in the presence of serial dilutions of IL-2 (0–1000 U/ml) cross-titrated with coated OKT3 mAb (0.01–10 µg/ml) (E and F). After 6 days, cell proliferation was monitored by pulsing the cells with BrdU as described in Materials and Methods. The assay was performed in duplicates and mean values are shown. A typical experiment of three is shown.

 
In a second set, we incubated CD4+CD25+ Treg cells in plates coated with the agonistic Abs to provide higher signaling strengths by forced TCR/CD3 and CD28 cross-linking. The agonistic anti-CD3 and anti-CD28 mAbs were immobilized on microtiter plates in cross-titrated concentrations as described in Materials and Methods. Upon incubation on coated plates, both CD4+CD25+ and CD4+CD25– T cells proliferate vigorously (Fig. 3, C and D). CD4+CD25+ Treg cells, however, have a significantly higher activation threshold for induction of proliferation than CD4+CD25– T cells. At high concentrations of immobilized Abs, BrdU incorporation of Treg cells and CD4+CD25– T cells was virtually similar, indicating that both T cell populations have nearly the same proliferative capacity under conditions of adequate stimulation.

Resting CD4+CD25– T cells can be activated by stimulation via TCR/CD3 in the presence of IL-2 without additional CD28 costimulation. Because CD4+CD25+ Treg cells express CD25 at high densities on the cell surface, we asked whether addition of IL-2 substitutes for CD28 costimulation in the induction of Treg cell proliferation. We incubated freshly isolated CD4+CD25+ Treg and CD4+CD25– T cells in the presence of increasing amounts of IL-2 (1–1000 U/ml) and of the solid-phase bound anti-CD3 mAb OKT3 in a cross-titration setting and recorded cell proliferation. CD4+CD25+ Treg cells did not proliferate in the presence of IL-2 alone in concentrations up to 1000 U/ml nor in the presence of solid-phase anti-CD3 mAb OKT3 plus IL-2 (Fig. 3, E and F). In contrast, CD4+CD25– T cells proliferated very efficiently upon incubation on anti-CD3 mAb-coated plates in the presence of 100 U/ml IL-2. Obviously, IL-2 cannot substitute CD28 costimulation to initiate proliferation when CD4+CD25+ T cells engage CD3/TCR.

Since the CD4+CD25+ T cell population contains a small subpopulation of cells with low or medium FoxP3 expression, we asked which cells of the CD4+CD25+ population proliferate in response to the respective stimuli. To resolve this issue, we labeled freshly isolated CD4+CD25– and CD4+CD25+ T cells with CFSE and stimulated these cells by incubation in plates coated with the agonistic anti-CD3 mAb OKT3 plus the anti-CD28 mAb 15E8 or in anti-CD3 mAb OKT3-coated plates in presence of IL-2 (1000 U/ml). For control, cells were incubated in medium or in the presence of IL-2 only. After 6 days, cells were recovered and stained simultaneously for CD25 and FoxP3 expression using the anti-FoxP3 mAb PCH101 (12). As summarized in Fig. 4, A and B, CD25 as well as FoxP3 expression were up-regulated in both CD4+CD25– and CD4+CD25+ T cells upon CD3 plus CD28 or IL-2 stimulation. This is in accordance to published observations that FoxP3 expression is up-regulated in both CD4+CD25+ cells and in CD4+CD25– T cells upon activation (13, 14, 15). Although the degree of FoxP3 up-regulation depends on the individual activating stimuli, FoxP3high and FoxP3low T cell populations remain clearly distinguishable upon flow cytometric analysis with adjusted region markers. Remarkably, FoxP3 expression in the CD4+CD25+ population is strongly up-regulated upon both combined CD3/CD28 and CD3/IL-2 stimulation, whereas CD25 expression is only slightly increased upon CD3/IL-2 stimulation. In the CD4+CD25– T cell population, in contrast, both CD3/CD28 and CD3/IL-2 stimulation increased CD25 expression to a similar extent.


Figure 4
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  		FIGURE 4. Proliferation of CFSE-labeled FoxP3+ and FoxP3– T cells. Freshly isolated CD4+CD25+ and CD4+CD25– T cells were labeled with CFSE and 2.5 x 104 cells/well were incubated in the presence or absence of IL-2 (1,000 U/ml) in microtiter plates that were either coated with OKT3 and 15E8 mAbs (10 µg/ml each) or OKT3 mAb alone (10 µg/ml) or incubated with coating buffer for control. After 6 days, cells were recovered, triplicates were pooled and stained simultaneously for CD25 and FoxP3 expression by a PE-conjugated anti-CD25 and an allophycocyanin-conjugated anti-FoxP3 mAb (PCH101), respectively, and analyzed by flow cytometry. A, Displays expression of CD25 and FoxP3. B, FoxP3 and CD25 expression as determined by the mean fluorescence intensity (MFI). C, Proliferation of CFSE-labeled FoxP3high and FoxP3low cells in the CD4+CD25– and CD4+CD25+ T cell population that were stimulated under identical conditions. D, Numbers of proliferating FoxP3+ and FoxP3– cells in the CD4+CD25– and CD4+CD25+ T cell populations, respectively. A typical experiment of three is shown.

 
To identify the proliferating T cells upon stimulation, we recorded CFSE-labeled, CD4+CD25–, and CD4+CD25+ T cells with respect to FoxP3 expression (Fig. 4, C and D). Upon stimulation with solid-phase agonistic anti-CD3 and anti-CD28 mAbs, both FoxP3high and FoxP3low cells of the CD4+CD25+ T cell population proliferated vigorously. Proliferation of CD4+CD25+ cells was similar to that of CD4+CD25– T cells. Remarkably, the ratio of proliferating FoxP3high and FoxP3low T cells compared with nonproliferating cells within the CD4+CD25+ cell population remained nearly unaltered, indicating a similar proliferative capacity of FoxP3high and FoxP3low cells under these conditions. Upon stimulation with immobilized anti-CD3 mAb in the presence of IL-2, in contrast, nearly no proliferation of FoxP3high cells of the CD4+CD25+ cell population was recorded and FoxP3low cells proliferated weakly, whereas CD4+CD25– T cells proliferated with similar efficiencies upon CD3 plus IL-2 stimulation as upon CD3/CD28 stimulation. Moreover. these data confirm that CD4+CD25+ FoxP3high T cells do not respond with proliferation upon CD3 plus IL-2 stimulation, whereas CD4+CD25– T cells do. To exclude that identification of Treg cells via FoxP3 expression is dependent on the Ab used, we tested the anti-FoxP3 mAbs PCH101 and 3G3 (16) in parallel for tracking of CFSE-labeled cells. As summarized exemplarily in Fig. 5, both Abs detect the same clearly distinguishable population, although the anti-FoxP3 mAb PCH101 has a higher background than the anti-FoxP3 mAb 3G3 at the same cytometer settings (Fig. 5, B and C). Accordingly, the quadrant markers are adjusted for appropriate comparison of FoxP3high and FoxP3low T cell populations. These data clearly demonstrate that detection of CD4+CD25+FoxP3high T cells is independent of the anti-FoxP3 Ab used.


Figure 5
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  		FIGURE 5. Identification of Treg cells by different anti-FoxP3 mAbs. CD4+CD25+ and CD4+CD25– T cells were isolated from the peripheral blood by magnetic cell sorting procedures and analyzed for CD4 and CD25 expression as described in Materials and Methods (A). Isolated CD4+CD25+ and CD4+CD25– T cells were analyzed for CD25 and FoxP3 expression by intracellular staining using a PE-conjugated anti-CD25 mAb and the allophycocyanin-conjugated anti-FoxP3 Abs PCH101 and 3G3 as described in Materials and Methods (B). Freshly isolated CD4+CD25+ and CD4+CD25– T cells were labeled with CFSE and 2.5 x 104 cells/well were incubated in microtiter plates that were coated with OKT3 and 15E8 mAbs (10 µg/ml each). After 6 days, cells were recovered, triplicates were pooled and stained for FoxP3 expression by the allophycocyanin-conjugated anti-FoxP3 mAbs PCH101 and 3G3, respectively, and analyzed by flow cytometry. The number of cells in quadrants is expressed as percentage of total cells (C). A typical experiment of two is shown.

 
We recorded secretion of the cytokines IFN-{gamma}, IL-2, and IL-10 into the culture supernatants of proliferating CD4+CD25+ Treg and CD4+CD25– T cells, respectively, upon appropriate stimulation (Fig. 6). CD3/CD28 stimulation activated CD4+CD25– T cells to secrete high amounts of the Th1 cytokines IFN-{gamma} and IL-2 as well as high amounts of IL-10, whereas CD4+CD25+ Treg cells secreted lower amounts of IFN-{gamma} and IL-10 and, remarkably, no IL-2. These data are in accordance with the reported cytokine pattern of activated Treg cells (17). Upon CD3 stimulation in the presence of IL-2, CD4+CD25– T cells secreted similar amounts of IFN-{gamma} and IL-10 compared with CD3/CD28 stimulation, whereas CD4+CD25+ Treg cells that did not proliferate under these conditions, secreted low amounts of IFN-{gamma} but high amounts of IL-10. Moreover, extensive consumption of added IL-2 by Treg cells is unlikely to account for lack in proliferation of CD4+CD25+ T cells under these conditions because added IL-2 was still detectable in the added amounts after incubation for 7 days (Fig. 6B).


Figure 6
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  		FIGURE 6. Cytokine secretion of CD4+CD25+ and CD4+CD25– T cells upon stimulation. Freshly isolated CD4+CD25+ and CD4+CD25– T cells (2.5 x 104 cells/well) were incubated for 7 days in the presence or absence of IL-2 (1000 U/ml) in microtiter plates that were coated with OKT3 mAb and 15E8 mAb (10 µg/ml each), OKT3 mAb alone (10 µg/ml), or coating buffer for control. Culture supernatants were removed and analyzed for secretion of IFN-{gamma} (A), IL-2 (B), and IL-10 (C), respectively, by ELISA. The assay was performed in triplicates and mean values ± SEM are shown. A typical experiment of three is shown.

 
Since CD3 engagement plus CD28 costimulation induced proliferation of CD4+CD25+FoxP3high T cells, we asked whether endogenous IL-2, that may be present in background concentrations, may sustain proliferation of these cells. We labeled freshly isolated CD4+CD25– and CD4+CD25+ T cells with CFSE and incubated these cells on plate-bound agonistic anti-CD3 mAb OKT3 plus the anti-CD28 mAb 15E8 in the presence or absence of a neutralizing anti-IL-2 mAb. As controls, cells were incubated in the presence of the solid-phase bound anti-CD3 mAb OKT3 (10 µg/ml) plus IL-2 (50 U/ml). Upon activation, cells were recovered and stained for FoxP3 expression and proliferation of CFSE-labeled FoxP3high and FoxP3low cells was recorded. As summarized in Fig. 7, CD4+CD25+FoxP3high T cells proliferated upon combined CD3/CD28 stimulation even in the presence of the neutralizing anti-IL-2 Ab. Remarkably, addition of the neutralizing anti-IL-2 mAb did not block anti-CD3/CD28-induced proliferation of neither CD4+CD25–FoxP3low nor CD4+CD25+FoxP3high T cells. For comparison, OKT3 mAb plus IL-2-induced proliferation of CD4+CD25–FoxP3low T cells was efficiently blocked by the anti-IL-2 mAb.


Figure 7
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  		FIGURE 7. Neutralization of IL-2 does not abrogate proliferation of CD4+CD25+FoxP3high T cells upon CD3/CD28 stimulation. Freshly isolated CD4+CD25+ and CD4+CD25– T cells were CFSE labeled and 2.5 x 104 cells/well were incubated in the presence or absence of IL-2 (50 U/ml) in microtiter plates that were coated with OKT3 and 15E8 mAbs (10 µg/ml each), OKT3 mAb alone (10 µg/ml), or incubated with coating buffer for control. To neutralize IL-2, which is added or might be produced by the cells, a neutralizing anti-IL-2 mAb or an isotype control mAb (20 µg/ml each) was added. After 6 days, cells were recovered, triplicates pooled and stained for FoxP3 expression using an allophycocyanin-conjugated anti-FoxP3 mAb, and analyzed by flow cytometry. FoxP3high and FoxP3low populations were defined as demonstrated in Fig. 4C and gates for analyses were set appropriately. A–H, Histograms of gated FoxP3high and FoxP3low populations. I, The number of proliferating FoxP3high and FoxP3low cells under different stimulatory conditions are displayed. A typical experiment of three is shown.

 
Since IL-2 did not activate resting CD4+CD25+ Treg cells to enter proliferation, we asked whether IL-2 has an impact on the viability of CD4+CD25+ Treg cells in vitro. Freshly isolated CD4+CD25+ and CD4+CD25– T cells were incubated for 7 days with and without IL-2 or, alternatively, were activated by incubation in plates coated with the agonistic anti-CD3 mAb plus CD28 mAb in the presence or absence of IL-2. Annexin V staining indicating apoptotic cells was determined (Fig. 8). CD4+CD25+ Treg cells exhibited a substantially higher rate of spontaneous apoptosis than CD4+CD25– T cells. In the presence of added IL-2, however, the number of apoptotic CD4+CD25+ Treg cells decreased dramatically. This is in accordance with data published earlier (18). Activation by CD3/CD28 stimulation, in contrast, prevented spontaneous apoptosis of CD4+CD25+ Treg cells as efficiently as IL-2. IL-2 in addition to CD3/CD28 stimulation had only minor effects on the prevention of Treg cell apoptosis. Taken together, the data demonstrate that IL-2 rather prevents apoptosis than induces proliferation of resting CD4+CD25+ Treg cells. Induction of Treg cell proliferation, however, is strictly dependent on CD3/TCR engagement along with strong CD28 costimulation which cannot be substituted by IL-2.


Figure 8
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  		FIGURE 8. IL-2 prevents spontaneous apoptosis of CD4+CD25+ Treg cells. Freshly isolated CD4+CD25+ and CD4+CD25– T cells (2.5 x 104 cells/well) were incubated for 7 days in the presence or absence of IL-2 (500 U/ml) in microtiter plates that were either coated with the anti-CD3 mAb OKT3 plus the anti-CD28 mAb 15E8 (each 5 µg/ml) or with PBS as control. The cells were harvested and apoptotic cells were identified by staining with FITC-conjugated annexin V and 7-aminoactinomycin D as described in Materials and Methods. The assay was performed in triplicates and mean values ± SEM are shown. A typical experiment of two is shown.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Although the activation of CD4+CD25– T cells has been elucidated in detail, little is known about the requirements for the activation of human Treg cells to induce their expansion or to prevent their apoptosis. Consequently, current protocols to amplify Treg cells from the peripheral blood are less effective and efficient. Biotechnological procedures are still not available to amplify functionally active Treg cells to sufficient numbers that allow adoptive T cell transfer for clinical applications. Activation of Treg T cells, moreover, is extremely difficult because Treg cells isolated from the peripheral blood are functionally anergic and remain in a resting state under conventional stimulatory conditions (19).

Treg cells express a high-avidity TCR against self-Ags, suggesting that these cells can be triggered by highly intense signaling via the TCR-CD3 complex upon encounter with Ag (5). Due to the dual signaling dogma for activation of resting T cells, the primary TCR/CD3 signal must be accompanied by a costimulatory signal, prototypes of which are CD28 (20, 21) and IL-2 (22), to induce complete activation resulting in T cell proliferation (20, 23, 24). We therefore systematically explored the activation requirements of resting human CD4+CD25+ Treg cells by triggering via TCR/CD3 in various strengths simultaneously with increasing CD28 costimulation or adding IL-2. This systematic approach revealed the following fundamental aspects: 1) CD4+CD25+ and CD4+CD25– T cells differ in their stimulatory requirements to initiate cellular activation, particularly in the strengths of the TCR/CD3 and the costimulatory signal; 2) the stimulatory conditions that induce proliferation of CD4+CD25– T cells are not sufficient to induce proliferation of Treg cells; 3) CD28 costimulation of very high strength in combination with intense TCR/CD3 stimulation is indispensable to induce activation and proliferation of CD4+CD25+ Treg cells; and 4) despite high levels of CD25 expression in Treg cells, IL-2 alone even in high concentrations cannot substitute for CD28 cosignaling to induce proliferation. IL-2, however, efficiently prevents apoptosis of human Treg cells in vitro.

Once activated via CD3 plus strong CD28 signals, CD4+CD25+FoxP3high Treg cells proliferate in vitro as efficiently as do CD4+CD25– T cells even in the presence of a neutralizing anti-IL-2 Ab. Despite the overall up-regulation of CD25 and FoxP3 even in the CD4+CD25– population, CD25+FoxP3high Treg cells conserve their phenotype and remain distinguishable via FoxP3 expression from the other CD4+ subtype cells independent of the utilized FoxP3 detecting Ab. Moreover, we exclude that contaminating CD4+CD25– cells may account for our observations because 1) isolated CD4+CD25+ T cells do not proliferate in the presence of added IL-2 and the absence of high CD28 costimulation as do CD4+CD25– T cells, 2) FoxP3high cells in the CFSE-stained CD4+CD25+ population proliferate only upon CD3/CD28 stimulation but not upon CD3/IL-2 stimulation, and 3) a neutralizing anti-IL-2 Ab does not abrogate CD3/CD28-induced proliferation of CD4+CD25+FoxP3high T cells. The data moreover clearly demonstrate that human peripheral Treg cells have a higher activation threshold than their CD4+CD25– counterpart T cells, but are not per se anergic to TCR/CD3-mediated activation. The requirement of a strong CD28 costimulatory signal for initiating proliferation of Treg cells is sustained by the observation that a CD28 superagonistic Ab can increase the number of peripheral Treg cells in a mouse model in vivo (10).

Whereas combined CD3/CD28 signaling results in autocrine IL-2 secretion and in increased proliferation of CD4+CD25– T cells, CD4+CD25+ Treg cells do not produce IL-2 and cannot use added IL-2 to promote their proliferation, thus remaining dependent on a strong CD28 costimulatory signal. Treg cells, in contrast, consume IL-2 that prevents their apoptosis. Moreover, IL-2 consumption is thought to contribute, at least in part, to the execution of their suppressive activities toward proliferating responder cells (25). This is partly in contrast to the reported role of IL-2 for the maintenance of Treg cells in vivo (7, 8). Our study, however, demonstrates that IL-2 is required to prevent spontaneous apoptosis of Treg cells, thereby contributing merely indirectly to maintain their repressor functions. Taken together, CD28 costimulation and IL-2 obviously exhibit different roles toward Treg cells, since strong CD28 costimulation along with TCR/CD3 signaling contributes to execute Treg cell proliferation and other effector functions, whereas IL-2 sustains survival of Treg cells by preventing spontaneous apoptosis. This, however, does not exclude that consumption of IL-2 by Treg cells helps, in addition, to repress proliferation of CD4+CD25– T cells.

Our findings have significant consequences for the basic understanding of activation and hemostasis of human CD4+CD25+ Treg cells and, consequently, for a number of immunotherapeutic strategies. Due to their potential in limiting a dysregulated immune response, there is growing interest to use Treg cells for the treatment of acute autoimmune diseases and transplant rejection. Because the number of Treg cells in the periphery is low, the cells must be efficiently and selectively expanded and activated ex vivo before therapeutic application. The elucidation of the activation conditions for human Treg cells now makes the development of efficient expansion protocols for clinical applications feasible. In contrast, a number of cellular immunotherapeutic regimens require the ex vivo expansion of specific effector cells which, however, is frequently accompanied by culture-associated T cell defects (26, 27). Because coexpansion of Treg cells may account for these defects (28), protocols for amplification of effector CD4+CD25– T cells should be modified in the way that stimulatory conditions below that of Treg cells are applied to avoid simultaneous activation and expansion of Treg cells. Amplification of effector T cells without activation of Treg cells is likely to increase the efficacy of an adoptively transferred T cell immune response. Moreover, the identification of the activation threshold opens the possibility to expand human Treg cells with the aim to limit an ongoing immune response by adaptive transfer of ex vivo-amplified Treg cells.


   Acknowledgments
 
We thank Birgit Hops, Frank Steiger, and Petra Hofmann for excellent technical assistance.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
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 grants from Deutsche Krebshilfe, Deutsche Forschungsgemeinschaft, Wilhelm Sander-Stiftung, Fortune program of the Medical Faculty of the University of Cologne, and European Commission through the ATTACK project. Back

2 Address correspondence and reprint requests to Dr. Andreas Hombach, Klinik I für Innere Medizin, Labor für Tumorgenetik, Universität zu Köln, Kerpener Strasse 62, D-50924 Köln, Germany. E-mail address: andreas.hombach{at}uk-koeln.de Back

3 Abbreviation used in this paper: Treg, regulatory T cell. Back

Received for publication November 30, 2006. Accepted for publication September 19, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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