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Immunomodulation

Transient expression of FOXP3 in human activated nonregulatory CD4+ T cells


Jun Wang, Andreea Ioan-Facsinay, Ellen I. H. van der Voort, Tom W. J. Huizinga and Ren E. M. Toes
Department of Rheumatology, Leiden University Medical Center, Leiden, The Netherlands Foxp3 plays a key role in CD4+CD25+ Treg cell function in mice and represents a specific marker for these cells. Despite the strong association between FOXP3 expression and regulatory function in fresh human T cells, little is known about the dynamics of endogenous FOXP3 expression and its relation to the suppressive function in activated human T cells. Here, we addressed the dynamics of FOXP3 expression during human CD4+ T cell activation by plate-bound anti-CD3 Ab as well as the relationship between its expression and regulatory function at the single-cell level. Our data show that FOXP3 is expressed in a high percentage of activated T cells after in vitro stimulation of human CD4+CD25 cells. FOXP3 expression is strongly associated with hyporesponsiveness of activated T cells, but is not directly correlated with their suppressive capabilities, as we demonstrate that it is also expressed in activated nonsuppressive T cells. However, in this nonsuppressive T cell population, FOXP3 expression is transient, while it is stably expressed in activated T cells that do display suppressive function, and in natural CD4+CD25++ Treg cells. These data indicate that expression of endogenous FOXP3, in humans, is not sufficient to induce regulatory T cell activity or to identify Treg cells.
Supporting information for this article is available at http://www.wiley-vch.de/contents/jc_2040/2007/36435_s.pdf See accompanying commentary: http://dx.doi.org/10.1002/eji.200636929
Received 27/6/06 Revised 2/10/06 Accepted 17/10/06 [DOI 10.1002/eji.200636435]

Key words: Cell activation Human T cells Transcription factors

Introduction
Human FOXP3 (Foxp3 in mice) is a member of the forkhead or winged-helix family of transcription factors. FOXP3/Foxp3 was originally reported to be the causative gene for an X-linked multi-organ autoimmune/inflammatory disease in humans and mice. Mutations in the human FOXP3 gene cause the chronic wasting disease
Correspondence: Dr. Ren E. M. Toes, Department of Rheumatology C1-R, Leiden University Medical Center, Albinusdreef 2, 2300 RC, Leiden, The Netherlands Fax: +31-71-5266752 e-mail: R.E.M.Toes@lumc.nl Abbreviations: APC: allophycocyanin pb-anti-CD3: platebound anti-CD3 SCD25: 1-wk in vitro stimulated CD4+CD25 T cells SCD25++: 1-wk in vitro stimulated CD4+CD25++ Treg cells
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termed immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX), which is characterized by high incidences of autoimmune diseases including type 1 diabetes, thyroiditis, inflammatory bowel disease and allergic disease such as atopic dermatitis and food allergy [13]. Moreover, the Foxp3mutant scurfy mice develop similar X-linked multi-organ pathology accompanying massive lymphocytic infiltration and uncontrolled activation of CD4+ T cells [48]. Immunological similarities between IPEX/scurfy and abnormalities produced by depletion of CD4+CD25+ Treg cells [911] triggered intensive studies on the role of Foxp3 in the development and function of CD4+CD25+ Treg cells in mice [1214]. Until now, Foxp3 represents the first specific marker for murine CD4+CD25+ Treg cells since it is reported to be exclusively expressed by
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Treg but not by activated nonregulatory T cells. Furthermore, gene transfer of Foxp3 converts naive murine CD4+CD25 T cells towards a regulatory phenotype [12, 13]. Together, these results indicate that Foxp3 is a master regulatory gene for cell lineage commitment and function of CD4+CD25+ Treg cells in mice. As described for murine CD4+CD25+ T cells, CD4+CD25+ Treg cells isolated from human peripheral blood are hyporesponsive and are thought to play a crucial role in the control of T cell-mediated autoimmunity by suppressing the proliferation and cytokine production of other T cells [1517]. They express high levels of FOXP3, whereas CD4+CD25 T cells do not [1823]. However, contrasting findings have been reported on the dynamics and functional implications of FOXP3 expression in activated human CD4+CD25+ T cells. For example, up-regulation of FOXP3 expression at the bulk T cell level as measured by RT-PCR and/or Western blotting after activation of CD4+CD25 T cells has been observed in some studies [1821], but not in others [22]. Likewise, some studies presented a strict correlation between FOXP3 expression at the bulk T cell population and the display/acquisition of regulatory T cell function [18, 19], whereas other studies indicate that FOXP3 expression in human T cells does not necessarily associate with immune regulation [21, 24]. Although the studies described above provided important insights into the expression and role of FOXP3 in human Treg cell function, it is difficult to ascertain the degree to which de novo FOXP3 expression occurs because the observed increase in FOXP3 expression might also result from expansion of the small population of pre-existing FOXP3+CD25 T cells [23]. It has recently been shown, at the single-cell level, that in vitro activation of human CD4+CD25 T cells can induce de novo FOXP3 expression [23, 24]. High expression levels of FOXP3 were associated with a hyporesponsive state, whereas intermediate FOXP3 expression did not seem to influence production of pro-inflammatory cytokines [24]. However, whether FOXP3 expression did correlate with the ability of activated human FOXP3-expressing cells to suppress the function of other T cells was not investigated. More insight into the relationship between FOXP3 expression and regulatory function of human T cells is important as FOXP3 is widely used to detect the presence of Treg cells in human autoimmune diseases [2527], tumors [28, 29] and infections [30]. Moreover, these studies will provide additional information on the role of FOXP3 in the acquisition of a regulatory phenotype by human T cells. Here, we show that in humans, FOXP3 is expressed in an unexpectedly high percentage of activated T cells after in vitro stimulation of CD4+CD25 cells. These FOXP3-expressing T cells are hyporesponsive to TCR
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stimulation. However, FOXP3 expression is not directly correlated with suppressive capabilities as FOXP3 is transiently expressed in activated nonsuppressive T cell populations. These data indicate that expression of endogenous FOXP3 in humans might not be sufficient to identify Treg cells, especially in patients with ongoing immune responses.

Results
Expression of FOXP3 in resting human CD4+ T cells In order to characterize the FOXP3 protein expression in resting human T cells, PBMC were purified from healthy donors and endogenous FOXP3 protein was detected by flow cytometry. Because the expression of FOXP3 is mainly restricted to the CD4+CD25+ Treg subset in mice and humans [1214, 18, 19, 22, 31], we stained the cells with anti-CD4 and anti-CD25 Ab to correlate the FOXP3 expression with CD25. As expected, 93.3% (79.495.5)

Figure 1. Expression of FOXP3 in resting human CD4+ T cells. (A) PBMC were isolated, surface-stained with anti-CD4 and anti-CD25 Ab, and intracellularly stained with anti-human FOXP3 Ab. FOXP3 expression was monitored by flow cytometry and results were expressed as percentage of FOXP3-positive cells within each gate. The inserts indicate gates 14: CD4+, CD4+CD25 (CD25), CD4+CD25int (CD25int) and CD4+CD25++ (CD25++) T cells. Each circle represents one healthy individual, and median values for each gate are indicated by a horizontal line. (B) FACS-sorted CD4+CD25 (CD25) and CD4+CD25++ (CD25++) T cells were stimulated either alone or mixed at 1 : 1, 2 : 1, 4 : 1 ratio (104 cells/well) and stimulated with 1 lg/ mL PHA in the presence of 5 104 cells/well feeders. Proliferation after 5 days was determined by [3H]thymidine incorporation. Results are expressed as means SEM of triplicate cultures. One out of six independent experiments is shown.
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of CD4+CD25++ Treg cells and only 14.2% (6.825.3) of CD4+CD25int and 1.8% (0.93.4) of CD4+CD25 T cells stained positive for FOXP3 (Fig. 1A). In line with previous reports [15, 17], only CD4+CD25++ Treg, but not the CD4+CD25 T cells, were hyporesponsive and suppressive (Fig. 1B). Thus, in resting human CD4+ T cells, FOXP3 is predominantly expressed in CD4+CD25++ Treg cells and its expression strongly correlates with regulatory activity. Expression of FOXP3 in activated human CD4+ T cells To investigate whether nonregulatory T cells can upregulate FOXP3 expression after activation, and to determine which proportion of the activated cells

express FOXP3, highly purified CD4+CD25 T cells were stimulated with plate-bound anti-CD3 (pb-anti-CD3) Ab and soluble anti-CD28 Ab in the presence of IL2. At the indicated times (Fig. 2A), cells were collected and processed for analysis of CD25 and FOXP3 expression. After 1 day of activation, 10% of the cells expressed FOXP3. This percentage increased further to approximately 50, 70 and 90% on days 3, 5 and 7, respectively (Fig. 2A, B). Notably, most FOXP3-expressing cells were restricted to the CD25+ T cell population (Fig. 2A). As controls, CD4+CD25 T cells cultured only with IL-2 remained negative for both CD25 and FOXP3 (Fig. 2B), while CD4+CD25++ Treg cells expressed even higher levels of FOXP3 after a 1-wk stimulation (Fig. 2D). On a per cell analysis, 1-wk-stimulated CD4+CD25 cells (Fig. 2A) expressed higher levels of FOXP3 in compar-

Figure 2. Expression of FOXP3 in activated human CD4+ T cells. FACS-sorted CD4+CD25 (A, B, and left graph in E) or CD4+CD25++ (D and right graph in E) T cells were activated with pb-anti-CD3 and soluble anti-CD28 Ab in the presence of exogenous IL-2. After the indicated times, cells were collected and processed for analysis of FOXP3 and/or CD25 expression as described in Materials and methods. (A, D) Histogram graph (upper panel in A) showing the FOXP3 (open black)/isotype (filled gray) staining, or representative flow cytometric plots (D and lower panel in A) showing CD25 vs. FOXP3 expression in cells before and after activation of CD4+CD25 (A) or CD4+CD25++ (D) T cells. (E) After 5 days of activation, cells were intracellularly co-stained with PE-labeled antiFOXP3 (PCH101) and APC-conjugated anti-FOXP3 Ab (236A/E7; eBioscience). The quadrants for FOXP3 were based on the isotype control antibody and the upper right numbers indicate the percentage of cells in each quadrant. (B) Line graph of the changes over time in expression of CD25 and FOXP3 after activation of CD4+CD25 T cells. Results are expressed as means SEM of three independent experiments with different donors. (C) CFSE-labeled CD4+CD25 T cells were stimulated with pb-anti-CD3 and soluble anti-CD28 Ab in the absence or presence of exogenous IL-2. Intracellular FOXP3 expression was analyzed 4 days later using flow cytometry.
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ison to freshly isolated CD4+CD25++ Treg cells, but lower levels compared to activated CD4+CD25++ Treg cells (Fig. 2D). This high expression of FOXP3 in both activated human CD4+CD25 and CD4+CD25++ T cells was further confirmed by co-staining the cells with another FOXP3-specific Ab (236A/E7) which recognizes a different epitope (Fig. 2E). The high FOXP3 expression in activated human CD4+CD25 T cells was unlikely to be due to exogenous TGF-b possibly present in the pooled human serum batch used [32], as activation of T cells in serum-free medium led to a similar percentage of FOXP3+ T cells (supplementary Fig. 1A). From these experiments, we conclude that FOXP3 is expressed in most CD4+CD25 T cells after 1-wk of activation with pb-anti-CD3 and soluble anti-CD28 Ab, and that its expression correlates with cell surface CD25 expression. To determine the kinetics of FOXP3 expression and to investigate the effects of IL-2 on its expression, we next analyzed FOXP3 expression in activated T cells in relation to cell division. CFSE-labeled CD4+CD25 T cells were stimulated with pb-anti-CD3 and soluble anti-CD28 Ab in the presence or absence of IL-2, and the

expression of FOXP3 was evaluated 4 days later by flow cytometry. In line with the results shown in Fig. 2A and B, FOXP3 was rapidly induced after the start of cell division, irrespective of the addition of exogenous IL-2 (Fig. 2C). However, addition of IL-2 during activation resulted not only in a higher percentage of FOXP3+ T cells (76% vs. 20%), but also in a T cell population that expressed remarkably higher levels of FOXP3 (highFOXP3-expressing cells in top left part of Fig. 2C, 19.3% vs. 0.6%). Together, these data indicate that FOXP3 is induced rapidly after T cell activation, which is further boosted by the presence of exogenous IL-2. Proliferative capacity and cytokine profile of activated CD4+FOXP3+ T cells Human CD4+CD25++ Treg cells are hyporesponsive and produce relatively high levels of IL-10 but low levels of IFN-c [17]. Since more than 85% of the 1-wk in vitro stimulated CD4+CD25 T cells (SCD25) expressed FOXP3 (Fig. 2A, B), permitting to obtain a relatively homogenous population of endogenous FOXP3-expres-

Figure 3. Proliferative capacity and cytokine production profile of activated CD4+FOXP3+ T cells. Cryopreserved autologous CD4+ (CD4+), 1-wk-stimulated CD4+CD25 (SCD25) or CD4+CD25++ (SCD25++) T cells (2.5 104 cells/well) were activated with PHA (1 lg/mL) and feeders (5 104 cells/well) in the absence or presence of 50 U/mL exogenous IL-2. (A) Cell proliferation was determined by [3H]thymidine uptake 5 days later. Results are expressed as means SEM. One representative experiment is shown (left), whereas the relative proliferations (normalized to the corresponding proliferation of cryopreserved autologous CD4+ T cells) of cells from five different donors are summarized on the right. (B) Culture supernatants were collected after 36 or 96 h and analyzed by cytometric bead assay to determine amounts of IL-2, IFN-c, TNF-a and IL-10. Results are representative of three different donors. ND: non-detectable; NT: not tested.
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sing cells, we wished to investigate whether expression of FOXP3 in activated CD4+CD25 T cells is associated with a phenotype similar to CD4+CD25++ Treg cells. Both stimulated CD4+CD25++ (SCD25++) and SCD25 T cells were hyporesponsive upon activation by PHA (Fig. 3A) and anti-CD3 Ab (data not shown) at concentrations that induced autologous CD4+ T cells to proliferate vividly. This hyporesponsiveness was reversed by the addition of exogenous IL-2 in all donors tested (Fig. 3A). In addition to the hyporesponsive phenotype, the cytokine levels produced by these FOXP3-expressing cells were also reduced. A significantly reduced level of IL-2 was observed in culture supernatants of FOXP3expressing SCD25 cells in comparison to cultures containing autologous CD4+ T cells (Fig. 3B). Moreover, although both SCD25++ Treg and SCD25 T cells were impaired in their capacity to produce IFN-c, TNF-a and IL-10 compared to autologous CD4+ T cells, SCD25 T cells produced more IFN-c and TNF-a but less IL-10 than SCD25++ Treg cells (Fig. 3B). Furthermore, in the presence of exogenous IL-2, SCD25 T cells secreted large amounts of IFN-c and TNF-a, whereas SCD25++ Treg cells only produced considerable levels of IL-10 (Fig. 3B). Therefore, the cytokine production profile of SCD25 T cells was distinct from that of conventional Treg cells, indicating that these two T cell populations are not alike. Suppressive activity of human activated CD4+FOXP3+ T cells We next tested the FOXP3-expressing SCD25 T cells for their capacity to inhibit T cell responses in vitro. Autologous CD4+ T cells were activated with PHA or anti-CD3 Ab (data not shown) and feeders in the absence or presence of SCD25 T cells. In all experiments, autologous CD4+ T cells were added at a 1 : 1 ratio as a negative control to exclude the possibility that inhibition was due to competition for space/nutrients in the coculture wells. As shown in Fig. 4A, FOXP3-expressing SCD25 T cells did not suppress the proliferation of autologous CD4+ T cells in six of nine donors, whereas SCD25++ Treg cells potently suppressed proliferation in all the donors by 82.6 8.3% on average. Furthermore, in one of these six individuals, we also purified CD25++ SCD25 T cells, but only marginal suppression by these cells (12% inhibition) was detected (data not shown). To confirm that the SCD25 T cells used in the proliferation assays described above did not suppress the proliferation of autologous CD4+ T cells, we also labeled the CD4+ T cells with CFSE and independently assessed their proliferation. As shown in Fig. 4B, co-culture with control (CFSE-unlabeled CD4+ T cells) or SCD25 T cells did not affect proliferation of CFSE-labeled CD4+ T cells, whereas addition of SCD25++ Treg cells potently did.
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It has been shown in mice that Treg cells may suppress effector cell function but not proliferation [33]. We therefore examined whether FOXP3-expressing SCD25 T cells were capable of suppressing IFN-c production of the target cells. Consistent with the results described above, addition of SCD25 cells at a 1 : 1 ratio did not inhibit IFN-c secretion (Fig. 4C), while addition of SCD25++ Treg cells suppressed IFN-c production by an

Figure 4. FOXP3-expressing human activated CD4+ T cells are not suppressive in six out of nine donors. (A) Cryopreserved autologous CD4+ T cells (2.5 104 cells/well) were activated with PHA (1 lg/mL) and feeders (5 104 cells/well) in the absence or presence of SCD25 T cells at a 4 : 1, 2 : 1 or 1 : 1 (responders/suppressors) ratio. Autologous CD4+ or SCD25++ T cells were added at a 1 : 1 ratio as negative or positive controls, respectively. Proliferation after 5 days was determined by [3H]thymidine incorporation. Results are expressed as means SEM of at least triplicate cultures. The upper right inserts show the percentage of FOXP3+ cells in SCD25 (left) or SCD25++ (right) T cells. (B) In parallel experiments, proliferation of CFSE-labeled autologous CD4+ T cells, alone or in the presence of a 1 : 1 ratio of CD4+, SCD25 or SCD25++ T cells, was analyzed by flow cytometry after 96 h. (C) Culture supernatants were collected after 96 h and analyzed to determine the amount of IFN-c by ELISA. Numbers represent the percentage of inhibition in (A) and (C) or percent of undivided cells in (B). Results are representative of six independent experiments.
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average of 80.2 9.4%. Together, we conclude that FOXP3-expressing SCD25 T cells from these six different donors do not display suppressive abilities in vitro. In contrast, FOXP3+ SCD25 T cells from three other donors not only inhibited the proliferation of autologous CD4+ T cells (Fig. 5A), but also suppressed their division (data not shown) and IFN-c production (Fig. 5B), although the inhibition was less profound than that of the SCD25++ Treg cells (66.5% vs. 82.6% in proliferation and 45.3% vs. 80.2% in IFN-c production at a 1 : 1 ratio). This inconsistent suppressive ability between donors supported the recent findings with FOXP3transduced cells, in which ectopic FOXP3 expression did not result in detectable suppressive activity in four out of seven experiments [21]. Collectively, these data indicate that FOXP3+ activated CD4+ T cells from all donors are hyporesponsive (Fig. 3A), but their in vitro suppressive capacity varies. Transient expression of FOXP3 in activated nonsuppressive human CD4+ T cells The unexpectedly high percentage of FOXP3+ cells in recently activated CD4+CD25 T cells (>85%;

Fig. 2A, B) prompted us to investigate whether the FOXP3 expression is stably induced. After 1 wk of stimulation, activated FOXP3+ cells were rested in IL-2 for another 7 days, and CD25/FOXP3 expression on/in cells was subsequently analyzed by flow cytometry. The percentage of FOXP3-expressing T cells dramatically decreased in activated nonregulatory T cells (87% vs. 23%, donor 1 in left panel of Fig. 6A), faster than the down-regulation of cell surface CD25 expression (94.6% vs. 71.2%). In contrast, suppressive SCD25 cells (donor 2 in middle panel of Fig. 6A) as well as SCD25++ Treg cells (right panel of Fig. 6A) retained their CD25 and FOXP3 expression after the 1-wk rest, despite a slight decrease in the expression levels. Next, we also determined the suppressive function of these cells. Cells stably expressing FOXP3 were still suppressive after the 1-wk rest, inhibiting proliferation even stronger than before resting (Fig. 6B; donor 2). Similar results were obtained with SCD25++ Treg cells

Figure 5. Expression of FOXP3 in human activated CD4+ T cells is associated with the acquisition of suppressive activity in three individuals. Cells were cultured as described in the legend to Fig. 4. (A) Proliferation was determined by [3H]thymidine uptake 5 days later. The upper right inserts show the percentage of FOXP3+ cells in SCD25 (left) or SCD25++ (right) T cells. (B) Culture supernatants were collected and analyzed by ELISA to determine amounts of IFN-c after 96 h. Numbers represent the percent suppression compared with CD4+ T cell alone. Results are expressed as means SEM of at least triplicate cultures and represent three independent experiments.
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Figure 6. Transient expression of FOXP3 in activated nonsuppressive human CD4+ T cells. FACS-sorted CD4+CD25 and CD4+CD25++ T cells were activated for 1 wk as described in Fig. 2, after which they were rested in complete medium supplemented with 100 U/mL IL-2 for another 7 days. (A) Representative flow cytometric plots showing CD25 versus FOXP3 expression in cells before (D7, upper panel) and after (D14, lower panel) resting of activated CD4+CD25 (CD25, left and middle panels) or CD4+CD25++ (CD25++, right panel) T cells. The quadrants for FOXP3 were based on the isotype control Ab, and the upper right numbers indicate the percentage of cells in each quadrant. (B) Suppressive ability of the cells before and after resting was measured as described in the legend to Fig. 4. Percent inhibition of proliferation is shown and results are expressed as means SEM of at least triplicate cultures.
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(data not shown). A marginal suppression by the cells that were nonsuppressive before resting was detected (Fig. 6B; donor 1). Taken together, these results indicate that FOXP3 is transiently expressed by activated CD4+CD25 T cells that do not obtain suppressive ability, whereas a stable FOXP3 expression is associated with the acquisition of regulatory T cell function.

Discussion
Immunological self tolerance, i.e. unresponsiveness to self antigens, is generated in the thymus by deletion of self-reactive lymphocytes, but also in the periphery through the control of their activation and expansion. As a key mechanism of such peripheral self tolerance, a small specialized subset of CD4+ T cells, identified by the expression of CD25, has the ability to suppress the activation and expansion of self-reactive T cells that have escaped thymic selection or have arisen de novo [9, 34]. The molecular mechanisms governing CD4+CD25+ Treg cell development and function are currently under intensive investigation due to their importance in controlling immune responses. Recently, Foxp3 has been demonstrated to program the development and function of Treg cells, and represents a specific marker of murine CD4+CD25+ Treg cells [1214]. Intriguingly, the dynamics of FOXP3 expression appears to differ between mice and humans. For example, unlike the murine counterpart, human FOXP3 is reported to be significantly up-regulated in activated conventional T cells [1821]. However, because in these studies FOXP3 expression was analyzed in the bulk of T cells either by Western blotting [18, 19], RT-PCR [20] or both [21], it is unclear whether the observed increase in FOXP3 mRNA and/or protein expression is a consequence of a few cells expressing high levels of FOXP3 or results from a moderate FOXP3 expression in a large population of activated cells. Here, we investigated FOXP3 expression in activated T cells at the single-cell level by using recently developed FOXP3-specific Ab. Our results demonstrate that the FOXP3+ activated T cells are most likely derived from FOXP3 conventional T cells because most CD4+CD25 T cells rapidly acquire FOXP3 expression after activation (i.e., rapidly after the start of cell division) (Fig. 2AC, E). These results are consistent with recent findings describing that the FOXP3 promoter region is in an open conformation and accessible to the transcription machinery in human CD4+CD25 T cells. Because the FOXP3 promoter contains binding sites for NF-AT and AP-1 [23] and has been reported to interact directly with NF-AT or NFjB [35], which are well-known mediators of T cell activation, it is conceivable that FOXP3 gene expression
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is regulated in a similar way as other immune-related genes that are controlled in an NF-AT/AP-1-dependent manner [23]. On the other hand, FOXP3 is able to bind to the forkhead-binding sites located within promoter regions, including promoter regions of cytokine genes such as IL-2 [35, 36]. In this way, FOXP3 is able to suppress the effector function of T cells as, e.g., the production of IL-2 and IFN-c [35, 36]. Thus, FOXP3 is likely to play a role in the negative feedback loop of T cell activation, by inhibiting cytokine production through binding to forkhead-binding sites in their promoters. The putative role of FOXP3 in this negative feedback loop is also in line with our findings that addition of exogenous IL-2 during activation enhanced both the frequency of FOXP3-expressing cells as well as the expression levels within cells (Fig. 2C). Thus, IL-2 not only promotes proliferation of T cells, but is also likely to up-regulate FOXP3 expression through increasing NFAT production [37] and/or via a STAT-dependent mechanism [38], thereby counteracting T cell responses. In contrast to the previous studies reporting that less than 30% of the CD4+CD25 T cells expressed FOXP3 after activation [23, 24], our data show that FOXP3 can be expressed in most human activated CD4+CD25 T cells (Fig. 2AC, E). This difference most likely relates to the stimulus given, as stimulation by pb-anti-CD3 Ab in the presence of exogenous IL-2 leads to a higher FOXP3 expression compared to stimulation with soluble anti-CD3 Ab (supplementary Fig. 1). The high percentage of FOXP3+ activated cells (>85%) enabled us to investigate the relationship between endogenous FOXP3 expression and suppressive function in individual cells. However, although the percentages of SCD25 T cells that produce FOXP3 as well as the FOXP3 expression levels were comparable in all nine donors (Fig. 46), a clear discrepancy in regulatory abilities between donors was noted (Fig. 4, 5). This discrepancy was not due to the age of the donors as we could not detect a relation between the age of the donors and the outcome of suppressive functions (supplementary Table 1). Likewise, we do not consider it likely that the presence of a small number of FOXP3+ T cells before stimulation is responsible for the inter-individual differences, as their percentages were similar in all donors (<3%; Fig. 2B) and, more importantly, even spiking of the CD25 T cell population with 3% of CD25++ Treg cells directly after purification did not convert nonsuppressive donors to suppressive donors (supplementary Fig. 2). Therefore, the differences between donors could be donor intrinsic, as also evidenced by the fact that analogous results were obtained with a donor in two independent experiments (data not shown). Similar findings have been reported recently by showing that ectopic expression of FOXP3 in human CD4+ T cells did not result in acquisition of consistent and significant suppressive activity [21].
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Together, these data suggest that FOXP3 alone is not sufficient for the generation of bona fide Treg cells [21, 39], as FOXP3 expression in conventional T cells is associated with hyporesponsiveness and reduced cytokine production (Fig. 3), but not necessarily correlated with suppressive activity in vitro (Fig. 4, 5). It is therefore likely that FOXP3 acts as a transcriptional repressor of various immune response genes in human T cells rather than as a master switch gene for Treg cell development. As the expression levels of FOXP3 in nonsuppressive activated human CD4+ T cells following a 1-wk stimulation were at least as high as those of the freshly isolated CD4+CD25++ Treg cells (Fig. 2A, D), it seems that FOXP3 expression levels do not correlate with regulatory function. Indeed, a stronger suppressive ability but decreased FOXP3 expression levels were observed in cells after a 1-wk rest (Fig. 6). However, in contrast to the relatively stable expression of FOXP3 in Treg cells and suppressive activated T cells, it is only transiently up-regulated in activated nonsuppressive T cells (Fig. 6), indicating that only cells that stably express FOXP3 during activation are regulatory. Moreover, the increased suppressive capacity of rested cells compared to freshly activated ones (Fig. 6B) could also explain the apparent discrepancy with other published data in which activated CD4+CD25 T cells were rested for 310 days before being used in experiments, resulting in a suppressive phenotype of these cells [18]. Although some insights into the molecular mechanisms underlying FOXP3 induction in human T cells have been recently achieved [23], the factors that stabilize FOXP3 expression in activated T cells are not yet known. Furthermore, it is tempting to speculate that other proteins in addition to FOXP3 may also be required for human T cells to acquire regulatory function because even FOXP3-transduced human CD4+ T cells can be nonsuppressive [21] and Foxp3 controls Treg cell function through cooperation with NF-AT in mice [40]. FOXP3 is now widely used to identify Treg cells due to its correlation with suppressive activity in freshly isolated human CD4+ T cells in healthy individuals [22, 31]. Additionally, the presence of FOXP3 expression is also used as an argument for the presence of Treg cells in autoimmunity [2527], as well as in patients with tumors [28, 29] or infections [30]. However, our data on the presence of nonregulatory FOXP3+ activated T cells emphasize that FOXP3 is not a specific marker for human Treg cells since it is conceivable that transient expression of FOXP3 following T cell activation also occurs in vivo [41], especially in patients with ongoing immune responses [27, 42]. In conclusion, our data indicate that the expression pattern of FOXP3 differs between mice and humans, as FOXP3 is also transiently expressed in human activated
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nonregulatory T cells, arguing that FOXP3, in humans, is not sufficient to induce regulatory T cell activity or to identify Treg cells. Materials and methods
Culture medium Human T cells were cultured in complete medium consisting of IMDM (Cambrex) supplemented with 10% heat-inactivated pooled human serum (the same batch in all experiments), 100 U/mL penicillin, 100 lg/mL streptomycin (Cambrex) and 2 mM GlutaMAX (Invitrogen). Cell isolation Human peripheral blood was obtained from healthy blood bank donors after informed consent in accordance with procedures approved by the local human ethical committee. PBMC were prepared by centrifugation over Ficoll-Hypaque gradients and CD4+ T cells were purified by negative selection with the CD4+ T cell isolation kit (Miltenyi Biotec). CD4+ T cells were then labeled with allophycocyanin (APC)conjugated anti-CD4 Ab and PE-coupled anti-CD25 Ab (BD Biosciences) for 30 min at 4 C. Cells were washed, and CD25 or the highest 12% of CD25++ T cells were sorted via a FACS Vantage cell sorter (Becton Dickinson, CA) with greater than 95% purity. The analysis and sort gates were restricted to the small lymphocyte gate as determined by their characteristic forward and side scatter properties. CD4-depleted PBMC, irradiated with 4500 rad were used as feeders in the proliferation/suppression assay. Activation of human T cells in vitro Purified CD4+CD25 or CD4+CD25++ T cells were activated with 5 lg/mL pb-anti-CD3 Ab (OKT-3; BD Biosciences) and 1 lg/mL soluble anti-CD28 Ab (CLB-CD28/1; Sanquin), in the presence of 50 or 300 (for CD4+CD25++) U/mL IL-2. Cells were restimulated, if necessary, as described above. In some cases, after extensive washing, activated T cells were rested in complete medium supplemented with 100 U/mL IL-2 before further experiments. FACS analysis of FOXP3 expression Cells were first surface-stained with anti-CD4-APC, anti-CD25PE or anti-CD25-FITC Ab (BD Biosciences); after fixation and permeabilization, cells were incubated with FITC/PE-conjugated anti-human FOXP3 Ab (PCH101; eBioscience) or rat IgG2a j isotype control according to the manufacturer's instructions. Proliferation and suppression of T cells Cells were plated at 2.5 104 cells/well in 96-well plates with 1 lg/mL PHA and 5 104 cells/well feeders in the presence or absence of 50 U/mL IL-2. Cells were pulsed with [3H]thymidine (0.5 lCi/well) on day 4 and proliferation
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Immunomodulation
receptor alpha-chains (CD25). Breakdown of a single mechanism of selftolerance causes various autoimmune diseases. J. Immunol. 1995. 155: 11511164. 10 Itoh, M., Takahashi, T., Sakaguchi, N., Kuniyasu, Y., Shimizu, J., Otsuka, F. and Sakaguchi, S., Thymus and autoimmunity: Production of CD25+CD4+ naturally anergic and suppressive T cells as a key function of the thymus in maintaining immunologic self-tolerance. J. Immunol. 1999. 162: 53175326. 11 Asano, M., Toda, M., Sakaguchi, N. and Sakaguchi, S., Autoimmune disease as a consequence of developmental abnormality of a T cell subpopulation. J. Exp. Med. 1996. 184: 387396. 12 Hori, S., Nomura, T. and Sakaguchi, S., Control of regulatory T cell development by the transcription factor Foxp3. Science 2003. 299: 10571061. 13 Fontenot, J. D., Gavin, M. A. and Rudensky, A. Y., Foxp3 programs the development and function of CD4+CD25+ regulatory T cells. Nat. Immunol. 2003. 4: 330336. 14 Fontenot, J. D., Rasmussen, J. P., Williams, L. M., Dooley, J. L., Farr, A. G. and Rudensky, A. Y., Regulatory T cell lineage specification by the forkhead transcription factor foxp3. Immunity 2005. 22: 329341. 15 Dieckmann, D., Plottner, H., Berchtold, S., Berger, T. and Schuler, G., Ex vivo isolation and characterization of CD4+CD25+ T cells with regulatory properties from human blood. J. Exp. Med. 2001. 193: 13031310. 16 Ng, W. F., Duggan, P. J., Ponchel, F., Matarese, G., Lombardi, G., Edwards, A. D. and Isaacs, J. D. et al., Human CD4+CD25+ cells: A naturally occurring population of regulatory T cells. Blood 2001. 98: 27362744. 17 Levings, M. K., Sangregorio, R. and Roncarolo, M. G., Human CD25+CD4+ T regulatory cells suppress naive and memory T-cell proliferation and can be expanded in vitro without loss of function. J. Exp. Med. 2001. 193: 12951302. 18 Walker, M. R., Kasprowicz, D. J., Gersuk, V. H., Benard, A., Van Landeghen, M., Buckner, J. H. and Ziegler, S. F., Induction of FoxP3 and acquisition of T regulatory activity by stimulated human CD4+CD25 T cells. J. Clin. Invest. 2003. 112: 14371443. 19 Walker, M. R., Carson, B. D., Nepom, G. T., Ziegler, S. F. and Buchner, J. H., De novo generation of antigen-specific CD4+CD25+ regulatory T cells from human CD4+CD25 cells. Proc. Natl. Acad. Sci. USA 2005. 102: 41034108. 20 Morgen, M. E., van Bilsen, J. H., Bakker, A. M., Heemskerk, B., Schilham, M. W., Hartgers, F. C., Elferink, B. G. et al., Expression of FOXP3 mRNA is not confined to CD4+CD25+ T regulatory cells in humans. Hum. Immunol. 2005. 66: 1320. 21 Allan, S. E., Passerini, L., Bacchetta, R., Crellin, N., Dai, M., Orban, P. C., Ziegler, S. F. et al., The role of 2 FOXP3 isoforms in the generation of human CD4+ Tregs. J. Clin. Invest. 2005. 115: 32763284. 22 Yagi, H., Nomura, T., Nakamura, K., Yamazaki, S., Kitawaki, T., Hori, S., Maeda, M. et al., Crucial role of FOXP3 in the development and function of human CD25+CD4+ regulatory T cells. Int. Immunol. 2004. 16: 16431656. 23 Mantel, P. Y., Ouaked, N., Ruckert, B., Karagiannidis, C., Welz, R., Blaser, K. and Schmidt-Weber, C. B., Molecular mechanisms underlying FOXP3 induction in human T cells. J. Immunol. 2006. 176: 35933602. 24 Gavin, M. A., Torgerson, T. R., Houston, E., Deroos, P., Ho, W. Y., StrayPedersen, A., Ocheltree, E. L. et al., Single-cell analysis of normal and FOXP3-mutant human T cells: FOXP3 expression without regulatory T cell development. Proc. Natl. Acad. Sci. USA 2006. 103: 66596664. 25 de Kleer, I. M., Wedderburn, L. R., Taams, L. S., Patel, A., Varsani, H., Klein, M., de Jager, W. et al., CD4+CD25bright regulatory T cells actively regulate inflammation in the joints of patients with the remitting form of juvenile idiopathic arthritis. J. Immunol. 2004. 172: 64356443. 26 Ruprecht, C. R., Gattorno, M., Ferlito, F., Gregorio, A., Martini, A., Lanzavecchia, A. and Sallusto, F., Coexpression of CD25 and CD27 identifies FoxP3+ regulatory T cells in inflamed synovia. J. Exp. Med. 2005. 201: 17931803. 27 Kriegel, M. A., Lohmann, T., Gabler, C., Blank, N., Kalden, J. R. and Lorenz, H. M., Defective suppressor function of human CD4+CD25+ regulatory T cells in autoimmune polyglandular syndrome type II. J. Exp. Med. 2004. 199: 12851291.

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was assessed 18 h later using a liquid scintillation counter. To test for suppressive capacity, cryopreserved autologous CD4+ T cells were stimulated as described above without the addition of exogenous IL-2. Stimulated CD4+CD25 T cells were added, and suppression was assessed by determining [3H]thymidine incorporation as well as by measuring the amount of IFN-c present in the supernatants. In some cases, autologous CD4+ T cells were also labeled with CFSE (2.5 lM; Invitrogen) and cultured in the absence or presence of stimulated CD4+CD25 T cells at a 1 : 1 ratio. After 96 h, the amount of CFSE dye dilution was analyzed by FACS. Autologous CD4+ and in vitro activated CD4+CD25++ Treg cells were included as negative and positive controls in all experiments, respectively. Cytokine detection To determine the amount of IFN-c in suppression assays, a capture ELISA was performed on supernatants collected 4 days after T cell stimulation. To determine cytokine concentrations in proliferation assays, Th1/Th2 cytometric bead assays (BD Biosciences) were used to analyze culture supernatants after 36 and 96 h, following the protocol provided by the manufacturer. The beads were analyzed on a FACSCalibur (Becton Dickinson) using the CBA software purchased from BD Pharmingen.

Acknowledgements: This work was supported by an NWO VIDI grant to R.E.M.T. The authors have no financial conflict of interest.

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