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Keywords:

  • FOXP3;
  • Human;
  • Treg

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and discussion
  5. Concluding remarks
  6. Materials and methods
  7. Acknowledgements
  8. References

FOXP3 is required for the development of Treg and its expression is often used as a surrogate marker of functional suppression. However, it is now known that activated human T effector cells can also express FOXP3 without acquiring regulatory activity. To more closely examine the requirements for FOXP3 to reprogram human T cells into Treg, we developed a conditionally active form of FOXP3 and show here that full acquisition of Treg phenotype and function is strictly dependent on the amount of active FOXP3 a T cell expresses. In addition, the phenotypic and functional alterations induced by FOXP3 are only fully manifested following prolonged induction of protein activity. Induction of FOXP3 activity does not upregulate EBI3 or p35 mRNA, providing evidence that secretion of IL-35 does not substantially contribute to the suppressive mechanism of human Treg. These data represent the first formal evidence that FOXP3 acts as a quantitative regulator rather than a simple molecular switch for Treg.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and discussion
  5. Concluding remarks
  6. Materials and methods
  7. Acknowledgements
  8. References

FOXP3 is a forkhead family transcription factor involved in the development of CD4+CD25+ Treg 1. In mice and humans, deletion of FoxP3 results in the absence of functional Treg in the periphery and the development of systemic autoimmunity, and ectopic expression of Foxp3 in mouse CD4+ T cells converts T effector (Teff) cells into functional Treg 1. Although these data suggest an essential role for FOXP3 in the development of Treg, its expression also appears to be necessary for controlling their function. For example, ablation of Foxp3 in adult mice, which have fully developed Treg, leads to the development of autoimmune inflammation 2. Furthermore, attenuated expression of Foxp3 in mouse Treg abrogates their suppressor function but leaves other aspects of the Treg phenotype, such as anergy and suppression of cytokine production, intact 3.

Other findings indicate that additional factors may be required for commitment to and stabilization of the Treg lineage 4–6. For example, several genes previously thought to be direct targets of Foxp3 are now known to be transcriptionally co-regulated with it 5. Furthermore, although Foxp3 is important for normal Treg function, other upstream events are important for defining and shaping the Treg lineage 4, 6. We and others have recently shown that in order to convert human CD4+ T cells into potent and stable Treg, FOXP3 must be expressed constitutively at high levels that do not fluctuate with the state of T-cell activation 7–10. Thus, FOXP3 might act as a quantitative regulator rather than a “master switch” of regulatory activity in human T cells. To directly test this hypothesis, we developed a conditionally active form of FOXP3, the activity of which could be controlled in a dose- and time-dependent manner, and investigated the consequences of its expression in human CD4+ T cells.

Results and discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and discussion
  5. Concluding remarks
  6. Materials and methods
  7. Acknowledgements
  8. References

Development of a conditionally active form of FOXP3

Conditionally active transcription factors can be made by fusion to the hormone-binding domain of the estrogen receptor (ER), which leads to translation of an inactive fusion protein 11, 12; on exposure to 4-hydroxytamoxifen (4HT), there is a rapid and dose-dependent induction of function. The activating effects of 4HT are reversed within 16 h of withdrawal, as the drug is rapidly metabolized in vitro11. In order to test whether fusion of the ER to FOXP3 was sufficient to create a conditionally active form of the protein, the FOXP3–ER fusion cDNA was transferred into a bi-directional lentivirus that encodes a truncated version of the nerve growth factor receptor (ΔNGFR) as a marker gene 7 (Fig. 1A). Naïve CD4+CD25CD45RO T cells were transduced with control or FOXP3–ER lentivirus, and populations were purified on the basis of ΔNGFR expression. Control ΔNGFR-transduced cells expressed a moderate amount of endogenous activation-induced FOXP3 7, 9, whereas FOXP3–ER-transduced cells expressed levels of FOXP3 that were equivalent to those in T cells transduced with wild-type FOXP3 or ex vivo Treg (Fig. 1B).

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Figure 1. Characterization of transduced human CD4+ T cells expressing conditionally active FOXP3. (A) Lentiviral expression constructs: control ΔNGFR, FOXP3-ER, and wild-type FOXP3. (B) FOXP3 expression in transduced ΔNGFR+ T cells in the resting state compared with ex vivo CD4+CD25+ Treg. Ex vivo and expanded T-cell lines are gated differently due to differences in fluorescence-minus-one controls between freshly isolated and cultured cells. FOXP3–ER cells were cultured in the presence of 150 nM 4HT for 12 days prior to staining. (C) Transduced 293T cells were left untreated or treated for 2 h with 4HT. Overlays of confocal and DIC images of 293T cells stained with Hoechst (blue) and anti-FOXP3-PE (red). (D) Analysis of CD25 and CD127 expression in the resting state. Histograms show a representative experiment, and graphs show averaged data derived from four different donors. ΔMFI indicates the fold-difference in the MFI of indicated populations compared with ΔNGFR-transduced T cells. (E) Analysis of CD25 expression following exposure to increasing amounts of 4HT for 7 or 12 days. Histogram depicts representative data from four experiments at 12 days; graph depicts the linear correlation between the average fold increase in MFI and the concentration of 4HT. (F) The fold-expansion of transduced T cells over 12 days was determined by cell counting. Data shown are pooled from four independent experiments. Error bars in (D–F) represent the SD of pooled data.

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Under normal conditions, FOXP3 is constitutively located in the nucleus of T cells. To assess whether fusing FOXP3 to the ER altered its sub-cellular localization, we used confocal and differential interference contrast (DIC) microscopy (Fig. 1C). These studies were performed in 293T cells since FOXP3 is known to localize in the nucleus of these cells, and their large cytoplasm allowed for better visualization of nuclear translocation 13. In the absence of 4HT, FOXP3–ER-transduced cells had a strong emission signal for FOXP3 in the cytoplasm and nuclear region. In contrast, after culture in 4HT for 2 h, FOXP3 was exclusively localized to the nucleus. We also investigated whether 4HT affected the stability of the FOXP3–ER protein by analyzing the MFI of FOXP3 in transduced cells cultured with or without 150 nM 4HT. After 24 h, there was a 1.70±0.05-fold increase in FOXP3–ER expression compared with untreated cells (p=0.003, n=9, data not shown), consistent with previous reports with other ER fusion proteins 11, 14. At times longer than 24 h, the expression did not increase further. Thus, the activity of the FOXP3–ER fusion protein can be rapidly regulated by altering its sub-cellular localization and expression 15.

To determine whether conditionally active FOXP3–ER was functional, we assessed the expression of known transcriptional targets of FOXP3 7. Transduced T cells were expanded for 12 days in the presence or absence of 4HT, and assayed for CD25 and CD127 expression (Fig. 1D). In the absence of 4HT, FOXP3–ER- and ΔNGFR-transduced T cells expressed equivalent levels of CD25 and CD127. In the presence of 4HT, FOXP3–ER-transduced cells upregulated CD25 and downregulated CD127 to levels that were equivalent to those in cells transduced with wild-type FOXP3, which are phenotypically and functionally indistinguishable from ex vivo Treg 7. We also investigated whether the activity of FOXP3–ER was dose-dependent by analyzing CD25 expression in transduced cells that were exposed to increasing amounts of 4HT. We observed a strong linear correlation between the expression of CD25 and the concentration of 4HT after both 7 and 12 days (7 days, R2=0.988; 12 days, R2=0.999; Fig. 1E). These data confirm that both the dose and the duration of 4HT exposure directly affect the transcriptional activity of the FOXP3–ER fusion protein.

In addition to altering the expression of cell-surface molecules, over-expression of FOXP3 results in induction of hyporesponsiveness 7. To further test the functionality of FOXP3–ER, we determined the expansion of the transduced T-cell lines over 12 days. In the absence of 4HT, FOXP3–ER-transduced T cells expanded equivalently to ΔNGFR-transduced cells (Fig. 1F), whereas addition of 4HT led to reduced expansion of the FOXP3–ER-transduced T cells (p=0.037, n=4 donors, t=12 days) to a level similar to that of T cells expressing wild-type FOXP3. Thus, FOXP3–ER acts as a conditionally active protein and can induce naïve T cells to take on a Treg phenotype when activated by 4HT.

FOXP3 suppresses Th1 and Th2 cytokines in a time- and dose-dependent manner

Expression of FOXP3 in CD4+ T cells results in suppressed cytokine production 7, 8, and FOXP3 is known to directly repress the expression of several cytokine genes 16. To define whether the amount of active FOXP3 directly correlated with the degree of cytokine suppression and to examine the temporal nature of this effect, we stimulated transduced T cells for different amounts of time and in the absence or presence of different concentrations of 4HT. FOXP3–ER-transduced cells displayed a dose-dependent decrease in the production of IL-2, IFN-γ, TNF-α, IL-10, IL-4, and IL-5 upon addition of 4HT at the time of TCR activation (t=0) (Fig. 2A). To examine whether inducing FOXP3–ER activity prior to TCR activation augmented its inhibitory capacity, we also analyzed cytokine production following either short-term (t=16 h) or long-term (t=12 days) pre-treatment with 4HT. Pre-incubation with 4HT significantly increased the degree of cytokine inhibition in FOXP3–ER-transduced T cells, with the most potent effects observed after long-term culture (p⩽0.035, n⩾5 when cytokine production was compared between T cells incubated with 4HT at t=0 and those cultured in the drug for t=12 days). Addition of 4HT to FOXP3–ER-transduced T cells had no effect on TGF-β production (data not shown), confirming our previous findings 7. Although IL-10 and TGF-β are not required for in vitro suppression by human Treg 17, they are involved in the suppression of mucosal inflammation by mouse Treg 18. Since neither IL-10 nor TGF-β production was induced by FOXP3, additional factors may therefore be required to stimulate the production of these cytokines in vivo. Overall, these data indicate that the full capacity of FOXP3 to inhibit cytokine production likely involves molecular and/or transcriptional changes that occur over a prolonged period of time. Moreover, the strict dose dependence of this effect supports our previous conclusion that high and constitutive levels of active FOXP3 are required to convert naïve T cells into Treg 7.

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Figure 2. Inhibition of cytokine production by FOXP3 is dose- and time-dependent. (A) Transduced T cells were exposed to the indicated concentration of 4HT either at the time of the assay (t=0), or 16 h or 12 days prior to stimulation with anti-CD3/anti-CD28. Supernatants were collected after stimulation for 24 h (IL-2) or 48 h (all other cytokines) and analyzed using a Th1/Th2 cytometric bead assay. Plots are representative of data from a minimum of three donors and error bars represent SD. (B) cDNA isolated from resting, transduced, or ex vivo T cells was analyzed for expression of the indicated mRNA species, and mRNA levels were normalized to GAPDH expression in each sample and expressed relative to activated macrophages (MΦ). Graphs show pooled data from three independent experiments, and error bars represent the SD of pooled data.

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It has been reported that the secretion of IL-35 is an important mechanism of suppression mediated by mouse Treg 19, 20, but whether this cytokine is produced by human Treg was unknown. We therefore examined the expression of EBI3 and p35, which encode the two subunits of IL-35, in transduced T cells, ex vivo Treg, and Teff cells. Unlike murine Treg, ex vivo human Treg did not express significant levels of EBI3 mRNA (Fig. 2B), and levels of p35 did not differ between ex vivo Treg and Teff cells. In addition, neither levels of EBI3 nor levels of p35 mRNA were affected by addition of 4HT in the FOXP3–ER-transduced T cells. Activated macrophages, which are known to express EBI3 and p3519, 21, served as a positive control. These data suggest that IL-35 may not play a major role in the suppressive mechanism of human Treg.

Induction of hyporesponsiveness by FOXP3 is time- and dose-dependent

To determine whether the capacity of FOXP3 to induce hyporesponsiveness was dose- and time-dependent, we assayed the proliferation of transduced T cells in the presence of increasing concentrations of 4HT added at the time of TCR stimulation. The inhibitory capacity of FOXP3–ER was dose-dependent, and at 150 nM 4HT, proliferation was suppressed to a level equivalent to that of T cells transduced with wild-type FOXP3 (Fig. 3A).

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Figure 3. Induction of T-cell hyporesponsiveness and suppressive capacity by FOXP3 is dose- and time-dependent. (A) Transduced T cells were tested for their ability to proliferate in response to immobilized anti-CD3 in the presence of increasing amounts of 4HT added at the time of the assay (t=0). (B) FOXP3–ER-transduced T cells were stimulated with a 1:1 ratio of irradiated APC and soluble anti-CD3 and exposed to increasing amounts of 4HT either at the time of the assay (t=0), or 16 h or 12 days prior. (C) Transduced T cells were tested for their ability to respond to immobilized anti-CD3 (1 μg/mL) in the presence or absence of IL-2 (100 U/mL). 4HT (150nm)was added at the time of the assay (t=0) or prior to assay as indicated. (D–F) Ex vivo CD4+CD25 responder T cells were stimulated with a 1:1 ratio of APC and anti-CD3 in the presence or absence of transduced T cells. 4HT was added at the time of the experiment at the concentrations indicated in (D and F) and for (E) at 150 nM for the times indicated. In (G) cells were expanded in 4HT for 12 days, or expanded in 4HT, followed by withdrawal (WD) of 4HT from culture medium for the indicated times prior to testing for their capacity to suppress IFN-γ secretion at a 1:1 or 1:2 ratio (suppressors to responders). Ex vivo CD4+CD25+ Treg were included as a positive control. Cellular proliferation was determined by thymidine incorporation and IFN-γ secretion was determined using a cytometric bead array. (A–F) Representative of a minimum of three experiments; (G) representative of five experiments. Error bars represent the SD from triplicate wells.

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To further investigate the kinetic requirements for the suppressive effect of FOXP3, we compared the proliferative capacity of FOXP3–ER-transduced T cells pre-incubated with 4HT (for either 16 h or 12 days) or exposed to 4HT at the time of the experiment (t=0). Pre-incubation of FOXP3–ER-transduced T cells with 4HT resulted in more pronounced hyporesponsiveness, which increased with long-term exposure to the drug (Fig. 3B). Interestingly, addition of low-dose 4HT (5 nM) at the time of the assay had a minimal effect on proliferation, whereas long-term culture in the same concentration resulted in marked hyporesponsiveness. The anti-proliferative effects of active FOXP3–ER did not compromise viability, as anergy was fully reversed upon addition of IL-2 (Fig. 3C). Thus, FOXP3 induces T-cell hyporesponsiveness in a time- and dose-dependent manner.

High levels of FOXP3 activity are required to induce maximum suppressive capacity

Addition of 4HT at the time of an in vitro suppression assay caused FOXP3–ER-transduced T cells to inhibit the proliferation of ex vivo CD4+CD25 responder cells in a dose-dependent manner (Fig. 3D) with an average of 45±20% (n=4) suppression at 150 nM 4HT. Importantly, pre-induction of FOXP3 activity prior to the assay resulted in enhanced suppressive capacity (Fig. 3E). Similar results were obtained upon analysis of IFN-γ production: at a 1:1 ratio FOXP3–ER-transduced cells inhibited IFN-γ production by ex vivo responder T cells by only 13±22% when 4HT was added at the time of the assay (t=0), whereas pre-incubation with 4HT for 12 days resulted in 76±9% inhibition (p=0.007, n=4) (Fig. 3F). 4HT affected neither the proliferation of the responder T cells nor the suppressor function of ex vivo CD4+CD25+ Treg (Fig. 3D). Thus, functional reprogramming is induced only upon long-term and high expression of functional FOXP3.

A final question was whether expression of FOXP3 was required solely as a lineage specification factor and/or to also maintain suppressive function in fully differentiated Treg. To address this question, we cultured FOXP3–ER cells in 4HT for 12 days and then withdrew 4HT for different times prior to the assay. Withdrawal of 4HT resulted in rapid loss of suppressor function, and after only 24 h without 4HT, the suppressive capacity of the FOXP3–ER cells was significantly different from that of FOXP3–ER cells maintained in 4HT (p=0.004, n=5; Fig. 3G). In line with the observation that 4HT increased the stability of the FOXP3–ER protein, withdrawal of 4HT resulted in a gradual decline in the intensity of FOXP3–ER expression and returned to baseline after 72 h (data not shown). Together, these data indicate that continual FOXP3 activity is required to maintain suppressive capacity in FOXP3-transduced Treg.

Concluding remarks

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and discussion
  5. Concluding remarks
  6. Materials and methods
  7. Acknowledgements
  8. References

FOXP3 was originally thought to be exclusively expressed in functional Treg, and all FOXP3+ cells were thought to be suppressive. We have shown here that full acquisition of the Treg phenotype and function is strictly dependent of the amount of active FOXP3 a T cell expresses. In addition, alterations induced by FOXP3 are only fully manifested following prolonged induction of protein activity, suggesting that transcriptional reprogramming likely requires new protein synthesis and molecular changes that enhance or reinforce the Treg phenotype. These events might include physical interactions with other transcriptional factors 22, alterations of chromatin structure at specific loci 23, and/or induced expression of other molecules that are required for suppression. Our data support previous findings that activation-induced expression of FOXP3 is not sufficient to re-program human Teff cells into Treg 9, 10 and that only upon high and constitutive expression of FOXP3 are the Treg phenotype and function recapitulated 7. It cannot be ruled out, however, that long-term, low-level FOXP3 expression might also be sufficient to induce a regulatory phenotype.

Since FOXP3 activity is directly correlated with regulatory function, even small reductions in FOXP3 expression or activity could markedly affect Treg function in vivo. Indeed, IPEX can be caused by mutations in, or upstream of, FOXP3 that reduce but do not abolish mRNA expression 24. In addition, a study of multiple sclerosis patients detected a significant decrease in FOXP3 expression and suppressive function in Treg from patients compared with healthy controls 25. Enhancing the expression or molecular function of FOXP3 might be a promising strategy to increase the suppressive function of Treg in vivo. In line with this idea, it has recently been demonstrated that augmenting the acetylation, and thus the molecular function, of Foxp3 by treatment with the histone deacetylase inhibitor Trichostatin A leads to enhanced Treg function in vivo26. Thus, reduced FOXP3 expression might contribute to the development of autoimmunity, whereas targeted manipulation of its expression and post-translational modification might be an effective way to modulate the immune response.

In conclusion, using a conditionally active FOXP3 protein, we have clearly defined that the capacity of FOXP3 to reprogram human T cells into Treg is dose- and time-dependent. These data indicate that simply classifying T cells as FOXP3+ or FOXP3 is not sufficient to draw conclusions regarding suppressive function. Expression levels of FOXP3 must be carefully quantified and compared with ex vivo Treg over the course of T-cell activation before extrapolations on the functional status of T cells can be made. The conditionally active FOXP3–ER protein will also be a valuable tool to further study the cellular and molecular phenotype of Treg generated by FOXP3 transduction and to regulate their function in vivo.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and discussion
  5. Concluding remarks
  6. Materials and methods
  7. Acknowledgements
  8. References

Construction and production of lentiviral vectors

Human FOXP3a was fused at the C-terminus to the ER. The ER contained a point mutation to prevent binding to estrogen, but allow interaction with the estrogen analog 4HT (Sigma-Aldrich) 12. The FOXP3–ER fusion protein was expressed in a bidirectional lentiviral vector 7.

Cell purification

Peripheral blood was obtained from healthy volunteers following approval by the University of British Columbia Clinical Research Ethics Board and obtaining written informed consent. Ex vivo CD4+CD25hi Treg, CD4+CD25CD45RO naïve T cells, and CD3-depleted APC were purified from PBMC 7. Activated macrophages were prepared by culturing the adherent fraction of PBMC in GM-CSF (Stemcell Technologies) for 4 days, followed by 4 h of activation with LPS (0.1 ng/mL, Sigma-Aldrich) and IFN-γ (10 ng/mL, R&D Systems).

Lentiviral transduction and culture of CD4+ T cells

Naïve T cells were activated with anti-CD3 (1 μg/mL OKT3, Orthoclone) and autologous irradiated APC in complete medium (X-VIVO 15 (Cambrex) with 5% pooled AB human serum (Cambrex), and penicillin/streptomycin (Invitrogen)), containing rhIL-2 (100 U/mL, Chiron) and rhIL-7 (10 ng/mL, BD Biosciences) 7. After 16 h, lentivirus was added at a multiplicity of infection of 10. Transduced T cells were purified and expanded as described previously 7. Based on titration data, 4HT solubilized in ethanol was added at 150 nM unless otherwise indicated.

Microscopy, image acquisition, and image processing

Transduced 293T cells were purified on the basis of ΔNGFR expression and co-stained with Hoechst (Sigma-Aldrich) and anti-FOXP3-PE (clone 236A/E7, eBiosciences). Confocal and DIC images were acquired with a Leica AOBS SP2 laser scanning confocal microscope using a Leica 63×/1.4 Plan-Apochromat oil immersion objective and Leica Confocal TCS SP2 acquisition software. Volocity software (Improvisions) was used to overlay images and enhance contrast.

Proliferation, suppression, and phenotypic analyses

A total of 5×104 T cells/well were activated either by immobilized anti-CD3 (1 μg/mL) or a 1:1 ratio of APC (irradiated 5000 rads) with 1 μg/mL of soluble anti-CD3 in the presence or absence of IL-2 (100 U/mL). Proliferation was assessed after 72 h (immobilized anti-CD3 assay) or 96 h (APC/anti-CD3 assay) by [3H]thymidine (1 μCi/well, GE Life Sciences) incorporation. Suppressive capacity was determined as described previously 7. Staining for cell-surface markers ΔNGFR, CD25, CD127 (all BD Pharmingen) was carried out prior to intracellular staining for FOXP3 (clone 236A/E7, eBiosciences). Samples were acquired on a BD FACSCanto and analyzed with FCS Express Pro Software Version 3 (De Novo Software). Cytokine production following activation with anti-CD3 (10 μg/mL) and soluble anti-CD28 (1 μg/mL) was performed as described previously 7. cDNA was analyzed for EBI3, p28, and p35 expression using the following primers: EBI3 sense: 3′-AGCACATCATCAAGCCCGAC-5′; EBI3 antisense: 3′-GCTCCCTGACGCTTGTAACG-5′; p35 sense: 3′-CTCCTGGACCACCTCAGTTTG-5′; p35 antisense: 3′-CGGCCCTCAGCAGGTTTT-5′. FOXP3 and GAPDH mRNA were quantified as described previously 7. Samples were run in triplicate, normalized to GAPDH, and fold-changes in mRNA relative to activated macrophages were determined.

Statistics

All analyses for statistically significant differences were performed with one-tailed paired Student's t test; p values of less than 0.05 were considered significant.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and discussion
  5. Concluding remarks
  6. Materials and methods
  7. Acknowledgements
  8. References

The paper was supported by grants from the Canadian Institutes for Health Research (III68855) and StemCell Technologies. Core support for lentivirus production and flow cytometry was funded by the Immunity and Infection Research Centre MSFHR Research Unit. M.K.L. holds a Canada Research Chair in Transplantation and is an MSFHR Scholar. S.E.A. holds an MSFHR Senior Graduate Studentship award and a CIHR Canada Graduate Scholarship Doctoral Award. A.N.M. holds a CIHR/MSFHR Transplantation Training Program award, an MSFHR Junior Graduate Studentship award, and a CIHR Masters Award. We thank Paul Orban for critical reading of the paper.

Conflict of interest: The authors declare no financial or commercial conflict of interest.

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  2. Abstract
  3. Introduction
  4. Results and discussion
  5. Concluding remarks
  6. Materials and methods
  7. Acknowledgements
  8. References
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