Prostaglandin E₂ Induces FOXP3 Gene Expression and T Regulatory Cell Function in Human CD4⁺ T Cells

Cell isolation and culture

Human CD4⁺C25⁺ T reg cells and CD4⁺CD25⁻ T cells were purified from enriched buffy coat from healthy volunteers using the T reg cell isolation kit (Miltenyi Biotec) and the AutoMACS cell sorter (Miltenyi Biotec). An institutional review board approval was obtained, and all donors signed informed consent. Cell purity assessed by flow cytometry was ≥92%. Before being used in different experimental conditions, purified CD4⁺CD25⁺ and CD4⁺CD25⁻ T cell fractions were preincubated for 24 h in X-Vivo 15 (BioWhittaker), 10% FBS, and 1% human serum AB (Gemini) (24) with or without 16,16-dimethyl-PGE₂ (Cayman Chemical; 13 or 26 μM). In some experiments, CD4⁺CD25⁻ T cells (2 × 10⁶) were preincubated for 18–24 h with 2 ml of undiluted tumor supernatant obtained from H157 (human squamous cell carcinoma; American Type Culture Collection) NSCLC, genetically modified to express COX-2 sense (S), COX-2 antisense (AS), or control empty vector (CV) (25). A 2.0-kb cDNA fragment of human COX-2 (provided by Dr. H. Herschman, University of California, Los Angeles, CA) was cloned in S and AS orientations in the retroviral vector pLNCX (BD Clontech) as previously described (25). For each cell line, an ∼10-fold higher level of COX-2 expression and PGE₂ production was noted in COX-2 S compared with parental controls or CV (25). To neutralize the activity of the PGE₂ contained in the COX-2 S supernatant, anti-PGE₂ (10 μg/ml; provided by J. Portanova, Searle, St. Louis, MO) was added, and a mouse IgG (10 μg/ml; Sigma-Aldrich) was used as a control Ab. T cell activation was performed by incubation of both CD4⁺CD25⁻ T cells and CD4⁺C25⁺ T reg cells in plate-bound anti-CD3 (1 μg/ml)-coated, 96-well plates (BD Biosciences) from 24 to 120 h. Alternatively, T cells were activated in the presence of PMA (20 ng/ml; Sigma-Aldrich) and ionomycin (1 μg/ml; Sigma-Aldrich).

Flow cytometric analysis

CD4⁺CD25⁻ T cells and CD4⁺CD25⁺ T reg cells, pretreated with or without PGE₂ and cultured with or without anti-CD3 stimulation, were resuspended in PBS/2% FBS and labeled with human anti-CD25 PE (Miltenyi Biotec) and human anti-CD4 FITC Abs or with control isotype (all from BD Biosciences) at the indicated time points. Acquisition was performed using a Life Science Research flow cytometer or a SCAN X instrument (BD Biosciences) using CellQuest software (BD Biosciences) at University of California-Los Angeles Jonsson Comprehensive Cancer Center Flow Cytometry Core Facility. Data analysis was performed with CellQuest software.

In vitro proliferation assay

T cell proliferation was assessed by BrdU incorporation using a BrdU ELISA colorimetric assay (Roche). Briefly, magnetic bead-purified human CD4⁺CD25⁻ T cells or CD4⁺C25⁺ T reg cells were pretreated with PGE₂ (13 or 26 μM) for 24 h. PGE₂ was removed by washing before starting the proliferation assay. To rule out the possibility that residual PGE₂ would be transferred to the second culture, we performed preliminary experiments in which T cells were pretreated with PGE₂ (26 μM; Cayman Chemical; catalogue no. 14010) for 12–18 h. The presence of PGE₂ in the supernatant was then assessed by a specific enzyme immunoassay (PGE₂ EIA kit; Cayman Chemical) after cell washing. The latter procedure was determined to be effective in removing >99% of the added PGE₂ (data not shown). Both CD4⁺CD25⁻ and CD4⁺C25⁺ T cell populations, with or without PGE₂ pretreatment, were then cultured at a 1:1 ratio with autologous CD4⁺CD25⁻ responder T cells (1 × 10⁵) in anti-CD3 plate-bound, 96-well plates. In some experiments IL-2 (Proleukin; biological activity, 18 × 10⁶ IU/1.1 mg; Chiron) or IL-7 (biological activity, 2 × 10⁶ U/mg; PeproTech) was added to the cells. The concentrations of IL-7 and IL-2 used in the proliferation assays (2 ng/ml equivalent to 4 U/ml and 20 IU/ml corresponding to 1.56 ng/ml, respectively) were determined based on previous reports (26, 27) and our own preliminary studies. In four independent experiments, the T cell proliferations expressed as OD values of BrdU incorporation on day 5 of culture were 0.95 ± 0.3 in the presence of IL-2 (20 IU/ml) and 0.93 ± 0.06 in the presence of IL-7 (2 ng/ml). Thus, the concentrations of IL-2 and IL-7 used were comparable based on the capacity of both cytokines to induce a similar degree of proliferation of CD3-activated CD4⁺ T cells in vitro. After 5 days in culture, cells were pulsed with BrdU and 4 h later were assessed for BrdU incorporation. To analyze cell contact-independent inhibition, 3 × 10⁵ untreated or PGE₂-treated CD4⁺CD25⁻ T cells and untreated or PGE₂-treated CD4⁺CD25⁺ T reg cells were separated from mitomycin C (50 μg/ml; Sigma-Aldrich)-treated, allogeneic-dendritic cell-stimulated CD4⁺CD25⁻ responder T cells (3 × 10⁵) by 3-μm Transwell inserts (Costar; Corning) in 24-well plates. T cell proliferation was then measured after a 5-day culture period by analysis of BrdU incorporation. Results are expressed as the percentage of proliferation of responder T cells alone.

Real-time PCR for FOXP3

For quantitative real-time analysis, RNA was extracted, and cDNA was prepared with a kit (Invitrogen Life Technologies) according to the manufacturer’s instructions. Human FOXP3 mRNA expression was quantified using the SYBR Green quantitative PCR kit (Finnzymes) in the iCycler (Bio-Rad) and was corrected with human β-actin housekeeping control amplifications. Amplification was conducted in a total volume of 20 μl for 40 cycles of 15 s at 95°C, 20 s at 60°C, and 30 s at 72°C. Samples were run in triplicate, and their relative expression was determined by normalizing the expression of each target to β-actin and then comparing this normalized value to the normalized expression in a reference sample to calculate a fold change value.

For FOXP3, primers were: forward, 5′-CAA GTT CCA CAA CAT GCG AC-3′; and reverse, 5′-ATT GAG TGT CCG CTG CTT CT-3′. FOXP3 primers were synthesized by Integrated DNA Technologies. For β-actin, primers were: forward, 5′-GATGAGATTGGCATGGCTTT-3′; and reverse, 5′-CACCTTCACCGTTCCAGTTT-3′.

FOXP3 promoter cotransfection of Jurkat T cells and dual luciferase assay

A 350-bp fragment (FOXP3-SubD) containing the FOXP3/scurfin gene intron 0 upstream from the initiation codon, located within the 65,825–65,475 bp region of human chromosome X, multiple clone map pII.23, was inserted into a firefly luciferase reporter vector (pGL3-basic; Promega) as previously described (28). FOXP3-SubD and Renilla luciferase gene CV (pRL-TK Renilla) were then cotransfected into Jurkat T cells using the SuperFect Transfection Reagent (Qiagen). PGE₂ (13 μM), PMA (20 ng/ml) plus ionomycin (1 μg/ml) (PI), and PMA, ionomycin, and PGE₂ were added separately to Jurkat T cells 24 h after transfection for an additional 18 h. Firefly and Renilla luciferase activities were measured using the dual luciferase reporter assay system (Promega) and a single-sample luminometer (Bacterial Systems; GEM Biomedical). Relative luciferase activity was calculated as the ratio of firefly to Renilla luciferase.

Western blot analysis for FOXP3

FOXP3 protein expression was analyzed in the lysate of Jurkat T cells (5.0 × 10⁵ cells). The protein concentration in the cell lysate was determined using a bicinchoninic acid assay (Pierce). Protein-normalized aliquots of the cell lysate (25 μg) were electrophoresed on an 8% SDS-polyacrylamide gel and transferred onto nitrocellulose membranes. FOXP3 was immunodetected with 1/5000 rabbit anti-human FOXP3 polyclonal Ab (Abcam), followed by incubation with 1/500 HRP-conjugated donkey anti-rabbit Ig (Santa Cruz Biotechnology). Immunoblots were developed using an ECL detection system (Supersignal West Pico chemiluminescence; Pierce), followed by autoradiography. Equal protein loading was confirmed by immunodetecting the membranes with anti-GAPDH Ab (Advanced Immunochemical). Relative protein quantification was determined by computerized densitometric analysis using Scion Image software (version 1.62c).

Statistics

The p values were calculated using unpaired Student’s t test. Values ≤0.05 were considered significant.

PGE₂ enhances CD4⁺CD25⁺ T regulatory function and confers T regulatory activity in CD4⁺CD25⁻ T cells

Previous studies have focused on the direct effect of PGE₂ in mediating suppression of lymphocyte proliferation, highlighting the role of calcium-dependent mechanisms (29, 30). In the current study we first investigated whether exogenous PGE₂ was able to augment the suppressive activity of CD4⁺CD25⁺ T reg cells. Purified CD4⁺CD25⁺ T reg cells demonstrated significant enhancement of their inhibitory function after incubation with PGE₂ for 24 h in vitro (Fig. 1⇓A). Treatment of CD4⁺CD25⁺ T reg cells with escalating doses of PGE₂ (13 or 26 μM) inhibited T cell proliferation in a dose-dependent pattern (Fig. 1⇓A). The T cell proliferations, expressed as OD values of BrdU incorporation, obtained from seven different donors were 1.04 ± 0.17 (CD4⁺CD25⁻), 0.68 ± 0.16 (CD4⁺CD25⁺), and 0.34 ± 0.14 (CD4⁺CD25⁺+ PGE₂; 26 μM). During the 5-day culture period of the assay, we examined the T cell proliferative response by light microscopy. Wells containing stimulator T cells not treated with PGE₂ displayed numerous homogeneously distributed cell clusters, indicative of ongoing proliferation. In contrast, wells containing stimulator T cells pretreated with PGE₂ consistently showed significantly fewer proliferating clusters (data not shown). The OD values of BrdU incorporation reflected the above observations. Accumulating evidence indicates that T cells with regulatory features can be generated in vitro (6, 7, 8, 9, 10, 11, 12, 13). In the present study, preincubation of CD4⁺CD25⁻ T cells with PGE₂ (13 or 26 μM) conferred regulatory T cell function (Fig. 1⇓B). In fact, the PGE₂-treated CD4⁺CD25⁻ stimulator cells inhibited the CD3-activated proliferation of autologous CD4⁺CD25⁻ responder T cells. The levels of inhibition demonstrated by CD4⁺CD25⁻ T cells exposed to PGE₂ were similar to those induced by untreated CD4⁺CD25⁺T reg cells (Fig. 1⇓B). The T cell proliferations, expressed as OD values of BrdU incorporation, obtained from three different donors were 1.26 ± 0.39 (CD4⁺CD25⁻) and 0.65 ± 0.40 (CD4⁺CD25⁻ plus PGE₂ (26 μM)). Addition of IL-7 (2 ng) partially overcame the PGE₂-mediated suppression and reversed the CD4⁺CD25⁺ T reg cell-mediated inhibition of proliferation (Fig. 1⇓C). The T cell proliferations, expressed as OD values of BrdU incorporation, obtained from three different donors were 0.93 ± 0.06 (CD4⁺CD25⁻), 0.72 ± 0.17 (CD4⁺CD25⁻ plus PGE₂ (26 μM)), 0.89 ± 0.07 (CD4⁺CD25⁺), and 0.69 ± 0.22 (CD4⁺CD25⁺ plus PGE₂ (26 μM)). In contrast, IL-2 (20 IU/ml) did not overcome the PGE₂-induced or the CD4⁺CD25⁺ T reg cell-induced suppression (Fig. 1⇓D). The T cell proliferations, expressed as OD values of BrdU incorporation, obtained from three different donors were 0.95 ± 0.29 (CD4⁺CD25⁻), 0.46 ± 0.25 (CD4⁺ CD25⁻ plus PGE₂ (26 μM)), 0.64 ± 0.36 (CD4⁺CD25⁺), and 0.31 ± 0.16 (CD4⁺CD25⁺ plus PGE₂ (26 μM)). In assays that directly assessed CD4⁺CD25⁺ T reg cell proliferation, bothIL-7 (≥2 ng/ml) and a high concentration of IL-2 (≥100 IU/ml) demonstrated the capacity to induce proliferation of T reg cells (data not shown) in agreement with previous reports (31, 32). Interestingly, when CD4⁺CD25⁻ responder T cells were separated by Transwell inserts from PGE₂-treated stimulator cells (CD4⁺CD25⁻ or CD4⁺CD25⁺), both PGE₂-treated populations maintained the capacity to inhibit T cell proliferation (Fig. 1⇓E). These data taken together suggest that the PGE₂-induced T regulatory function does not require cell contact. We have previously found that PGE₂ is a potent inducer of lymphocyte IL-10 gene transcription (15), and PGE₂ has also been reported to increase TGF-β expression (33, 34, 35). However, neither anti-IL-10 nor anti-TGF-β neutralizing Abs were able to reverse the PGE₂-induced inhibitory activity mediated by CD4⁺CD25⁻ or CD4⁺CD25⁺ T reg cells (data not shown). These findings suggest the contribution of additional soluble factors other than IL-10 or TGF-β to PGE₂-induced cell-contact independent T regulatory cell function.

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FIGURE 1.

A, PGE₂ enhances T reg cell function. Purified CD4⁺CD25⁺ and CD4⁺CD25⁻ cells, precultured with or without PGE₂, were mixed with autologous CD4⁺ cells in anti-CD3-bound, 96-well plates for 5 days. B, PGE₂ induces T reg cell activity in CD4⁺CD25⁻ T cells. CD4⁺CD25⁺ and CD4⁺CD25⁻ cells were precultured with increasing concentrations of PGE₂ before a 5-day proliferation assay. C and D, IL-7, but not IL-2, overcomes suppression of T cell proliferation. IL-7 (C) or IL-2 (D) was added on day 1 to the proliferation assay described above. E, PGE₂ mediates T reg cell activity without the requirement for cell contact. Both untreated and PGE₂-treated CD4⁺CD25⁺ and CD4⁺CD25⁻ cells were separated from dendritic cell-activated CD4⁺CD25⁻ T cells by Transwell inserts. In all experiments, cell proliferation was measured by BrdU incorporation. Results are expressed as the mean (±SD) of one representative experiment of at least three performed in triplicate from different donors.

Down-regulation of CD25 expression in PGE₂-treated CD4⁺T cells

Because T reg cells are identified as a CD4⁺CD25^brightT cell subset (36), we analyzed whether the PGE₂-treated CD4⁺CD25^dim starting population acquired the same phenotype of naturally occurring, thymic-derived CD4⁺CD25^bright T reg cells. As shown in Table I⇓, CD25 was differentially expressed in magnetic bead-purified CD4⁺CD25^dim and CD4⁺CD25^brightT cells. Indeed, with (Fig. 2⇓A and Table II⇓) or without PGE₂ preincubation (data not shown), CD4⁺CD25^dim up-regulated CD25 as early as 24 h after stimulation with anti-CD3 plate-bound Ab (Fig. 2⇓A). These T cells maintained high levels of CD25 expression throughout a 96-h culture period (Fig. 2⇓A). However, when anti-CD3 Ab-activated T cells were pretreated with PGE₂, CD25 expression declined in both CD4⁺CD25^dim T cells (Fig. 2⇓B, bottom left, and Table II⇓) and CD4⁺CD25^bright T reg cells (Fig. 2⇓B, bottom right, and Table III⇓), compared with their counterparts without PGE₂ exposure (Fig. 2⇓, B and C, upper panels, and Tables II⇓ and III⇓). Despite decreased CD25 expression, suppressive T reg function was maintained, as shown in Fig. 1⇑. Other markers indicative of the T reg phenotype, including glucocorticoid-induced TNFR family-related protein, CTLA-4, human latency-associated peptide of TGF-β1, lymphocyte activation Ag 3, PD-1, CD103, CD122, and CCR4, were either not altered or were down-regulated by PGE₂ (data not shown).

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FIGURE 2.

PGE₂ down-regulates CD25 expression in CD3-activated CD4⁺ lymphocytes. A, PGE₂-treated, CD3-activated CD4⁺CD25^dim T cells up-regulate CD25 expression. Double-positive CD4⁺CD25⁺ cells were measured in unstimulated (top panel) and anti-CD3-stimulated (bottom panel), PGE₂-treated CD4⁺CD25⁻ T cells. Density plots of a representative experiment of four performed are shown. A, The number inserted in the upper right quadrant indicates the percentage of double-positive cells (CD4⁺CD25⁺). PGE₂ decreased CD25 expression in both CD4⁺CD25^dim (B) and CD4⁺CD25^bright starting populations (C). CD25 was measured in anti-CD3-stimulated, CD4⁺CD25^dim T cells and in CD4⁺CD25^bright T reg cells without (−; top panel) or with (+) PGE₂ (26 μM; bottom panel) at the indicated time points. Results were obtained from a donor different from that in A. In the histograms, dotted lines represent the isotype control, and bold lines indicate surface marker staining. B, The numbers inserted indicate the mean fluorescent intensity (MFI) of CD25 expression. Results are representative of one experiment of three performed.

PGE₂ induces FOXP3 in CD4⁺CD25⁻ T cells and up-regulates its expression in CD4⁺CD25⁺ T reg cells

Because FOXP3 is specifically expressed in naturally occurring T reg cells and programs their development and function (1, 2, 3, 4, 5), we determined whether PGE₂ had an impact on the induction of FOXP3 gene expression in both CD4⁺CD25⁺ T reg and CD4⁺CD25⁻T cells. As expected, purified, peripheral blood-derived, CD3-activated CD4⁺CD25⁺ T reg cells expressed high levels of FOXP3 mRNA compared with CD4⁺CD25⁻ T cells (Fig. 3⇓A). Furthermore, a 24-h preincubation with PGE₂ (26 μM) resulted in >4-fold up-regulation of FOXP3 (Fig. 3⇓A). When CD4⁺CD25⁻ T cells were cultured under the same stimulatory conditions in the presence of PGE₂ (26 μM), FOXP3 mRNA expression was also significantly induced, consistent with the acquisition of a T reg cell suppressive function (Fig. 3⇓B). Thus, PGE₂ facilitated the differentiation of CD4⁺CD25⁻ T cells into a T cell population endowed with inhibitory properties and FOXP3 mRNA expression.

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FIGURE 3.

PGE₂ induces FOXP3 mRNA expression in both CD4⁺CD25⁺ T reg cells and CD4⁺CD25⁻ T cells. Purified CD4⁺CD25⁺ T reg cells (A) and CD4⁺CD25⁻ T cells (B) were incubated without (−) or with (+) PGE₂ (26 μM) for 24 h. C, Alternatively, CD4⁺CD25⁻ T cells were cultured for 24 h with medium alone or in tumor supernatant from COX-2 S (with or without anti-PGE₂ Ab (10 μg/ml) or mouse IgG control Ab (10 μg/ml)), COX-2 AS, or CV-transfected H157. T cell FOXP3 mRNA expression was quantified after 48-h activation with plate-bound anti-CD3 Ab by real-time PCR as described in Materials and Methods. In CD4⁺CD25⁺ T reg cells treated with or without PGE₂ (A), the fold increase in FOXP3 mRNA was relative to FOXP3 mRNA expression in CD4⁺CD25⁻. In PGE₂-treated CD4⁺CD25⁻ cells (B), the fold change in FOXP3 mRNA was relative to that in untreated CD4⁺CD25⁻ cells. In CD4⁺CD25⁻ T cells cultured with gene-modified H157 supernatant (C), the fold change in FOXP3 was relative to that in CD4⁺CD25⁻ cells in medium alone. Results are expressed as the mean (±SE) of one experiment of at least three performed in triplicate from different donors. Asterisks indicate statistical significance compared with the control value.

Both preclinical and clinical studies have suggested that T reg cells play a role in suppressing the host immune response to malignancy. In particular, recent reports have demonstrated enhanced T reg cell activity in cancer patients (17, 18, 19, 20, 21, 22). Among the malignancies associated with augmented T reg cell function are tumors known to overexpress COX-2 and secrete high levels of PGE₂ (17, 18, 19, 20, 21, 22). Thus, to determine the importance of tumor-derived PGE₂, we used COX-2 gene-modified NSCLC cells as an experimental model for the tumor site, which is often enriched for PGE₂. For this purpose we genetically modified NSCLC cell lines to express COX-2 S, COX-2 AS, or empty CV, as described in Materials and Methods and as we have previously reported (25). Using this in vitro model we preincubated CD4⁺CD25⁻ T cells with tumor supernatant from H157 COX-2-gene modified NSCLC cell lines. FOXP3 gene expression was quantified in CD4⁺CD25⁻ cells after 24-h activation with anti-CD3 plate-bound Ab. A 24-h exposure to H157 COX-2 S supernatant significantly induced FOXP3 gene expression in CD4⁺CD25⁻ T cells. In contrast, COX-2 AS and CV supernatant had no effect on FOXP3 gene expression. Addition of anti-PGE₂ Ab to COX-2 S supernatant blocked the induction of FOXP3 mRNA expression to the levels demonstrated in the AS or CV supernatants (Fig. 3⇑C). Control Ab did not impact the COX-2 S tumor supernatant-mediated induction of FOXP3 mRNA expression (Fig. 3⇑C).

PGE₂ up-regulates FOXP3 mRNA and protein expression and induces FOXP3 promoter activity

To determine the mechanisms underlying PGE₂-dependent regulation of FOXP3, we analyzed the expression of FOXP3 mRNA and protein levels in untreated or PGE₂-treated (13 and 26 μM) Jurkat T cells in the presence or the absence of TCR-activating stimuli. We found that PGE₂ up-regulated FOXP3 mRNA levels in both TCR-activated and non-activated Jurkat T cells (Fig. 4⇓, A and C). Consistent with our findings in PBL, PGE₂ induced the greatest increase in FOXP3 in TCR-stimulated Jurkat T cells. Western blot analysis showed a similar pattern of expression (Fig. 4⇓B). Non-activated Jurkat T cells, in fact, expressed FOXP3 protein that was significantly up-regulated by both PGE₂ concentrations tested (13 and 26 μM). However, maximum FOXP3 up-regulation was obtained in Jurkat T cells stimulated with PGE₂ (26 μM) and activated with PMA plus ionomycin (Fig. 4⇓B). Finally, in promoter reporter assays, Jurkat T cells transfected with a FOXP3 promoter construct (FOXP3-SubD) demonstrated significantly enhanced FOXP3 promoter activity in response to PGE₂ and costimulatory signals (Fig. 4⇓C). This suggests that PGE₂ operates to induce FOXP3 at the level of mRNA transcription.

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FIGURE 4.

PGE₂ enhances FOXP3 mRNA expression, gene transcription, and protein. A, Jurkat T cells were activated for 18 h as indicated. FOXP3 mRNA expression was quantified by real-time PCR as described in Materials and Methods. B, Top panel, FOXP3 protein was analyzed by Western blot after stimulation for 18 h as indicated. B, Bottom panel, Densitometric analysis of FOXP3 expression of the Western blot. One representative result of two independent experiments performed is shown. C, PGE₂ induces FOXP3 promoter activity. Jurkat T cells cotransfected with FOXP3 promoter and firefly luciferase reporter gene constructs (FOXP3-SubD) or with Renilla luciferase gene (CV) were stimulated for 18 h as indicated. FOXP3 promoter expression was analyzed by a dual luciferase reporter assay system, and relative luciferase activity was expressed as the ratio of firefly to Renilla luciferase. A representative experiment of three performed is shown.