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Lung Cancer Research Program of the Jonsson Comprehensive Cancer Center and Department of Medicine, David Geffen School of Medicine, University of California, Los Angeles, CA 90095; and Veterans Affairs Greater Los Angeles Healthcare System, Los Angeles, CA 90073
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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PGE2 has diverse effects on the regulation and activity of CD4+ T cells, including modulation of proliferation (13). Elevated tumor cyclooxygenase-2 (COX-2) and PGE2 levels have been described in several malignancies, where they have been implicated in suppression of antitumor immunity (15, 16). In particular, the CD4+CD25+ T reg cell population has been found to be significantly increased in PBL and within the tumor-infiltrating lymphocytes of epithelial cancers, including non-small cell lung cancer (NSCLC) (17, 18, 19, 20, 21, 22, 23). Given that COX-2 overexpression and PGE2 overproduction are highly associated with the progression of this malignancy (16), we hypothesized that PGE2 could contribute to the tumor-induced immunosuppression through modulation of T reg cell function. In this study we demonstrate that PGE2 enhances the inhibitory capacity of in vitro purified human CD4+CD25+ T reg cells and induces a suppressive regulatory phenotype in CD4+CD25– T cells. The PGE2-dependent acquisition of T reg cell function was correlated with induction of FOXP3 gene and protein expression. The enhanced inhibitory activity of PGE2-treated CD4+CD25+ T reg cells was also associated with significant up-regulation of FOXP3. This report highlights novel roles for PGE2 in controlling the generation and function of T reg cells.
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Materials and Methods |
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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 x 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 x 105) in anti-CD3 plate-bound, 96-well plates. In some experiments IL-2 (Proleukin; biological activity, 18 x 106 IU/1.1 mg; Chiron) or IL-7 (biological activity, 2 x 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 x 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 x 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 
-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 -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 -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 x 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.
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Results |
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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. 1A). 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. 1A). 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. 1B). 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. 1B). 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. 1C). 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. 1D). 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. 1E). 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- expression (33, 34, 35). However, neither anti-IL-10 nor anti-TGF- 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- to PGE2-induced cell-contact independent T regulatory cell function.
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Because T reg cells are identified as a CD4+CD25brightT cell subset (36), we analyzed whether the PGE2-treated CD4+CD25dim starting population acquired the same phenotype of naturally occurring, thymic-derived CD4+CD25bright T reg cells. As shown in Table I, CD25 was differentially expressed in magnetic bead-purified CD4+CD25dim and CD4+CD25brightT cells. Indeed, with (Fig. 2A and Table II) or without PGE2 preincubation (data not shown), CD4+CD25dim up-regulated CD25 as early as 24 h after stimulation with anti-CD3 plate-bound Ab (Fig. 2A). These T cells maintained high levels of CD25 expression throughout a 96-h culture period (Fig. 2A). However, when anti-CD3 Ab-activated T cells were pretreated with PGE2, CD25 expression declined in both CD4+CD25dim T cells (Fig. 2B, bottom left, and Table II) and CD4+CD25bright T reg cells (Fig. 2B, 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-1, 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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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. 3A). Furthermore, a 24-h preincubation with PGE2 (26 µM) resulted in >4-fold up-regulation of FOXP3 (Fig. 3A). 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. 3B). 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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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. 4B). 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. 4B). 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. 4C). This suggests that PGE2 operates to induce FOXP3 at the level of mRNA transcription.
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Discussion |
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A specific subset of T reg cells bearing a CD4+CD25+ T cell phenotype has now been the focus of extensive investigation (1, 2, 3, 4, 5, 35). These T cells, endowed with distinct immunomodulatory properties, are acknowledged as fundamental in the prevention of autoimmunity and may be important in preventing effective immune responses in malignancy (17, 18, 19, 20, 21, 22). Although naturally occurring CD4+CD25+ T reg cells develop directly from CD4+ precursors in the thymus, a body of work has now demonstrated the induction of cells with regulatory function in the periphery from naive CD4+ T cells in response to a variety of stimuli (6, 7, 8, 9, 10, 11, 12).
In this study we report a novel mechanism of PGE2-induced immunosuppression that occurs through the modulation/induction of human T reg cell function. Our results show that PGE2 not only enhances the suppressive capacity of in vitro purified CD4+CD25+ T reg cells, but also induces a CD4+CD25– T cell population to develop T reg cell function. Analysis of this newly induced T reg cell-like population revealed that PGE2 exposure conferred acquisition of FOXP3 expression consistent with the phenotypic feature seen in naturally occurring T reg cells. The original CD25dim population became CD25bright upon anti-CD3 activation. However, PGE2 exposure markedly down-regulated the surface expression of CD25 in these CD3-stimulated CD4+CD25– T cells that typically developed CD25high expression upon stimulation. The down-regulation of CD25, the 
-chain of the high affinity IL-2R, was also evident in the purified CD4+CD25+ T reg cells exposed to the same concentration of PGE2. Constitutive expression of CD25 commonly has been used to define CD4+CD25+ T reg cells and IL-2 has been implicated as an important modulator of T reg activity, in vivo (39, 40). Our finding of PGE2-mediated down-regulation of IL-2R is consistent with previous studies showing decreased IL-2 production and IL-2R expression in TCR-activated, PGE2-stimulated T cells (37, 38, 42, 43). Thus, the PGE2-mediated down-regulation of IL-2R may limit typical autocrine and paracrine IL-2-dependent function, such as the TCR-mediated proliferative response. Despite the reduced IL-2R expression, both PGE2-treated CD4+CD25+ and CD4+CD25–T cell subsets retained immunosuppressive capacities and exerted their suppressive functions in a cell contact independent manner.
The biochemical signals generated by the IL-2R are similar to those of the IL-7R (44). The receptors for these cytokines in fact use shared components such as the common 
-chain that appears to be essential for their mitogenic signals (44, 45). It has been shown that IL-2 signaling via STAT5 is required for T reg cell development, and yet this transcription factor is activated by IL-7 (45). In contrast, it has also been shown that, as members of the -chain family, IL-2R and IL-7R activate distinct signal-transducing factors, which may be differentially recruited to the receptor complex by their ligand-specific unit (45, 46). In the current study, addition of exogenous IL-7 overcame the suppressive capacity of naturally occurring CD4+CD25+ T reg cells and partially neutralized the inhibition of proliferation mediated by the PGE2-induced regulatory T cells (CD4+CD25– and CD4+CD25+). Exogenous IL-2, however, had no ability to counteract the observed suppressed proliferation. The PGE2-induced IL-2R down-regulation may affect T cell IL-2 responsiveness, and perhaps higher concentrations of IL-2 may be required to reverse the inhibitory function of both PGE2-treated CD4+CD25– and CD4+CD25+ T reg cells. Alternatively, IL-2 and IL-7 may activate different signal transduction pathways, ultimately leading to different outcomes (43, 44, 45, 46). Additional studies will be required to define the differences in response to IL-2 and IL-7.
PBL-derived T reg cells have been categorized into T regulatory 1 or Th3 based on their pattern of cytokine secretion, because they have been shown to preferentially produce IL-10 or TGF-, respectively (46). However, there is as yet no clear consensus regarding their phenotype or their ontogeny (5, 46). Our findings indicate that compared with untreated CD4+CD25– T cells, PGE2-induced T reg-like cells secrete lower levels of IL-10 and similar levels of TGF- (data not shown). Neither of these cytokines appeared to significantly contribute to the in vitro regulatory function, because Ab-blocking studies could not reverse the inhibitory effect of either PGE2-treated CD4+CD25+ or CD4+CD25– T cells (data not shown). Taken together, our data indicate that PGE2-induced CD4+CD25– T cells share common features of both thymic and peripheral T reg cells (4, 5, 47, 48).
The transcription factor FOXP3 exerts a key role in the development and function of T reg cells (1, 2, 3, 4, 5, 46). This separates FOXP3 from other T reg cell-associated markers such as CD25, glucocorticoid-induced TNFR family-related protein, and CTLA-4, which may be more generally expressed by activated T cells (35, 36). In our study, PGE2-dependent T reg differentiation was indeed associated with induction of the transcription factor FOXP3, whose expression was also significantly up-regulated by PGE2 in purified CD4+CD25+ and CD4+CD25– T cells. Consistent with previous reports in which TGF- was investigated (6, 7, 8), PGE2-mediated T reg cell differentiation and FOXP3 induction were evident predominantly in the presence of costimulation by either anti-CD3 or allogeneic dendritic cells (data not shown). Using a variety of approaches, several studies are now modeling the differentiation of cells with T regulatory function arising in the periphery (6, 7, 8, 9, 10, 11, 12, 48). For example, recent studies in mice and humans, have demonstrated that CD4+CD25– T cells treated with TGF- in the presence of appropriate costimulation lead to T reg cell differentiation with the capacity for suppressive function in vitro (6, 7). It is unclear whether these extrathymic T reg cells represent altered states of differentiation or belong to a unique T cell lineage (48).
Our findings suggest a role for PGE2 in modulating T reg cell function and differentiation. In conditions of PGE2 overproduction, such as described in several malignancies (17, 18, 19, 20, 21, 22), increased T reg cell differentiation and function could contribute to tumor-induced immunosuppression. Consistent with this concept, malignancies such as lung cancer that have previously been demonstrated to have high COX-2 expression and PGE2 production (16, 49) have also been noted to have increased T reg cell number and function within the PBL as well as the tumor-infiltrating lymphocyte populations (17, 18, 19, 20, 21, 22). Extending these previous reports, we found that NSCLC cell lines overexpressing COX-2 were able to induce FOXP3 gene expression in CD4+CD25– T cells in a PGE2-dependent manner. We have recently reported that these relationships are operative in vivo in murine lung cancer models (50). Thus, taken together, our data indicate that regulation of PGE2 production may, in turn, modulate T reg cell development and function. These findings suggest new therapeutic strategies targeting COX-2/PGE2 in the prevention and treatment of cancer. This could include addition of COX-2 inhibitor treatment to immune-based therapies.
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Acknowledgments |
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Disclosures |
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Footnotes |
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1 This work was supported by the University of California-Los Angeles Lung Cancer Specialized Program of Research Excellence P50CA90388, National Institutes of Health Grant R01CA85686, Medical Research Funds from the Department of Veteran Affairs, the Research Enhancement Award Program in Cancer Gene Medicine, and the Tobacco-Related Disease Research Program of University of California. 
2 Address correspondence and reprint requests to Dr. Steven M. Dubinett, Lung Cancer Research Program, David Geffen School of Medicine, University of California, Los Angeles, Room 37-131 Center for Health Sciences, 10833 Le Conte Avenue, Los Angeles, CA 90095. E-mail address: sdubinett{at}mednet.ucla.edu
3 Abbreviations used in this paper: T reg cell, regulatory T cell; AS, antisense; COX-2, cyclooxygenase-2; CV, control empty vector; FOXP3, forkhead/winged helix transcription factor gene; NSCLC, nonsmall cell lung cancer; S, sense.
Received for publication December 23, 2004. Accepted for publication April 29, 2005.
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 S. Klein-Hessling, T. Bopp, M. K. Jha, A. Schmidt, S. Miyatake, E. Schmitt, and E. Serfling Cyclic AMP-induced Chromatin Changes Support the NFATc-mediated Recruitment of GATA-3 to the Interleukin 5 Promoter J. Biol. Chem., November 7, 2008; 283(45): 31030 - 31037. [Abstract] [Full Text] [PDF]
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 A. Bosco and P. G. Holt Genome-wide expression profiling of T-cells in childhood wheeze Eur. Respir. J., November 1, 2008; 32(5): 1138 - 1140. [Full Text] [PDF]
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H. J. McSorley, Y. M. Harcus, J. Murray, M. D. Taylor, and R. M. Maizels Expansion of Foxp3+ Regulatory T Cells in Mice Infected with the Filarial Parasite Brugia malayi J. Immunol., November 1, 2008; 181(9): 6456 - 6466. [Abstract] [Full Text] [PDF]
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 C. Chizzolini, R. Chicheportiche, M. Alvarez, C. de Rham, P. Roux-Lombard, S. Ferrari-Lacraz, and J.-M. Dayer Prostaglandin E2 synergistically with interleukin-23 favors human Th17 expansion Blood, November 1, 2008; 112(9): 3696 - 3703. [Abstract] [Full Text] [PDF]
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L. Jarvinen, L. Badri, S. Wettlaufer, T. Ohtsuka, T. J. Standiford, G. B. Toews, D. J. Pinsky, M. Peters-Golden, and V. N. Lama Lung Resident Mesenchymal Stem Cells Isolated from Human Lung Allografts Inhibit T Cell Proliferation via a Soluble Mediator J. Immunol., September 15, 2008; 181(6): 4389 - 4396. [Abstract] [Full Text] [PDF]
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 B. J. E. Raveney, C. Richards, M.-L. Aknin, D. A. Copland, B. R. Burton, E. Kerr, L. B. Nicholson, N. A. Williams, and A. D. Dick The B Subunit of Escherichia coli Heat-Labile Enterotoxin Inhibits Th1 but Not Th17 Cell Responses in Established Experimental Autoimmune Uveoretinitis Invest. Ophthalmol. Vis. Sci., September 1, 2008; 49(9): 4008 - 4017. [Abstract] [Full Text] [PDF]
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X. Feng, S. Kajigaya, E. E. Solomou, K. Keyvanfar, X. Xu, N. Raghavachari, P. J. Munson, T. M. Herndon, J. Chen, and N. S. Young Rabbit ATG but not horse ATG promotes expansion of functional CD4+CD25highFOXP3+ regulatory T cells in vitro Blood, April 1, 2008; 111(7): 3675 - 3683. [Abstract] [Full Text] [PDF]
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 J. Yokokawa, V. Cereda, C. Remondo, J. L. Gulley, P. M. Arlen, J. Schlom, and K. Y. Tsang Enhanced Functionality of CD4+CD25highFoxP3+ Regulatory T Cells in the Peripheral Blood of Patients with Prostate Cancer Clin. Cancer Res., February 15, 2008; 14(4): 1032 - 1040. [Abstract] [Full Text] [PDF]
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 T. Bryn, S. Yaqub, M. Mahic, K. Henjum, E. M. Aandahl, and K. Tasken LPS-activated monocytes suppress T-cell immune responses and induce FOXP3+ T cells through a COX-2-PGE2-dependent mechanism Int. Immunol., February 1, 2008; 20(2): 235 - 245. [Abstract] [Full Text] [PDF]
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I. Y. Lee, W. Cho, J. Kim, C.-S. Park, and J. Choe Human Follicular Dendritic Cells Interact with T Cells via Expression and Regulation of Cyclooxygenases and Prostaglandin E and I Synthases J. Immunol., February 1, 2008; 180(3): 1390 - 1397. [Abstract] [Full Text] [PDF]
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M. Yamashita, T. Shinohara, S. Tsuji, Q. N. Myrvik, A. Nishiyama, R. A. Henriksen, and Y. Shibata Catalytically Inactive Cyclooxygenase 2 and Absence of Prostaglandin E2 Biosynthesis in Murine Peritoneal Macrophages following In Vivo Phagocytosis of Heat-Killed Mycobacterium bovis Bacillus Calmette-Guerin J. Immunol., November 15, 2007; 179(10): 7072 - 7078. [Abstract] [Full Text] [PDF]
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C. Bergmann, L. Strauss, R. Zeidler, S. Lang, and T. L. Whiteside Expansion of Human T Regulatory Type 1 Cells in the Microenvironment of Cyclooxygenase 2 Overexpressing Head and Neck Squamous Cell Carcinoma Cancer Res., September 15, 2007; 67(18): 8865 - 8873. [Abstract] [Full Text] [PDF]
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 C. Prevosto, M. Zancolli, P. Canevali, M. R. Zocchi, and A. Poggi Generation of CD4+ or CD8+ regulatory T cells upon mesenchymal stem cell-lymphocyte interaction Haematologica, July 1, 2007; 92(7): 881 - 888. [Abstract] [Full Text] [PDF]
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 M. Yamashita, S. Tsuji, A. Nishiyama, Q. N. Myrvik, R. A. Henriksen, and Y. Shibata Differential subcellular localization of COX-2 in macrophages phagocytosing heat-killed Mycobacterium bovis BCG Am J Physiol Cell Physiol, July 1, 2007; 293(1): C184 - C190. [Abstract] [Full Text] [PDF]
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P. Sinha, V. K. Clements, A. M. Fulton, and S. Ostrand-Rosenberg Prostaglandin E2 Promotes Tumor Progression by Inducing Myeloid-Derived Suppressor Cells Cancer Res., May 1, 2007; 67(9): 4507 - 4513. [Abstract] [Full Text] [PDF]
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E. A. Wohlfert, F. C. Nichols, E. Nevius, and R. B. Clark Peroxisome Proliferator-Activated Receptor {gamma} (PPAR{gamma}) and Immunoregulation: Enhancement of Regulatory T Cells through PPAR{gamma}-Dependent and -Independent Mechanisms J. Immunol., April 1, 2007; 178(7): 4129 - 4135. [Abstract] [Full Text] [PDF]
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 J. Bodor, Z. Fehervari, B. Diamond, and S. Sakaguchi Regulatory T cell-mediated suppression: potential role of ICER J. Leukoc. Biol., January 1, 2007; 81(1): 161 - 167. [Abstract] [Full Text] [PDF]
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 A. Kicic, E. N. Sutanto, P. T. Stevens, D. A. Knight, and S. M. Stick Intrinsic Biochemical and Functional Differences in Bronchial Epithelial Cells of Children with Asthma Am. J. Respir. Crit. Care Med., November 15, 2006; 174(10): 1110 - 1118. [Abstract] [Full Text] [PDF]
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P. Grimbert, S. Bouguermouh, N. Baba, T. Nakajima, Z. Allakhverdi, D. Braun, H. Saito, M. Rubio, G. Delespesse, and M. Sarfati Thrombospondin/CD47 Interaction: A Pathway to Generate Regulatory T Cells from Human CD4+CD25- T Cells in Response to Inflammation J. Immunol., September 15, 2006; 177(6): 3534 - 3541. [Abstract] [Full Text] [PDF]
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Y. Shibata, J. Gabbard, M. Yamashita, S. Tsuji, M. Smith, A. Nishiyama, R. A. Henriksen, and Q. N. Myrvik Heat-killed BCG induces biphasic cyclooxygenase 2+ splenic macrophage formation--role of IL-10 and bone marrow precursors J. Leukoc. Biol., September 1, 2006; 80(3): 590 - 598. [Abstract] [Full Text] [PDF]
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M. Beyer and J. L. Schultze Regulatory T cells in cancer Blood, August 1, 2006; 108(3): 804 - 811. [Abstract] [Full Text] [PDF]
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M. Mahic, S. Yaqub, C. C. Johansson, K. Tasken, and E. M. Aandahl FOXP3+CD4+CD25+ Adaptive Regulatory T Cells Express Cyclooxygenase-2 and Suppress Effector T Cells by a Prostaglandin E2-Dependent Mechanism J. Immunol., July 1, 2006; 177(1): 246 - 254. [Abstract] [Full Text] [PDF]
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M. S. von Bergwelt-Baildon, A. Popov, T. Saric, J. Chemnitz, S. Classen, M. S. Stoffel, F. Fiore, U. Roth, M. Beyer, S. Debey, et al. CD25 and indoleamine 2,3-dioxygenase are up-regulated by prostaglandin E2 and expressed by tumor-associated dendritic cells in vivo: additional mechanisms of T-cell inhibition Blood, July 1, 2006; 108(1): 228 - 237. [Abstract] [Full Text] [PDF]
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J. P. J. J. Hegmans, A. Hemmes, H. Hammad, L. Boon, H. C. Hoogsteden, and B. N. Lambrecht Mesothelioma environment comprises cytokines and T-regulatory cells that suppress immune responses Eur. Respir. J., June 1, 2006; 27(6): 1086 - 1095. [Abstract] [Full Text] [PDF]
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S. G. Zheng, J. H. Wang, W. Stohl, K. S. Kim, J. D. Gray, and D. A. Horwitz TGF-beta Requires CTLA-4 Early after T Cell Activation to Induce FoxP3 and Generate Adaptive CD4+CD25+ Regulatory Cells J. Immunol., March 15, 2006; 176(6): 3321 - 3329. [Abstract] [Full Text] [PDF]
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K. Mimori, H. Ishii, H. Nagahara, T. Sudo, K. Yamashita, H. Inoue, G. F. Barnard, and M. Mori FHIT Is Up-Regulated by Inflammatory Stimuli and Inhibits Prostaglandin E2-Mediated Cancer Progression. Cancer Res., March 1, 2006; 66(5): 2683 - 2690. [Abstract] [Full Text] [PDF]
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Y. Shibata, R. A. Henriksen, I. Honda, R. M. Nakamura, and Q. N. Myrvik Splenic PGE2-releasing macrophages regulate Th1 and Th2 immune responses in mice treated with heat-killed BCG J. Leukoc. Biol., December 1, 2005; 78(6): 1281 - 1290. [Abstract] [Full Text] [PDF]
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