Published online before print February 12, 2008
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,1,1,
,2
* The Biotechnology Centre of Oslo,
Centre for Molecular Medicine Norway, Nordic EMBL Partnership, University of Oslo, Norway;
Department of Gastroenterological Surgery, Ullevaal University Hospital, and
Norwegian Institute of Public Health, Oslo, Norway
2Correspondence: The Biotechnology Centre of Oslo, University of Oslo, Gaustadalleen 21, N-0349 Oslo, Norway. E-mail: kjetil.tasken{at}biotek.uio.no
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: human • regulatory T cells (TR cells) • immunoregulation • cyclooxygenase • TGF-β • IL-10-IL17
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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TRadapt cells can be induced in vivo and in vitro from naive CD4+ T cells in various experimental conditions [4 , 15 16 17 ]. We have recently shown that continuous activation of human CD4+CD25– T cells leads to generation of CD4+CD25+FOXP3+ TRadapt cells, which express cyclooxygenase 2 (COX-2) and suppress effector T cells by a PGE2-dependent mechanism [18 ]. However, it is unclear how the generation of the suppressive phenotype is related to the differentiation of naive CD4+ T cells into effector and memory T cells. Here, we have characterized the differentiation profile, cytokine expression pattern, and expression of FOXP3 and COX-2 during the conversion of CD4+CD25– T cells into TRadapt cells. Our data demonstrate that acquisition of the regulatory phenotype occurs concomitantly with generation of CD4+ T cell effector functions and indicate that the suppressive activity is an inherent part of a normal immune response and regulates the local immune responsiveness.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Stimulation of T cells and generation of TRadapt cells
Conditions for optimal stimulation were titrated and optimized for each assay, and the effects of primary stimulation of PBMCs with Staphylococcoal enterotoxin B (SEB; Sigma-Aldrich, St. Louis, MO, USA) were comparable with stimulation with CD3/CD28 mAb. TRadapt cells were generated from PBMC, depleted of CD25+ cells, and incubated with 3 µg/ml SEB in complete medium for 1, 2, 4, or 7 days. At the end of each incubation period, CD4+CD25+ T cells were isolated and used in further experiments. In coculture suppression assays, where intracellular cytokines were measured, cells were activated with 10 µg/ml SEB, and for CFSE proliferation assays, 1 µg/ml SEB was used. For sorting or analysis of different daughter cell populations in CFSE dilution assays, cells were stimulated with soluble anti-CD3 (2.5 µg/ml) and anti-CD28 (0.5 µg/ml) or 2 µg/ml SEB. For ELISA of cytokines in supernatants, T cells were stimulated with plate-bound anti-CD3 (10 µg/ml) and soluble anti-CD28 (0.5 µg/ml) Upon restimulation, anti-CD3/CD28 was used to stimulate T cells independently of their Vβ repertoires.
Western blot analysis
Cells (2x107) were lysed in radioimmunoprecipitation assay lysis buffer, loaded onto one-dimensional SDS-polyacrylamide gels (Bio-Rad Laboratories AB, Sweden), subjected to electrophoresis, and transferred onto polyvinyldifluoride membranes (Millipore, Bedford, MA) by electroblotting. The membranes were blocked for 2 h at room temperature in a solution containing 25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.05% Tween-20, and 5% milk and incubated overnight at 4°C with monoclonal mouse anti-human COX-2 (1:5000, Cayman Chemical, Ann Arbor, MI, USA), polyclonal rabbit anti-human COX-2 (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA, USA), or polyclonal mouse anti-human FOXP3 (1:500, Abcam Ltd., Cambridge, UK). The membranes were washed in a solution containing 25 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 0.05% Tween-20. Immunoreactive proteins were visualized by Supersignal (Pierce) using HRP-conjugated secondary antibodies and subjected to autoradiography. The loading was based on quantification of total protein in the samples, and equal loading was confirmed by reprobing with mouse anti-human protein kinase C-
(PKC-; 1:4000, BD Biosciences, San Jose, CA, USA).
In vivo experiments in mice
The Local Animal Ethics Committee approved animal experiment protocols. BALB/cJBomTac mice were used for in vivo experiments at the Norwegian Institute of Public Health (Oslo). Spleens were removed from four donor mice per transfusion (48 mice total), and T cells were isolated using a mouse T cell-negative isolation kit (Dynal Invitrogen, Oslo, Norway). CD25+ T cells were depleted with a CD25 Microbead kit (Miltenyi Biotec) and pooled. CD25– T cells were stained with 2 µM CFSE and adoptively transfused into syngeneic acceptor mice (50x106 cells per mouse; n=12 mice). Eight mice were subsequently injected with 50 µg SEB i.p. to induce an immune response, and four mice were left untreated. Three SEB-treated mice were killed on Day 2, and five SEB-treated and four control mice were killed Day 4 post-SEB injection. T cells were isolated from spleens and analyzed for intracellular FOXP3 expression by flow cytometry.
Flow cytometry and antibodies
Cells were fixed in 4% paraformaldehyde and permeabilized in FACS-permeabilizing solution (BD BioSciences) prior to staining with CD8-allophycocyanin (APC), CD3-PerCP, CD25-PE, and anti-human IFN-
-PE (BD BioSciences; BD Biosciences PharMingen, San Diego, CA, USA). The cells were washed once in PBS containing 1% BSA prior to data acquisition on a flow cytometer (FACSCalibur, BD Biosciences) and analysis using FlowJo software (Tree Star, San Carlos, CA, USA). For analysis of cell surface markers, cells were stained with CD3-APC-CY7, CD4-PE-CY7, CD25-APC, CD28-PE, CD62 ligand (CD62L)-APC, CD45RO-APC, CD45RA-APC, CD95-APC, and CD127- and CD69-PE or -APC (BD BioSciences; BD Biosciences PharMingen). When stained with anti-human or anti-mouse FOXP3 Alexa 647, cells were fixed and permeabilized according to the manufacturer’s instructions (BD Biosciences). In experiments with human T cells, cells were further stained with CTLA4-PE or -APC after fixation and permeabilization as described above, and samples were analyzed on FACSCalibur or FACSAria (BD Biosciences).
Coculture experiments and suppression assays
TR cells were added in increasing ratios to autologous PBMC depleted of CD25+ cells to a total of 1 x 106 cells/ml and stimulated with 10 µg/ml SEB for 20 h. Brefeldin A (Sigma-Aldrich) was added at a final concentration of 5 µM for the last 5 h of incubation. In all coculture experiments, TR cells were prestained with 2 µM CFSE to allow separation of the TR cell population from the responding effector T cells. The cells were fixed, permeabilized, stained for cell surface proteins and intracellular IFN-, and analyzed by flow cytometry as described above.
CFSE proliferation assay and sorting of daughter cell populations
CD4+CD25– T cells (1x107 cells/ml) were labeled with 2 µM CFSE prior to stimulation with soluble anti-CD3 (2.5 µg/ml) and anti-CD28 (0.5 µg/ml) or 2 µg/ml SEB and incubated in complete medium for 4 days. The cells were stained for cell surface markers, and cell division was assessed by CFSE dilution. In some experiments, the different CD4+CD25+ T cell daughter cell populations representing cell division cycles were sorted by FACSAria gated on the CFSE signal intensity.
ELISA and BioPlex assay for cytokine detection
CD4+CD25– T cells or TRadapt cells (1x106 cells/ml) were stimulated with plate-bound anti-CD3 and anti-CD28 for 24 h, and cell-free supernatants were harvested and analyzed for IL-2, PGE2, and TGF-β1 by ELISA (R&D, London, UK), according to the manufacturer’s instructions. For measurements of TGF-β levels, the cells were incubated in serum-free medium. In addition, the supernatants were analyzed on BioPlex (Bio-Rad Laboratories AB) for assessment of IL-2, IL-17, IL-10, IL-12, IFN-, and TNF-, according to the manufacturer’s instructions.
Statistical analysis
Data are presented as mean ± SEM or SD and were analyzed by paired samples t-test using SPSS for Windows. Differences with two-sided P < 0.05 were considered significant.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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production, but surprisingly, the CD4+CD25+ T cells isolated on Day 7 were only weakly suppressive (Fig. 1A
1B
1C)
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Figure 1. CD4+CD25– T cells express FOXP3 and COX-2 and acquire suppressor properties after stimulation with SEB. (A) SEB-induced proliferation of CD4+ T cells in PBMC depleted of CD25+ cells, with or without coculture, with TRadapt cells induced for 2, 4, and 7 days from autologous donors. (B) SEB-induced, intracellular IFN- expression in CD4+ T cells in PBMC depleted of CD25+ cells, with or without coculture with TRadapt cells from autologous donors (induced for 2, 4, and 7 days, left panel) or with TRnat cells or TRadapt cells induced for 4 days (right panel). (C) Intracellular IFN- expression and CFSE proliferation assay of effector T cells, with or without TRadapt cells induced for 2 days, are shown (one representative of n=3 experiments). (D) PBMC depleted of CD25+ T cells was stimulated with SEB for 2–7 days. Western blot analysis shows temporal regulation of COX-2 and FOXP3 expression in CD4+CD25+ cells isolated from SEB-stimulated PBMC depleted of CD25 cells. PKC- is used as a control for equal loading of cell lysates. (E) Concentration of PGE2 in supernatants of purified TRadapt cells restimulated with anti-CD3/anti-CD28 for 24 h. TRadapt cells were induced by stimulation of CD25– T cells with SEB for 1, 2, 4, or 7 days. Day 0 represents CD4+CD25–, which were not induced with SEB prior to stimulation.
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We next examined whether the induced CD4+CD25+ TRadapt cells expressed typical surface markers found on TRnat cells. CTLA4 is expressed by activated T cells, but its expression is constitutively high on TRnat cells. In contrast, CD69 serves as an early activation marker and is not expressed by TRnat cells. We compared the expression of CTLA4 and CD69 on CD4+CD25+FOXP3+ and CD4+CD25+FOXP3– T cells after antigen stimulation at the indicated time-points (Fig. 2 ). CD69 and CTLA4 were expressed at higher levels in the CD4+CD25+FOXP3+ T cells than in the CD4+CD25+FOXP3– T cell population at Day 2. The difference in expression of CTLA4 between the two populations was little but still detectable after 4 days and absent at Day 7. In contrast, CD69 was down-regulated following 2 days of stimulation in the FOXP3+ T cell population.
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Figure 2. Cell-surface phenotype analysis of CD4+CD25+FOXP3+ T cells. Expression of CD69 and CTLA4 in CD4+CD25+FOXP3+ and CD4+CD25+FOXP3– cell populations was determined after SEB activation of PBMC depleted of CD25 for 2, 4, and 7 days. Cells at Day 0 were unstimulated. IgG1 and IgG2 were used as isotype controls (top, right panel). Data are representative of five independent experiments with cells from three different blood donors.
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, IFN-, and TGF-β. As shown in Figure 3
, the production of most of the cytokines was temporally regulated with a peak at Days 2–4, followed by a decrease at Day 7. Production of IL-10 and IL-12 differed from other cytokines by being highest at Day 1. At Day 7, all cytokines were produced at comparably low levels. Furthermore, this expression profile coincides with the production of PGE2. This indicates that the regulatory phenotype develops in parallel with the acquisition of effector functions. However, it is important to point out that the cultures on Days 4 and 7 may not be highly enriched in TRadapt cells, as FOXP3 levels are significantly lower at these time-points. Although isolation of TRadapt cells was based on CD25 expression, the correlation between CD25 and FOXP3 levels is not always absolute (Fig. 2
, left panels).
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Figure 3. Cytokine profile of TRadapt cells. Concentration of IL-2, IL-10, IL-12, IL-17, TGF-β, TNF-, and IFN- in supernatants of purified TRadapt cells and CD4+CD25– T cells restimulated with anti-CD3/CD28 for 24 h. TRadapt cells were induced by stimulation of CD4+CD25– T cells for 1, 2, 4, or 7 days with SEB. Day 0 represents CD4+CD25– that were not induced with SEB prior to stimulation for cytokine measurements. Data represent mean ± SEM of n = 3 experiments. #, P < 0.05, Day 0 versus Day 1; *, P < 0.05, Day 1 versus Day 2, 4, or 7; determined by paired samples t-test.
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expression in responding T cells from the autologous donor (Fig. 4
, lower, left panel). Although CD4+CD25– and CD4+CD25+ T cells were present within the nondividing cell population (Peak 0), only the CD4+CD25+ T cells were examined for suppressive activity. Interestingly, all four daughter populations efficiently suppressed the responder T cells. In contrast, the nondividing CD4+CD25+ T cells were not suppressive. Moreover, cells from Peak 0 produced high levels of IL-2 in contrast to the cells from Peak 4 that produced little or no IL-2 (Fig. 4
, lower, right panel). This indicates that suppressive activity is exclusively acquired in cells that undergo a proliferative immune response.
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Figure 4. Only proliferating CD4+CD25+ T cells acquire suppressive properties. CFSE-stained CD4+CD25– T cells were stimulated with soluble anti-CD3 and anti-CD28 for 4 days. Each generation of cells was isolated by cell-sorting based on fluorescence intensity. Sorted populations were next cocultured at a 1:1 ratio with autologous, SEB-stimulated, CD25-depleted cells. Intracellular IFN- levels were measured 20 h poststimulation. IL-2 levels in supernatants from CD4+CD25+ cells sorted from different generations and restimulated with anti-CD3/CD28 for 24 h were measured by ELISA.
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Figure 5. Phenotypic shift during proliferation of CD4+CD25– T cells. Expression of indicated markers was determined for each peak before and following activation for 4 days of CFSE-stained CD4+CD25– T cells with anti-CD3/CD28. Data are representative of a minimum of four independent experiments with cells from two different donors.
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Figure 6. Immune activation in mice leads to generation of TRadapt cells. (A) CD25– T cells were isolated from spleens from BALB/c mice and assessed for CD25 and FOXP3 expression. (B) The CD25-depleted T cells were next labeled with CFSE and adoptively transfused into syngeneic mice. FOXP3 expression was measured on Days 2 and 4 following injection of SEB (50 µg). Data from one representative experiment are shown. (C) Amalgamated data of SEB-treated animals (n=8, 2–4 days) and untreated mice (n=4, 4 days) are shown as mean ± SEM for FOXP3 expression (P<0.05).
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The concomitant secretion of IFN-, TNF-, IL-12, and IL-2 with acquisition of suppressive function supports the notion that effector function and the suppressive phenotype develop in parallel. However, as the cytokine expression profiles were obtained in supernatants from CD4+CD25+ T cells, the population may also contain subsets of cells that were not FOXP3+. This may explain the level of IL-17 in the supernatants, as it has recently been reported that the Th17 and TR cell lineages are functionally distinct [23
].
Interestingly, only dividing CD4+CD25+ T cells acquired immunosuppressive capacity, whereas cells that had not proliferated produced high levels of IL-2 and expressed lower levels of FOXP3 (Figs. 4 and 5) . Thus, suppressive activity appears to be acquired as an inherent part of a normal CD4+ T cell immune response, but not all activated cells become inhibitory. Taken together, it appears that CD4+ T cells that mount an immune response and undergo clonal expansion during T cell activation transiently attain inhibitory activity, whereas cells that do not proliferate produce IL-2 and may develop into memory T cells.
In conclusion, our study suggests that upon activation of CD4+CD25– T cells, FOXP3 and COX-2 are expressed, and the cells acquire a transient immunosuppressive phenotype that lasts for 2–7 days. However, the suppressive CD25+CD4+ T cells are exclusively found in the population that divides upon activation, whereas the nonproliferating cell population produces IL-2. Acquisition of immunosuppressive activity appears to develop in parallel to development of effector functions such as cytokine secretion and clonal expansion and may regulate and facilitate termination of an ongoing immune response.
Received May 25, 2007; revised January 8, 2008; accepted January 17, 2008.
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