Secretion Retinoic Acid Receptor-Mediated IL-2 of Machinery in Normal T Cells

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Retinoic Acid Stimulates the Cell Cycle Machinery in Normal T Cells: Involvement of Retinoic Acid Receptor-Mediated IL-2 Secretion This information is current as of September 29, 2021. Aase Ertesvag, Nikolai Engedal, Soheil Naderi and Heidi Kiil Blomhoff J Immunol 2002; 169:5555-5563; ; doi: 10.4049/jimmunol.169.10.5555 http://www.jimmunol.org/content/169/10/5555 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2002 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Retinoic Acid Stimulates the Cell Cycle Machinery in Normal T Cells: Involvement of Retinoic Acid Receptor-Mediated IL-2 Secretion1

Aase Ertesvag, Nikolai Engedal, Soheil Naderi, and Heidi Kiil Blomhoff2

The mechanisms whereby vitamin A stimulates the immune system are poorly understood. In the current study, we attempted to elucidate the potential mechanisms of action of all-trans retinoic acid (atRA) on proliferation of human T lymphocytes. We found that physiological levels of atRA potently augmented T cell proliferation when added in combination with common T cell- stimulating agents. This was reﬂected in a time- and concentration-dependent stimulation of the cell cycle machinery. The presence of atRA led to elevated levels of cyclin D3, -E, and -A, decreased levels of p27Kip1, increased activity of cyclin-dependent kinase 2, and enhanced phosphorylation of the retinoblastoma protein (pRB). The atRA-mediated changes in the cell cycle machinery Downloaded from were late events, appearing after 20 h of stimulation, indicating that the effects of atRA were indirect. atRA did not alter the expression of the high-afﬁnity IL-2R. However, the level of IL-2 secreted by T cells was strongly enhanced by atRA. rIL-2 was able to substitute for the effects of atRA on the cell cycle machinery and on DNA synthesis, and blocking the IL-2R markedly inhibited atRA-induced cell proliferation and pRB phosphorylation. A retinoic acid receptor (RAR)-selective agonist and 9-cis-RA had the same potency as atRA on T cell proliferation and IL-2 secretion, whereas a retinoid X receptor-selective agonist had only marginal

effects. Furthermore, a RAR-selective antagonist completely suppressed T cell proliferation and pRB phosphorylation induced by http://www.jimmunol.org/ atRA. Taken together, these results suggest that atRA stimulates the cell cycle machinery and proliferation of normal human T cells by increasing IL-2 secretion through mechanisms involving RARs. The Journal of Immunology, 2002, 169: 5555Ð5563.

itamin A plays an important role in the regulation of The effects of vitamin A are primarily mediated via its acid immune function (1, 2). Vitamin A deficiency is asso- derivatives, including all-trans- and 9-cis RA (atRA and 9cRA) V ciated with decreased resistance to infection (3) and both (2). These isomeric forms of RA are ligands of two families of specific and nonspecific responses are impaired. Supplementation nuclear RA receptors, the RA receptors (RARs) and the retinoid X of vitamin A generally improves immune function in vitamin A- receptors (RXRs), of which there are several isoforms (9, 10). In deficient animals and enhances immune responses to certain Ags the presence of atRA, RAR and RXR form heterodimers, and ac- by guest on September 29, 2021 in vitamin A-depleted children (4). In apparent contrast to the find- tivate transcription through binding to RA response elements near ings that vitamin A protects against infections, we previously re- target genes (11, 12). The existence of stable RA analogs specific ported that physiological levels of retinoids inhibit the prolifera- for RARs and RXRs, respectively (13), in addition to selective tion of normal peripheral blood B lymphocytes (5, 6), as well as B antagonists (14), permit elucidation of the receptor pathway in- cell precursors from bone marrow (7). In support of these obser- volved in vitamin A responses. vations, we recently showed that in B cells, retinoic acid (RA)3 In the body, T cells are activated by interactions between TCR inhibits the cell cycle machinery responsible for G1- to S-phase and Ags displayed on the surface of APCs, and this, together with transition (8). These observations suggested to us that rather the T a second costimulatory signal, triggers a cascade of events that cell compartment could be the major target for vitamin A action in culminates in IL-2 production which drives the cells to proliferate stimulating the specific part of the immune system. (15). Activation of T lymphocytes in vitro can be mimicked by a variety of natural and synthetic compounds such as anti-CD3- and anti-CD28 Abs, Con A, PHA, and 12-O-tetradecanoylphorbol 13- Department of Medical Biochemistry, Institute Group of Basic Medical Sciences, Faculty of Medicine, University of Oslo, Oslo, Norway acetate (TPA) (16, 17). The tumor-promoting phorbol ester TPA Received for publication February 7, 2002. Accepted for publication September activates T cells by mimicking diacylglycerol in activation of pro- 5, 2002. tein kinase C (PKC) (18) and thereby bypasses the need for Ag The costs of publication of this article were defrayed in part by the payment of page binding (19, 20). charges. This article must therefore be hereby marked advertisement in accordance Passage through the cell cycle is a highly regulated process in- with 18 U.S.C. Section 1734 solely to indicate this fact. volving sequential activation of a series of cell cycle control pro- 1 This work was supported by Norwegian Cancer Society, Norwegian Research Council, Freia Research Foundation, Jahre Research Foundation, Nansen Foundation, teins (21). Phosphorylation of the tumor suppressor gene product, and Rakel and Otto Kr. Bruun’s legacy. the retinoblastoma protein (pRB), appears to be a key event in 2 Address correspondence and reprint requests to Dr. Heidi Kiil Blomhoff, Depart- regulating the entry of cells into S phase (22). By phosphorylation ment of Medical Biochemistry, Institute Group of Basic Medical Sciences, Faculty of of pRB, transcription factors such as E2Fs are released from their Medicine, University of Oslo, P. O. Box 1112 Blindern, N-0317 Oslo, Norway. E- mail address: [email protected] complex with pRB leading to transcription of S-phase genes (23). Phosphorylation of pRB during mid- to late G is accomplished by 3 Abbreviations used in this paper: RA, retinoic acid; atRA, all-trans RA; 9cRA, 9-cis 1 RA; RAR, RA receptor; RXR, retinoid X receptor; TPA, 12-O-tetradecanoylphorbol mitogen-activated cyclin D- and cyclin E-associated kinases, and 13-acetate; PKC, protein kinase C; pRB, retinoblastoma protein; CDK, cyclin-depen- later by cyclin A-associated kinase (24). Activation of these cy- dent kinase; CKI, cyclin-dependent kinase inhibitor; TTNPB, 4-((E)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl)benzoic acid; BrdU, 5-bromo- clin-dependent kinases (CDKs) depends not only on assembly with 2Ј-deoxyuridine; PI, propidium iodide. cyclin, but also on the correct stoichiometric ratio between the

Copyright © 2002 by The American Association of Immunologists, Inc. 0022-1767/02/$02.00 5556 RA STIMULATES IL-2 SECRETION AND THE CELL CYCLE IN T CELLS kinases and certain kinase inhibitors such as p16Ink4, p21Cip1, and Cell cycle analysis Kip1 p27 , collectively termed cyclin-dependent kinase inhibitors The cell cycle distribution was assessed by pulse labeling the cells with (CKIs; Ref. 25). In B cells, we have previously observed that atRA BrdU 1 h before harvesting. The cells were fixed in 70% ethanol before induced rapid dephosphorylation of pRB concomitant with an in- staining the cells with a FITC-conjugated anti-BrdU Ab and PI (27). The creased level of p21Cip1 and decreased levels of cyclin E and cy- percentage of BrdU-positive cells and the cell cycle distribution of 10,000 clin A (8). cells was analyzed using a FACScan flow cytometer (BD Biosciences, San Jose, CA) according to the manufacturer’s procedure. In the current study, we have focused on peripheral blood T lymphocytes with the purpose of understanding how atRA regu- Western blot analysis lates proliferation in these cells. We demonstrate that atRA Total cell lysates were prepared by lysing 5 ϫ 106 cells for 20 min on ice strongly potentiates the proliferation of peripheral blood T cells by in Triton X-100 lysis buffer (50 mM Tris, pH 7.5, 250 mM NaCl, 0.1% stimulating the cell cycle machinery leading to G1- to S-phase Tween X-100, 10 ␮g of leupeptin/ml, 9.5 ␮g of aprotinin/ml, 35 ␮gof transition. Furthermore, our results indicate that atRA mediates PMSF/ml, 5 mM NaF, 0.1 mM orthovanadate, 10 mM ␤-glycerophos- these effects by increasing IL-2 secretion through mechanisms that phate). Cellular debris was removed by centrifugation at 4¡C, and the protein content of the supernatant was determined by Bradford analysis (Bio- involve RARs. Rad, Hercules, CA). Lysate corresponding to equal amounts of protein (35Ð70 ␮g) were mixed with 3ϫ Laemmli sample buffer (28) and boiled for 5 min. The samples were resolved on 7.5 (for pRB) or 10Ð12% (for Materials and Methods other proteins) polyacrylamide gel under reducing conditions, and the pro- Reagents and Abs teins were transferred onto a nitrocellulose membrane (Amersham) using a semidry transfer cell (Bio-Rad). After blocking in TBST (0.1% Tween 20) TPA, RA (all-trans and 9-cis), 4-((E)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetra- Downloaded from containing 5% nonfat dry milk for1hatroom temperature, the membrane methyl-2-naphthalenyl)-1-propenyl)benzoic acid (TTNPB), ionomycin, was incubated with 1 ␮g/ml of the indicated Ab in TBST for2hatroom Con A, 5-bromo-2Ј-deoxyuridine (BrdU), and propidium iodide (PI) were temperature. The membrane was then washed four times with TBST, in- purchased from Sigma-Aldrich (St. Louis, MO). SR11217 was provided by cubated with a 1/6000 dilution of HRP-linked secondary Ab (Bio-Rad), Dr. M. I. Dawson (Life Science Division, SRI International, Menlo Park, and the immunoreactive proteins were visualized with the ECL detection CA) and Ro 41-5253 was provided by Dr. M. Klaus (Hoffman-La Roche, system (Amersham). Basel, Switzerland). Dynabeads anti-CD3/CD28 T cell expander was obtained from Dynal Biotech (Oslo, Norway). The retinoid compounds were Kinase assays http://www.jimmunol.org/ dissolved in ethanol or DMSO, flushed with argon, and stored in light- proof containers at Ϫ20¡C. All experiments were performed in subdued For histone H1 kinase assays, whole cell extracts were prepared by incu- light. Controls were run using the same concentrations of diluents as bating 107 cells for 20 min on ice in Triton X-100 lysis buffer as described present in the retinoid solutions, and these concentrations of diluents had above, and the lysates were cleared by centrifugation at 4¡C. After pre- no effects. Specific mouse mAbs anti-CD3⑀ mAb OKT3 were purified from clearing with 20 ␮l of 1:1 slurry of protein G-Sepharose beads (Amersham hybridoma culture supernatants. Abs against the IL-2R were purified from Biosciences, Uppsala, Sweden) for 30 min at 4¡C, 400 ␮g of each lysate supernatants of the mouse hybridoma cell line 2A3A1H (American Type was immunoprecipitated with 2 ␮g of anti-CDK2 Ab. The immunocom- Culture Collection, Manassas, VA). Anti-cyclin E- (HE12), anti-cyclin A- plexes were adsorbed onto 30 ␮l of a 1:1 slurry of protein G-Sepharose (C-19), anti-CDK2- ((M2)-G), anti-cyclin D3- (D-7), and anti-p27Kip1- (C- beads for1hat4¡C, collected by centrifugation at 400 ϫ g for 5 min and 19) Abs were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). washed twice with lysis buffer, and once with kinase buffer (50 mM Tris,

FITC-conjugated anti-BrdU Ab and anti-pRB Ab (G3-254) were purchased pH 7.5, 10 mM MgCl2, 1 mM DTT, 2 mM EGTA, 1 mM NaF, 0.1 mM by guest on September 29, 2021 from BD PharMingen (San Diego, CA). FITC-conjugated anti-CD25- orthovanadate, 10 mM ␤-glycerophosphate). The beads were then resus- (IL-2R ␣-chain) and FITC-conjugated anti-IgG1 Abs were obtained from pended in 15 ␮l of kinase buffer containing 30 ␮M ATP, 5 ␮g of histone Diatec (Oslo, Norway). Human rIL-2 was purchased from R&D Systems H1 (Upstate Biotechnology, Lake Placid, NY), and 10 ␮Ci of [␥-32P] ATP (Minneapolis, MN). per reaction mixture and incubated for 30 min at 30¡C. Reactions were stopped by the addition of 7.5 ␮lof3ϫ Laemmli sample buffer (28). The T cell purification samples were boiled for 5 min, and subjected to SDS-PAGE. Following electrophoresis, gels were stained with Coomassie blue, dried, and sub- T cells were negatively selected from peripheral blood obtained from jected to autoradiography. healthy blood donors from the Blood Bank at Ullevaal Hospital (Oslo, Norway). Mononuclear cells were isolated by Ficoll-Hypaque (Lym- Flow cytometric analysis of IL-2R expression phoprep; Nycomed, Oslo, Norway) density gradient centrifugation according to Boyum et al. (26). To remove monocytes, the cells were diluted to Isolated lymphocytes were pelleted, washed with PBS containing 2% FBS, ϫ 6 ϫ 5 107 cells/ml in RPMI 1640 (Life Technologies, Grand Island, NY), and and resuspended to 5 10 cells/ml. Cells (5 10 ) were incubated with ␣ incubated with 70 mg/ml carbonyl iron (Sigma-Aldrich) for1hat37¡C. FITC-conjugated anti-CD25 (IL-2R -chain) Ab or FITC-conjugated anti- The monocytes that had phagocytosed carbonyl iron particles were then IgG1 Ab (isotype-matched control) for1hat4¡C. Cells were subsequently absorbed to a samarium cobalt magnet, and the remaining lymphocytes washed three times, resuspended in 0.5 ml PBS containing 2% FBS, and were recovered from the supernatant. PBLs were mixed with Dynabeads analyzed on a FACScan flow cytometer (BD Biosciences) according to the coated with anti-CD14 Ab (product no. 111.11), anti-CD19 Ab (product manufacturer’s procedure. no. 111.04), and anti-HLA class II Ab (product no. 111.23), all from Dynal Biotech, in a cell-bead ratio of 1:4. After incubation of the mixture on a IL-2 secretion assay rotating wheel for 45 min at 4¡C, the rosetted cells were attracted to a T cells (105 cells/0.2 ml) were cultured as described for assessment of samarium cobalt magnet and the T cells were recovered from the super- DNA synthesis. After 24 h, cells were centrifuged and the cell-free supernatant. The purity of the T cell population was analyzed by flow cytometry natants were stored at Ϫ80¡C. The IL-2 levels were determined in parallel using Abs against CD3. The average yield of CD3-positive T cells varied by ELISA according to the manufacturer’s procedure (R&D Systems). between 85 and 95%. Statistical analysis Cell culture and determination of DNA synthesis Statistical analysis was performed using the Wilcoxon-signed rank test. T cells were cultured at 1.5 ϫ 106 cells/ml in RPMI 1640 supplemented with 10% heat-inactivated FBS (Life Technologies), 2 mM glutamine, 125 Results U/ml penicillin, and 125 ␮g/ml streptomycin at 37¡C in a humidified in- atRA potentiates T cell proliferation cubator with 5% CO2. For measurement of DNA synthesis, cells were 5 cultured in microtiter plates at an initial density of 10 cells/0.2 ml. Cells To study the effect of retinoids on T cell proliferation, we used were pulsed with 0.2 ␮Ci of [3H]thymidine (Amersham, Buckinghamshire, U.K.) for the last 20 h of a 68-h incubation. The cells were then harvested atRA, which is the major active retinoid metabolite in target cells. on a cell harvester and counted on a liquid scintillation counter (Topcount; atRA is present in peripheral blood at a concentration of ϳ10 nM Packard Instrument, Meriden, CT). (29) and it has been shown that atRA can be directly taken up by The Journal of Immunology 5557

cells both in vitro and in vivo (30, 31). The major source of retinoids for target cells in vivo is however retinol bound to retinol- binding protein or retinylester in chylomicron particles (32). How- ever, we have previously shown that atRA could substitute for retinol bound to retinol-binding protein or retinylesters in their effects on normal B cells (5), and, therefore, we routinely use atRA as a source for retinoids in lymphocyte assays. Proliferation was assessed by measuring the incorporation of [3H]thymidine into DNA, a frequently used method to estimate cell proliferation in lymphocytes (33, 34). A range of common T cell activators (anti-CD3⑀ (OKT3), anti CD3 ϩ anti CD28 Abs, Con A, TPA ϩ ionomycin and TPA) was tested for atRA responses (Fig. 1A). atRA potentiated cell proliferation induced by all agents tested. The most pronounced and consistent response was obtained when using TPA as the stimulant. In 10 independent experiments using TPA as stimulant, atRA induced proliferation 3.5-fold ( p ϭ 0.005, data not shown). Therefore, we decided to use TPA as the T cell activator in the additional experiments. How-

ever, it should be noted that a smaller, but statistically signiﬁcant, Downloaded from effect of atRA on proliferation was observed also when using the more physiological T cell stimulant anti-CD3⑀ (OKT3). In six independent experiments, atRA induced a 1.8-fold stimulation ( p ϭ 0.046, data not shown). atRA potentiated DNA synthesis of TPA-stimulated T cells in a

concentration-dependent manner, the effect being noted at 1 nM, http://www.jimmunol.org/ but was optimal at a concentration of 1 ␮M (Fig. 1B). Concentra- tions of atRA above 1 ␮M are generally known to be toxic, and led to decreased [3H]thymidine incorporation (data not shown). In the absence of mitogens, atRA had no effect on proliferation (Fig. 1B). The stimulatory effect of atRA on proliferation was confirmed by flow cytometric analysis of cells after incorporation of BrdU and staining with PI. As shown in Fig. 1C, atRA markedly enhanced the number of cells in S phase. This was first noted after 32 h and increased with time. After 48 h, atRA had caused a 4-fold by guest on September 29, 2021 increase in the number of cells entering into S phase (Fig. 1C). Interestingly, the ability of atRA to enhance T cell proliferation depended on the time of atRA addition (Fig. 2). By adding atRA 8 h after the addition of TPA, the proliferative effect was already diminished, and when added 24 h after TPA, virtually no effect of atRA was seen (Fig. 2). This suggests that atRA might affect some

early event in the transition of T lymphocytes from G1- to S phase. To exclude the possibility of interference from contaminating cells in the T cell population, the effect of atRA in positively selected CD4-positive cells was examined. We observed that atRA

FIGURE 1. atRA increases DNA synthesis and S-phase entry. Human T cells were isolated as described in Materials and Methods. A, T cells (105/ 0.2 ml) were stimulated with anti-CD3⑀ Ab (OKT3; 1 ␮g/ml), anti-CD3/ CD28 T cell expander (␣-CD3 ϩ CD28; 0.125 ␮l/well), Con A (4 ␮g/ml), TPA (10Ϫ8 M) and ionomycin (I; 0.5 M), or TPA (10Ϫ8 M) in the presence or absence of atRA (1 ␮M). Cells were cultured in triplicates and [3H]thymidine incorporation was determined as described in Materials and Meth- ods. Incorporation of [3H]thymidine is expressed as the mean of fold induction compared with medium control Ϯ SEM of three independent experiments. B, T cells (105/0.2 ml) were stimulated with TPA (10Ϫ8 M) in the presence or absence of atRA at indicated concentrations (nanomoles per liter) as described in A. Incorporation of [3H]thymidine is expressed as the mean Ϯ SEM of four independent experiments. C, Cells (1.5 ϫ 106 cells/ml) were stimulated with TPA (10Ϫ8 M) in the presence or absence of atRA (1 ␮M). The kinetics of the effect of atRA on T cell DNA synthesis were determined by ﬂow cytometric analysis following labeling of the cells with BrdU for 1 h and staining with PI as described in Materials and Methods. The percentage of cells in S phase is indicated. One representative experiment of three is shown. 5558 RA STIMULATES IL-2 SECRETION AND THE CELL CYCLE IN T CELLS

FIGURE 4. Kinetics of atRA on pRB phosphorylation and expression ϫ 6 of cell cycle regulatory proteins in G1. T cells (1.5 10 cells/ml) were stimulated with TPA (10Ϫ8 M) in the presence or absence of atRA (1 ␮M) FIGURE 2. Effect of adding atRA at various time points. atRA (1 ␮M) or TTNPB (100 nM). Total cell lysates were prepared at the indicated times was added to cell cultures (105/0.2 ml) at the indicated times (hours) after (hours) and equal amounts of total protein (35 ␮g for pRB, 70 ␮g for Ϫ stimulation of the cells with TPA (10 8 M). [3H]Thymidine incorporation cyclins and p27Kip1) were subjected to Western blot analysis as described was measured as described in Materials and Methods. The vertical bars in Materials and Methods with Ab against the proteins indicated on the indicate the mean Ϯ SEM of four independent experiments. right. The lane indicated as 0 refers to a sample harvested before mitogenic stimulation of the cells. One representative experiment of four is shown. Downloaded from enhanced the proliferation of T cells to the same degree in this highly pure cell population as in negatively isolated T cells (data tions of increased levels of the cyclins and decreased level of not shown). A similar response was noted in T cell cultures de- p27Kip1, we conducted an in vitro CDK2 kinase assay using his- pleted of CD56-positive NK cells (data not shown), indicating that tone H1 as substrate. As shown in Fig. 5, TPA induced a low level NK cells do not interfere with the T cell response. of CDK2 activity, whereas atRA strongly increased the kinase atRA stimulates the cell cycle machinery in T cells leading to activity. http://www.jimmunol.org/ increased pRB phosphorylation As evident from Fig. 3, atRA stimulated the components of the cell cycle machinery in a concentration-dependent manner, with an Functional inactivation of pRB by phosphorylation at multiple optimal effect noted at 1 ␮M. However, the changes in the protein sites is required for G1- to S-phase transition (22). Phosphorylated levels were late events, ﬁrst appearing 20 h after atRA addition pRB proteins are visualized as slower-migrating forms of the pro- (Fig. 4). The time- and concentration-dependent effects of atRA on tein on SDS-polyacrylamide gels. As shown in Fig. 3, atRA en- the cell cycle parameters closely resembled that on pRB phosphor- hanced TPA-mediated phosphorylation of pRB after 32 h in a con- ylation, indicating a causative relationship between these events. centration-dependent manner. The effect was maximal at a However, the slow changes in the cell cycle protein levels induced

␮ by guest on September 29, 2021 concentration of 1 M. In contrast to the rapid effect of atRA on by atRA indicated that in T cells, atRA does not affect the cell pRB phosphorylation in B cells reported previously (8), the cycle machinery directly. changes in pRB phosphorylation in T cells appeared 20 h after atRA addition (Fig. 4). atRA increases the secretion of IL-2 without changing the Phosphorylation and inactivation of pRB are mediated by expression of its receptor CDKs. Activation of CDKs is the result of correct complex for- Due to the vital role of IL-2 in the growth and clonal expansion of mation with cyclins and CKIs, as well as phosphorylation and T cells (36), we examined the effect of atRA on IL-2R expression dephosphorylation of certain amino acids in the CDK proteins and IL-2 production. The IL-2R comprises three subunits: IL-2R␣ (35). By Western blot analysis, we demonstrated that atRA in- (CD25), IL-2R␤, and IL-2R␥, forming high-affinity receptors creased TPA-induced expression of cyclin D3, cyclin E, and cyclin when all three subunits are present (37). T lymphocytes can reg- A in a time-dependent manner, concomitant with reduced levels of ulate their responsiveness to IL-2 through transcriptional control of p27Kip1 (Fig. 4). atRA had no effect on the protein levels of cyclin Cip1 one of the components of the high-affinity receptor, namely the D2, CDK2 or p21 (data not shown). To confirm the implica- IL-2R␣ gene (38). Using flow cytometric analysis, we observed that TPA induced expression of the IL-2R ␣-chain after 24 h, but the expression of this subunit was not affected by atRA (Fig. 6). In contrast, atRA potently enhanced IL-2 protein secretion (Fig. 7A) and the effect was notable at 8 h (Fig. 7B). In atRA-treated cells,

FIGURE 3. atRA-mediated changes in pRB phosphorylation and expression of cell cycle regulatory proteins in G1. Isolated human T cells (1.5 ϫ 106 cells/ml) were stimulated for 32 h with TPA (10Ϫ8 M) in the FIGURE 5. atRA increases CDK2 activity. Mitogenic stimulation, presence or absence of atRA at the indicated concentrations (nanomoles atRA treatment, and harvesting of the cells were performed as described in per liter). Total cell lysates were prepared and equal amounts of total pro- the legend to Fig. 4. Total cell lysates were prepared and equal amounts of tein (35 ␮g for pRB, 70 ␮g for cyclins and p27Kip1) were subjected to whole cell extracts (400 ␮g of protein/sample) were immunoprecipitated Western blot analysis as described in Materials and Methods using Abs with Ab against CDK2, followed by the kinase assay using histone H1 as against the proteins indicated on the right. Hypophosphorylated pRB and substrate as described in Materials and Methods. The lane indicated as 0 h hyperphoshorylated pRB (ppRB) were detected using mAb speciﬁc for refers to a sample harvested before mitogenic stimulation of the cells. IP, pRB. One reproducible experiment of two is shown. immunoprecipitating Ab. One of three reproducible experiments is shown. The Journal of Immunology 5559 Downloaded from http://www.jimmunol.org/

FIGURE 6. Effect of atRA on the expression of CD25. T cells (1.5 ϫ 106 cells/ml) were stimulated with TPA (10Ϫ8 M) in the presence or ab- FIGURE 7. atRA increases IL-2 secretion. T cells (105/0.2 ml) were Ϫ sence of atRA (1 ␮M) for 24 h and were stained with FITC-conjugated stimulated with TPA (10 8 M) in the presence or absence of atRA (1 ␮M) anti-CD25 Ab as described in Materials and Methods. An isotype-matched for 24 h (A) or for the indicated times (hours) (B) as described in Materials irrelevant anti-IgG1 Ab was used as a negative control (top panel). The and Methods. The cell-free supernatants were examined for IL-2 secretion

ﬂuorescence intensity is given in logarithmic scale. One representative ex- as described in Materials and Methods. A, Vertical bars indicate the by guest on September 29, 2021 .p ϭ 0.041 ,ء ;periment of three is shown. mean Ϯ SEM of eight independent experiments

stimulated proliferation (Fig. 9A). As a control for the efficiency of the level of secreted IL-2 after 24 h varied between 0.02 and 6 the IL-2/IL-2R pathway inhibition, we showed that the IL-2R Ab ng/ml (T cells from eight donors were analyzed) reflecting the bio- completely blocked the potentiating effect of 1 ng/ml rIL-2 on logical diversity between human donors. The average level secreted at TPA-induced proliferation (Fig. 9A). Moreover, as shown by 24 h was 1.5 Ϯ 0.7 ng/ml (SEM, p ϭ 0.041, n ϭ 8; Fig. 7A). Western blot analysis, the IL-2R Ab markedly reduced the phosphorylation of pRB mediated by atRA (Fig. 9B). Again, the effect IL-2 mimics and an IL-2R-blocking Ab inhibits the of rIL-2 was completely blocked by the IL-2R blocking Ab (Fig. effects of atRA 9B). These results strongly indicate that induced production of Because atRA increased IL-2 production in T cells, it was of in- IL-2 has a vital role in atRA-stimulated T cell proliferation. terest to determine whether IL-2 could replace the effect of atRA on T cell proliferation. Addition of rIL-2 to TPA-stimulated T cells A RAR agonist mimics and a RAR-selective antagonist had the same effect as atRA on DNA synthesis (Fig. 8A), but on counteracts the effects of atRA on T cell proliferation average, a higher concentration of rIL-2 was needed to replace the atRA is generally believed to act through binding to ligand-depen- effect of atRA than the level of IL-2 usually induced by atRA. The dent transcription factors (39), although some reports have pro- amount of rIL-2 needed to mimic the proliferative effect of atRA posed other mechanisms for atRA-mediated effects (40, 41). To was donor-dependent and in the range of 0.25Ð10 ng/ml. The av- study the contribution of the various nuclear retinoid receptors to erage concentration of rIL-2 needed to restore the effect of atRA the proliferative response and IL-2 secretion of T cells, we as- was 4.9 Ϯ 0.9 ng/ml (SEM, n ϭ 10). To examine whether rIL-2 sessed the effect of a pan RAR agonist (TTNPB) and a RXR- could mimic the effects of atRA on the cell cycle machinery, we selective agonist (SR11217) in addition to 9-cis RA, which is a added rIL-2 to the TPA-stimulated cells and performed Western blot ligand for both RAR and RXR. TTNPB and 9-cis RA fully mim- analyses. Indeed, 5 ng/ml rIL-2 replaced the effect of atRA on pRB icked the effect of atRA on DNA synthesis, whereas the effect of phosphorylation and expression of cyclin E and cyclin A (Fig. 8B). SR11217 was only marginal (Fig. 10A). TTNPB was also tested on To confirm the role of IL-2 in atRA-mediated T cell prolifera- the various components of the cell cycle machinery and it was tion, we blocked the IL-2/IL-2R pathway by using an Ab against shown to fully mimic the potentiating effect of atRA (Fig. 4). We the IL-2R, and assessed its effects on atRA-stimulated prolifera- also determined whether the agonists could mimic the effects of tion. A significant reduction in [3H]thymidine incorporation was atRA on IL-2 secretion. TTNPB and 9-cis RA both strongly po- seen in the presence of the IL-2R blocking Ab (Fig. 9A). By com- tentiated TPA-induced secretion of IL-2, whereas again the effect parison, an isotype-matched irrelevant Ab had no effect on atRA- of SR11217 was marginal (Fig. 10B). 5560 RA STIMULATES IL-2 SECRETION AND THE CELL CYCLE IN T CELLS Downloaded from

FIGURE 9. Blocking IL-2R inhibits DNA synthesis and phosphoryla- http://www.jimmunol.org/ tion of pRB. A, T cells (105/0.2 ml) were stimulated with TPA (10Ϫ8 M) in the presence or absence of atRA (1 ␮M) or rIL-2 (1 ng/ml), with or FIGURE 8. IL-2 mimics the effect of atRA on DNA synthesis and cell without anti-IL-2R Abs (10 ␮g/ml) as indicated. Cells were cultured in cycle regulatory proteins. A, T cells (105/0.2 ml) were stimulated with TPA triplicates and [3H]thymidine incorporation (mean Ϯ SD) was determined Ϫ (10 8 M) in the presence or absence of atRA (1 ␮M) or rIL-2 (nanograms as described in Materials and Methods. One representative experiment of per milliliter) as indicated. DNA synthesis was measured as described in four is shown. ␣IL-2R, anti-IL-2R Ab; IrrAb, irrelevant Ab. B, T cells Materials and Methods. The vertical bars indicate the mean Ϯ SEM of 10 (1.5 ϫ 106 cells/ml) were stimulated for 32 h with TPA (10Ϫ8 M) in the independent experiments. B, T cells (1.5 ϫ 106 cells/ml) were stimulated presence or absence of atRA (1 ␮M) or rIL-2 (1 ng/ml) as indicated. Anti- Ϫ8 ␮ ␮ for 32 h with TPA (10 M) in the presence or absence of atRA (1 M) IL-2R Ab (10 g/ml) was added to the cell cultures as shown. Total cell by guest on September 29, 2021 or rIL-2 at indicated concentrations (nanograms per milliliter). Total cell lysates were prepared and equal amounts of total protein (30 ␮g) were lysates were prepared and equal amounts of total protein per sample were analyzed for hypophosphorylated pRB and hyperphoshorylated pRB analyzed for hypophosphorylated pRB and hyperphoshorylated pRB (ppRB) by Western blot analysis as described in Materials and Methods. (ppRB), cyclin E, and cyclin A by Western blot analysis as described in One representative experiment of three is shown. ␣IL-2R, anti-IL-2R Ab. Materials and Methods. The lane indicated as 0 refers to a sample harvested before mitogenic stimulation of the cells. One representative experiment of three is shown. (46). However, in none of these reports have the mechanisms been explored. In the current study, using normal peripheral T cell and stimulants such as anti-CD3 Ab alone, costimulation with anti- Finally, to verify the involvement of the RAR pathway, we used CD3 and CD28 Abs, Con A, or TPA and ionomycin, a potentiating a RAR-selective antagonist, Ro 41-5253. In increasing concentra- effect of atRA was observed, but the response varied in degree and tions, Ro 41-5253 inhibited DNA synthesis induced by TPA and was donor-dependent. A statistically significant effect of atRA was atRA (Fig. 11A). A complete inhibition was noted at a concentra- obtained using anti-CD3 Ab as a stimulant, but the most extensive tion of 10 ␮MofRo41Ð5253 (Fig. 11A), without affecting the effect of atRA was noted in T cells stimulated with suboptimal viability of the cells (analyzed by PI staining, data not shown). Ro doses of TPA. Therefore, we used this agent to examine the mech- 41-5253 at 10 ␮M also prevented atRA-induced pRB phosphory- anisms whereby atRA stimulates T cells. lation, inhibited the increase in the protein level of cyclin E, and The key regulatory event in the G1- to S-phase transition of the reversed the reduction of p27Kip1 induced by atRA (Fig. 11B). cell cycle is phosphorylation of pRB (22) and we found that atRA Taken together, these results suggest that the effects of atRA on cell markedly increased TPA-induced pRB phosphorylation. The effect proliferation and the cell cycle machinery are mediated through a of atRA on pRB phosphorylation was consistent with its stimula- RAR-dependent pathway. tory effect on the events known to direct phosphorylation of pRB, such as enhanced protein levels of cyclin D3, cyclin E, and cyclin Discussion A, reduced levels of p27Kip1, and increased CDK2 activity. Of note In the current study, we have shown that physiological levels of was the delayed effect of atRA on these cell cycle parameters. atRA stimulate the proliferation of human peripheral blood T cells Thus, in great contrast to the rapid and direct inhibitory effect of and we have examined the mechanisms involved. In particular, we atRA on the cell cycle machinery in peripheral blood B cells (8), have investigated the effects of atRA on the cell cycle machinery the effect of atRA on T cells appeared after a lag period of 20 h. and assessed the role of IL-2 in this process. Previous reports on This, taken together with the observation that atRA had to be the effect of vitamin A derivatives have indicated that retinoids added to T cells early during stimulation of the cells to enhance strengthen the cellular response of human thymocytes (42), of mu- proliferation, indicated to us that the effects of atRA on the cell rine spleen T lymphocytes (43Ð45), and of murine thymic T cells cycle machinery, and hence on proliferation, were indirect. The Journal of Immunology 5561 Downloaded from http://www.jimmunol.org/ FIGURE 11. A RAR-selective antagonist abolishes the effect of RA on proliferation and pRB phosphorylation. A, T cells (105/0.2 ml) were stimulated with TPA (10Ϫ8 M) in the presence or absence of atRA (10 nM) and Ro 41-5253 in increasing concentrations (micromoles per liter) as indi- 3 FIGURE 10. The RAR agonist TTNPB mimics the effect of atRA on cated. Cells were cultured in triplicates and [ H]thymidine incorporation cell proliferation and on IL-2 secretion. A, Cells (105/0.2 ml) were stimu- was determined as described in Materials and Methods. One representative Ϫ ϫ 6 lated with TPA (10 8 M) in the presence or absence of retinoids (100 nM) experiment of three is shown. B, T cells (1.5 10 cells/ml) were stim- Ϫ8 as indicated. Cells were cultured in triplicates and [3H]thymidine incorpo- ulated for 32 h with TPA (10 M) in the presence or absence of atRA (10 ␮ ration was determined as described in Materials and Methods. The results nM) with or without Ro 41-5253 (10 M). Total cell lysates were made represent the mean Ϯ SEM of seven independent experiments. B, T cells and Western blot analysis was performed as described in Materials and by guest on September 29, 2021 (105/0.2 ml) were stimulated with TPA (10Ϫ8 M) in the presence or ab- Methods. Ro, Ro 41-5253. One of two reproducible experiments is shown. sence of retinoids (100 nM) for 24 h as described in Materials and Meth- ods. The cell-free supernatants were examined for IL-2 as described in Materials and Methods. One representative experiment of three is shown. However, we did not observe any significant changes in these parameters in the presence of atRA (data not shown). Thus, at present, we have no indications which pathways other than IL-2/ Given the central role of IL-2 in T cell proliferation (36), we IL-2R can be involved in atRA-mediated T cell proliferation. tested whether atRA would increase TPA-induced IL-2 produc- An important finding in the current study was the different ef- tion. We detected only marginal secretion of IL-2 in the presence fects of atRA in T cells as compared with the effect we previously of TPA alone. Interestingly however, atRA potently enhanced IL-2 reported in B cells. Whereas the proliferation of B cells is potently secretion. Increased IL-2 mRNA synthesis has previously been inhibited by physiological levels of retinoids (5, 6), T cells are observed in T cells stimulated with anti-CD3 Ab and retinol (47). stimulated by atRA. This cannot be ascribed to the fact that a However, in this previous report, the level of IL-2 secreted was not different mitogen than TPA was used to stimulate B cells in the significantly elevated by retinol. By adding rIL-2 to TPA-stimu- latter study, because in the current study we found that atRA also lated T cells, we showed in the current study that IL-2 replaced the inhibited the proliferation of TPA-stimulated B cells (data not effects of atRA on both cell cycle regulation and on proliferation. shown). A clue to understanding the mechanisms underlying the However, the average concentration of rIL-2 (5 ng/ml) needed to contrasting effects was provided by our analysis of the cell cycle replace the effect of atRA was higher than the average level of IL-2 machinery in B cells vs T cells. The cell cycle machinery in B cells (1.5 ng/ml) that we measured was secreted by the cells, indicating was inhibited by atRA through decreased levels of cyclin E and -A that IL-2 cannot fully explain the effects of atRA. In contrast, it is and increased levels of p21Cip1 (8), whereas in T cells it was stim- possible that the assay we used to assess the amount of IL-2 se- ulated through increased levels of cyclin D3, -E, and -A and de- creted underestimates the actual level of IL-2 produced, due to creased levels of p27Kip1. Even more interesting was the different rapid internalization of secreted IL-2 once it binds its receptor kinetics whereby atRA affected the cell cycle machinery in B cells (48Ð50). The notion that IL-2 cannot fully explain the effect of vs T cells. Whereas atRA induced very rapid changes in the cell atRA was supported by experiments using a blocking IL-2R Ab. cycle machinery in B cells, the changes observed in T cells were The Ab totally inhibited rIL-2-mediated responses, whereas it slow and appeared to depend on the production of IL-2. In B cells, never completely blocked the effect of atRA on T cell proliferation the effects of atRA on the expression of p21Cip1, cyclin E, and and pRB phosphorylation. We have addressed the possibility that cyclin A were observed already after 30 min, and appeared to be atRA could induce expression of other cytokines known to drive T regulated at the level of transcription (8). In T cells, atRA did not cell proliferation and differentiation such as IFN-␥ and IL-4 (51). change the expression of p21Cip1, although it has been reported 5562 RA STIMULATES IL-2 SECRETION AND THE CELL CYCLE IN T CELLS that p21Cip1 possesses a RAR-responsive element in its promoter 8. Naderi, S., and H. K. Blomhoff. 1999. Retinoic acid prevents phosphorylation of (52). Thus, the effects of atRA on the cell cycle machinery in B pRB in normal human B lymphocytes: regulation of cyclin E, cyclin A, and p21Cip1. Blood 94:1348. cells appear to be direct, while the effects in T cells are indirect and 9. Giguere, V., E. S. Ong, P. Segui, and R. M. Evans. 1987. Identification of a may proceed via increased production of IL-2. An interesting ques- receptor for the morphogen retinoic acid. Nature 330:624. Cip1 10. Mangelsdorf, D. J., U. Borgmeyer, R. A. Heyman, J. Y. Zhou, E. S. Ong, tion is why p21 is induced by atRA in B cells but not in T cells. A. E. Oro, A. Kakizuka, and R. M. 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