QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: jpet.102.048496v1 305/3/1222    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Schafer, P. H. Articles by Stirling, D. I. Search for Related Content PubMed PubMed Citation Articles by Schafer, P. H. Articles by Stirling, D. I. Pubmed/NCBI databases Compound via MeSH Substance via MeSH Hazardous Substances DB THALIDOMIDE

INFLAMMATION AND IMMUNOPHARMACOLOGY

Enhancement of Cytokine Production and AP-1 Transcriptional Activity in T Cells by Thalidomide-Related Immunomodulatory Drugs

Celgene Corporation, Warren, New Jersey (P.H.S., A.K.G., M.A.L., R.S.C., H.-W.M., S.G., L.G.C., F.P., G.W.M., D.I.S.); and Department of Physiology and Biophysics, Faculty of Medicine, University of Calgary, Alberta, Canada (P.P.M.S., G.W.)

Received December 23, 2002; accepted March 7, 2003.

	Abstract

Top Abstract Materials and Methods Results Discussion References

 
CC-4047 (Actimid) and CC-5013 (Revimid) belong to a class of thalidomide analogs collectively known as the immunomodulatory drugs (IMiDs), which are currently being assessed in the treatment of patients with multiple myeloma and other cancers. IMiDs potently enhance T cell and natural killer cell responses and inhibit tumor necrosis factor-, interleukin (IL)-1, and IL-12 production from LPS-stimulated peripheral blood mononuclear cells. However, the molecular mechanism of action for these compounds is unknown. Herein, we report on the ability of the IMiDs to up-regulate production of IL-2 from activated human CD4⁺ and CD8⁺ peripheral blood T cells, production of IL-2 and IFN- from T helper (Th)1-type cells, and production of IL-5 and IL-10 from Th2-type cells. Elevation of IL-2 production from Jurkat T cells was observed as early as 6 h poststimulation and correlated with an increase in IL-2 promoter activity that was dependent upon the proximal but not the distal AP-1 binding site. The IMiDs enhanced AP-1-driven transcriptional activity 2- to 4-fold after 6 h of T cell stimulation, and their relative potencies for AP-1 activation correlated with their potencies for increased IL-2 production in Jurkat T cells and in CD4⁺ or CD8⁺ human peripheral blood T cells. The most potent of these IMiDs, CC-4047, had no effect on nuclear factor of activated T cells transcriptional activity, calcium signaling, or phosphorylation of extracellular signal-regulated kinase 1/2, c-Jun NH₂-terminal kinase 1/2, p38 mitogen-activated protein kinase, or c-Jun/Jun D in Jurkat T cells. These data suggest that IMiDs increase T cell cytokine production by potentiating AP-1 transcriptional activity.

To better define the impact of IMiDs on T cell responses, the effect of three IMiDs on various T cell populations was examined. IMiD treatment enhanced IL-2 production in both CD4⁺ and CD8⁺ subsets. These IMiDs increased Jurkat IL-2 production in response to anti-CD3 and anti-CD28 antibody in solution, in response to staphylococcal enterotoxin E (SEE) presented by Raji B cells, and in response to PMA and ionomycin, but not to any single stimulus alone. These IMiDs increased IL-2 and IFN- production by human Th1-type cells, as well as IL-5 and IL-10 production by Th2-type cells. Because a common feature of these cytokines is regulation by the transcription factor AP-1, the effect of IMiDs on AP-1 activity was examined. IMiDs were found to increase AP-1 transcriptional activity in a time- and dose-dependent manner. IL-2 production by Jurkat T cells correlated with an increase in IL-2 promoter activity that was almost completely dependent upon the proximal AP-1 binding site. The increased AP-1 activity was not blocked by the addition of anti-IL-2 neutralizing antibody, indicating that the increased AP-1 activity was not dependent on the increase in secreted IL-2. Calcium flux and NFAT activation was not affected by treatment with CC-4047, nor was phosphorylation of extracellular signal-regulated kinase (ERK)1/2, c-Jun N-terminal kinase (JNK)1/2, c-Jun/Jun D, or p38 MAPK. Furthermore, the elevated IL-2 production remained sensitive to pharmacological inhibitors of calcineurin, CD45 phosphatase, MEK1, PKC/, PKA, JAK2, and p38 MAPK. Together, these data indicate that these IMiDs potentiate T cell cytokine production by a mechanism that involves enhancement of AP-1 transcriptional activity, but without affecting many of the upstream signaling components involved in T cell stimulation.

	Materials and Methods

Top Abstract Materials and Methods Results Discussion References

 
Materials. IMiDs were synthesized by Celgene Corporation (Warren, NJ) as described previously (Muller et al., 1999). All IMiDs used herein are amino-substituted analogs of thalidomide. Cyclosporin A, PD98059, SB203580, and H-89 were purchased from Calbiochem-Novabiochem (La Jolla, CA). Rottlerin, AG-490, and RWJ60475(AM)₃ were obtained from BIOMOL Research Laboratories (Plymouth Meeting, PA). All compounds were dissolved in 100% DMSO (Sigma-Aldrich, St. Louis, MO) before further dilution in cell culture media. Final DMSO concentrations were kept at a constant 0.1% for all samples, including controls, unless otherwise stated.

Cell Lines. The mouse hybridoma clone OKT3 secreting anti-human CD3 monoclonal antibody was obtained from American Type Culture Collection (Manassas, VA). OKT3 antibody was purified from culture supernatants on HiTrap protein G columns (Amersham Biosciences Inc., Piscataway, NJ) according to manufacturer's instructions. Jurkat E6-1 leukemic T cells and Raji human Burkitt's lymphoma B cells were obtained from American Type Culture Collection and grown in RPMI 1640 medium (BioWhittaker, Walkersville, MD), 10% fetal bovine serum, 100 U/ml penicillin, 100 mg/ml streptomycin, and 2 mM L-glutamine.

T Cell Purification. CD4⁺ and CD8⁺ T cells were purified from human leukocytes obtained from Sera-Tec Biologicals (North Brunswick, NJ) by negative selection as described previously (Schafer et al., 1999b). CD4⁺ Th1 and Th2 cells were obtained by stimulation of human PBMCs with phytohemagglutinin-L and recombinant human (rh)IL-2 in the presence of either rhIL-12 plus anti-IL-4R antibody for Th1 conditions, or in the presence of rhIL-4 plus anti-IL-12 for Th2 conditions, followed by CD4⁺ T cell purification as described previously (Schafer et al., 1999a), except that major histocompatibility complex class II antibody-coated beads were not used, to avoid depletion of activated T cells.

Cytokine Production Assays. All samples were set up in duplicate in 96-well flat-bottom tissue culture-treated plates. T cells were plated at 2 to 3 x 10⁵ cells/well into plates that had been precoated with anti-CD3 monoclonal antibody OKT3 (at 5 µg/ml in 0.1 ml of phosphate-buffered saline for 4 h at 37°C and washed four times with complete medium to remove unbound antibody) and immediately treated with IMiDs at a constant final DMSO concentration of 0.1%. Supernatants were harvested after 1 to 3 days and assayed for IL-2, IFN-, IL-5, and IL-10 by ELISA (R & D Systems, Minneapolis, MN). Jurkat T cells (2 x 10⁵ cells/well) were pretreated with IMiDs for 1 h and then stimulated either with Raji cells (4 x 10⁴ cells/well) plus SEE (Toxin Technology, Sarasota, FL) (20 ng/ml) or with soluble OKT3 (1 µg/ml) and CD28 monoclonal antibody CD28.2 (BD Biosciences PharMingen, San Diego, CA) (10 µg/ml) plus F(ab')₂ goat anti-mouse IgG (Jackson Immunoresearch Laboratories, West Grove, PA) (30 µg/ml), or with PMA (1 ng/ml) (Sigma-Aldrich) plus ionomycin (1 µg/ml) (Sigma-Aldrich). Supernatants were harvested after 1 to 2 days and tested for IL-2 by ELISA. IL-2 IC₅₀ values (the concentration of compound required to inhibit 50% of IL-2 production) were calculated using Prism 3.02 (GraphPad Software Inc., San Diego, CA). For SB203580, IL-2 EC₁₅₀ values (the concentration of SB203580 required to elevate IL-2 production to 150% of stimulated levels) were calculated using Prism 3.02.

Transfections and Luciferase Reporter Assays. Jurkat T cells were transiently transfected in bulk and then aliquoted just before IMiD treatment and cell stimulation to eliminate possible sample variability due to differences in transfection efficiency. Cells were transfected with human IL-2 promoter constructs subcloned into the luciferase reporter plasmid pGL2 (a gift of Christopher Hughes, University of California at Irvine, Irvine, CA). The wild type IL-2 promoter (WT pIL-2-Luc), distal AP-1 mutant IL-2 promoter (mt dAP-1 pIL-2-Luc), and proximal AP-1 mutant IL–2 promoter (mt pAP-1 pIL-2-Luc) constructs have been described previously (Hughes and Pober, 1996). The AP-1 mutants each contain a three base pair mutation that eliminates AP-1 binding in vitro. The AP-1 and NFAT-driven plasmids pAP-1-Luc and pNFAT-Luc were obtained from Stratagene (La Jolla, CA). pAP-1-Luc contains seven tandem repeats of the AP-1 DNA binding sequence TGACTAA. pNFAT-Luc contains four tandem repeats of the NFAT binding sequence from the IL-2 gene promoter (-286 to -257). In all cases, 2 x 10⁶ Jurkat T cells were transfected with 4 µg of DNA and the transfection reagent DMRIEC (18 µl/sample) (Invitrogen, Carlsbad, CA) according to manufacturer's instructions. When indicated, the plasmid expressing MEKK (pFC-MEKK; Stratagene) was cotransfected at 50 ng/2 x 10⁶ cells along with pAP-1-Luc as a positive control for activation of the AP-1 pathway. Twenty-four hours after transfection, Jurkat cells were washed, aliquoted (2 x 10⁶ cells/sample), and pretreated with IMiDs for 1 h before stimulation with Raji B cells (1–2 x 10⁶ cells/ml) plus SEE (100 ng/ml). For IL-2 neutralization, anti-IL-2 antibody and mouse IgG1 (R & D Systems) were used at 10 µg/ml. Luciferase activity was assayed at the indicated times using LucLite luciferase lysis buffer and substrate (Packard Biosciences, Meridian, CT) and read on a TopCount plate reader (PerkinElmer Life Sciences, Boston, MA).

Western Blotting. Jurkat cells (2 x 10⁶/sample) were serum-starved overnight in AIM-V medium and then placed in 1.5-ml Eppendorf tubes in RPMI 1640 medium on the day of the experiment. CC-4047 (10 µM) or DMSO (0.1% final) was added to cells 1 h before stimulation with OKT3 (5 µg/ml final) and CD28.2 (10 µg/ml final). Cells were spun down for 6 s in a Microfuge and immediately lysed in 0.1 ml of lysis buffer containing 10 mM Tris-HCl, pH 8.0, 10 mM EDTA, 150 mM NaCl, 1% NP-40, 0.5% SDS, 1 mM dithiothreitol, 1 mM Na₃VO₄, plus Complete protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN) and then spun through a Qiashredder (QIAGEN, Valencia, CA) for 1 min and frozen on dry ice. Samples were diluted 1:1 with 2x SDS sample buffer (Invitrogen) containing 5% -mercaptoethanol and boiled 5 min. Approximately 50 µl of this mixture was loaded per lane on 10% Tris-glycine polyacrylamide gels (Invitrogen), electrophoresed, and transferred to polyvinylidene difluoride membranes (Invitrogen). Polyvinylidene difluoride membranes were blocked for 1 h at room temperature in TBST buffer (20 mM Tris, pH 7.6, 137 mM NaCl, 0.05% Tween 20) containing 5% bovine serum albumin, washed five times in TBST, and then blotted overnight at 4°C with phospho-ERK1/2 (Thr202/Tyr204), phospho-JNK1/2 (Thr183/Tyr185), phospho-p38 MAPK (Thr180/Tyr182) (New England Biolabs/Cell Signaling Technology, Beverly, MA), phospho-c-Jun/JunD (Ser73) (Upstate Biotechnology, Lake Placid, NY). Membranes were washed five times in TBST and incubated with anti-rabbit IgG (Santa Cruz Biotechnology Inc., Santa Cruz, CA) (1:10,000 dilution) if required for 30 min at room temperature, washed five times in TBST, and then developed using the ECL Plus chemiluminescent detection system (Amersham Biosciences Inc.). Western blot images were read in blue fluorescence mode on a Storm 840 PhosphorImager and analyzed using ImageQuant software (Amersham Biosciences Inc.).

Calcium Measurements. Jurkat T cells were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum; immediately before use, cells were incubated for 30 min at room temperature with 20 µM fluo-3AM (Molecular Probes, Eugene, OR) and 10 µM CC-4047 in a volume of 1 to 2 ml containing 10⁸ cells. Cells were diluted 20-fold with 150 mM NaCl, 1.5 mM CaCl₂, 3 mM KCl, 10 mM glucose, 20 mM HEPES buffer (pH 7.4), and 250 µM sulfinpyrazone, and sedimented for 4 min at 800g. Cells were washed once more and resuspended in the same medium with or without 10 µM CC-4047 at a cell density of 10⁸ cells/ml. Cells were left at room temperature for an additional 15 min to allow full deesterification of partially hydrolyzed fluo-3AM. For calcium measurements, cells were diluted 20-fold in the above-mentioned medium, placed in a thermostated cuvette equipped with a magnetic stirrer and fluorescence was measured in a SLM-Aminco series 2 luminescence spectrometer (SLM Instruments, Urbana, IL). The cells suspension was excited at 480 nm (8-nm bandwidth) and emission was measured at 530 nm (8-nm bandwidth); a 495-nm long pass filter was placed before the emission monochromator to minimize apparent fluorescence due to light from the excitation beam scattered by the cell suspension. Minimum and maximum fluorescence levels were obtained in the presence of 0.02% saponin and either 5 mM EDTA or 1.5 mM Ca²⁺, respectively.

Statistical Analysis. Two-tailed p values were calculated using a paired t test in Prism 3.02 (GraphPad Software Inc.).

	Results

Top Abstract Materials and Methods Results Discussion References

 
Increased IL-2 Production from CD4⁺ and CD8⁺ Human Peripheral Blood T Cells. To examine the effect of IMiDs on IL-2 production by T cell subsets, human peripheral blood T cells were separated into CD4⁺ and CD8⁺ populations by negative selection using antibody-coated magnetic beads to avoid preactivating the T cells. Cells were plated onto anti-CD3 (OKT3) antibody-coated 96-well plates and immediately treated with CC-4047 (6.4 nM–10 µM). Immobilized TCR ligands such OKT3 have been shown to provide a full T cell stimulus sufficient for IL-2 production and proliferation, even in the absence of costimulatory signals (Geppert and Lipsky, 1987; Luxembourg et al., 1998). Supernatants were harvested after 1, 2, or 3 days of stimulation and the IL-2 levels measured by ELISA. On day 1, a marginal increase in IL-2 production by both CD4⁺ and CD8⁺ was observed, but much larger increases were observed on days 2 and 3 (Fig. 1, A and B). In general, the CD4⁺ population produced more IL-2 than did the CD8⁺ population. The potencies of three IMiDs were therefore compared in 3-day stimulations of both the CD4⁺ and CD8⁺ subsets. All three compounds were slightly more potent in the CD4⁺ subset, with maximal IL-2 production achieved at slightly lower compound concentrations in the CD4⁺ cells than in the CD8⁺ cells. In both CD4⁺ and CD8⁺ cells, CC-4047 was the most potent IL-2-elevator, followed by CC-6032 and CC-5013. The IMiDs were found to increase IL-2 mRNA levels in human T cells by Northern blot analysis, RNase protection assay, and cDNA array hybridization (data not shown).

View larger version (31K): [in this window] [in a new window]   Fig. 1. IMiDs enhance IL-2 production from both CD4+ and CD8+ T cells in a time- and dose-dependent manner. A, CD4+ and B, CD8+ T cells were purified from human peripheral blood by negative selection and stimulated with immobilized anti-CD3 (OKT3) antibody for either 1, 2, or 3 days in the presence of titrated amounts of CC-4047. Compound was added only once at the beginning of the culture period. Separate groups of wells were used for each of the 3 days. DMSO concentrations were held constant at 0.1% for all samples. IL-2 levels were analyzed by ELISA. Values are the mean ± S.D. of duplicate samples from one experiment, representative of two experiments showing similar results. C, CD4+ T cells and D, CD8+ T cells were stimulated for 3 days in the presence of titrated amounts of CC-4047, CC-5013, or CC-6032. Values are the mean ± S.D. of samples from four experiments using blood from different donors. Maximal IL-2 was defined as the amount of IL-2 produced in the presence of 10 µM CC-4047, which ranged from 57 to 4400 pg/ml for CD4+ T cells and from 150 to 520 pg/ml for CD8+ T cells.

Increased Cytokine Production in Th1- and Th2-Type Human T Cells. Having observed elevation of IL-2 in freshly isolated T cells, the effect of IMiDs on differentiated Th1- and Th2-type T cells was next examined. CD4⁺ Th1 and Th2 cells were generated and stimulated with immobilized OKT3 for 3 days in the presence of titrated amounts of CC-4047 or CC-5013. Under these conditions, the Th1-polarized population produced large amounts of IL-2 and IFN- (10 –20 ng/ml), but little IL-5 and IL-10 (100 –200 pg/ml). The Th2-polarized population produced no IL-2, smaller amounts of IFN- (<4 ng/ml) and relatively large amounts of IL-5 and IL-10 (500 –1500 pg/ml). This cytokine expression profile is consistent with that of human Th1 and Th2 cells in previously published reports (Kelso et al., 1991; Palmer and van Seventer, 1997; Schafer et al., 1999a). IL-4 production was not detectable in either cell population. The IMiDs enhanced IL-2 and IFN- production from Th1 cells and also enhanced IL-5 and IL-10 production from Th2 cells (Fig. 2). CC-4047 was significantly more potent than CC-5013 at elevating IL-2, IL-5, and IL-10, and slightly more potent than CC-5013 at elevating IFN-.

View larger version (27K): [in this window] [in a new window]   Fig. 2. IMiDs increase cytokine production in polarized Th1-type and Th2-type human CD4+ T cells. Total human PBMC were stimulated for 17 days with PHA-L and rhIL-2, plus either rhIL-12 and anti-IL-4R antibody for Th1 conditions (A and B), or with rhIL-4 and anti-IL-12 antibody for Th2 conditions (C and D). CD4+ T cells were then purified by negative selection and stimulated with immobilized anti-CD3 antibody for 3 days in the presence of titrated amounts of CC-4047 or CC-5013. DMSO concentrations were held constant at 0.1% for all samples. IL-2 (A), IFN- (B), IL-5 (C), and IL-10 (D) levels were analyzed by ELISA. Values are the mean ± S.D. of duplicate samples from one experiment, representative of four experiments showing similar results. Under these conditions, Th1 cells produced <100 pg/ml IL-5 and <200 pg/ml IL-10, and Th2-polarized cells produced no detectable IL-2 and <4000 pg/ml IFN-.

Increased IL-2 Production in Jurkat T Cells. To better understand the mechanism of action of IMiDs in T cells, the effects of CC-4047 on IL-2 production by the human Jurkat leukemic T cell line was examined. Jurkat cells were stimulated with either the superantigen SEE, with the Raji B cell lymphoma, or with the combination of SEE plus Raji cells in the presence of titrated amounts of CC-4047. SEE is known to bind the TCR V8.1 expressed on Jurkat T cells and the 1 domain of major histocompatibility complex class II molecules on antigen-presenting cells, thus providing a TCR stimulus to the Jurkat cells (Hudson et al., 1993). Raji B cells provide a source of the costimulatory molecules B7-1 and B7-2, which bind to CD28 on the Jurkat cells (Yan et al., 1998). The combination of both SEE and Raji cells was required to elicit an IL-2 response, which was enhanced by CC-4047 (Fig. 3A). Significantly, CC-4047 was not able to substitute for either SEE or Raji B cells, because no IL-2 was made in the absence of either signal, regardless of the amount of CC-4047 present. A similar result was obtained using the combination of anti-CD3 and anti-CD28 antibodies (Fig. 3B), or the combination of the phorbol ester PMA and the calcium ionophore ionomycin (Fig. 3C). Therefore, CC-4047 did not act as a stimulatory or costimulatory signal by itself, but rather enhanced the T cell response to the combination of stimulus and costimulus. The enhancing effect of CC-4047 on IL-2 production stimulated by PMA and ionomycin (2-fold at 10 µM CC-4047) was much lower than the enhancing effect observed when either SEE plus Raji or anti-CD3 plus anti-CD28 antibodies were used (2-fold at approximately 0.1 µM CC-4047), revealing a 100-fold decrease in sensitivity to CC-4047 when PMA and ionomycin were used. This would suggest that CC-4047 more potently augments stimuli propagated from the T cell surface than those supplied directly to PKC and calcineurin. Therefore, the cell surface-directed stimuli were used for subsequent experiments. Using SEE plus Raji B cells, Jurkat IL-2 production was best enhanced by CC-4047, followed by CC-6032 and CC-5013 (Fig. 3D).

View larger version (29K): [in this window] [in a new window]   Fig. 3. IMiDs enhance IL-2 production in Jurkat T cells. Jurkat cells (2 x 105/well) were pretreated with titrated amounts of CC-4047 at constant 0.1% DMSO for 1 h, and then stimulated with SEE (20 ng/ml) and/or Raji B cells (4 x 104/well) for 2 days (A); anti-CD3 (1 µg/ml) and/or anti-CD28 (10 µg/ml) antibody plus cross-linking F(ab')2 fragments of goat anti-mouse IgG (30 µg/ml) for 2 days (B); PMA (1 ng/ml) and/or ionomycin (1 µg/ml) for 1 day (C). Values are the mean ± S.D. of duplicate samples from experiment, representative of two experiments showing similar results. D, Jurkat cells were pretreated for 1 h with titrated amounts of CC-4047, CC-5013, or CC-6032 for 1 h then stimulated for 2 days with Raji B cells and SEE as described above. Values are the mean ± S.D. from three independent experiments, with maximal IL-2 levels ranging from 1300 to 8900 pg/ml.

Increased IL-2 Promoter Activity. To determine the earliest time at which an IMiD-induced increase in IL-2 production could be observed, a time course analysis was performed. Jurkat T cells were stimulated with Raji B cells plus SEE in the presence or absence of CC-4047 (10 µM), and supernatants were harvested after various lengths of time and analyzed for IL-2 protein (Fig. 4A). IL-2 was detected as early as 4 h poststimulation, and by 6 h a clear effect of CC-4047 was observed, with a 46% increase in IL-2 compared with the DMSO control. At 24 and 30 h poststimulation, CC-4047 elevated IL-2 production by 69 and 76%, respectively, compared with the DMSO controls (Fig. 4A). This time-dependent increase in the effect of CC-4047 on IL-2 production was also observed in human peripheral blood T cells (Fig. 1, A and B), suggesting a possible amplification effect by the IMiD over time. However, because 6 h was the minimum time required to obtain a reproducible increase in IL-2 by CC-4047, this time point was used in subsequent transcriptional studies.

View larger version (17K): [in this window] [in a new window]   Fig. 4. CC-4047 increases IL-2 promoter activity. A, Jurkat T cells (2 x 105/sample) were pretreated for 1 h with either DMSO (0.1%) or CC-4047 (10 µM) then stimulated with Raji B cells (4 x 104/sample) plus SEE (20 ng/ml) Supernatants were harvested 2, 4, 6, 24, or 30 h poststimulation and assayed for IL-2 protein by ELISA. Data shown are the mean ± S.D. from duplicate samples. Similar results were obtained in three independent experiments. B, Jurkat T cells (2 x 106/sample) were transfected with the human IL-2 promoter luciferase expression construct (pIL-2-Luc) or the control luciferase vector (pGL2), pretreated with DMSO (0.1%) or CC-4047 (10 µM) for 1 h, and then stimulated with SEE (100 ng/ml) plus various amounts of Raji B cells (0, 1.25 x 105, 5 x 105, or 2 x 106/sample) for 6 h. Luciferase activity was measured as described under Materials and Methods. Data shown are the mean ± S.E.M. of three independent experiments. Statistically significant difference is indicated (***, p < 0.001). C, Jurkat T cells (2 x 106/sample) were transfected with the wild-type (WT), distal AP-1 mutant (mt dAP-1), or proximal AP-1 mutant (mt pAP-1) human IL-2 promoter luciferase expression constructs. Jurkat cells were then pretreated with DMSO (0.1%) or CC-4047 (10 µM) for 1 h and stimulated with SEE (100 ng/ml) plus Raji B cells (5 x 105/sample) for 6 h. Data shown are the mean ± S.E.M. of three independent experiments and were normalized to WT pIL-2-Luc activity in the presence of DMSO. Statistically significant differences are indicated (**, p < 0.01; ns, not significant).

To determine whether CC-4047 increased IL-2 protein levels by elevating IL-2 promoter activity, Jurkat cells were transfected with the human IL-2 promoter-luciferase expression construct pIL-2-Luc or the control luciferase expression vector pGL2, and then stimulated with Raji B cells plus SEE in the presence or absence of CC-4047 (10 µM) for 6 h. The number of Raji cells was titrated against a constant number of Jurkat cells to achieve a submaximal stimulation index. At a Raji to Jurkat ratio of 1:4, CC-4047 significantly increased IL-2 promoter activity by an average of 41% compared with the DMSO control. Transcriptional activity of the pGL2 vector control was minimal, and no significant effect of CC-4047 on pGL2 activity was observed (Fig. 4B). This 41% increase in IL-2 promoter activity by CC-4047 correlated well with the observed 46% increase in IL-2 protein levels at the same 6-h time point (Fig. 4A).

The cytokines IL-2, IFN-, IL-5, and IL-10, all up-regulated by IMiDs, have all been found to be transcriptionally controlled by the transcription factor AP-1 (Jain et al., 1992; Sweetser et al., 1998; Mori et al., 1999; Gollnick et al., 2001). On the IL-2 promoter there are two AP-1 binding sites: the distal AP-1 site (position -179 to -185) and the proximal AP-1 site (position -145 to -152) (Hughes and Pober, 1996). Mutation of the proximal AP-1 site was found to reduce IL-2 promoter activity more than any other cis-regulatory element, including NFAT, NF-B, and Oct binding sites (Hughes and Pober, 1996). Indeed, an IL-2 promoter construct with a mutated proximal AP-1 site produced very little luciferase activity compared with the wild-type IL-2 promoter or a promoter mutated at the distal AP-1 site (Fig. 4C). Mutation of the proximal AP-1 site eliminated the 42% enhancement of IL-2 promoter activity caused by CC-4047, whereas mutation of the distal AP-1 site still permitted CC-4047 to elevate IL-2 promoter activity by 30%. Therefore, the enhancing effect of CC-4047 on IL-2 promoter activity is dependent upon the proximal but not the distal AP-1 binding site.

Up-Regulation of AP-1 Transcriptional Activity. The increase in IL-2 production in Jurkat T cells seemed to correlate with an increase in IL-2 promoter activity, which was almost completely dependent on the proximal AP-1 binding site. Therefore, the effect of IMiDs on AP-1 transcriptional activity in Jurkat cells was examined. Jurkat cells were transfected with a luciferase reporter gene under the transcriptional control of a promoter containing a heptamer repeat of the AP-1 binding sequence (pAP-1-Luc). Twenty-four hours later, Jurkat cells were stimulated with SEE and Raji B cells in the presence or absence of titrated amounts of CC-4047, and the luciferase activity was assayed 6 h later. Stimulation of Jurkat cells with SEE and Raji cells caused an increase in AP-1 promoter activity that was enhanced by CC-4047 in a dose-dependent manner, with a maximal enhancement of 4-fold at 1 µM CC-4047 (Fig. 5A). Treatment of Jurkat cells with CC-4047 in the absence of the SEE and Raji stimulus did not result in any AP-1 transcriptional activity. Cotransfection of Jurkat cells with the plasmid encoding MEKK1, which activates the JNK pathway and thus serves as a positive control for maximal AP-1 activation, caused a much larger increase in AP-1 activity than the SEE plus Raji B cell stimulus, indicating that the IMiD-enhanced AP-1 promoter activity was not yet approaching maximal levels that might saturate the detection system.

View larger version (27K): [in this window] [in a new window]   Fig. 5. IMiDs enhance AP-1 transcriptional activity in Jurkat T cells. A, Jurkat cells (2 x 106/sample) were transfected in bulk with the pAP-1-Luciferase reporter construct, divided into samples, pretreated with titrated amounts of CC-4047 or DMSO (0.01% final) for 1 h, and then stimulated with Raji B cells (1–2 x 106/sample) and SEE (100 ng/ml) for 6 h. Cells were lysed and luciferase activity measured on a TopCount plate reader. Values are the mean ± S.D. of duplicate samples from one experiment, representative of three experiments. B, time course comparison of AP-1 and NF-AT transcriptional activation in the presence or absence of 10 µM CC-4047 or 0.1% DMSO. Values are the mean ± S.D. of samples from two experiments. C, AP-1 transcriptional activity in Jurkat cells stimulated with Raji B cells and SEE in the presence of either an IgG1 isotype control antibody (10 µg/ml) or an anti-IL-2 neutralizing antibody (10 µg/ml), in the presence of 250 nM CC-4047 or 0.0025% DMSO for 6 h. Values are the mean ± S.D. of duplicate samples from one experiment, representative of two experiments. D, AP-1 activity in Jurkat cells stimulated with Raji B cells and SEE in the presence of 10 µM CC-4047, CC-5013, CC-6032, or 0.1% DMSO. Values are the mean ± S.D. of samples from two experiments.

To examine the time course and specificity of the effect of CC-4047 on AP-1 activity, Jurkat cells were transfected with either the pAP-1-Luc construct or the pNFAT-Luc construct, and then stimulated with SEE and Raji B cells for 4, 6, 24, or 30 h in the presence or absence of 10 µM CC-4047. Although SEE plus Raji-induced AP-1 activity continued to increase throughout the course of the 30-h experiment, CC-4047 caused maximal enhancement in AP-1 activity at 6 to 24 h. (Fig. 5B). Meanwhile, NFAT transcriptional activity reached a maximum at 6 h and was not affected by CC-4047. Therefore, the enhancing effect of CC-4047 on AP-1 activity is selective and not the result of a general up-regulation in transcriptional activity.

In addition to measuring AP-1 transcriptional activity by luciferase reporter assay, AP-1 DNA binding activity was assessed by electromobility shift assay. Nuclear extracts from CC-4047-treated Jurkat cells, stimulated with a combination of anti-CD3 and anti-CD28 antibodies or with a combination of PMA and ionomycin, yielded increased amounts of AP-1 DNA binding activity, relative to Jurkat cells stimulated in the absence of CC-4047. This increased AP-1 DNA binding activity was observed as early as 1 h poststimulation (F. Payvandi, manuscript in preparation).

IL-2 triggers cell cycle progression and prevents apoptosis in T cells by a mechanism that involves activation of signal transducer and activator of transcription 3 and 5, not via AP-1 activation (Iacobelli et al., 1999). Nevertheless, to eliminate the possibility that the increase in AP-1 activity caused by CC-4047 might be an indirect result of higher IL-2 production, Jurkat AP-1 activity was assessed in the presence of an anti-IL-2 neutralizing antibody. CC-4047 increased AP-1 activity approximately 3-fold, even in the presence of anti-IL-2 antibody, an effect similar to that observed in the presence of an isotype control antibody (Fig. 5C).

To study the relative potencies of IMiDs in the potentiation of AP-1 activity, Jurkat cells were stimulated with SEE and Raji cells for 6 h in the presence of 10 µM CC-4047, CC-5013, or CC-6032. The most potent compound was CC-4047, which caused a 4-fold increase, followed by CC-6032 and CC-5013, which caused 3-fold and 2-fold increases in AP-1 activity, respectively (Fig. 5D). The IMiDs order of potency for AP-1 activation was equivalent to the order of potency for IL-2 elevation.

No Effect on ERK, JNK, p38 MAPK, or Jun Phosphorylation. AP-1 consists of a dimer of Fos and Jun proteins that are primarily regulated by the ERK and JNK pathways, respectively (for review, see Foletta et al., 1998). Stimulation of the TCR/CD3 complex alone results in rapid phosphorylation of ERK1 and ERK2 (p44 and p42 MAPK, respectively), which in turn phosphorylate the transcription factor Elk-1, causing increased c-Fos expression. Stimulation of the TCR/CD3 complex in conjunction with CD28 triggers phosphorylation of JNK1 and JNK2, which in turn phosphorylate c-Jun and activates the AP-1 complex. Therefore, the effect of CC-4047 on phosphorylation of ERK1/2 and JNK1/2 was investigated. Stimulation of Jurkat cells with anti-CD3 and anti-CD28 antibodies triggered phosphorylation of ERK1/2 and JNK1/2 that occurred within 5 min and gradually decreased over time, as detected by Western blot using phospho-specific ERK1/2 and JNK1/2 antibodies. However, the degree of ERK or JNK phosphorylation at each time point was not increased by the presence of 10 µM CC-4047 (Fig. 6). No ERK or JNK phosphorylation was observed after 6 h of stimulation (data not shown). In vitro kinase assays of JNK1 and ERK2 immunoprecipitated from CC-4047-treated Jurkat cells also showed that there was no effect of CC-4047 on the kinase activity of these enzymes (data not shown). In addition to serving as a costimulus for JNK activation, CD28 signaling can also activate p38 MAPK, although p38 MAPK activity is not required for IL-2 production (Schafer et al., 1999a). Phosphorylation of p38 MAPK reached maximum levels at 30 min poststimulation but was not affected by CC-4047 (Fig. 6). An analysis of Jun phosphorylation using antibodies specific for phosphorylated c-Jun and JunD showed maximal phosphorylation after 30 min of stimulation. However, CC-4047 had no significant effect on the degree of c-Jun/JunD phosphorylation at any time point (Fig. 6). No c-Jun/JunD phosphorylation was detected after 6 h of stimulation (data not shown).

View larger version (59K): [in this window] [in a new window]   Fig. 6. CC-4047 does not affect phosphorylation of ERK1/2, JNK1/2, p38 MAPK, or c-Jun/JunD. Jurkat T cells were pretreated either with DMSO (0.1%) or with CC-4047 (10 µM) for 1 h then stimulated with anti-CD3 and anti-CD28 antibodies for the indicated period of time. Cells were rapidly lysed and homogenized, and then electrophoresed and subjected to Western blotting using anti-phospho-ERK1/2 (Thr202/Tyr204), phospho-JNK1/2 (Thr183/Tyr185), phospho-p38 MAPK (Thr180/Tyr182), or phospho-c-Jun/JunD (Ser73) antibody. Results are representative of two experiments.

No Effect on Calcium Signaling. A relatively early event during T cell stimulation is the Ca²⁺ flux that results from TCR ligation and inositol triphosphate (IP₃) production (Corado et al., 1990). Increased Ca²⁺ signaling has been implicated in the mechanism of action of the T cell costimulatory compound tucaresol (Hall and Rhodes, 2001). Therefore, the effect of CC-4047 on Ca²⁺ signaling in Jurkat cells was examined. Jurkat cells were pretreated with 10 µM CC-4047 and stimulated with various concentrations of anti-CD3 antibody. Free cytoplasmic Ca²⁺ was measured via fluorescence of the calcium-sensitive probe fluo-3. Anti-CD3 antibody induced a rise in free cytosolic Ca²⁺ with two components: the initial rise of free Ca²⁺ due to inositol triphosphate-induced Ca²⁺ release from internal stores, and the sustained increase in free Ca²⁺ is due Ca²⁺ influx via store-operated calcium channels in the plasma membrane. The initial rise in free cytoplasmic calcium levels rose within 60 s after stimulation and slowly declined thereafter (Fig. 7). The initial Ca²⁺ rise and sustained Ca²⁺ components can be separated by carrying out the experiment illustrated in Fig. 5 in Ca²⁺-free medium. In that case, anti-CD3 antibody resulted in a transient rise in free cytosolic Ca²⁺; subsequent addition of calcium to the medium resulted in a large calcium influx via store-operated channels (data not shown). Regardless of anti-CD3 antibody concentration, 10 µM CC-4047 had no significant effect on the initial Ca²⁺ rise, or on the sustained Ca²⁺ level (Fig. 7). Minor differences in the Ca²⁺ measurement between the control and CC-4047-treated samples are within experimental error for this method of detection. These data are consistent with the observed lack of an effect of CC-4047 on NFAT transcriptional activity (Fig. 5), which is regulated by the Ca²⁺-sensitive phosphatase calcineurin (Clipstone and Crabtree, 1992).

View larger version (21K): [in this window] [in a new window]   Fig. 7. Effect of CC-4047 on calcium flux in response to T cell stimulation. Jurkat cells loaded with fluo-3 were placed in a thermostatted cuvette containing 2 ml 150 mM NaCl, 1.5 mM CaCl2, 3 mM KCl, 10 mM glucose, 20 mM HEPES buffer (pH 7.4), and 250 µM sulfinpyrazone. Cells were stimulated at time 0 with the indicated concentrations of anti-CD3 antibody (OKT3) and changes in free cytosolic Ca2+ were monitored as described under Materials and Methods. Temperature: 25°C. Similar results were obtained in another six independent experiments.

Effect of Calcineurin, CD45, MEK1, PKC, PKA, JAK2, and p38 MAPK Inhibitors. Pharmacological inhibitors have been used to define the roles of many kinases and phosphatases in the signal transduction processes contributing to IL-2 production in T cells. To help define the mechanism of action of IMiDs, the ability of various pharmacological inhibitors to block the CC-4047-mediated enhancement in IL-2 was assessed. Jurkat cells were pretreated with 10 µM CC-4047, and then stimulated with SEE and Raji B cells in the presence of agents known to modulate IL-2 production: the calcineurin phosphatase inhibitor cyclosporin A (Liu et al., 1991); a cell-permeable analog of the CD45 phosphatase inhibitor RWJ60475 (Beers et al., 1997); the MEK1 inhibitor PD98059 (Dumont et al., 1998); the PKC/ inhibitor Rottlerin (Khoshnan et al., 2000; Solomou et al., 2001); the PKA inhibitor H-89 (Chijiwa et al., 1990); the JAK2 inhibitor AG-490 (Saemann et al., 2000), and; the p38 MAPK inhibitor SB203580 (Schafer et al., 1999a). Although treatment of Jurkat cells with 10 µM CC-4047 caused more than a 2.5-fold increase in IL-2 production compared with control cells, the various inhibitors had equivalent potency in both the CC-4047-treated and control cells. Cyclosporin A, RWJ60475(AM)₃, PD98059, Rottlerin, H-89, and AG-490 all inhibited IL-2 production with essentially the same IC₅₀ value whether CC-4047 was present or not (Table 1). The p38 MAPK inhibitor SB203580, which has been shown to elevate IL-2 production in Th1 cells by approximately 50% (Schafer et al., 1999a), was found here to also elevate IL-2 production in Jurkat cells with approximately equivalent EC₁₅₀ in either the presence or absence of CC-4047 (Table 1). CC-4047, therefore, elevates IL-2 production in Jurkat cells by a mechanism that does not circumvent any of these kinases or phosphatases.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
The IMiDs constitute a novel class of immunomodulatory drugs displaying a wide range of biological activities that are highly dependent on the particular cell type and stimulus used. In general, these compounds inhibit production of proinflammatory cytokines derived from monocytes, but are stimulatory to T cells. Herein, we have shown that IMiDs increase IL-2 production by both CD4⁺ and CD8⁺ T cells, with a slightly more potent effect on the CD4⁺ subset (Fig. 1). These results differ somewhat from those reported for thalidomide, which was found to increase IL-2 production, IFN- production, and proliferation to a greater degree in CD8⁺ cells than in CD4⁺ cells (Haslett et al., 1998). This difference may be due to differences in T cell purification procedures. In the previous study, T cells were isolated by positive selection, whereas in the present study they were isolated by negative selection to avoid potential activation caused by ligation of CD4 or CD8 molecules. Using negative T cell selection techniques, thalidomide was found to have very little IL-2-enhancing activity in either CD4⁺ or CD8⁺ cells (data not shown) and was found to enhance proliferation of CD4⁺ and CD8⁺ cells with approximately equivalent potency (Davies et al., 2001).

IMiDs have been referred to as costimulatory to T cells because they augment the classical responses of proliferation and cytokine production when administered along with a typical laboratory T cell stimulus, such as anti-CD3 antibody (Davies et al., 2001). Because it is possible to fully activate T cells using TCR ligands immobilized on a surface without the requirement for a costimulus such as anti-CD28 antibody or cytokines (Geppert and Lipsky, 1987; Luxembourg et al., 1998), in this situation IMiDs are not acting as true costimulatory signals. Using Jurkat cells, both a TCR ligand (in the form of SEE or soluble anti-CD3 antibody) and a costimulatory signal (in the form of Raji B cell surface or soluble anti-CD28 antibody) were required to stimulate IL-2 production, regardless of how much CC-4047 was present (Fig. 3, A and B). Similarly, no amount of CC-4047 was able to substitute for either the PKC activation signal provided by PMA or the calcineurin activation signal provided by ionomycin (Fig. 3C). This indicates that CC-4047 does not provide a costimulatory signal per se, but rather merely amplifies the signals provided from other sources.

The IMiDs augmented production of IL-2, IFN-, IL-5, and IL-10 by polarized Th1 and Th2-type CD4⁺ T cells (Fig. 2), indicating that their immunomodulatory activity is not restricted to primary T cell responses alone. It has previously been shown that CC-4047 decreased IL-10 production from anti-CD3 antibody-stimulated PBMCs (Corral et al., 1999). In the present study, however, IL-10 production from differentiated Th2 cells was enhanced by CC-4047 (Fig. 2D). This difference may be caused by the presence of a cytokine in the PBMC culture not produced by Th2 cells, IL-12, which is known to inhibit IL-10 production (Oswald et al., 1994). Thus, the inhibition of IL-10 production observed upon CC-4047-treatment of anti-CD3-stimulated PBMCs may have mediated by increased IL-12 production (Corral et al., 1999). Because the IMiDs augmented both Th1 and Th2 cytokine production from CD4⁺ cells that had already been polarized into those phenotypes, it is unclear from these studies whether IMiDs would direct a primary immune response toward either a Th1- or a Th2-type response in vivo. Relevant to this issue, treatment of mice with CC-4047 in the CT26 autologous colorectal tumor vaccination model conferred greater resistance of the animals to subsequent challenge with the CT26 tumor cells (Dredge et al., 2002). In this model, splenic T cells from CC-4047-treated animals produced higher amounts of the Th1 cytokines IL-2 and IFN- when challenged with CT26 cells in vitro. However, levels of Th2 cytokines from CC-4047-treated mouse splenocytes were not measured in that system. Stimulation of naïve splenocytes in vitro with anti-CD3 antibodies and CC-4047 caused an elevation of IL-2, IFN-, and granulocyte-macrophage colony-stimulating factor, and a decrease in IL-4 and IL-10 compared with CD3-stimulated splenocytes (Dredge et al., 2002). Therefore, in a naïve T cell population, it seems that CC-4047 drives a Th1 response. However, given the findings of the present study, one would predict that in an already developed Th2 response, an IMiD might further augment Th2 cytokine production. This hypothesis has not yet been tested.

CC-4047 caused an increase in IL-2 promoter activity that correlated well with the magnitude of the increased IL-2 protein production (41– 46% at 6 h poststimulation; see Fig. 4, A and B). This increase required the presence of an intact proximal AP-1 binding site, because mutation of that site resulted in a dramatic loss of promoter activity and no subsequent enhancement upon addition of CC-4047 (Fig. 4C). The proximal AP-1 site is the single most important cis- regulatory element on the IL-2 promoter (Hughes and Pober, 1996). In contrast, mutation of the distal AP-1 binding site caused a small but reproducible increase in IL-2 promoter activity, as previously reported (Hughes and Pober, 1996), and the mutated distal AP-1 site still allowed a significant increase in activity upon addition of CC-4047 (Fig. 4C). Therefore, the proximal but not the distal AP-1 site is involved in the IMiD response. IMiDs also increase IL–2 mRNA levels in T cells (F. Payvandi, manuscript in presentation).

The IMiDs were found to significantly enhance AP-1 transcriptional activity in Jurkat T cells, with relative potencies correlating with their abilities to increase IL-2 production by Jurkat cells and purified human CD4⁺ and CD8⁺ T cells. The magnitude of the increased AP-1 activity caused by CC-4047 (4-fold at 6 h poststimulation) was severalfold higher than the observed increase in IL-2 promoter activity (41%). This difference may be due to the fact that the AP-1-driven luciferase construct contains seven tandem repeats of the AP-1 binding site. An increase in AP-1 DNA-binding activity upon treatment of stimulated T cells with IMiDs was also observed using the technique of DNA electromobility shift assay, and the specific AP-1 subunits involved in the IMiD response is the subject of current investigation (F. Payvandi, manuscript in preparation).

The stimulatory effect of thalidomide on T cell proliferation was found to be dependent on the induction of endogenous IL-2 production, and not vice versa, because anti-IL-2 antibody abrogated the proliferative effect of thalidomide (Haslett et al., 1998). Using flow cytometry, CC-4047 was shown to increase intracellular IL-2 and IFN- production by splenic T cells, on a per cell basis, from mice treated with this IMiD (Dredge et al., 2002). In the current study, CC-4047 increased Jurkat IL-2 production as early as 6 h poststimulus, which coincided both in magnitude and timing with the CC-4047 increase in AP-1-dependent IL-2 promoter activity (Fig. 4). Furthermore, the CC-4047-induced increase in AP-1 transcriptional activity was not blocked by anti-IL-2 neutralizing antibody, indicating that increased IL-2 production was not the cause of the potentiated AP-1 activity (Fig. 5C). Together, these observations provide strong evidence that the increased IL-2 expression in response to IMiDs is not dependent on increased cell proliferation, but rather that increased AP-1 activity drives IL-2 production, which in turn augments T cell proliferation.

It should be noted that the effects of IMiDs on IL-2 production seem to be amplified over time. In peripheral blood CD4⁺ T cells, for example, CC-4047 increased IL-2 from 50 pg/ml up to100 pg/ml after 24 h, up to 300 pg/ml after 48 h, and up to 600 pg/ml after 72 h (Fig. 1A). Similarly, in Jurkat cells, CC-4047 increased IL-2 production by 46, 69, and 76% at 6, 24, and 30 h, respectively (Fig. 4B). Meanwhile, the effect of CC-4047 on AP-1 activity peaked at 6 to 24 h, and then declined by 30 h (Fig. 5B). This suggests that although increased AP-1 activity may be responsible for the immediate increase in IL-2 production, it is possible that there are other IMiD effects at later time points after 24 h. Possibilities include indirect effects on other transcription factors as a result of exposure to higher levels of IL-2 or other cytokines.

It has been reported that the cAMP-specific phosphodiesterase type 4 (PDE4) inhibitor rolipram has the ability to augment AP-1 activity in T cells, despite a concomitant decrease in IL-2, IFN-, IL-5, and IL-10 production and inhibition of NFAT transcriptional activity (Jimenez et al., 2001). However, the IMiDs do not inhibit PDE4 enzymatic activity in vitro (Muller et al., 1999), do not cause inhibition of the aforementioned cytokines (Fig. 2), and do not inhibit NFAT transcriptional activity (Fig. 5B). Therefore, inhibition of PDE4 function in the cell is not a likely explanation for their observed effect on AP-1 activity. It has also been reported that thalidomide can inhibit NF-B activation in TNF--stimulated Jurkat cells (Keifer et al., 2001; Majumdar et al., 2002). However, the IMiDs do not inhibit NF-B activation in response to CD3 and CD28 stimulation in Jurkat T cells (F. Payvandi, manuscript in preparation), and the enhancing effect of IMiDs on T cell cytokine production cannot be explained by a mechanism that involves inhibition of NF-B activity.

In summary, these data indicate that potentiation of AP-1 activity is at least one key step in the mechanism of action of IMiDs in T cells. Surprisingly, treatment of T cells with CC-4047 did not enhance activation of any of the other signaling events examined, including calcium signaling (Fig. 7), activation of ERK1/2, JNK1/2, p38 MAPK, or c-Jun/Jun D (Fig. 6), or expression of Fos and Jun proteins (data not shown). The lack of an effect on calcium flux or on ERK2 activation differentiates IMiDs from the T cell stimulatory agent tucaresol, which forms Schiff bases on T cell surface amines. Tucaresol has been shown to trigger ERK2 phosphorylation in the absence of other T cell signals (Chen et al., 1997) and to prime T cells for enhanced Ca²⁺ signaling upon CD3 stimulation (Hall and Rhodes, 2001). Given the lack of any effect of IMiDs on the aforementioned signal transduction steps preceding AP-1 activation, IMiDs may have the inherent ability to stabilize the AP-1/DNA complex, or there may an AP-1 transcriptional coactivator that is up-regulated by IMiD treatment. Whatever the underlying mechanism of action may be, it is clear that a variety of T cell responses are up-regulated by the IMiDs. This may provide an unprecedented clinical opportunity to augment T cell responses against a wide variety of agents including viruses, bacteria, parasites, and tumors.

	Footnotes

 
This work was supported by Celgene Corporation.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

DOI: 10.1124/jpet.102.048496.

ABBREVIATIONS: IMiD, immunomodulatory drug; TNF-, tumor necrosis factor-; LPS, lipopolysaccharide; PBMC, peripheral blood mononuclear cell; IL, interleukin; MM, multiple myeloma; NK, natural killer; SEE, staphylococcal enterotoxin E; PMA, phorbol 12-myristate 13-acetate; NFAT, nuclear factor of activated T cells; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; JAK2, Janus kinase 2; DMSO, dimethyl sulfoxide; Th, T helper; PKC, protein kinase C; PKA, protein kinase A; rhIL, recombinant human interleukin; ELISA, enzyme-linked immunosorbent assay; MEK1, mitogen-activated protein kinase/ERK kinase 1; MEKK1, mitogen-activated protein kinase kinase kinase 1; TBST, Tris-buffered saline-Tween 20; PDE4, phosphodiesterase type 4; PD98059, 2'-amino-3'-methoxyflavone; SB203580, 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole; H-89, N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide HC1; AG-490, N-benzyl-3,4-dihydroxybenzylidenecyanoacetamide; RWJ-60475 (AM₃), 2-(4-bromophenoxy)-5-nitrophenylhydroxymethylphosphonic acid Tris acetoxymethyl ester.

Address correspondence to: Dr. Peter H. Schafer, Celgene Corporation, 7 Powder Horn Dr., Warren, NJ 07059. E-mail: pschafer{at}celgene.com

	References

Top Abstract Materials and Methods Results Discussion References

 

Beers SA, Malloy EA, Wu W, Wachter MP, Gunnia U, Cavender D, Harris C, Davis J, Brosius R, Pellegrino-Gensey JL, et al. (1997) Nitroarylhydroxymethylphosphonic acids as inhibitors of CD45. Bioorg Med Chem 5: 2203-2211.[Medline]

Chen H, Hall S, Heffernan B, Thompson NT, Rogers MV, and Rhodes J (1997) Convergence of Schiff base costimulatory signaling and TCR signaling at the level of mitogen-activated protein kinase ERK2. J Immunol 159: 2274-2281.[Abstract/Free Full Text]

Chijiwa T, Mishima A, Hagiwara M, Sano M, Hayashi K, Inoue T, Naito K, Toshioka T, and Hidaka H (1990) Inhibition of forskolin-induced neurite outgrowth and protein phosphorylation by a newly synthesized selective inhibitor of cyclic AMP-dependent protein kinase, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89), of PC12D pheochromocytoma cells. J Biol Chem 265: 5267-5272.[Abstract/Free Full Text]

Clipstone NA and Crabtree GR (1992) Identification of calcineurin as a key signalling enzyme in T-lymphocyte activation. Nature (Lond) 357: 695-697.[CrossRef][Medline]

Corado J, Le Deist F, Griscelli C, and Fischer A (1990) Inositol 1, 4, 5-trisphosphate- and arachidonic acid-induced calcium mobilization in T and B lymphocytes. Cell Immunol 126: 245-254.[Medline]

Corral LG, Haslett PA, Muller GW, Chen R, Wong LM, Ocampo CJ, Patterson RT, Stirling DI, and Kaplan G (1999) Differential cytokine modulation and T cell activation by two distinct classes of thalidomide analogues that are potent inhibitors of TNF-. J Immunol 163: 380-386.[Abstract/Free Full Text]

Davies FE, Raje N, Hideshima T, Lentzsch S, Young G, Tai YT, Lin B, Podar K, Gupta D, Chauhan D, et al. (2001) Thalidomide and immunomodulatory derivatives augment natural killer cell cytotoxicity in multiple myeloma. Blood 98: 210-216.[Abstract/Free Full Text]

Dredge K, Marriott JB, Todryk SM, Muller GW, Chen R, Stirling DI, and Dalgleish AG (2002) Protective antitumor immunity induced by a costimulatory thalidomide analog in conjunction with whole tumor cell vaccination is mediated by increased Th1-type immunity. J Immunol 168: 4914-4919.[Abstract/Free Full Text]

Dumont FJ, Staruch MJ, Fischer P, DaSilva C, and Camacho R (1998) Inhibition of T cell activation by pharmacologic disruption of the MEK1/ERK MAP kinase or calcineurin signaling pathways results in differential modulation of cytokine production. J Immunol 160: 2579-2589.[Abstract/Free Full Text]

Foletta VC, Segal DH, and Cohen DR (1998) Transcriptional regulation in the immune system: all roads lead to AP-1. J Leukoc Biol 63: 139-152.[Abstract]

Geppert TD and Lipsky PE (1987) Accessory cell independent proliferation of human T4 cells stimulated by immobilized monoclonal antibodies to CD3. J Immunol 138: 1660-1666.[Abstract]

Gollnick SO, Lee BY, Vaughan L, Owczarczak B, and Henderson BW (2001) Activation of the IL-10 gene promoter following photodynamic therapy of murine keratinocytes. Photochem Photobiol 73: 170-177.[CrossRef][Medline]

Hall SR and Rhodes J (2001) Schiff base-mediated co-stimulation primes the T cell-receptor-dependent calcium signalling pathway in CD4 T cells. Immunology 104: 50-57.[Medline]

Haslett PA, Corral LG, Albert M, and Kaplan G (1998) Thalidomide costimulates primary human T lymphocytes, preferentially inducing proliferation, cytokine production and cytotoxic responses in the CD8+ subset. J Exp Med 187: 1885-1892.[Abstract/Free Full Text]

Hideshima T, Chauhan D, Shima Y, Raje N, Davies FE, Tai YT, Treon SP, Lin B, Schlossman RL, Richardson P, et al. (2000) Thalidomide and its analogs overcome drug resistance of human multiple myeloma cells to conventional therapy. Blood 96: 2943-2950.[Abstract/Free Full Text]

Hudson KR, Robinson H, and Fraser JD (1993) Two adjacent residues in staphylococcal enterotoxins A and E determine T cell receptor V specificity. J Exp Med 177: 175-184.[Abstract/Free Full Text]

Hughes CC and Pober JS (1996) Transcriptional regulation of the interleukin-2 gene in normal human peripheral blood T cells. Convergence of costimulatory signals and differences from transformed T cells. J Biol Chem 271: 5369-5377.[Abstract/Free Full Text]

Iacobelli M, Rohwer F, Shanahan P, Quiroz JA, and McGuire KL (1999) IL-2-mediated cell cycle progression and inhibition of apoptosis does not require NF-B or activating protein-1 activation in primary human T cells. J Immunol 162: 3308-3315.[Abstract/Free Full Text]

Jain J, Valge-Archer VE, and Rao A (1992) Analysis of the AP-1 sites in the IL-2 promoter. J Immunol 148: 1240-1250.[Abstract]

Jimenez JL, Punzon C, Navarro J, Munoz-Fernandez MA, and Fresno M (2001) Phosphodiesterase 4 inhibitors prevent cytokine secretion by t lymphocytes by inhibiting nuclear factor-B and nuclear factor of activated T cells activation. J Pharmacol Exp Ther 299: 753-759.[Abstract/Free Full Text]

Keifer JA, Guttridge DC, Ashburner BP, and Baldwin AS Jr (2001) Inhibition of NF-B activity by thalidomide through suppression of IB kinase activity. J Biol Chem 276: 22382-22387.[Abstract/Free Full Text]

Kelso A, Troutt AB, Maraskovsky E, Gough NM, Morris L, Pech MH, and Thomson JA (1991) Heterogeneity in lymphokine profiles of CD4+ and CD8+ T cells and clones activated in vivo and in vitro. Immunol Rev 123: 85-114.[Medline]

Khoshnan A, Bae D, Tindell CA, and Nel AE (2000) The physical association of protein kinase C theta with a lipid raft-associated inhibitor of B factor kinase (IKK) complex plays a role in the activation of the NF-B cascade by TCR and CD28. J Immunol 165: 6933-6940.[Abstract/Free Full Text]

Liu J, Farmer JD Jr, Lane WS, Friedman J, Weissman I, and Schreiber SL (1991) Calcineurin is a common target of cyclophilin-cyclosporin A and FKBP-FK506 complexes. Cell 66: 807-815.[CrossRef][Medline]

Luxembourg AT, Brunmark A, Kong Y, Jackson MR, Peterson PA, Sprent J, and Cai Z (1998) Requirements for stimulating naive CD8+ T cells via signal 1 alone. J Immunol 161: 5226-5235.[Abstract/Free Full Text]

Majumdar S, Lamothe B, and Aggarwal BB (2002) Thalidomide suppresses NF-B activation induced by TNF and H₂O₂, but not that activated by ceramide, lipopolysaccharides, or phorbol ester. J Immunol 168: 2644-2651.[Abstract/Free Full Text]

Marriott JB, Clarke IA, Dredge K, Muller G, Stirling D, and Dalgleish AG (2002) Thalidomide and its analogues have distinct and opposing effects on TNF- and TNFR2 during co-stimulation of both CD4+ and CD8+ T cells. Clin Exp Immunol 130: 75-84.[CrossRef][Medline]

Marriott JB, Muller G, Stirling D, and Dalgleish AG (2001) Immunotherapeutic and antitumour potential of thalidomide analogues. Expert Opin Biol Ther 1: 675-682.[CrossRef][Medline]

Mori A, Kaminuma O, Mikami T, Inoue S, Okumura Y, Akiyama K, and Okudaira H (1999) Transcriptional control of the IL-5 gene by human helper T cells: IL-5 synthesis is regulated independently from IL-2 or IL-4 synthesis. J Allergy Clin Immunol 103: S429-S436.[CrossRef][Medline]

Muller GW, Chen R, Huang SY, Corral LG, Wong LM, Patterson RT, Chen Y, Kaplan G, and Stirling DI (1999) Amino-substituted thalidomide analogs: potent inhibitors of TNF- production. Bioorg Med Chem Lett 9: 1625-1630.[CrossRef][Medline]

Oswald IP, Caspar P, Jankovic D, Wynn TA, Pearce EJ, and Sher A (1994) IL-12 inhibits Th2 cytokine responses induced by eggs of Schistosoma mansoni. J Immunol 153: 1707-1713.[Abstract]

Palmer EM and van Seventer GA (1997) Human T helper cell differentiation is regulated by the combined action of cytokines and accessory cell-dependent costimulatory signals. J Immunol 158: 2654-2662.[Abstract]

Richardson PG, Schlossman RL, Weller E, Hideshima T, Mitsiades C, Davies F, LeBlanc R, Catley LP, Doss D, Kelly K, et al. (2002) Immunomodulatory drug CC-5013 overcomes drug resistance and is well tolerated in patients with relapsed multiple myeloma. Blood 100: 3063-3067.[Abstract/Free Full Text]

Saemann MD, Bohmig GA, Osterreicher CH, Staffler G, Diakos C, Krieger PM, Horl WH, Stockinger H, and Zlabinger GJ (2000) Suppression of primary T cell responses and induction of alloantigen-specific hyporesponsiveness in vitro by the Janus kinase inhibitor tyrphostin AG490. Transplantation 70: 1215-1225.[CrossRef][Medline]

Schafer PH, Wadsworth SA, Wang L, and Siekierka JJ (1999a) p38 mitogen-activated protein kinase is activated by CD28-mediated signaling and is required for IL-4 production by human CD4+CD45RO+ T cells and Th2 effector cells. J Immunol 162: 7110-7119.[Abstract/Free Full Text]

Schafer PH, Wang L, Wadsworth SA, Davis JE, and Siekierka JJ (1999b) T cell activation signals up-regulate p38 mitogen-activated protein kinase activity and induce TNF- production in a manner distinct from LPS activation of monocytes. J Immunol 162: 659-668.[Abstract/Free Full Text]

Solomou EE, Juang YT, and Tsokos GC (2001) Protein kinase C- participates in the activation of cyclic AMP-responsive element-bind