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The Journal of Immunology, 2008, 180, 1490-1498
Copyright © 2008 by The American Association of Immunologists, Inc.
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TGF-β Inhibits IL-2 Production and Promotes Cell Cycle Arrest in TCR-Activated Effector/Memory T Cells in the Presence of Sustained TCR Signal Transduction1

Lopamudra Das* and Alan D. Levine2,*,{dagger}

* Department of Medicine and {dagger} Departments of Pathology, Pharmacology, and the Case Comprehensive Cancer Center, School of Medicine, Case Western Reserve University, Cleveland, OH 44106


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
TGF-β signaling is critical for controlling naive T cell homeostasis and differentiation; however, the biological and biochemical changes induced by TGF-β in effector/memory T cells are poorly defined. We show that although TGF-β inhibits effector/memory peripheral blood T lymphoblast proliferation and IL-2 production, the intensity and kinetics for TCR-induced global tyrosine phosphorylation are markedly increased compared with that in untreated cells or naive T cells. After TCR ligation, tyrosine phosphorylation of proximal tyrosine kinases and docking proteins like linker for activation of T cells is maintained for >30 min in TGF-β-primed cells compared with untreated cells where phosphorylation of these targets returned to basal levels by 10 min. Extended phosphorylation of linker for activation of T cells in treated peripheral blood T selectively prolongs ERK 1/2 signaling and phospholipase C-{gamma}1 activation leading to increased Ca2+ flux. A kinase/phosphatase imbalance could not account for extended phosphorylation as CD45R, SHP-1, and SHP-2 expression remains unaltered. The contradiction between prolonged signal transduction and inhibition of proliferation is partially explained by the observation that TGF-β priming results in ERK 1/2-independent p21 induction and decreased cyclin D1 expression leading to accumulation of T cells in G0/G1 phases of the cell cycle and cell cycle arrest. Despite inhibition of T cell function by TGF-β priming, TCR and cytokine signaling pathways are intact and selectively extended, suggesting that suppression in the effector/memory T cell is mediated by reprogramming signal transduction, rather than its inhibition as in the naive T cell.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Adaptive immunity is regulated by a careful balance between the protective naive, effector, and memory T cell population and regulatory T cells (Treg)3 that function to limit an ongoing response and prevent autoimmunity. Among the various populations of Treg, expression of the immunosuppressive cytokine TGF-β1 is one mechanism to inhibit an immune response (1). TGF-β regulates diverse immune functions ranging from tumor rejection to immune tolerance and suppression of autoimmune disorders (2). For example, experimental allergic encephalomyelitis is an autoimmune disease model characterized by inflammation and demyelination in the CNS (3). TGF-β treatment markedly inhibits the activation and proliferation of myelin basic protein-specific lymph node cells, resulting in a significant reduction in CNS damage and expression of such cell surface activation markers as lymphocyte function-associated Ag-1 and class II MHC molecules (4). These and similar findings have identified TGF-β as a possible molecular target in the human demyelinating disease multiple sclerosis (5, 6). Other reports demonstrate that systemic administration of TGF-β profoundly antagonizes the development of polyarthritis in susceptible rats by reducing inflammatory cell infiltration and joint erosion (7). Moreover, metallothionein suppresses collagen-induced arthritis via induction of TGF-β and down-regulation of proinflammatory mediators (8). Although these observations provide insight into the systemic, multidimensional immunoregulatory effects of TGF-β, little is known about the mechanisms by which an individual TGF-β-suppressed leukocyte fails to initiate or continue its assigned immune function.

Of particular interest within the discipline of Treg is their ability to suppress, or in some models anergize, a naive vs effector/memory T cell response. TGF-β is involved in both the development and the effector function of murine CD4+ Tregs (1). For instance, adoptive transfer of CD4+CD25+ T cells into Helicobacter hepaticus-infected T cell-reconstituted RAG2–/– mice inhibits both adaptive T cell-mediated and innate immune responses via a TGF-β-dependent mechanism (9). The primary effect of TGF-β on naive T cells is to inhibit T cell proliferation and IL-2 production (10), yet it was also reported that TGF-β interferes with T cell cytokine polarization under conditions in which T cell proliferation is normal (11). In addition, in naive rat T cells TGF-β inhibited the activation of a variety of tyrosine kinases that are induced after TCR engagement (12). Although these reports implicate TGF-β in the suppressor function against naive T cells, far less is understood about TGF-β regulation of effector/memory T cells.

TGF-β is believed to target multiple signaling pathways to inhibit proliferation directly, by blocking the transcription of cell cycle genes such as cyclins and stimulating the expression of cyclin-dependent kinase inhibitors (CDKIs) in organotypic slice cultures from rat brain (13), as well as indirectly by modulating the signal transduction pathways initiated from the TCR (12, 14, 15). Investigations into the molecular mechanisms of TGF-β interference in naive TCR-mediated signal transduction have shown that upon TCR ligation, TGF-β interferes with Tec kinase phosphorylation and Ca2+ mobilization (16). When T cells are stimulated through the TCR, a well-characterized sequence of biochemical events is initiated in which T cell-specific protein tyrosine phosphorylation marks a predominant feature (17, 18, 19, 20). Activation of protein tyrosine kinases (PTKs) following TCR engagement results in the tyrosine phosphorylation of the critical docking protein linker for activation of T cells (LAT), which mediates the recruitment of enzymes like phospholipase C (PLC)-{gamma}1 and adaptors such as growth factor receptor binding protein 2 (Grb-2) and Src homology 2 (SH2)-domain-containing leukocyte protein of 76 kDa. These proximal activation events initiate parallel second messenger pathways, including calcium mobilization (through PLC-{gamma}1) and the Ras/MAPK pathway (via Grb-2) (21), which lead to the transcriptional activation of multiple genes resulting in T lymphocyte proliferation, differentiation, and effector function.

The absence of existing information on the molecular events initiated by TGF-β to block effector/memory T cell responses to Ag led us to investigate the modulation of signal transduction emanating from the TCR after priming this T cell subset with this immunosuppressive cytokine. As is reported for naive T cells, we establish that TGF-β also inhibits proliferation, blocks IL-2 secretion, and induces CDKI expression resulting in G0/G1 cell cycle arrest in effector/memory T cells activated by TCR/CD3 cross-linking. Contrary to expectation, however, priming with TGF-β extends the duration of TCR-proximal protein tyrosine phosphorylation and selected second messengers that include the PLC-{gamma}1/Ca2+ and ERK/MAPK pathway. Extended signaling is not a consequence of reduced dephosphorylation, as expression levels of the three major phosphatases CD45R, SHP-1, and SHP-2 remain unchanged in TGF-β-treated cells. Preincubation of TGF-β-treated cells with the MEK 1/2 inhibitor U0126 did not prevent the induction of CDKI expression, eliminating a possible role for sustained ERK 1/2 activity in the induction of p21, as reported for other cell types. Although TGF-β suppresses TCR-induced proliferation of effector T cells by inhibiting IL-2 production, exogenously added IL-2-induced proliferation is not suppressed by TGF-β. Despite the block in T cell responses caused by TGF-β priming of human effector PBT, the TCR- and IL-2-signaling pathways are intact and selectively extended, suggesting that the suppressed T cell modulates, rather than inhibits, its TCR- and IL-2R-dependent signaling pathways.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Cells and reagents

PBMCs were purified from venous blood of healthy donors by Ficoll-Hypaque density separation (Sigma-Aldrich). T lymphoblasts were prepared by 0.5% PHA (Invitrogen Life Technologies) stimulation for 48 h in the presence of 5 ng/ml IL-2 (R&D Systems) in RPMI 1640, 10% heat-inactivated FCS (both from BioWhittaker), and 25 mM HEPES (Invitrogen Life Technologies), and thereafter carried in 5 ng/ml IL-2 for >8 days to obtain a population of 95% pure CD3+CD45RO+ peripheral blood T (PBT). As assessed by flow cytometry, 8-day-old effector/memory PBT contained >95% CD3+ cells that stained positively for CD45RO+. The Institutional Review Board of University Hospital Case Medical Center approved this study.

The following rabbit Abs were purchased: anti-GAPDH (Trevigen), -phospho-Src-Y416 (which recognizes phospho-Lck-Y394), -phospho-Lck Y505, -phospho-Zap70-Y319, -phospho-LAT-Y191, -phospho-LAT-Y171, -phospho-MEK1, -phospho-Raf, -phospho-ERK 1/2, -phospho-STAT5, -phospho-JNK, -phospho-p38, -cyclin D1, -SHP-2, and mouse anti-p21 (all obtained from Cell Signaling Technology). Mouse anti-SHP-1 was purchased from BD Transduction Laboratories. Anti-CD28 Ab was purchased from Ancell and anti-IL-2R{alpha} Ab was obtained from Labvision. HRP-conjugated monoclonal anti-phosphotyrosine (PY20; BD Transduction Laboratories) and HRP-conjugated secondary Abs (Santa Cruz Biotechnology) were used for detection. Cytokines TGF-β, IL-7, and IL-15 were purchased from R&D Systems and GM-CSF from PeproTech. The pharmacological MEK 1/2 inhibitor U0126 was obtained from Cell Signaling Technology.

T cell stimulation

Eight-day-old IL-2-expanded peripheral T cells were removed from IL-2 and incubated with or without 3 ng/ml TGF-β for 72 h. Thereafter untreated and TGF-β-treated PBT were washed two times with RPMI 1640 containing 25 mM HEPES, resuspended in 100 µl of the same medium at a concentration of 5 x 107 cells/ml, and incubated at 37°C for 5 min. Cells were stimulated via the TCR/CD3 complex with cross-linking sheep anti-mouse F(ab')2 (10 µg/ml; Sigma-Aldrich) and CD3 Ab (10 µg/ml OKT3; Ortho Diagnostic Systems) or with the cytokines IL-2, IL-7, IL-15, and the growth factor GM-CSF (5 ng/ml) at 37°C for the times indicated, followed by addition of 100 µl 2x Laemmli sample buffer to stop the reaction. Samples are boiled for 10 min. Unstimulated cells were incubated with only sheep anti-mouse F(ab')2 to serve as control for TCR cross-linking.

Immunoblotting

Proteins were separated by SDS-PAGE on a 10% gel under reducing conditions and electrotransferred to nitrocellulose membranes (Invitrogen Life Technologies) in a buffer consisting of 20 mM Tris-HCl, 150 mM glycine, and 20% methanol. After transfer membranes were incubated at room temperature for 1 h in blocking buffer (5% nonfat milk, 0.1% Tween 20 in PBS), primary and secondary Abs were diluted in blocking buffer as recommended by the manufacturer and incubated with the membranes for 1 h at room temperature with six washes (0.1% Tween 20 in PBS) in between. Phosphotyrosine analysis was performed as follows. Membranes were blocked 1 h at room temperature in an alternate blocking buffer (3% BSA, 0.1% Tween 20 in PBS). HRP-conjugated PY20 Ab was diluted 1/1000 in the alternate blocking buffer and incubated with the membranes for 1 h at room temperature. Detection of HRP-conjugated Abs was performed using West Pico Supersignal (Pierce). Chemiluminescence for all membranes was detected using BioMax MR Film (Kodak). Band intensity was determined by Bio-Rad Gel Doc XR and quantified using Bio-Rad Quantity One 1D analysis software.

Fura-2 cytosolic Ca2+ measurements

A total of 3 ng/ml TGF-β was added to an 8-day-old culture of PBT lymphoblasts and incubated for 72 h as described earlier. After washing two times with a basal salt solution containing 125 mM NaCl, 5 mM KC1, 1 mM MgCl2, 1.5 mM CaCl2, 25 mM HEPES (pH 7.4), 5 mM glucose, and 2 mg/ml BSA (ICN Immunobiologicals), cytosolic Ca2+ was measured using the fluorescent dye fura-2 following previous methods (22). Washed cell suspensions containing 2 x 106 cells/ml were incubated with 1 µM acetomethoxy ester of fura-2 (Molecular Probes) for 45 min at 37°C. The cells were then harvested by centrifugation, washed, and allowed to equilibrate for 2 min before stimulation with cross-linker and OKT3 at 10 µg/ml each. Ca2+ measurements were made at 37°C with continual stirring in a custom-designed spectrofluorometer (Johnson Foundation, University of Pennsylvania, Philadelphia, PA).

Proliferation assay

Day 8 PBT lymphoblasts were washed from IL-2 and cultured in a T75 flask in the absence or presence of 3 ng/ml TGF-β for 24 h. Treated and control cells were activated for 48 h in the presence or absence of TGF-β, as appropriate, at 2 x 105 cells/well with either 25 ng/ml IL-2 or in 1 µg/ml anti-CD3 coated, 96-well U-bottom tissue-culture plates (Falcon). A total of 1 µg/ml soluble anti-CD28 was added to anti-CD3-treated cells at culture initiation. To measure proliferation, [3H]thymidine (0.5 µCi/well; New England Nuclear) was added for the final 6 h of incubation. The cells were harvested onto filter mats with a Tomtec cell harvester (Wallac) and [3H] incorporation into DNA was determined using a scintillation counter (Wallac). Proliferation was calculated using mean values from triplicate cultures.

Cytokine analysis

Conditioned medium was harvested at 48 h from PBT stimulated as described above. A sandwich ELISA (BD Pharmingen) was used to detect levels of IFN-{gamma} and IL-2 secreted in the presence of a 1 µg/ml blocking Ab to the IL-2R. Cytokine detection and analyses were performed with a multiwell plate reader (Molecular Devices) and the Soft Max Pro 4.3 LS computer analysis software.

Flow cytometry

Surface markers on effector/memory PBT were detected by incubation with the appropriate mAbs, followed by fixation in 4% paraformaldehyde. To detect intracellular forkhead transcription factor (FoxP3) and CTLA4, cells were permeabilized and fixed with Perm Fix buffer using the FoxP3 staining kit from eBioscience and then stained with other appropriate mAbs. Samples were analyzed using a LSR II flow cytometer (BD Biosciences). The following mAbs were used: anti-FoxP3-FITC, anti-CD45RO-FITC, CD3-, CD4-, CD8-, and CTLA4-allophycocyanin obtained from Caltag Laboratories.

Analysis of cell cycle distribution

Propidium iodide staining of cellular DNA was performed and quantified as described previously (23). Briefly, cells were fixed in 70% ethanol and treated with 0.04 mg/ml RNase A (Sigma-Aldrich) in 20 mM Tris (pH 7.5), 250 mM sucrose, 5 mM MgCl2, and 0.37% Nonidet P-40 (Sigma-Aldrich). Cellular DNA was stained with 25 µg/ml propidium iodide (Sigma-Aldrich) in 0.05% sodium citrate and quantified by flow cytometry with the Elite ESP or Epics XL flow cytometers (Beckman Coulter) using UV and/or 488-nm excitation and band pass filters optimized for individual fluorochromes. Cell cycle analysis was performed using Winlist and Modfit software (Verity Software House).

Statistical analysis

Where indicated, statistical analysis was performed using ANOVA or a two-sided paired t test with type I error of 0.05 and 80% power.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Priming effector PBT with TGF-β reduces their level of proliferation and IL-2 secretion induced by TCR/CD3 cross-linking

TGF-β is reported to inhibit the proliferation and IL-2 production of naive T cells, and as such has been proposed to be a major contributor to T cell tolerance and immune suppression (24). Because far less is known about the effects of TGF-β on tissue-derived effector/memory T cells, we established a model of >95% CD3+CD45RO+ T lymphoblasts by growing blood-derived T cells in IL-2 for 8 days. The purity of these cells was confirmed in unreported experiments by magnetic separation using a negative selection method as described (25). To evaluate the functional effects of TGF-β on these cells, PBT lymphoblasts were washed from IL-2 and preincubated with and without 3 ng/ml TGF-β for 24 h, and subsequently stimulated with anti-CD3 or -CD3 plus anti-CD28 for an additional 48 h in the presence or absence of TGF-β. When compared with control PBT, TGF-β treatment inhibits proliferation by >80% (Fig. 1A). Similarly, when control and TGF-β-treated T cells were stimulated in the presence of an anti-IL-2R Ab, to eliminate the consumption of IL-2 by the PBT, TGF-β priming inhibited IL-2 cytokine secretion by 65% compared with that of untreated controls (Fig. 1B). Consistent with other reports of TGF-β as an immunosuppressive and inhibitory cytokine, exposure of effector PBT to TGF-β inhibits their biological response to TCR engagement.


Figure 1
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  		FIGURE 1. Priming PBT lymphoblasts with TGF-β reduces their proliferative capacity and IL-2 production after TCR engagement. PBT treated with TGF-β for 72 h and untreated controls were activated with plate-bound anti-CD3 (1 µg/ml OKT3) alone or with soluble anti-CD28 (1 µg/ml) for the final 48 h of culture. A, [3H]Thymidine was added for the last 6 h of culture. B, Conditioned medium was collected from triplicate wells after 48 h of activation to measure IL-2 production by ELISA. Neutralizing anti-IL-2R Ab (1 µg/ml) was added to each well to block the consumption of secreted IL-2. Results are expressed as mean ± SD; *, p < 0.005 compared with controls stimulated under the same conditions. These data are representative of eight consecutive donors.

 
TGF-β priming of effector PBT extends the duration of TCR-induced global tyrosine phosphorylation without a concurrent increase in tyrosine phosphatase expression

As TGF-β priming inhibited proliferation and IL-2 production in response to TCR engagement, we assumed that TGF-β priming would similarly reduce the early biochemical events associated with cross-linking the TCR/CD3 complex. PBT lymphoblasts were incubated with or without TGF-β for 72 h, then stimulated through the TCR for 1, 5, 15, or 30 min, and the lysates were assessed for global phosphotyrosine by immunoblotting. Surprisingly, TGF-β-treated PBT showed a more intense and sustained induction of tyrosine phosphorylation for longer than 30 min compared with that seen with untreated PBT (Fig. 2A), without a detectable increase in PTK levels themselves (Fig. 3). Based on the m.w. markers and immunoprecipitation in some cases (data not shown), we identified some of the tyrosine phosphorylated proteins as PLC-{gamma}1, Zap70, p56Lck, LAT, and proteins migrating in the molecular range of ITAM containing CD3{epsilon} and TCR-{zeta}. Sustained phosphorylation of these kinases and adaptor proteins may result from reduced expression of protein tyrosine phosphatases (PTP). We therefore examined the levels of key PTPs, SHP-1, SHP-2, and CD45R, known to regulate membrane proximal signaling (26). TGF-β treated cells stimulated through the TCR showed no detectable change in cell surface expression of CD45R (Fig. 2B) using flow cytometry or expression of SHP-1 and SHP-2, as determined by immunoblot (Fig. 2C). It thus appears that perturbation in the balance of kinases and phosphatases is not a cause for sustained signaling.


Figure 2
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  		FIGURE 2. TGF-β priming of effector PBT extends the duration of TCR-induced global tyrosine phosphorylation without altering phosphatase levels. PBT were left untreated or pretreated with 3 ng/ml TGF-β for 72 h. A, Cells (5 x 106) were washed from TGF-β and stimulated for the indicated times with anti-CD3 and cross-linking Ab (10 µg/ml each). Immunoblots were probed for tyrosine phosphorylation (upper panel), phosphorylation of the ITAMs on CD3{epsilon} and TCR-{zeta} (middle), and GAPDH as a loading control (bottom). Expression levels of tyrosine phosphatases were compared between unprimed and 72 h TGF-β primed PBT by: flow cytometry for CD45R (B) and immunoblot for SHP-1 and SHP-2 (C).

 

Figure 3
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  		FIGURE 3. TGF-β priming of effector PBT sustains the proximal signaling cascade. PBT were incubated in the presence or absence of 3 ng/ml TGF-β for 72 h and activated as described in the legend of Fig. 2. Samples were immunoblotted for phospho-Lck-Y505 or -Y394, phospho-Zap70-Y319, phospho-LAT-Y191 or -Y171. Immunoblotting for total Lck, total Zap70, total LAT. and GAPDH expression was performed to control for uniform protein loading. These blots are representative of eight donors.

 
Enhancement of membrane proximal signal transduction emanating from the TCR by TGF-β

Having determined that TCR engagement of TGF-β-primed PBT lymphoblasts results in extended global protein tyrosine phosphorylation, we investigated many of the specific kinases and adaptor proteins known to mediate the apical steps of TCR/CD3 signal transduction. Upon TCR engagement, the signaling cascade is initiated with autophosphorylation at Y394 and further activation of the Src kinase Lck, which in turn phosphorylates the ITAMs on the CD3 complex. Phosphorylated ITAMs on TCR-{zeta} act as docking sites for the Syk kinase Zap70, which is subsequently phosphorylated, thus activated by Lck. Activated Zap70 is believed to regulate the subsequent combination of second messenger pathways by selectively phosphorylating tyrosines on the docking protein LAT. Two specific phosphorylated tyrosine residues on LAT act as docking sites for other SH2-containing adaptor proteins: phosphotyrosine 171 recruits both Gads-SH2 and Vav guanine nucleotide exchange factor, while phosphotyrosine 191 recruits Grb-2, thereby initiating the Ras-Raf-MEK 1/2-ERK 1/2 pathway. Extracts from TCR-stimulated TGF-β-primed and control PBT were fractionated by SDS-PAGE and immunoblotted for phosphorylation of each of the tyrosines discussed. As was observed for global tyrosine phosphorylation, TGF-β priming of effector PBT reprograms the TCR-signaling pathway, such that phosphorylation of each tyrosine on these various proteins is increased and sustained for at least an additional 30 min (Fig. 3). These findings indicate that the entire membrane proximal TCR-signaling cascade is energized in TGF-β-primed PBT lymphoblasts. However, this conclusion must be tempered by the additional observation that phosphorylation of Y505, a residue that contributes to inhibition of Lck activity, is also increased and temporally extended upon TGF-β priming, compared with controls. Together, these results suggest that the overall spatiotemporal regulation of TCR signal transduction has been altered by TGF-β exposure.

Selective modulation of a specific MAPK pathway by TGF-β priming

Three MAPK branches emanate from the TCR: the ERK pathway that is activated by Ras via the Raf group of MKKK, the p38 and JNK MAPK pathways, which are activated via both MEKK-1 and Rho family GTPases. Increased and prolonged phosphorylation of Y191 on LAT in TGF-β-primed PBT suggests that Grb2 docking to LAT and activation of the ERK 1/2 component of the MAPK pathways should be similarly extended. TGF-β-primed PBT showed a robust increase in phosphorylation of ERK 1/2 that extends well beyond 30 min compared with that seen in untreated cells (Fig. 4A). Enhanced and sustained ERK 1/2 activity corresponds to a similar increase in c-Raf and MEK 1/2 activity, as measured by protein phosphorylation. To investigate the specificity of this response, evaluation of p38 and JNK kinase activation triggered by TCR engagement revealed no difference in signal intensity or kinetics between TGF-β-treated vs control PBT (Fig. 4B). Together, these results point to the selectivity of TGF-β to up-regulate one MAPK pathway.


Figure 4
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  		FIGURE 4. Selective, sustained activation of one MAPK pathway in response to TCR cross-linking after pretreatment of PBT lymphoblasts with TGF-β. TGF-β-treated or untreated effector PBT were stimulated as described before. Samples were immunoblotted for: A, phospho-Raf, phospho-MEK 1/2, and phospho-ERK 1/2 to delineate the activity of one pathway and, B, phospho-p38 and phospho-JNK to evaluate the activity of the two other MAPK pathways. As appropriate, the membranes were reprobed for total ERK expression and GAPDH to control for equal protein loading.

 
TGF-β priming of PBT lymphoblasts up-regulates PLC-{gamma}1 activity and Ca2+ mobilization

Prolonged phosphorylation of LAT at two sites (Y171 and Y191), and possibly others, suggests that additional second messenger pathways that dock to LAT may also be extended by TGF-β priming of effector T cells. One important pathway emanating from LAT-Y171 is initiated by PLC-{gamma}1, which serves to mobilize Ca2+ from its stores. TGF-β priming prolongs activation of CD3-induced phosphorylation of PLC-{gamma}1, compared with untreated PBT, indicating again that distinct second messenger pathways are activated in treated cells (Fig. 5A). To confirm this observation, we examined signals downstream of PLC-{gamma}1 activation, focusing on calcium mobilization (Fig. 5B). TGF-β pretreatment leads to greater cytosolic Ca2+ transients within 30 s of OKT3 cross-linking than that of untreated PBT, indicating that TGF-β triggers increased TCR-induced Ca2+ mobilization, and is likely to enhance the nuclear recruitment of NFAT.


Figure 5
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  		FIGURE 5. Increased activation of TCR-induced PLC-{gamma}1-dependent Ca2+ influx in TGF-β-primed PBT lymphoblasts. A, Tyrosine phosphorylation of PLC-{gamma}1. TGF-β-treated or untreated effector PBT were stimulated and immunoblotted for phospho-PLC-{gamma}1, with GAPDH serving as the loading control. B, TCR-induced calcium mobilization. TGF-β-treated and untreated cells were loaded with the Ca2+-indicator dye, fura-2, and stimulated with anti-CD3 and cross-linking Ab (10 µg/ml each). Fura-2 fluorescence emission ratio was monitored as a function of time after addition of the stimulus. The cytosolic Ca2+ transients induced by TCR/CD3 stimulation are graphically superimposed to facilitate comparison.

 
TGF-β effects on TCR signal transduction are not generalized to cytokine signaling

To investigate whether TGF-β prolongation of TCR-induced tyrosine phosphorylation in PBT lymphoblasts is reflected in other signaling pathways, TGF-β-treated PBT were stimulated with several cytokines including IL-2, IL-7, or IL-15. These cytokines partially transduce a signal by phosphorylation and nuclear translocation of the STAT5 transcription factor. Immunoblot analysis revealed that the intensity and kinetics of STAT5 phosphorylation were indistinguishable between untreated control and TGF-β-primed PBT for all cytokines tested (Fig. 6A), demonstrating selectivity in TGF-β enhancement of the CD3/TCR-signaling pathway. Given that TCR ligation in TGF-β-primed PBT led to a robust increase and prolongation in ERK 1/2 phosphorylation (Fig. 4), we investigated other T cell stimuli that signal through ERK 1/2. Stimulation of PBT with IL-2, GM-CSF, or IL-15 activated ERK 1/2 (Fig. 6B), which is in keeping with reports of ERK being a component of the signaling pathway for each cytokine (27, 28). Of the three cytokines tested, only stimulation with IL-2 resulted in enhanced ERK 1/2 phosphorylation in TGF-β-treated cells compared with that in controls. This observation led us to investigate the ability of TGF-β to inhibit IL-2-driven proliferation. T cell proliferation in response to exogenously added IL-2 was not inhibited by TGF-β (Fig. 6C), possibly due to the finding that TGF-β acts cooperatively with TCR engagement to increase IL-2R{alpha} expression (29). Together, these results indicate that TGF-β selectively modulates the TCR signal transduction pathway, while leaving many other pathways intact. Furthermore, its antiproliferative effect is partially mediated by inhibiting IL-2 production but not function, as an exogenous supply of IL-2 can reverse TGF-β-mediated inhibition.


Figure 6
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  		FIGURE 6. Cytokine-signaling pathways and IL-2-driven proliferation remain intact after TGF-β exposure. Control and 72 h TGF-β-primed PBT lymphoblasts were stimulated with the following cytokines: IL-2 (25 ng/ml), IL-7 (10 ng/ml), IL-15 (10 ng/ml), or GM-CSF (10 ng/ml) for the times indicated. Samples were analyzed by SDS-PAGE and immunoblotted for: A, phospho-STAT5 and, B, phospho-ERK 1/2. Immunoblots of total STAT5 and total ERK 1/2 protein expression are shown to serve as protein loading controls. C, TGF-β-treated (for 72 h at 3 ng/ml) and untreated PBT were activated with IL-2 (25 ng/ml) for the final 48 h and [3H]thymidine was added for the last 6 h of culture to measure proliferation. Data in C is expressed as mean ± SD (n = 3). *, p < 0.001 vs TGF-β treated, anti-CD3/-CD28 stimulated T cells.

 
Prolonged TCR signaling corresponds with increased expression of cell cycle inhibitors, resulting in cell cycle arrest, in an ERK-independent manner

TGF-β inhibition of autocrine IL-2 production after TCR cross-linking may partially explain the reduction in thymidine incorporation detailed in Fig. 1. Yet, it remains unclear how prolonged tyrosine phosphorylation of membrane proximal events in TCR signal transduction and selective, sustained ERK phosphorylation might directly affect cell cycle regulation. We therefore investigated the cell cycle distribution of TGF-β-primed T cells using flow cytometric analysis of propidium iodide stained cells. Sixty-one percent of TCR-activated, TGF-β-treated T cells remain in G0/G1 compared with 45% in control cells (Table I), indicating that TGF-β priming promotes the accumulation of cells in the resting phase. Because altered expression of cell cycle kinases and inhibitors is indicative of cell cycle withdrawal into G0/G1, the expression level of key regulatory molecules responsible for initiating or inhibiting the various phases of the cell cycle in PBT exposed to TGF-β was determined. In TGF-β-primed effector T cells TCR ligation leads to sustained ERK 1/2 activation for at least 2 h (Fig. 7A), which temporally overlaps with an 8-fold increase in the ratio of p21 to cyclin D1 expression relative to controls (Fig. 7B; p < 0.001).


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  		Table I. Accumulation of TGF-β -primed effector/memory T cells in G0/G1 phase

 

Figure 7
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  		FIGURE 7. TGF-β priming inhibits TCR-induced cell cycle progression by increasing the ERK-independent p21 to cyclin D1 ratio. PBT were treated or untreated with TGF-β for 72 h and activated with cross-linked anti-CD3 for the final 2 h. Levels of: phospho-ERK 1/2 (A) and cyclin D1 and p21 (B) were analyzed by immunoblot and quantified by densitometry. Data are expressed as mean ± SD (n = 3). *, p < 0.001 compared with controls at 120 min; #, p < 0.001 compared with TGF-β-treated cells at 0 min using an ANOVA analysis. C, TGF-β-treated and control cells were preincubated with 20 µM U0126 for 30 min before TCR cross-linking for 2 h. p21 levels were analyzed by immunoblot.

 
The Ras/Raf/MEK/ERK MAPK pathway is broadly recognized to regulate cell cycle progression in diverse cell types, including T lymphocytes (30). Furthermore, it was recently reported that sustained, strong activation of the ERK 1/2 pathway induces cell cycle arrest in intestinal epithelial and HeLa cells, leading to transcriptional down-regulation of cyclin D1 and up-regulation of p21 and p27 (23, 31). Thus, the selective enhancement of ERK 1/2 phosphorylation by TGF-β priming suggests that this biochemical event may directly regulate p21 or cyclin D1 expression in T cells as well. We therefore prevented sustained ERK 1/2 signaling in TCR-activated PBT primed with TGF-β by pretreating the cells for 30 min with the widely accepted pharmacological inhibitor of MEK 1/2, U0126. As shown earlier, both the unphosphorylated (lower band) and phosphorylated (upper band) forms of p21 are up-regulated in TGF-β-primed PBT 2 h after TCR stimulation (Fig. 7C). No change in the levels of p21 in U0126-treated cells was detected. These results demonstrate that while sustained ERK 1/2 activation parallels cell cycle arrest, TCR-mediated induction of p21 and down-regulation of cyclin D1 after TGF-β priming is ERK 1/2 independent. Thus, an alternate, as yet undefined second messenger pathway initiated by prolonged membrane proximal TCR signal transduction must suppress cell cycle progression in a TGF-β-primed effector T cell.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Tregs have been implicated in such diverse responses as immune tolerance, autoimmunity, T cell homeostasis, and mucosal immunity (32, 33, 34, 35, 36), yet the molecular changes in the suppressed T cell have not been fully defined. TGF-β is one of many effector mechanisms for natural Treg function (37) and plays an even broader role in immune tolerance because it enhances the activity and development of the Treg population itself (38). Although the signaling events initiated by the TGF-βR are well-characterized in hemopoietic and nonhemopoietic lineages, surprisingly little biochemical information defines how a TGF-β-targeted effector/memory T lymphocyte is suppressed. The findings in this current study with human effector/memory T cells suggest that earlier reports in naive rodent T cells showing that TGF-β-mediated immune suppression leads to the total interruption of TCR signal transduction, potentially through the activation of PTPs (12 ,16), may not be universal. In this report, we demonstrate that selective molecular events following TCR engagement are enhanced and sustained in an effector T lymphocyte exposed to the immunoregulatory cytokine TGF-β. Moreover, we show that TGF-β-mediated inhibition of T cell proliferation involves both direct and indirect mechanisms. TGF-β priming inhibits TCR-induced cell cycle progression directly by increasing the CDKI:cyclin ratio within hours after T cell activation, as well as indirectly by preventing TCR-driven IL-2 production during the subsequent days, thus denying the T cell population of its critical growth factor.

The mechanisms through which Treg exert their suppressive functions are not fully understood, partly due to the diverse mediators they draw upon (39). Current opinion holds that two mechanisms account for Treg-mediated suppression of T cells: cytokines and cell-cell contact. CD4+CD25+ T cells secrete TGF-β and IL-10 and express high levels of cell surface TGF-β, which may mediate cell contact-dependent immunosuppression (1). Depending on the cell subset and its surrounding environment, the effects of TGF-β on T cells are multifactorial and include stimulation as well as inhibition of proliferation, differentiation, survival, effector function, and cytokine production (40, 41). Our findings in effector/memory T cells extend earlier studies on naive T cells by showing that TGF-β priming inhibits TCR-activated proliferation and IL-2 production, but not IL-2-induced proliferation (42). Recently, it was reported that TGF-β inhibits human Ag-specific CD4 T cell proliferation without modulating the cytokine response (11). In agreement with this observation we find IFN-{gamma} synthesis is unchanged in TGF-β-treated PBT (data not shown), although IL-2 production, which was not studied in the earlier report, is decreased. Whereas in vitro induction of Ag-specific regulatory T cells from naive murine CD4+ T cells is characterized by secretion of IL-10 (43), we determined that TGF-β priming of effector/memory human T cells upon activation does not induce IL-10 (data not shown).

The means by which TGF-β suppresses the overall immune response has often been interpreted as TGF-β directly suppressing the T cell (44). Alternatively, TGF-β may activate the T cell and redirect its function to yield the net effect of overall immune suppression. A recent report shows that cooperation of TCR engagement with TGF-β results in increased IL-2R{alpha} expression, providing a mechanism by which TGF-β may enhance the development of Treg (29). TGF-β in concert with TCR-mediated activation differentiates naive murine CD4+ T cells into Tregs, characterized by the induction of FoxP3 and acquisition of immunosuppressive activity (45). In humans, however, while FoxP3 expression is induced with TGF-β and TCR stimulation in naive CD4+FoxP3– T cells, it is not accompanied by a suppressive Treg phenotype (46). Using intracellular flow cytometry, we observed in our model that TGF-β marginally induces FoxP3 and CTLA4 expression in human CD3+CD45RO+ T cells in the presence of TCR activation (data not shown), in agreement with this recent report (46) that TGF-β does not induce human effector memory T cells to express an immunosuppressive phenotype. Together, these findings indicate that TGF-β does exert stimulatory effects, particularly on naive T cells (47) by synergizing with IL-2 to prevent apoptosis, promote effector functions (48, 49), and generate CD8+ T cells (50). Although TGF-β is most commonly considered an anti-inflammatory cytokine, it plays a critical role as a differentiation factor for a lineage of T cells producing the proinflammatory cytokine IL 17 (51). Furthermore, mice defective in TGF-β signaling lack Th17 cells and do not develop experimental autoimmune encephalomyelitis (52). These reports underscore the dual importance of TGF-β in regulating T cell biology.

The outcome of TCR-mediated signaling is dictated by the nature of the stimulus, the cell’s prior history, and environmental milieu. For example, the healthy intestinal lamina propria T cell is characterized by a relative state of hyporesponsiveness to TCR ligation (53). After TCR cross-linking a lamina propria T cell is weakly proliferative and secretes 5- to 10-fold less cytokine than its effector/memory circulating equivalent (54, 55). This mucosal phenotype is particularly intriguing in that a Th3 cell, which secretes TGF-β, has been proposed to regulate oral tolerance (56) and chronic intestinal inflammation (39, 57). In addition, we have previously reported that engagement of different members of the β1 integrin family modulates either TCR-induced proliferation or activation induced cell death (25). These and many other examples, which include CD28 and ICOS costimulation (58, 59), indicate that the TCR complex can initiate a variety of distinct signaling pathways that determine the specific outcome of Ag recognition. We therefore propose that classical immune suppression by TGF-β is more accurately depicted as a means to fine tune the signals emanating from the TCR rather than to block signaling and function altogether. In support of this novel perspective, we show in this report that a TGF-β-suppressed effector T cell, upon stimulation through its TCR, has enhanced and more sustained receptor proximal tyrosine phosphorylation compared with untreated T cells, while other signaling pathways initiated by IL-7, IL-15, and GM-CSF are intact in the TGF-β-suppressed T cell, IL-2-induced proliferation is normal, and IL-2R-mediated signaling is actually intensified and extended.

After TCR engagement, all three MAPK second messenger pathways are activated (60). The character of a resulting cellular response is partially regulated by the intensity and kinetics of MAPK signaling. For example, sustained strong activation of the ERK 1/2 pathway induces cell cycle arrest in intestinal epithelial and HeLa cells, leading to transcriptional down-regulation of cyclin D1 and up-regulation of p21 and p27 (23, 31). Prolonged ERK 1/2 activation is also associated with either senescence or apoptosis in fibroblasts and differentiation in neurons and PC12 cells (61). In our model, TGF-β-primed TCR-activated effector/memory T cells exhibit selective, enhanced, and sustained activation of the ERK 1/2 pathway that is accompanied by G0/G1 cell cycle arrest and an increase in p21:cyclin D1 ratio. However, our results demonstrate that the induction of p21 in TGF-β-treated TCR-activated PBT is independent of ERK 1/2 activation, suggesting that the observed effects on cell cycle progression established by TGF-β priming after TCR engagement is a complex process involving currently undefined pathways.

Sustained signal transduction emanating from the TCR may be mediated by a variety of molecular alterations including increased generation of reactive oxygen species (ROS) (62), elevated PTK activity, decreased PTP activity (26), reduced activity of negative regulators, and changes in gene expression, among others. Many of these possibilities were eliminated in a series of experiments not presented here. For instance, a reducing environment within the cytosol of an intestinal mucosal T cell accounts for decreased generation of ROS after TCR engagement, increased phosphatase activity, and the hyporesponsive nature of the cells (62). In keeping with this notion, we investigated whether ROS-mediated inhibition of phosphatases might result in the sustained kinase activity observed in this model. However, preincubation of TGF-β-primed cells with the anti-oxidant NAC, an inhibitor of ROS accumulation, did not antagonize prolonged signaling (data not shown). We also show that TGF-β-induced prolonged signaling in TCR-activated cells is not a consequence of reduced phosphatase expression that normally opposes TCR-coupled kinase activity, as SHP-1, SHP-2, and CD45R levels remain unchanged. Similarly, after TGF-β priming, we observed no significant change in the expression of the PD-1/PD-L1-negative costimulatory pathway (Ref. 63 ; data not shown) which might have explained the inhibition of proliferation and IL-2 secretion in the presence of enhanced signal transduction. Therefore, rather than blocking signal transduction emanating from the TCR, TGF-β-mediated effector/memory T cell immune suppression enhances this signaling via a novel pathway. Thus, while the antiproliferative effect of Treg exposure is the most visible biological outcome, our results indicate that modulation of TCR signaling will bring about an alternate functional state in the suppressed T cell target, which precipitates G0/G1 cell cycle arrest and inhibits IL-2 production.


   Acknowledgments
 
We thank all the members of the Levine laboratory in taking turns to carry the primary T cell culture; Melanie Campbell for technical assistance; Drs. Zhenfeng Zhang and Pingfu Fu for help with statistical evaluation; Dr. George Dubyak in assisting us to carry out calcium experiments and using the spectrofluorometer in his laboratory; Dr. Mary Laughlin for allowing us to use her equipment in the proliferation assays; Drs. Kevin Cooper and Christopher King for use of their Abs to perform the flow cytometric analysis; and the Case Comprehensive Cancer Center for their assistance with the flow cytometers.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by grants from the National Institutes of Health (DK-54213 and AI-53188; to A.D.L.) and the Crohn’s and Colitis Foundation of America (to L.D.). Back

2 Address correspondence and reprint requests to Dr. Alan D. Levine, Department of Medicine, School of Medicine, Case Western Reserve University, Biomedical Research Building 425, 10900 Euclid Avenue, Cleveland, OH 44106-4952. E-mail address: alan.levine{at}case.edu Back

3 Abbreviations used in this paper: Treg, regulatory T cell; CDKI, cyclin-dependent kinase inhibitor; LAT, linker for activation of T cells; PLC, phospholipase C; SH2, Src homology 2; PTK, protein tyrosine kinase; PTP, protein tyrosine phosphatase; Grb, growth factor receptor-binding protein; PBT, peripheral blood T cell; ROS, reactive oxygen species. Back

Received for publication June 28, 2007. Accepted for publication November 12, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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