TGF-β is an ubiquitous cytokine that plays a pivotal role in the maintenance of self-tolerance and prevention of immunopathologies. Under steady-state conditions, TGF-β keeps naive T cells in a resting state and inhibits Th1 and Th2 cell differentiation. Because rapid generation of Th1 and Th2 effector cells is needed in response to pathogen invasion, how do naive T cells escape from the quiescent state maintained by TGF-β? We hypothesized that stimulation by strong TCR agonists might interfere with TGF-β signaling. Using both primary mouse CD4+ T cells and human Jurkat cells, we observed that strong TCR agonists swiftly suppress TGF-β signaling. TCR engagement leads to a rapid increase in SMAD7 levels and decreased SMAD3 phosphorylation. We present evidence that TCR signaling hinders SMAD3 activation by inducing recruitment of TGF-βRs in lipid rafts together with inhibitory SMAD7. This effect is dependent on protein kinase Cθ, a downstream TCR signaling intermediary, as revealed by both pharmacological inhibition and expression of dominant-negative and constitutively active protein kinase Cθ mutants. This work broadens our understanding of the cross-talk occurring between the TCR and TGF-β signaling pathways and reveals that strong TCR agonists can release CD4 T cells from constitutive TGF-β signaling. We propose that this process may be of vital importance upon confrontation with microbial pathogens.

Transforming growth factor-β is the cytokine expressed constitutively at highest levels in lymphoid and nonlymphoid organs where it regulates T cell development, homeostasis, and differentiation (1, 2). TGF-β exists in three isoforms in mammals: TGF-β1, -β2, and -β3. TGF-β1 is the most abundant, universally expressed, and widely studied isoform. TGF-β is secreted as a latent protein complex that requires activation for biologic activity (3). Once activated, TGF-β stimulates cellular responses by inducing the assembly of heteromeric complexes containing two ubiquitously expressed cell surface receptors, the type I and type II TGF-βRs, both of which contain a serine/threonine protein kinase in their intracellular domains. Once bound to TGF-β, TGF-βRII recruits, binds, and transphosphorylates TGF-βRI, thereby stimulating its protein kinase activity. The activated TGF-βRI then recruits and phosphorylates the transcription factors, SMAD2 and SMAD3 (SMAD2/3), which then bind to the common SMAD4, translocate into the nucleus, and interact in a cell-specific manner with transcription factors, coactivators, and corepressors to regulate the transcription of TGF-β–responsive genes (410). Even though the TGF-β pathway can transduce signals via noncanonical SMAD-independent mechanisms (6), TGF-β signaling in T cells is mediated mainly if not exclusively via the canonical SMAD2/3-dependent pathway (11, 12).

The TGF-β pathway is very complex and is regulated at many levels (6). For instance, SMAD7 is a competitive inhibitor of SMAD2/3. SMAD7 can also induce the degradation of internalized TGF-βR complexes by recruiting SMURF2 (13). TGF-βR complexes can be internalized either through clathrin-dependent or lipid raft/caveolae-dependent endocytosis pathways. The former promotes signaling, whereas the latter leads to the rapid SMAD7-mediated degradation and reduced signaling (14). TGF-β signaling can also be inhibited in the cytosol by phosphatases that reduce the amount of active SMAD2/3 (15) and in the nucleus by corepressors that interfere with association of coactivators and recruitment of histone deacetylase to the transcription site (15).

TGF-β plays a pivotal role in maintaining self-tolerance and preventing the development of immunopathology (2, 16, 17). TGF-β signaling in T cells is also essential for establishment of tolerance to allografts (18, 19). Mice deficient in TGF-β rapidly develop a lethal multifocal CD4+ T cell-dependent autoimmune disease (20, 21). Furthermore, expression of a dominant-negative (DN) TGF-βRII or deletion of TGF-βRII specifically in T lymphocytes leads to autoimmunity with loss of naive T cells and accumulation of effector/memory T cells (22, 23). The pleiotropic effects of TGF-β on various T cell subsets are complex and context dependent (2, 16, 24). Under steady-state conditions, TGF-β promotes the survival of naive CD4+ T cells and keeps them in a resting state (2, 25). Accordingly, freshly isolated naive CD4+ T cells contain significant amounts of phosphorylated SMAD2 and SMAD3 proteins and express several TGF-β–dependent transcripts (25). TGF-β signaling prevents T cell proliferation and production of IL-2 (26, 27). Moreover, TGF-β inhibits Th1 and Th2 cell differentiation, whereas it contributes to differentiation of Th17 cells and maintenance of induced CD4+Foxp3+ regulatory T cells (2).

The fact that TGF-β is abundant in lymphoid and extralymphoid tissues and that constitutively active (CA) TGF-β signaling keeps naive T cells in a resting state brings about a fundamental question. Because brisk generation of Th1 and Th2 effector T cells is needed in response to pathogen invasion, how do naive T cells escape from the quiescent state maintained by TGF-β? A parsimonious explanation would be that stimulation by strong TCR agonists interferes with TGF-β signaling. Using both primary murine CD4+ T cells and Jurkat cells, we demonstrate in this paper that TGF-β/SMAD–dependent signaling and transcription are rapidly inhibited following activation with mAbs to CD3 and CD28. Most notably, the amount of phosphorylated SMAD3 decreases, and several TGF-β transcriptional targets are downregulated. Mechanistically, we identified protein kinase C (PKC)θ as a key TCR signaling intermediary that actively inhibits TGF-β signaling, probably by associating with TGF-βR complexes that are then recruited in lipid rafts.

C57BL/6(SJL)-Tg(SMAD binding element [SBE]/Tk-luc)7Twc/J mice, obtained from The Jackson Laboratory (Bar Harbor, ME), were housed under specific pathogen-free conditions in the animal care facilities of the Institute of Research on Immunology and Cancer and were used between 7 and 15 wk of age. These transgenic mice, referred to as SBE-Luc mice, express luciferase in response to activation of the Smad2/3-dependent signaling pathway. All work involving mice was conducted under protocols approved by the Comité de Déontologie de l’Expérimentation sur des Animaux de l’Université de Montréal (Montreal, Quebec, Canada). Human T lymphocyte Jurkat cell lines E6-1 (ATCC TIB-152), J.CaM1.6 (Lck kinase activity-deficient Jurkat cells, ATCC CRL-2063), and Jp116 (Zap70-deficient Jurkat cells provided by Dr. A. Veillette, Institut de Recherches Cliniques de Montréal, Montréal, Quebec, Canada) were cultured in RPMI 1640 medium containing 10% FBS, penicillin, streptomycin, and glutamine (Invitrogen Life Technologies, Carlsbad, CA).

Recombinant human TGF-β1 was obtained from PeproTech (Rocky Hill, NJ). Mowiol 4-88 reagent was from Calbiochem (Gibbstown, NJ). TRIzol reagent and Tris were from Invitrogen (Carlsbad, CA). Methyl-β-cyclodextrin (MβCD), Triton X-100, glycerol, KH2PO4, acetyl CoA, ATP, MgCl2, Brij-58, DTT, EDTA, sucrose, NaCl, and dynasore were from Sigma-Aldrich (St. Louis, MO). d-Luciferin was from Xenogen (Hopkinton, MA). DAPI and protein G-Sepharose beads were from Roche Diagnostics (Indianapolis, IN). SDS and Tween 20 were from Bio-Rad (Hercules, CA).

For T cell stimulation, we used mAbs specific for mouse (from BD Biosciences, San Jose, CA) or human (eBioscience, San Diego, CA) CD3 and mouse or human CD28 (both from eBioscience). Stimulation with anti-CD3 and anti-CD28 mAbs was performed as described previously (28, 29). Immunoprecipitation (IP) was done using the mouse anti–TGF-βRII (clone E-6) from Santa Cruz Biotechnology (Santa Cruz, CA). For immunofluorescence studies, we used Abs against the following molecules: mouse-SMAD2/3 from Abcam (Cambridge, MA); TCRβ-APC, CD4-APCCy7, CD8-PECy7, and CD44-FITC from BD Biosciences; CD62L-PECy5 from eBioscience; and rabbit IgG-Alexa 488, mouse IgG-Alexa 647, and mouse IgG-Alexa 555 from Invitrogen-Molecular Probes (Eugene, OR). For immunoblotting studies, we used Abs against goat Ig-HRP, mouse Ig-HRP, actin, PKCα (clone Y124), SMAD3, SMAD7, SMAD2, SMAD2-Ser465/Ser467, PPM1A (clone p6c7), SMURF2, TGF-βRI, and SnoN from Abcam; and PKCθ, clathrin H chain, Lck, Akt, and rabbit Ig-HRP from Cell Signaling Technology (Danvers, MA). Ab against SMAD3-Ser423/Ser425 was from Novus Biologicals (Littleton, CO) and Ab to TGIF (clone H-1) from Santa Cruz Biotechnology.

PKC vectors (CA-PKCα, DN-PKCα, CA-PKCθ, and DN-PKCθ) were gifts from Dr. J.-G. Lehoux (Université de Sherbrooke, Sherbrooke, Quebec, Canada) (30). SBE-FLuc vector was provided by Dr. T. Wyss-Coray (Stanford University, Palo Alto, CA) (31), and pCDNA3.1-PTEN-eGFP (Addgene plasmid 13039) was supplied by Dr. A. Ross (University of Massachusetts, Worcester, MA). pCDNA3.1-RLuc and pCDNA3.1-FLuc vectors were gifts from Drs. D. Lamarre and S. Meloche (Université de Montréal), respectively. Transfection of Jurkat cells was done using TransIT-Jurkat transfection reagent (Mirus Bio, Madison, WI), according to the manufacturer’s protocol. Cells were used 24–48 h later. Double and triple vector cotransfections were done using the Ingenio Electroporation solution (Mirus Bio) and 0.4-cm cuvettes (Bio-Rad), according to the manufacturer’s protocol. Cells were used 48–72 h later.

Cells were incubated (or not) with TGF-β (10 ng/ml) for 1 h, then stimulated (or not) with mAbs to CD3 and CD28 (1 and 0.5 μg/ml, respectively) and cultured for 5 h before cell lysis (lysis buffer: 1% [v/v] Triton X-100, 10% [v/v] glycerol, 5 mM Tris, 10 mM MgCl2, and 1 mM DTT). Luminescence activity was evaluated on a LUMIstar plate-reader from BMG Labtech (Fisher Scientifique, Nepean, Ontario, Canada) following incubation of cell lysates with a Luciferin Mix (1:2) (4 mM KH2PO4, 80 mM Tris, 15 mM MgCl2, 120 μM acetyl-CoA, 24 mM DTT, 1 mM ATP, and 0.5 mM luciferin mixed [1:1] with lysis buffer) for 1 min. In experiments with primary T cells from SBE-Luc transgenic mice, luciferase activity was expressed as a ratio of treated relative to untreated cells. In experiments with Jurkat cells, cells transiently transfected with either SBE-FLuc or pCDNA3.1-Fluc vector were treated and analyzed in parallel. Luciferase activity driven by SBE-Fluc was then normalized to that of luciferase activity in cells transfected with the pCDNA3.1-Fluc vector. pCDNA3.1 vectors contain the human CMV immediate-early promoter/enhancer, which permits efficient, high-level expression of recombinant proteins. Results obtained with that experimental protocol were validated using a dual-luciferase reporter assay (SBE-FLuc/pCDNA3.1-RLuc) performed as suggested by the manufacturer (Promega, Madison, WI) (Supplemental Fig. 1).

Approximately 1–4 × 108 cells treated for 30 min were washed twice in PBS and then lysed on ice with 1 ml lysis buffer containing 1%(w/v) Brij-58 in 50 mM Tris, 150 mM NaCl, 2 mM EDTA, 5 mM NaF, and 2 mM NaVO4, plus a mixture of protease inhibitors (complete mixture, EDTA free; Roche Diagnostics). Cell lysate was added 1:1 volume to 80% (w/v) sucrose and then a 5–30% discontinuous sucrose gradient was layered on top. Typically, 3–6 ml 35% sucrose and 1–2 ml 5% sucrose were layered on a 2-ml sample in 80% sucrose in a 10-ml centrifuge tube (Beckman Coulter, Brea, CA). Samples were centrifuged at 100,000 × g at 4°C for 20 h using a SW41 swinging bucket rotor (Beckman Coulter). Fractions of 0.5–1 ml (typically 10–11 fractions in total) were collected from the top of gradient tubes and used for SDS-PAGE analysis. Immunoblot analysis with cholera toxin subunit B-HRP was performed to identify lipid raft rich fractions (GM1 ganglioside positive). The relative proportion of each protein in the lipid raft rich fractions was done by quantifying band intensity in each fraction, using the Fujifilm MultiGauge 3.0 software (Fujifilm, Tokyo, Japan), and dividing the intensity of bands found in ganglioside-rich regions by the sum of intensities of the bands found in all fractions. Fold enrichment was calculated by dividing the relative proportion of each protein in the lipid-rich raft (LRR) fractions from treated versus untreated cells.

Quantitative PCR (qPCR) analyses were performed on Jurkat cells and sorted naive-phenotype CD4+ T cells (CD4+CD62L+CD44) as described previously (29, 32). In brief, total RNA of purified cells was extracted and reverse transcribed. Gene expression level was determined by qPCR using TaqMan primer and probe sets. Mouse hypoxanthine phosphoribosyltransferase (Hprt) was used as endogenous control gene.

For intracellular staining, cells were allowed to adhere to glass slides coated with poly-l-lysine (Sigma-Aldrich) for 30 min at 37°C in a humidified atmosphere with 5% CO2. After culture for 1 h in the presence of TGF-β1 (10 ng/ml), mAbs to CD3 and CD28 (1 and 0.5 μg/ml, respectively) were added and cells were incubated for 30 min. Cells were washed twice with PBS and fixed in cold methanol (−20°C) for 15 min. After PBS wash, cells were stained with Ab to SMAD2/3 (mouse, 1/100) for 1 h, then labeled with goat Ab to mouse IgG-Alexa647 and DAPI (1 μM). Slides were covered with Mowiol solution and coverslip and kept at 4°C for microscope analysis.

For surface staining, cells were treated with TGF-β1 (10 ng/ml) and stained with Ab to TGF-βRI (rabbit, 1/50) and αβTCR (mouse, 1/100) at 4°C for 30 min, washed with PBS, then incubated with TGF-β1 and secondary Abs to rabbit IgG-Alexa488 and mouse IgG-Alexa555 for 1 h at 4°C. Cells were finally treated with TGF-β1 and Abs to CD3 and CD28 for 30 min, fixed with 2% paraformaldehyde, washed with PBS, and stained with DAPI. Cell suspensions were then laid in glass bottom petri dishes (MatTek Cultureware, Ashland, MA) with an overlaid coverslip. Immunofluorescence confocal microscopy was performed with a LSM 510 Meta inverted microscope with ×40, ×63, and ×100 objectives and analysis was done with the LSM510 version 3.2 software (Carl Zeiss, Jena, Germany) and Metamorph version 7.5 software (Molecular Devices, Downingtown, PA). Colocalization coefficient and weighted colocalization coefficient were calculated with the LSM510 software.

Jurkat cells were lysed in SDS lysis buffer (50 mM Tris, 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 1% Triton X-100, 5 mM NaF, and 2 mM NaVO4, plus a mixture of protease inhibitors) and lysates were cleared by centrifugation at 13,000 rpm for 15 min at 4°C. Protein concentrations were determined using Bio-Rad protein assay (Bio-Rad). Equal amounts of total protein (20–200 μg) were separated on 8% SDS-PAGE followed by transfer to polyvinylidene difluoride membranes (GE Healthcare, Piscataway, NJ). After 30 min blocking in 2% milk in TBST (25 mM Tris-HCl [pH 8.0], 150 mM NaCl, and 0.05% Tween 20), Western blotting was done by overnight incubation at 4°C in the presence of indicated Abs (33). Protein staining of membrane was revealed by incubation with specific secondary Abs for 3 h at room temperature and then with ECL Advance Western blotting Detection kit (GE Healthcare). The relative level of each protein was quantified using the Fujifilm MultiGauge 3.0 software and normalized against the actin or calnexin band.

For IP studies, cells were lysed in IP buffer (50 mM Tris, 150 mM NaCl, 1 mM EDTA, and 1% Triton X-100), and 400 μg protein lysates was prewashed with 25 μl protein G-Sepharose beads for 1 h. Then, 5 μg of the IP-specific Ab to TGF-βRII was added to the cell lysates. After 1 h, 25 μl protein G-Sepharose beads was added for overnight incubation. Protein-Ab-bead complexes were centrifuged, washed in IP buffer, and diluted in SDS lysis buffer before heating at 95°C for 5 min. After removal of beads by centrifugation, half of the volume left was used for SDS-PAGE.

The Mann-Whitney rank test was used for analysis of qPCR results. For other data, the means of normally distributed data were compared using the Student t test, with a p value of <0.05 considered significant (*). Data are presented as mean ± SD.

Translocation of activated SMAD2 and SMAD3 from the cytoplasm to the nucleus is a hallmark of ongoing TGF-β signaling (6, 34). We therefore used confocal microscopy to evaluate the impact of TCR stimulation on TGF-β–driven SMAD2/3 accumulation in the nucleus. Primary mouse CD4+ T cells were stained with DAPI (nuclear staining) and anti-SMAD2/3 Ab. T cell stimulation with anti-CD3 and anti-CD28 mAbs abrogated TGF-β–induced accumulation of SMAD2/3 in the nucleus (Fig. 1A, 1B). To assess whether the decreased nuclear accumulation of SMAD2/3 would translate into a decrease in SMAD-dependent transcriptional output, we used cells from transgenic mice expressing a reporter gene containing 12 SBE repeats fused to the firefly luciferase (SBE-Fluc mice). Treatment of spleen-derived T cells with TGF-β led to a 5-fold increase in the luciferase activity, as previously described in astrocytes (31). Stimulation with anti-CD3 and anti-CD28 mAbs led to a 60% decrease in the luminescence output (Fig. 1C). These data show that TCR signaling in primary mouse T lymphocytes exposed to TGF-β suppresses translocation of SMAD2/3 in the nucleus and SMAD-mediated transcription.

FIGURE 1.

T cell activation inhibits TGF-β signaling in primary mouse CD4+ T cells. A and B, TCR stimulation inhibits SMAD2/3 accumulation in the nucleus. C57BL/6 mouse CD4+ T cells were stimulated or not with TGF-β for 1 h, then treated with mAbs to CD3 and CD28 or PBS for 30 min. Cells were fixed and stained with anti-SMAD2/3 Ab (red), followed by nuclear staining with DAPI (blue), and analyzed by confocal microscopy (original magnification ×63). Cells with no or minimal accumulation of SMAD2/3 in the nucleus have a red cytoplasm and blue nucleus; high SMAD2/3 activity (accumulation in the nucleus) leads to colocalization of red and blue (magenta). A, Global results based on analysis of >160 cells/condition are depicted. *p < 0.05; **p < 0.01. B, Representative cells (arrows in left panels) are enlarged in right panels. C, TCR stimulation inhibits SBE-mediated luciferase translation. Relative luciferase activity in CD4+ T cells from SBE-Fluc mice stimulated or not with TGF-β for 1 h, then treated with mAbs to CD3 and CD28 or PBS for 4 h. n = 3. *p < 0.05. D and E, TCR stimulation downregulates expression of TGF-β–related transcripts. qPCR analysis of sorted naive CD4+ T cells normalized to Hprt levels. Cells were stimulated with TGF-β for 1 h, then treated with mAbs to CD3 and CD28 for 5 h (D) or 23 h (E). Results are expressed as a percentage to gene expression in control condition (horizontal line at 100%). In the control condition, cells were cells were treated neither with TGF-β nor mAbs to CD3 and CD28. n = 3. *p < 0.05.

FIGURE 1.

T cell activation inhibits TGF-β signaling in primary mouse CD4+ T cells. A and B, TCR stimulation inhibits SMAD2/3 accumulation in the nucleus. C57BL/6 mouse CD4+ T cells were stimulated or not with TGF-β for 1 h, then treated with mAbs to CD3 and CD28 or PBS for 30 min. Cells were fixed and stained with anti-SMAD2/3 Ab (red), followed by nuclear staining with DAPI (blue), and analyzed by confocal microscopy (original magnification ×63). Cells with no or minimal accumulation of SMAD2/3 in the nucleus have a red cytoplasm and blue nucleus; high SMAD2/3 activity (accumulation in the nucleus) leads to colocalization of red and blue (magenta). A, Global results based on analysis of >160 cells/condition are depicted. *p < 0.05; **p < 0.01. B, Representative cells (arrows in left panels) are enlarged in right panels. C, TCR stimulation inhibits SBE-mediated luciferase translation. Relative luciferase activity in CD4+ T cells from SBE-Fluc mice stimulated or not with TGF-β for 1 h, then treated with mAbs to CD3 and CD28 or PBS for 4 h. n = 3. *p < 0.05. D and E, TCR stimulation downregulates expression of TGF-β–related transcripts. qPCR analysis of sorted naive CD4+ T cells normalized to Hprt levels. Cells were stimulated with TGF-β for 1 h, then treated with mAbs to CD3 and CD28 for 5 h (D) or 23 h (E). Results are expressed as a percentage to gene expression in control condition (horizontal line at 100%). In the control condition, cells were cells were treated neither with TGF-β nor mAbs to CD3 and CD28. n = 3. *p < 0.05.

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To define more precisely the impact of TCR signaling on TGF-β–regulated transcription, we performed qPCR analyses of 11 components and transcriptional targets of the TGF-β pathway (4, 31). We compared transcript levels in sorted primary naive-phenotype CD4+ T cells (CD4+CD62L+CD44) cultured for 6 or 24 h in the presence of TGF-β with or without anti-CD3/CD28 stimulation. Ten of 11 TGF-β–related genes were differentially expressed at one or both time points (Fig. 1D, 1E), the sole exception being Tgif1. Downregulation of Smad3 and Smad7 was most obvious at 6 h while that of Smad2, Ski, and Cxcr4 was more pronounced at 24 h. Upregulation of Myc and Myb was significant after 6 and 24 h, respectively. Downregulation of Smurf2, Tob1, and Smad4 was of similar magnitude at both time points. We conclude that TCR signaling rapidly downregulates expression of core components and targets of the canonical TGF-β pathway.

To define more precisely the chain of events linking TCR stimulation with inhibition of TGF-β signaling, we used the Jurkat human T cell line whose TCR signaling pathway has been characterized extensively (35). Jurkat cells were transfected with the SBE-Fluc vector or a control pCDNA3.1-Fluc vector, and SBE-mediated activity was calculated as a ratio of SBE-Fluc over pCDNA3.1-Fluc luminescence. Again, using this in vitro model, we observed that anti-CD3/CD28 stimulation suppressed TGF-β signaling (Fig. 2). This inhibition was dose and time dependent (Fig. 2B, 2C). Furthermore, it was quite brisk and extensive, reaching ~65% after 2 h and 80% after 5 h (Fig. 2B, 2C).

FIGURE 2.

T cell activation inhibits TGF-β–mediated signaling in a time- and dose-dependent manner. Jurkat cells were transiently transfected with SBE-FLuc reporter vector or pCDNA3.1-Fluc control vector. A, Jurkat cells were stimulated or not with TGF-β for 1 h, then treated with mAbs to CD3 (1 μg/ml) and CD28 (0.5 μg/ml) or PBS for 5 h. Histograms represent the ratio of luciferase activity in cells transfected with SBE-FLuc versus pCDNA3.1-Fluc vector. n = 3. *p < 0.05. B and C, The SBE-FLuc/pCDNA3.1-Fluc luminescence ratio of cells treated only with TGF-β was fixed at 100% and used to normalize the relative luminescence in cells treated with mAbs to CD3 and CD28. B, Jurkat cells were treated as in A, except that doses of mAb to CD3 ranged from 0 to 2.5 μg/ml. C, Jurkat cells were treated as in A, except that mAbs to CD3 and CD28 were added for 0–5 h. n = 3. *p < 0.05.

FIGURE 2.

T cell activation inhibits TGF-β–mediated signaling in a time- and dose-dependent manner. Jurkat cells were transiently transfected with SBE-FLuc reporter vector or pCDNA3.1-Fluc control vector. A, Jurkat cells were stimulated or not with TGF-β for 1 h, then treated with mAbs to CD3 (1 μg/ml) and CD28 (0.5 μg/ml) or PBS for 5 h. Histograms represent the ratio of luciferase activity in cells transfected with SBE-FLuc versus pCDNA3.1-Fluc vector. n = 3. *p < 0.05. B and C, The SBE-FLuc/pCDNA3.1-Fluc luminescence ratio of cells treated only with TGF-β was fixed at 100% and used to normalize the relative luminescence in cells treated with mAbs to CD3 and CD28. B, Jurkat cells were treated as in A, except that doses of mAb to CD3 ranged from 0 to 2.5 μg/ml. C, Jurkat cells were treated as in A, except that mAbs to CD3 and CD28 were added for 0–5 h. n = 3. *p < 0.05.

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Shuttling of phosphorylated SMAD2/3 from the cytosol to the nucleus is the cornerstone of TGF-β signaling. To investigate how TCR signaling could hamper accumulation of nuclear SMAD2/3 (Fig. 1A, 1B), we assessed by Western blot analysis the expression of core components of the TGF-β pathway and the phosphorylation state of SMAD2 and SMAD3 under three conditions: in the presence of TGF-β, anti-CD3/CD28, or both (Fig. 3). When compared with protein levels found in the presence of TGF-β alone, concurrent anti-CD3/CD28 stimulation had two conspicuous effects. First, although the amount of SMAD3 was unchanged, that of phospho-SMAD3 was decreased drastically. In contrast, TCR signaling did not impinge on the levels of phospho-SMAD2. Second, anti-CD3/CD28 stimulation upregulated expression of SMAD7. Notably, upregulation of SMAD7 protein expression by anti-CD3/CD28 stimulation was observed in the presence or absence of TGF-β. Of note, similar to what was seen with primary cells (Fig. 1D, 1E), anti-CD3/CD28 stimulation reduced Smad7 mRNA expression in TGF-β–treated Jurkat cells (Fig. 3C). SMAD7 is known to promote degradation of TGF-βR complexes and to inhibit SMAD2/3 phosphorylation (13). These results indicate that TCR signaling hinders a proximal event in TGF-β signaling: the accumulation of phospho-SMAD3.

FIGURE 3.

TCR stimulation increases expression of SMAD7 protein and decreases levels of phosphorylated SMAD3. A and B, Jurkat cells were treated or not with TGF-β for 1 h. Abs to CD3 and CD28 were added for 30 min where indicated. Cell lysates were immunoblotted with the indicated Abs. A, One representative out of three independent experiments is shown. B, Densitometric analysis of Western blots was conducted using cellular actin as a relative internal standard. Histograms represent the mean and SD for three independent experiments. *p < 0.05. C, qPCR analysis of Smad7 mRNA expression in Jurkat cells normalized to Hprt levels. Cells were stimulated with TGF-β for 1 h, then treated or not with mAbs to CD3 and CD28 for 5 h. In the control condition, cells were treated neither with TGF-β nor mAbs to CD3 and CD28. n = 3. *p < 0.05.

FIGURE 3.

TCR stimulation increases expression of SMAD7 protein and decreases levels of phosphorylated SMAD3. A and B, Jurkat cells were treated or not with TGF-β for 1 h. Abs to CD3 and CD28 were added for 30 min where indicated. Cell lysates were immunoblotted with the indicated Abs. A, One representative out of three independent experiments is shown. B, Densitometric analysis of Western blots was conducted using cellular actin as a relative internal standard. Histograms represent the mean and SD for three independent experiments. *p < 0.05. C, qPCR analysis of Smad7 mRNA expression in Jurkat cells normalized to Hprt levels. Cells were stimulated with TGF-β for 1 h, then treated or not with mAbs to CD3 and CD28 for 5 h. In the control condition, cells were treated neither with TGF-β nor mAbs to CD3 and CD28. n = 3. *p < 0.05.

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To identify components of the TCR signaling cascade that might be responsible for inhibition of the TGF-β pathway, we tested various pharmacological inhibitors on Jurkat cells transfected with the SBE-Fluc construct (the source, specificity, and usage of pharmacological inhibitors are shown in Table I). Cells treated with TGF-β alone produced high amounts of luciferase but concurrent CD3/CD28 stimulation inhibits TGF-β–induced luciferase expression (Figs. 2, 4A). We asked whether specific inhibitors could block the effect of CD3/CD28 stimulation and thereby “rescue luciferase expression”. Inhibition of the proximal kinase Lck (by herbimycin A or Lck inhibitor) rescued SBE-mediated luminescence output (Fig. 4A, 4B). We took advantage of the availability of Jurkat cell lines deficient in Lck activity (J.CaM1.6) or Zap70 protein (Jp116) to validate our pharmacological inhibition data. In these two cell lines, CD3/CD28 stimulation did not inhibit TGF-β signaling (Fig. 4C), thus confirming that both Lck and Zap70 proximal kinases were necessary for inhibition of TGF-β signaling. We tested inhibitors of numerous downstream targets of Lck and Zap70. Inhibition of AKT, calcineurin, Gαi, Jnk, MAPK, mTOR, NF-κB, PI3K, and Ras had no effect (Fig. 4A, 4B). However, inhibition of PKC (by bisindolylmaleimide IV [BimIV] and Go6976) rescued SBE-mediated luminescence output (Fig. 4A, 4B). These results suggest that PKC is the downstream target of Lck and Zap70, which is responsible for blocking TGF-β signaling upon TCR stimulation.

Table I.
Pharmacological inhibitors of TCR signaling used in this work
ReagentMain TargetDose UsedManufacturer
Akt inhibitor IV AKT 1 μM Calbiochem 
BimIV PKC and protein kinase A 100 nM Calbiochem 
Calcineurin autoinhibitory peptide Calcineurin 50 μM Calbiochem 
Go6976 PKC 5 μM Calbiochem 
GW5074 RAF 20 nM Sigma-Aldrich 
Herbimycin A LCK 5 μM Calbiochem 
Lck inhibitor LCK 25 nM Calbiochem 
Ly294002 PI3K 10 μM Calbiochem 
NF-κB activation inhibitor NF-κB 20 nM Calbiochem 
PD98059 MEKK 25 μM Calbiochem 
Pertussis toxin Gαi 100 ng/ml Sigma-Aldrich 
Rapamycin mTOR 200 nM Sigma-Aldrich 
SB203580 p38 5 μM Calbiochem 
Sp600125 JNK 100 nM Calbiochem 
U0126 MEK1 and MEK2 25 μM Calbiochem 
Wortmannin PI3K 100 nM Calbiochem 
ReagentMain TargetDose UsedManufacturer
Akt inhibitor IV AKT 1 μM Calbiochem 
BimIV PKC and protein kinase A 100 nM Calbiochem 
Calcineurin autoinhibitory peptide Calcineurin 50 μM Calbiochem 
Go6976 PKC 5 μM Calbiochem 
GW5074 RAF 20 nM Sigma-Aldrich 
Herbimycin A LCK 5 μM Calbiochem 
Lck inhibitor LCK 25 nM Calbiochem 
Ly294002 PI3K 10 μM Calbiochem 
NF-κB activation inhibitor NF-κB 20 nM Calbiochem 
PD98059 MEKK 25 μM Calbiochem 
Pertussis toxin Gαi 100 ng/ml Sigma-Aldrich 
Rapamycin mTOR 200 nM Sigma-Aldrich 
SB203580 p38 5 μM Calbiochem 
Sp600125 JNK 100 nM Calbiochem 
U0126 MEK1 and MEK2 25 μM Calbiochem 
Wortmannin PI3K 100 nM Calbiochem 
FIGURE 4.

CD3-CD28–mediated TGF-β inhibition is PKC dependent. A and B, We tested various pharmacological inhibitors of TCR signaling in SBE-Fluc–transfected Jurkat cells treated with TGF-β and mAbs to CD3 and CD28. Inhibitors and their target and concentration used are listed in Table I. In this assay, inhibitors that block the effect of CD3/CD28 stimulation rescue luciferase expression. A, Schematic representation of the TCR signaling pathway. Components of the TCR signaling pathway are circled in green when their inhibition rescued luciferase expression and in red when it did not. B, Results from three independent experiments with pharmacological inhibitors of TCR signaling. *p < 0.05; **p < 0.01; ***p < 0.005. C, SBE-Fluc–transfected Jurkat cells (wild-type clone E6.1; Lck activity-deficient J.CaM1.6 or Zap70-deficient Jp116) were treated with TGF-β and mAbs to CD3 and CD28 (n = 3). A, Adapted and reprinted with permission from Lin and Weiss (36).

FIGURE 4.

CD3-CD28–mediated TGF-β inhibition is PKC dependent. A and B, We tested various pharmacological inhibitors of TCR signaling in SBE-Fluc–transfected Jurkat cells treated with TGF-β and mAbs to CD3 and CD28. Inhibitors and their target and concentration used are listed in Table I. In this assay, inhibitors that block the effect of CD3/CD28 stimulation rescue luciferase expression. A, Schematic representation of the TCR signaling pathway. Components of the TCR signaling pathway are circled in green when their inhibition rescued luciferase expression and in red when it did not. B, Results from three independent experiments with pharmacological inhibitors of TCR signaling. *p < 0.05; **p < 0.01; ***p < 0.005. C, SBE-Fluc–transfected Jurkat cells (wild-type clone E6.1; Lck activity-deficient J.CaM1.6 or Zap70-deficient Jp116) were treated with TGF-β and mAbs to CD3 and CD28 (n = 3). A, Adapted and reprinted with permission from Lin and Weiss (36).

Close modal

Two of eight PKC isotypes expressed in T lymphocytes are involved in TCR signaling: PKCα and PKCθ (37, 38). To better ascertain a role for PKC and to identify the specific isotype responsible for inhibition of TGF-β signaling, we proceeded with co-IP studies. Our Western blot analyses revealed that TCR signaling hindered a proximal event in TGF-β signaling: the accumulation of phospho-SMAD3 (Fig. 3). Because SMAD3 is phosphorylated by TGF-βR complexes, we precipitated these complexes using an anti–TGF-βRII Ab. As expected, Western blot analysis of co-IP proteins revealed the presence of TGF-βRI and SMAD7 (Fig. 5A), two proteins known to be associated to TGF-βRII. The salient finding was that PKCθ but not PKCα co-IP with TGF-βR complexes. Of note, Lck, also highlighted by our pharmacological inhibitor screen as a potential TGF-β pathway inhibitor (Fig. 4), was not recovered with TGF-βR complexes. Thus, only PKCθ was found to be associated to TGF-βR complexes.

FIGURE 5.

TCR-mediated inhibition of TGF-β signaling is PKCθ dependent. A, PKCθ interacts with TGF-βRII. Jurkat cell lysates were IP with anti–TGF-βRII Ab, followed by Western blotting with the indicated Abs. Input total lysate (right) and IP (left) lanes were revealed in the same conditions. One representative out of three independent experiments is shown. Numbers depict representative densitometry normalized to the densitometry of total lysates from untreated cells. B, Relative luciferase activity in Jurkat cells transfected with SBE-Fluc and one of the following vectors: CA-PKCα, DN-PKCα, CA-PKCθ, DN-PKCθ, or empty control vector. Cells expressing a CA-PKC or empty vector were treated for 6 h with TGF-β, and cells expressing a DN-PKC or empty vector were treated with TGF-β for 1 h, then stimulated with mAbs to CD3 and CD28 for 5 h. n = 4. *p < 0.05.

FIGURE 5.

TCR-mediated inhibition of TGF-β signaling is PKCθ dependent. A, PKCθ interacts with TGF-βRII. Jurkat cell lysates were IP with anti–TGF-βRII Ab, followed by Western blotting with the indicated Abs. Input total lysate (right) and IP (left) lanes were revealed in the same conditions. One representative out of three independent experiments is shown. Numbers depict representative densitometry normalized to the densitometry of total lysates from untreated cells. B, Relative luciferase activity in Jurkat cells transfected with SBE-Fluc and one of the following vectors: CA-PKCα, DN-PKCα, CA-PKCθ, DN-PKCθ, or empty control vector. Cells expressing a CA-PKC or empty vector were treated for 6 h with TGF-β, and cells expressing a DN-PKC or empty vector were treated with TGF-β for 1 h, then stimulated with mAbs to CD3 and CD28 for 5 h. n = 4. *p < 0.05.

Close modal

We next transfected Jurkat cells with SBE-Fluc and one of the following constructs: 1) CA-PKCα, 2) CA-PKCθ, 3) DN-PKCα, or 4) DN-PKCθ. Double-transfected cells were then treated with TGF-β with or without anti-CD3/CD28 stimulation. Induction (CA) or inhibition (DN) of PKCα had no or minimal effects (Fig. 5B). However, in the presence of TGF-β alone, CA-PKCθ expression mimicked the effect of CD3/CD28 stimulation and reduced TGF-β signaling (luciferase production) (Fig. 5B). Furthermore, transfection with DN-PKCθ abrogated the effect of TCR activation on TGF-β signaling. We conclude that PKCθ activation is necessary and sufficient to explain the inhibition of TGF-β signaling upon TCR activation.

What is the mechanism of action of PKCθ? PKCθ has a direct role in NF-κB and NFAT activation (39, 40). However, we reasoned that this effect should not be instrumental in inhibition of TGF-β signaling because inhibition of NF-κB activity or of calcineurin (the coactivator of NFAT) could not rescue TGF-β signaling in TCR-stimulated Jurkat cells (Fig. 4). Another important role of PKCθ is in the internalization and recycling of TCR complexes (41, 42). Like TCR signaling, TGF-β signaling is regulated by receptor internalization (43). We therefore hypothesized that TGF-βR complexes could be internalized along with, or by the same mechanism as, TCRs. We first sought to characterize the plasma membrane distribution of TGF-βRs treated with TGF-β in the presence or absence of anti-CD3/CD28 stimulation. TCR binding to agonists induces capping of the TCRs that accumulate at one pole of the cell together with other receptors such as IFN-γR (44, 45). Using confocal microscopy, we found that following TCR and TGF-β stimulation, TCRs and TGF-βRI accumulated at the same pole of the cell (Fig. 6A). This resulted in a significantly increased colocalization coefficient between TGF-βRI and TCRs (Fig. 6B). Hence, TCR engagement led to copolarization of TCRs and TGF-βRs.

FIGURE 6.

TCR stimulation induces copolarization of TCRs and TGF-βRs in lipid rafts. A and B, Jurkat cells were stimulated with TGF-β for 1 h and then treated with mAbs to CD3 and CD28 or PBS for 30 min. Cells stained with Abs to TCR (red), TGF-βRI (green), and DAPI (blue) were analyzed by confocal microscopy (original magnification ×100). n = 3. *p < 0.05. Global evaluation of TCR/TGF-βR colocalization is depicted in B. C and D, TCR stimulation induces accumulation of TGF-βRI and SMAD7, but not SMAD3, in LRR regions. Jurkat cell lysates were fractionated on a sucrose gradient, and 0.5–1 ml fractions was Western blotted with the indicated Abs. Eleven fractions were loaded (F1–F11), F1 being the top and F11 the bottom fraction. Cells were stimulated with TGF-β for 1 h and then treated or not with mAbs to CD3 and CD28 for 30 min. C, Western blots from one representative out of five experiments are shown. Fold enrichment represents the ratio of the densitometric intensity in LRR fractions relative to the densitometric intensity in clathrin positive regions. D, Histograms depict the proportion of LRR-associated protein in treated relative to untreated cells. n = 4. *p < 0.05. E, Lipid raft disruption restores TGF-β–dependent luciferase activity. Luciferase activity in Jurkat cells transfected with SBE-Fluc and CA-PKCθ (black bars) or empty vector (gray bar). Cells were treated for 6 h with TGF-β in the presence of dynasore (80 μM), MβCD (1 mM), or DMSO. n = 4. *p < 0.05.

FIGURE 6.

TCR stimulation induces copolarization of TCRs and TGF-βRs in lipid rafts. A and B, Jurkat cells were stimulated with TGF-β for 1 h and then treated with mAbs to CD3 and CD28 or PBS for 30 min. Cells stained with Abs to TCR (red), TGF-βRI (green), and DAPI (blue) were analyzed by confocal microscopy (original magnification ×100). n = 3. *p < 0.05. Global evaluation of TCR/TGF-βR colocalization is depicted in B. C and D, TCR stimulation induces accumulation of TGF-βRI and SMAD7, but not SMAD3, in LRR regions. Jurkat cell lysates were fractionated on a sucrose gradient, and 0.5–1 ml fractions was Western blotted with the indicated Abs. Eleven fractions were loaded (F1–F11), F1 being the top and F11 the bottom fraction. Cells were stimulated with TGF-β for 1 h and then treated or not with mAbs to CD3 and CD28 for 30 min. C, Western blots from one representative out of five experiments are shown. Fold enrichment represents the ratio of the densitometric intensity in LRR fractions relative to the densitometric intensity in clathrin positive regions. D, Histograms depict the proportion of LRR-associated protein in treated relative to untreated cells. n = 4. *p < 0.05. E, Lipid raft disruption restores TGF-β–dependent luciferase activity. Luciferase activity in Jurkat cells transfected with SBE-Fluc and CA-PKCθ (black bars) or empty vector (gray bar). Cells were treated for 6 h with TGF-β in the presence of dynasore (80 μM), MβCD (1 mM), or DMSO. n = 4. *p < 0.05.

Close modal

TCRs recruited at one pole of the cell during formation of immunological synapses localize in LRR regions (46). TGF-βR complexes can be found both outside and inside LRR regions, and this distribution has been shown to influence TGF-β–mediated signaling (14). Using sucrose gradient fractionation of cell lysates, we compared the content of LRR regions (ganglioside positive) and clathrin-positive fractions from Jurkat cells treated with TGF-β with or without CD3/CD28 stimulation. We found that SMAD3 was equally distributed among the two regions, and its distribution did not change upon addition of TCR signaling (Fig. 6C). In contrast, TCR signaling enriched by 2- to 3-fold the abundance of SMAD7 and TGF-βRI in LRR regions (Fig. 6C, 6D). Hence, TCR activation led to accumulation of TGF-βRI and the inhibitory SMAD7, but not of SMAD3, in LRR regions. Localization of TGF-βR complexes in LRR is known to cause degradation of TGF-βRs and to downregulate TGF-β–mediated signaling (14). Increased physical proximity of TCRs and TGF-βRs may be instrumental in enhancing interaction between the two signaling pathways, and their localization in lipid rafts might confer similar membrane and internalization dynamics.

To directly test the importance of LRR regions in the TCR-mediated inhibition of TGF-β signaling, we used inhibitors of lipid raft and clathrin vesicle formation. MβCD disrupts lipid rafts whereas dynasore inhibits dynamin, which is essential for clathrin-dependent coated vesicle formation (47). Because lipid raft destabilization hampers response to TCR agonists, we could not use CD3/CD28 stimulation in the following experiments. Because the effect of TCR signaling on the TGF-β pathway is mediated by PKCθ (Fig. 5), we used CA-PKCθ vector transfection to circumvent the need for surface TCR stimulation. Jurkat cells transfected with SBE-Fluc and CA-PKCθ constructs were stimulated with TGF-β in the presence of dynasore or MβCD and then assessed for luciferase expression. Although dynamin inhibition had no effect, lipid raft disruption totally abrogated the effect of PKCθ on TGF-β signaling (Fig. 6E). These data show that lipid rafts integrity is crucial for the PKCθ-dependent inhibition of TGF-β signaling.

The copious amount of TGF-β present in lymphoid organs promotes constitutive TGF-β signaling and keeps naive T cells in a resting state. This constitutive inhibition of T cell activation could hamper compulsory responses to foreign Ags. Our work shows that strong TCR agonists rapidly suppressed TGF-β signaling in human and mouse CD4+ T cells. Stimulation with mAbs to CD3 and CD28 led to a decrease in levels of phospho-SMAD3 with concomitant increase in SMAD7 after 30 min (Fig. 3A), downregulation of TGF-β–induced translation after 1–2 h (Fig. 2C), and decreased expression of TGF-β target genes after 6–24 h (Fig. 1D, 1E). Downregulation of TGF-β signaling was dependent on lipid raft integrity and correlated with copolarization of TGF-βR and TCR complexes and accumulation of TGF-βRI and inhibitory SMAD7 in LRR regions (Fig. 6). Activation of PKCθ by TCR agonists was necessary and sufficient to explain the inhibition of TGF-β signaling upon TCR activation. Indeed, the inhibition was abrogated by PKC inhibitors (BimIV and Gö6976) and by a DN PKCθ construct and was reproduced by a CA form of PKCθ (Figs. 4, 5). Our findings are consistent with the fact that PKCθ is a key molecule in modulating T cell activation versus anergy (48) and that PKCθ effects are opposite to those of TGF-β. As PKCθ relays a subset of CD28 signals during T cell activation, absence of PKCθ raises the threshold for T cell activation and facilitates tolerance induction (4850).

Phosphorylation of SMAD2 and SMAD3 by activated TGF-βRs is a pivotal event in the initiation of TGF-β signal transduction (6). We found that TCR activation caused a rapid and selective decrease in levels of phospho-SMAD3 (Fig. 3). Further studies are needed to understand why levels of phospho-SMAD2 were not affected. Our favorite hypothesis hinges on the differential effect of Smad anchor for receptor activation (SARA) on SMAD2 and SMAD3 (14). SARA anchors SMAD2 but not SMAD3 in clathrin vesicles that do not contain SMAD7 (14, 51, 52). We therefore postulate that in this way, SARA selectively hinders interactions between SMAD2 and the inhibitory SMAD7 upon TCR activation. Nevertheless, it is clear that SMAD2 and SMAD3 have nonredundant effects and are regulated differently (53). For instance, insulin-like growth factor I downregulates TGF-β signaling by suppressing phosphorylation of SMAD3 but not SMAD2 (54, 55). Furthermore, evidence suggests that regulation of T cell activation versus tolerance is mediated primarily by SMAD3 (12, 56). Our data support an emerging model in which TCR signaling hinders SMAD3 activation by inducing recruitment of TGF-βRs in LRR regions together with inhibitory SMAD7 (Fig. 6). In addition, TCR activation led to a brisk upregulation of SMAD7 protein level (Fig. 3). At later time points after TCR activation, levels of Smad7 transcripts were decreased both in primary mouse lymphocytes (Fig. 1D, 1E) and human Jurkat cells, probably because of autoinhibitory feedback mechanisms. SMAD7 is both a TGF-β inhibitor and a TGF-β transcriptional target. Although the SMAD7 protein inhibits TGF-β signaling, decreased TGF-β activity downregulates Smad7 transcription (57, 58). Our hypothesis that SMAD7 is instrumental in inhibition of the TGF-β pathway by TCR agonists is coherent with studies showing that high T cell levels of SMAD7 correlate with inflammation and autoimmunity, whereas downregulation of SMAD7 is associated with tolerance and induction of regulatory T cells (5962). Furthermore, our model dovetails well with evidence that internalization of TGF-βRs located in LRR vesicles leads to SMAD7-dependent degradation of TGF-βRs and inhibition of SMAD3 phosphorylation (14).

Our work broadens our understanding of the cross-talk occurring between the TCR and TGF-β signaling pathways. TGF-β regulates TCR signaling at many levels. Thus, TGF-β blocks TCR-induced activation of the Tec kinase Itk, calcium ion influx in T cells, and the activation of the transcription factor NFAT (2). The present work shows that strong TCR agonists can free CD4+ T cells from constitutive TGF-β signaling. We propose that this process may be of vital importance upon confrontation with microbial pathogens.

We thank Dr. André Veillette for sound advice. We are grateful to the staff of the following core facilities at the Institute for Research in Immunology and Cancer for outstanding support: animal facility, bioimaging, flow cytometry, and genomics.

Disclosures The authors have no financial conflicts of interest.

This work was supported by grants from the Fonds de la Recherche en Santé du Québec (Groupe de recherche transdisciplinaire sur l'étude des prédicteurs du rejet) and the Leukemia and Lymphoma Society of Canada. M.G. and J.-S.D. are supported by the Cole Foundation and the Canadian Institutes for Health Research, respectively. M.-J.H. is the holder of the Shire Chair in Nephrology, Transplantation, and Renal Regeneration at the Université de Montréal. C.P. holds a Canada Research Chair in Immunobiology. The Institute for Research in Immunology and Cancer is supported in part by the Canadian Center of Excellence in Commercialization and Research, the Canada Foundation for Innovation, and the Fonds de la Recherche en Santé du Québec.

The online version of this article contains supplemental material.

Abbreviations used in this paper:

     
  • BimIV

    bisindolylmaleimide IV

  •  
  • CA

    constitutively active

  •  
  • DN

    dominant-negative

  •  
  • FLuc

    firefly luciferase

  •  
  • Hprt

    hypoxanthine phosphoribosyltransferase

  •  
  • IP

    immunoprecipitation

  •  
  • LRR

    lipid-rich raft

  •  
  • MβCD

    methyl-β-cyclodextrin

  •  
  • qPCR

    quantitative PCR

  •  
  • SARA

    Smad anchor for receptor activation

  •  
  • SBE

    SMAD binding element.

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