TGF-ß signaling of human T cells is modulated by the ancillary TGF-ß receptor endoglin
Carsten B. Schmidt-Weber1,
Michelle Letarte2,
Steffen Kunzmann1,
Beate Rückert1,
Carmelo Bernabéu3 and
Kurt Blaser1
1 Swiss Institute of Allergy and Asthma Research (SIAF), Obere Strasse 22, CH-7270 Davos, Switzerland
2 Hospital for Sick Children and Department of Immunology, University of Toronto, 555 University Avenue, Toronto M5G1X8, Canada
3 Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Ramiro de Maeztu 9, 28040 Madrid, Spain
Correspondence to: C. B. Schmidt-Weber; E-mail: csweber{at}siaf.unizh.ch
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Abstract
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Transforming growth factor beta (TGF-ß) inhibits T cell activation and alters differentiation of naive T cells into effector cells. Although four main cell-surface proteins can interact with TGF-ß, only the signaling receptors type I (TGF-ßR type I) and type II (TGF-ßR type II) have so far been described on T cells. The aim of the present study was to investigate the expression of the ancillary receptor endoglin (CD105) by T cells and its role in TGF-ß-mediated signal transduction and function. CD105 expression was analyzed on resting and activated human CD4+ T cells by flow cytometry, western blot, immunoprecipitation, proliferation and SMAD-responsive reporter gene assays. CD4+ T cells constitutively expressed CD105 in memory T cells and partially also in naive T cells; however, surface expression is regulated and is increased following TCR engagement, which induced serine/threonine phosphorylation of CD105. In contrast to the suppressive signal mediated by the TGF-ß, cross-linking of CD105 substantially enhanced T cell proliferation, indicating that CD105 by itself mediates signal transduction. Furthermore, CD105 cross-linking induced SMAD-independent signaling via ERK kinase phosphorylation. The present study demonstrates that CD105 is expressed on the surface by activated CD4+ T cells and CD3 regulated by post-translational means. Furthermore, CD105 acts as a regulatory receptor, counteracting TGF-ß-mediated suppression.
Keywords: cytokine receptor, T lymphocytes, tolerance/suppression/anergy
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Introduction
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The transforming growth factor beta (TGF-ß) cytokine inhibits the function of T and B cells and of cells of the innate immune system (1). The important role of TGF-ß in the adaptive immune system is underlined by the finding that animals expressing a dominant negative TGF-ßR type II [TGF-ßR type II (TßRII)] specifically in T cells develop a severe autoimmune disease, characterized by uncontrolled T cell activation (2). Furthermore, T cells engineered to produce latent TGF-ß can control Th2-induced airway hyperreactivity and inflammation (3). Over-expression of SMAD7, which targets the TGF-ß-signaling receptors to ubiquitin-mediated degradation pathways, had the opposite effect (4, 5). However, it is currently not clear how TGF-ß mediates suppression of T cells. TßRII, a serine/threonine kinase, binds TGF-ß, and recruits the type I receptor [TGF-ßR type I (TßRI)] in the complex, activating it by phosphorylation. TßRI is a serine/threonine kinase, which phosphorylates the signaling intermediates SMAD2 or SMAD3 enabling the complex formation with the common SMAD4 and translocation into the nucleus. The SMAD2/34 complex interacts with other transcription factors to bind DNA elements that will induce or down-regulate expression of the target genes (6). The molecular mechanisms underlying TGF-ß-mediated T cell suppression are still unclear. In non-hematopoietic cells, TGF-ß down-regulates G1 and G2 cyclin-dependent kinases and cyclins in terms of both kinase activity and protein amount. TGF-ß also inhibits phosphorylation of the protein retinoblastoma tumor-suppressor (pRb) (7).
Recent publications showed that SMAD-independent mechanisms are capable of down-regulating elements (8), which participate in the phosphorelay system of the cell, such as the mitogen-activated protein kinase (MAPK) ERK 1/2 (9). The ERK kinase is known to act on p90RSK, a regulator of cell cycle progression (10). Other SMAD-independent mechanisms include the transforming growth factor-ß-activated kinase (TAK-1, a MAPK kinase kinase family member), mitogen-activated protein kinase kinase kinase (MEKK) 1, the protein phosphatase A2 (PP2A), Rho and TRIP-1, a phosphorylation substrate of the TßRII (8). The PP2A phosphatase coupled to TGF-ß receptor interacting protein-1 (TRIP-1) associates with TßRI (11, 12).
The potential role of the ancillary receptors betaglycan and endoglin (CD105) in mediating SMAD-dependent and -independent pathways are poorly understood. Both molecules act as accessory molecules allowing the binding of TGF-ß family members (-ß1, -ß2 and -ß3 for betaglycan and -ß1 and -ß3 for CD105) to the receptor complex (1315). CD105 does not bind the ligand on its own but only in association with TßRII and is therefore considered an ancillary or accessory receptor (14, 15). It is predominantly expressed on endothelial cells (13), but is also detected on various hematopoietic cells (1618), including monocytes (19) and B-lineage cells (18, 20, 21). Mutations in the CD105 gene lead to hereditary hemorrhagic telangiectasia type 1, an autosomal dominant disorder associated with frequent nose bleeds and arteriovenous malformations (2226). Over-expression of CD105 in a monocytic line was shown to inhibit several effects that are normally induced by TGF-ß1, like down-regulation of c-myc, stimulation of fibronectin synthesis and cellular adhesion (27). Anti-sense oligonucleotides targeted at CD105 enhanced the suppressive activity of TGF-ß in endothelial cells (28), but inhibited TGF-ß action in placental trophoblasts (29). Thus, CD105 represents an accessory receptor, which can both negatively and positively modulate TGF-ß responsiveness. In the present study, we demonstrate CD105 expression in T cells and its effect on TGF-ß-mediated suppression of T cell proliferative responses.
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Methods
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Cell culture
PBMCs were isolated from healthy donors. The interphase cells of the Ficoll (Biochrom KG, Berlin, Germany) density gradients were washed and CD4+ T cells were purified using CD4-Dynal magnetic beads and Detach-a-Bead antibodies (Dynal, Hamburg, Germany) as previously described (30). The cell purity of the CD4 population was >95%, as initially tested by flow cytometry.
Cell cultures were carried out in serum-free AIM-V medium (Life Technologies, Basel, Switzerland). For T cell activation, 2 x 106 CD4+ T cells were stimulated on plastic plates previously coated with anti-CD3 [OKT3; American Type Tissue Collection, Manassas, VA, USA; 1 µg ml1] and/or anti-CD28 (15E8, CLB, Amsterdam, The Netherlands; 2 µg ml1). The 44G4 hybridoma was previously described (16); the IgG fraction was purified using protein-G affinity chromatography. For 5-bromo-2-deoxyuridine (BrdU) incorporation, cells were pulsed for the last 45 min of culture and processed as suggested by the supplier (BrdU Flow Kit, BD Bioscience, Basel, Switzerland).
For RNA isolation, 5 x 106 Dynal-bead (Dynal)-purified CD4+ T cells were lysed with RNeasy lysis buffer (Qiagen, Hamburg, Germany) following overnight stimulation using plate-bound anti-CD3 and anti-CD28.
Flow cytometry
Surface phenotyping of peripheral blood lymphocytes of healthy volunteers was performed using Coulter Q-Prep (Beckman Coulter, Nyon, Switzerland) whole-blood-lysed technique followed by PFA fixation. The blood cells were stained with PE-conjugated anti-CD105 mAb (Immunotech, Marseille, France), PCY5- or FITC-conjugated anti-CD4 mAb (Beckman Coulter), ECD-labeled CD45RO, FITC-labeled CD45RA, ECD-labeled anti-CD62L mAb (Beckman Coulter), PCY5-labeled anti-CD8 mAb (Beckman Coulter), FITC-labeled V
9 TCR (Immunotech) and PCY5-conjugated anti-CD25 mAb (BD Bioscience, Heidelberg, Germany). Isotype controls contained the same amount of matched fluorophore Ig isotype from the same supplier. For intracellular staining, T cells were fixed and permeabilized using the saponin-based Cytofix system according to the manufacturer's protocol (BD Bioscience) and stained for intracellular CD105 expression using the PE-conjugated anti-CD105 mAb (Immunotech). Flow cytometry analysis was performed with an EPICS XL-MCL (Beckman Coulter).
Quantitative real-time PCR
Total RNA was isolated from 3 x 106 CD4+ T lymphocytes using the RNeasy mini kit (Qiagen) according to the manufacturer's protocol. The RNA was eluted in 50 µl water. Approximately 4 µg total RNA (10 µl) was reverse transcribed using the TaqMan Reverse Transcription Reagents kit (Roche/Applied Biosystems, Rotkreuz, Switzerland) with random hexamers as primer following the recommendation of the supplier in a total volume of 30 µl. The validated CD105 (Hs00164438_m1; TaqMan® Gene Expression Assays; Applied Biosystems) primer was spanning exonintron borders to eliminate amplification of contaminations of genomic DNA. The EF-1
gene is not increasing following T cell activation and was used as the housekeeping gene (5' EF-1
forward primer 5'-CTG AAC CAT CCA GGC CAA AT-3', EF-1
reverse primer 5'-GCC GTG TGG CAA TCC AAT-3'). Accumulation of the PCR products was detected in real time by monitoring the probe cleavage-induced mobilization of the reporter dye. The prepared cDNAs were amplified using an UNG-containing PCR mastermix (Perkin Elmer/Applied Biosystems) according to the recommendations of the manufacturer in a total volume of 25 µl in an ABI PRISM 7000 Sequence Detection System (Applied Biosystems).
Relative quantification and calculation of the confidence interval was performed using the comparative 
CT method. All amplifications were carried out in triplicates.
Transfections and reporter gene assays
CD4+ T cells were purified as described above and kept in serum-free AIM-V medium overnight. Cells were washed in PBS and re-suspended in 100 µl NucleofectorTM reagent (Amaxa Biosystems, Köln, Germany) as previously described (31). Human CD105 expression plasmid or empty control vectors (2 µg) (27) and/or 1 µg of the TGF-ß-sensitive (CAGACA)4-luciferase reporter gene plasmid (32) or a nuclear factor of activated T cells (NFAT) responsive element (Stratagene, Basel, Switzerland) were added to the cells and transferred to an electroporation cuvette. Cells were electroporated using the U-15 program of the NucleofectorTM (Amaxa Biosystems) and immediately transferred into pre-warmed AIM-V medium. Cells were seeded into 24-well plates and TGF-ß (1 ng ml1; R&D Systems, Wiesbaden, Germany) or phorbol myristate acetate (PMA) (10 ng ml1) as positive control for the NFAT-reporter element was added. Cells were harvested after 19 h and subjected to luciferase assays (Steady-Glo, Promega, Madison, WI, USA) and Bradford protein quantification (BioRad, Hercules, CA, USA) as described by the manufacturer. To visualize the transfection efficacy, T cells were transfected with 1 µg GFP-plasmid (BD Bioscience) per 100 µl NucleofectorTM solution. Besides GFP, we also used the mouse MHCII surface antigen (H-2Kk) as control and did not observe any effect on SMAD-reporter activity. On an average, 45% GFP-positive T cells were detected. A transfection rate of 60% was achieved with PHA-pre-activated cells, which were used for TGF-ß presentation.
Proliferation assays
Samples in triplicate, containing 5 x 104 CD4+ T cells per well, were incubated in 96 flat-bottom plates, which were previously coated with 1 µg ml1 anti-CD3 mAb and/or anti-CD105 (recognizing the extracellular domain of endoglin; 44G4) (33) or anti-TßRII (E6; Santa Cruz Inc., CA, USA) or a matched isotype control. In soluble conditions anti-CD105 or a matched isotype control (IgG1, BD Bioscience) was added to the culture at 1 µg ml1. Cells were cultured for 3 days, pulsed for the last 16 h with 1 µCi [3H]thymidine (Hartmann, Braunschweig, Germany) and harvested on glass fiber filters using an automated multi-sample harvester (LKB, Pharmacia-Wallac, Turku, Finland). Filters were transferred in sample bags with liquid scintillation fluid and analyzed using a ß-scintillation counter (Pharmacia-Wallac).
Immunoprecipitation and immunoblotting
For immunoprecipitation, 3 x 106 CD4+ T cells were lysed in PBSTriton X-100 buffer (1%; Sigma, Buchs, Switzerland) supplemented with 1 mM sodium orthovanadate, 10 µg ml1 aprotinin and 10 µg ml1 leupeptin. The lysates were incubated overnight with 1 µg anti-CD105 mAb (44G4). Protein-G sepharose (Sigma) was used for precipitation. After 4 h, the beads were washed four times with PBS, re-suspended in dithiothreitol (DTT)-containing loading buffer (NuPAGE; Invitrogen) and heated to 70°C for 10 min and the eluants were loaded next to a protein-mass ladder (Magicmark, Invitrogen) on a NuPAGE 412% Bis-Tris gel (Invitrogen). The proteins were electroblotted onto a PVDF membrane (Amersham Life Science, Dübendorf, Switzerland) and detected as described in the figures. For blot purposes an anti-CD105 mAb was used, which recognizes denaturated peptides (P3D1, Chemicon, Temecula, CA, USA). Anti-phospho-ERK 1/2 (12D1) was purchased from Alexis (Lausen, Switzerland), the pan-ERK mAb from BD Biosciences, the rabbit anti-Phosphoserine or rabbit anti-Phosphothreonine from Zymed (San Francisco, CA, USA) and anti-pSer473-AKT from Cell Signaling (Beverly, MA, USA). The blots were visualized with a LAS 1000 camera (Fuji, Urdorf, Switzerland). For estimation of protein quantity, photographs were taken with incremental exposure times. Accumulated signals were analyzed using AIDA software (Raytest, Urdorf, Switzerland).
Statistical analysis
Comparisons of CD45RA/RO-expressing cells and anti-CD3-treated and anti-CD3- and CD105-treated conditions were analyzed using the Wilcoxon signed rank test.
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Results
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CD105 expression by lymphocytes
CD105 expression in peripheral blood cells was analyzed by flow cytometry. As described previously (19, 25), CD105 is expressed on CD14+ monocytes (30 ± 15.6%; data not shown). It is also found on some CD19+ (12.0 ± 2.9%) and CD20+ B cells (15.5 ± 4.5%; data not shown) and was equally distributed among
ß or 
T cells (data not shown). CD105 was found on half of the V
9 TCR-expressing T cells. The CD8+(dim) T cells express 12.9 ± 3.5% CD105. Among the CD4+ T cell subsets, CD105 was significantly more frequent among CD45RO+ memory T cells (49.3 ± 9.5%; P
0.01; Fig. 1A), compared with CD4+CD105+CD45RA+ T cells (28.3 ± 4.8%). The ratio RO/RA is very constant among investigated individuals (1.5 ± 0.36) and also the ratio of CD105+CD45RA/RO-expressing cells was similar to the ratio of the whole CD4+ T cell population (Fig. 1A). A large proportion of the CD25high-expressing regulatory T cells also expressed CD105 (69.2 ± 13%; Fig. 1B) and to a lesser extent with HLA-DR and VLA-4 (data not shown). CD105 expresses to 31.8 ± 8.6% the lymphoid homing receptor CD62L (L-selectin). The CD62L-expressing cells can be divided in two populations discriminated by their extent of the CD62L expression. One population expresses more abundantly CD105, but slightly less CD62L (Fig. 1C, green dots, 6.4 ± 3%) and another population, which expresses CD62L more abundantly (Fig. 1C, magenta, 3.1 ± 5%).

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Fig. 1. Surface expression of CD105 on T cells. CD105 was measured in blood using flow cytometry and lymphocytes were gated on the basis of forward-side scatter distribution (AC). Co-expression of the naive (CD45RA) and memory (CD45RO) T cells (A), CD25+ regulatory T cells (B) and L-selectin (CD62L; C) was analyzed. The cross in panel A indicates the percentages of the quadrants (black: CD4+, red: CD4+CD105+). Cells were gated for CD4 (black dots) and CD105 (red dots, panel A), for CD105 (red dots, panel B) or for CD4 (blue dots, panel C). Distinct populations in panel C are shown in magenta and indicate CD105low and CD62Lhigh, whereas the green dots highlight the CD105high and CD62Llow cells. The frequency of the cell populations among six healthy donors is shown on the scattergrams on the right-hand side. Statistical significance is indicated by asterisks.
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The highest degree of co-expression was found among CD25+, therefore CD105 surface expression was analyzed following T cell activation. Purified CD4+ T cells were activated with plate-bound anti-CD3 and/or -CD28. Isolated T cells partially loose or internalize CD105 for yet unknown reason, compared with conditions in blood. Incubation with anti-CD28 alone (Fig. 2A) or medium (Fig. 2D) did not alter the intensity of CD105+ on T cells, whereas anti-CD3 stimulation clearly increased CD105+ (Fig. 2C). The stimulation was maximal at 6 h and corresponded to a 2-fold increase in the intensity of CD105+ on CD4+ T cells with anti-CD3 (from 10 to 20%) and a 3.2-fold increase (10 to 32%) with anti-CD3/CD28 (Fig. 2B and D). At later time points (24 h), cell death in 5% of the cells was observed mainly in the non-dividing population, decreasing CD105 expression (data not shown). CD105 up-regulation could also be observed on naive CD45RA+ T cells. Flow cytometry analysis of the cell cycle using BrdU incorporation and 7-AAD DNA staining revealed that CD105 expression was restricted to T cells in the S-phase of the cell cycle (Supplementary Data 1, available at International Immunology Online).

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Fig. 2. CD105 surface expression upon activation. CD105 expression of purified, resting CD4+ T cells and anti-CD28 (A), anti-CD3/28 (B) or anti-CD3 (C) stimulated CD4+ T cells after 0, 3, 6, 8 and 16 h incubation is shown. Part D of the figure shows the time course of the changes. The experiment (AD) is representative of three independent experiments.
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The TCR-signaling pathways can be mimicked by calcium ionophores (ionomycin) and protein kinase C (PKC) activators such as PMA. CD105 could only be up-regulated with PMA, but not with ionomycin (data not shown), suggesting that the CD105 display is a target of the PKC pathway.
Mechanism of CD105 regulation
T cell stimulation did not affect CD105 mRNA expression as detected by real-time PCR, whereas IL-2 mRNA expression increased as expected 3 h following stimulation of the T cells by plate-immobilized anti-CD3 mAb antibodies (Fig. 3A). In line with the stimulation-induced surface display of CD105, intracellular staining revealed a decrease of CD105 expression (Fig. 3B). Furthermore, intracellular CD105 expression was found in 25% of the resting T cells, thus fitting the number of cells expressing CD105 on their surface upon activation. The analysis of intracellular CD105 among memory and naive T cells revealed that CD105 T cells occur only among naive, but not among CD45RO+ memory T cells (Fig. 3C). The immunoprecipitated and immunoblotted CD105 protein was detected as the expected homodimer of 180190 kDa (Fig. 3D and F). The blots carrying anti-CD105 precipitates were sequentially stripped and reprobed with anti-pSer-, pThr- and pTyr-specific antibodies. pSer detection was most efficiently visualized on DTT-free blots. CD105 was serine (Supplementary data 2, available at International Immunology Online) and threonine phosphorylated within 5 min upon anti-CD3 stimulation of the T cells and persisted up to 36 h (Fig. 3D). However, pTyr phosphorylation was only detected in very low amounts and was not induced by CD3 stimulation. To verify that CD3 engagement and not CD3-mediated TGF-ß release is responsible for CD105 phosphorylation, TGF-ß was exogenously added to the culture. It failed to induce CD105 phosphorylation (Fig. 3E). Cross-linking of CD105 itself, which is expressed constitutively by 10% of the T cells also induced CD105 pThr phosphorylation, whereas addition of the cross-linking antibody (data not shown) or non-cross-linked CD105 did not induce pThr phosphorylation (Fig. 3F). Anti-CD3 and anti-CD105 together did not show pThr phosphorylation, which was significantly above the levels of the individual stimuli alone (Fig. 3F).

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Fig. 3. CD105 expression is constitutive and subject to post-translational modification. CD105 mRNA expression (closed circles) was quantified following anti-CD3/CD28 stimulation along with IL-2 mRNA expression (open squares) as positive control (A). T cells which were resting (light gray) or stimulated (dark gray) for 6 h were permeabilized and stained for intracellular CD105 expression or with respective isotype controls (dotted lines). T cells negative for CD105 expression are mainly found in naive (CD45RA) but not in memory T cells (CD45RO) as it is shown in panel C by intracellular CD105 and surface CD45RA/RO staining. For phosphorylation analyses CD4+ T cells were stimulated with anti-CD3 or anti-CD105 mAb or a matched isotype control (IC) and cross-linked with anti-mouse mAb, lysed after 10 min and immunoprecipitated (IP) with anti-CD105 as indicated in the figure. Immunoblots (IB) were sequentially probed with anti-phosphothreonine or anti-CD105 antibodies (D). The effect of anti-CD3 versus exogenously added isotype control + TGF-ß (10 min) is shown in part E of the figure. The blot shown in part F of the figure was generated as described in part E. The blots are representative of three independent experiments.
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Effect of CD105 on T cell proliferation
To address the functional impact of CD105 on TGF-ß-mediated suppression of T cell proliferation, TGF-ß was added in increasing amounts to CD3-stimulated T cells with or without soluble anti-CD105 mAb. The anti-CD3 concentration was previously titrated to only give half-maximal stimulation, since TGF-ß as well as other suppressive cytokines are ineffective in suppressing T cells stimulated with maximal anti-CD3 doses (34). Interestingly, TGF-ß did not suppress T cell proliferation in a dose-dependent manner. At low concentrations TGF-ß slightly enhanced T cell proliferation and suppressed proliferation at higher concentrations, starting from 1 ng ml1 (Fig. 4A). This enhancing effect was dramatically enhanced when anti-CD105 mAb was added along with TGF-ß, and was only observed at low concentrations of TGF-ß. Also, TGF-ß-mediated suppression was neutralized by anti-CD105 mAb to a concentration of up to 5 ng ml1. Soluble anti-CD105 mAb without the addition of TGF-ß showed only a minor enhancing effect on T cell proliferation. Similar observations were made using plate-bound anti-CD105 and (soluble) TGF-ß. In contrast, plate-bound anti-CD105 significantly enhanced anti-CD3-induced proliferation by 3-fold in the experiment illustrated in Fig. 4(B and C). In line with the addition of the natural ligand (TGF-ß), this effect was maximal at low concentrations (0.25 µg ml1 plate-bound CD105; Fig. 4B). Since the natural ligand interacts with all three receptors, the suppressive effect of TGF-ß was also mimicked by anti-TßRII mAb. In contrast to the enhancing effect of anti-CD105, cross-linking of the TßRII, using a mAb of the same isotype, dramatically suppressed T cell proliferation in a dose-dependent manner. The isotype control alone or together with anti-CD105 did not show any difference compared with medium alone. This suppression could be partially antagonized by anti-CD105 particularly at lower concentrations of anti-TßRII. The stimulation index estimated for several experiments indicated a significant difference (P
0.01, n = 8) between anti-CD105/anti-CD3-treated cells versus anti-CD3 alone (Fig. 4C).

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Fig. 4. CD105 enhances CD3-induced proliferation and antagonizes TGF-ß- or TßRII-mediated suppression. T cell proliferation was induced by plate-immobilized anti-CD3 mAb and TGF-ß was added in increasing concentration in the presence or absence of soluble anti-CD105 mAb (A). Plate-immobilized anti-CD3 and/or anti-CD105 mAb were used to stimulate CD4+ T cells for 3 days (B). The anti-CD3-induced versus the anti-CD3/CD105-induced effect was analyzed in eight independent donors (C) and increases are shown as fold increase in relation to normalized starting values. Statistical significance (P 0.01) is indicated by asterisks. Error bars indicate the error of the mean of triplicate measurements. Part A and B of the figure are representative of three independent experiments.
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Effect of CD105 on SMAD activation
To assess the effect of CD105 on SMAD activation, CD105 was transiently over-expressed in freshly isolated CD4+ T cells. Over-expression of irrelevant genes (GFP) or empty expression vectors showed a 1.7-fold increase upon addition of TGF-ß compared with medium-treated cells (Fig. 5A). Anti-CD3 or anti-CD105 alone did not induce reporter activity (Supplementary data 3A, available at International Immunology Online). CD105 over-expression was verified by flow cytometry (Fig. 5B) and decreased TGF-ß-induced reporter gene activity, compared with TGF-ß-treated cells over-expressing GFP (Fig. 5A). Over-expression of CD105 without addition of TGF-ß did not alter reporter gene activity (data not shown). Since over-expression of receptor elements may alter the integrity or properties of heteromeric TGF-ßR complex, we also used soluble or plate-bound anti-CD105 mAb to interfere with SMAD-dependent reporter activity (Fig. 5C). However, neither soluble nor plate-bound anti-CD105 reduced TGF-ß-induced reporter gene activity of CD4+ T cells. The stimulation of plate-immobilized anti-CD3 and/or anti-CD105 mAb did not suppress, but rather enhanced, SMAD-dependent reporter gene activity. Anti-CD105 engagement did not affect the SMAD- or NFAT-reporter activity in CD4+ T cells (Supplementary data 3, available at International Immunology Online).

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Fig. 5. Effect of CD105 over-expression on SMAD-responsive reporter gene activity. CD105 was over-expressed in CD4+ T cells and surface expression was verified (B). T cells were transfected with a SMAD-responsive luciferase-promoter construct along with CMV-promoter-driven GFP as control (A, open bars) or CMV-driven CD105 (A, black column) and the reporter activity was measured in relative light units (RLU). T cells were transfected with SMAD-responsive luciferase-promoter construct and pre-stimulated with anti-CD3 (30 min) and treated with mAb as indicated in the figure. Error bars indicated the error of the mean of triplicate measurements. Panel C shows T cells transfected with the reporter gene only and stimulated with mAb as indicated in the figure. The experiment is representative of three independent experiments.
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Anti-CD105 participates actively in signal transduction
Data obtained from reporter and proliferation assays suggest that CD105 itself can lead to downstream signaling. Although SMAD-independent signaling pathways were previously described, it is currently unclear how the individual receptors are mediating these signals. As expected, anti-CD3 stimulation induced ERK 1/2 phosphorylation (5-fold, lane 2, Fig. 6); this was further enhanced by anti-CD105 (7-fold, lane 4, Fig. 6). Anti-CD105 alone induced low levels of phosphorylated ERK 1/2 (2-fold, lane 3, Fig. 6). TGF-ß showed slightly reduced ERK 1/2 phosphorylation (lane 5, Fig. 6) compared with CD3 stimulation alone. Anti-TßRII did not increase ERK phosphorylation (data not shown). Anti-CD105 cross-linking did not induce any AKT phosphorylation as it was demonstrated by reprobing the blots with anti-pSer-AKT mAb (data not shown), whereas anti-CD3-stimulated cells showed very strong pSer-AKT phosphorylation.

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Fig. 6. Effect of CD105 cross-linking on CD4+ T cells. T cells were treated with medium (lane 1), anti-CD3 (lane 2; 1 µg ml1), anti-CD105 mAb (lane 3; 1 µg ml1), anti-CD3 and anti-CD105 (lane 4), TGF-ß and anti-CD105 (lane 5; 1 ng ml1) and TGF-ß alone (lane 6; 1 ng ml1). The antibodies were all cross-linked with anti-mouse Ig mAb (1 µg ml1) as indicated in the figure for 10 min. Western blots were performed (IB) with anti-phospho-ERK 1/2 mAb. The lower blot shows the detection with an anti-pan-ERK antibody. The bar graph shows a densitometric analysis of serial exposures of the phospho-ERK 1/2 blot shown above (C). The experiment is representative of three independent experiments.
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Discussion
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The present study demonstrates that TGF-ß-mediated suppression of T cells is actively counteracted by TCR-regulated CD105. The TGF-ß co-receptor CD105 is constitutively present in most of the memory T cells and in 30% of the naive T cells. However, surface expression is not permanent but is decreasing in culture and can be induced by TCR engagement. Serine/threonine phosphorylation coincides with CD105 up-regulation, while CD105 protein and mRNA remain constant, confirming by alternate means that the TCR engagement as well as CD105 cross-linking acts on CD105 by post-translational mechanisms. The mechanisms of CD105 surface display are still unclear. Cells lacking CD105 intracellularly were found only among naive T cells, suggesting that CD105 expression is acquired during differentiation into mature CD45RO+ T cells, as it was demonstrated for TGF-ß-induced CD105 expression in differentiating monocytes (27). A high proportion of CD105 expression was found on regulatory, CD25-expressing cells, which are known to have suppressive capacity on CD25 T cells. However, the functional role of CD105 in the suppression of CD25+ T cells in vitro remained inconclusive. The co-expression of CD105+ T cells with CD62L (L-selectin) is of particular interest, since it shows that potentially lymph node homing T cells differ in their composition of TGF-ßRs and thus in their responsiveness to this cytokine. Furthermore, two distinct populations of CD105- and CD62L-expressing cells could be identified; however, their origin and function is yet unknown.
The regulation of CD105 via TCR-mediated activation confirms our previous observations (35, 36) and those of others (37, 38) that elements of the TGF-ßR complex and signaling cascade are subjected to TCR-dependent regulation. Therefore, TGF-ß signaling has to be seen in an immunological context, namely, the antigen-dependent events including antigen uptake, processing, presentation and co-stimulation by antigen-presenting cells.
The differences in CD105 regulation raise the question of whether CD105 can functionally affect TGF-ß-mediated signal transduction. The current study demonstrates that TGF-ß as well as cross-linking of TßRII suppresses T cell proliferation. This result is in line with previous studies, showing that mice expressing a dominant negative TßRII in T cells cannot be suppressed by TGF-ß (2) and develop an inflammatory, multi-organ, autoimmune disease. This result also suggests that antibodies can be used to mimic TGF-ß-mediated signal transduction and therefore may allow to study the function of CD105 in relation to the TßRII. However, it should be noted that engaging CD105 with a surrogate ligand is simplifying the function of this receptor, since CD105 also interacts with the activin pathway and betaglycan (39) and may also play a role in binding of TGF-ß to T cell surfaces (40). The current study shows that TGF-ß rather enhances proliferation at low concentrations, which is further enhanced by anti-CD105 mAb. Dose-dependent effects are likely to be dependent on CD105, which is known to enhance cellular affinity to TGF-ß by
2-fold (15). Cross-linking of CD105 has an antagonistic effect on TGF-ß- and TßRII-mediated suppression and enhances T cell proliferation either when TGF-ß is present or if CD105 molecules are cross-linked. Therefore, CD105 appears to transmit signals into the cell and to have an effect exceeding the expected increase in TGF-ß affinity of the TGF-ßR complex (15). The anti-suppressive effect of CD105 on TGF-ß signaling was also observed for U937 cells (27) and endothelial cells (28). The anti-CD105 mAb did not increase the degree of cell death; however, the applied cell culture condition did not induce growth factor-withdrawal-mediated cell death. Current investigations are focused on the effect of CD105 on the anti-apoptotic capacity of TGF-ß following IL-2 withdrawal. Over-expression of CD105 inhibited the induction of a SMAD2/3-responsive reporter element, which essentially argues for an antagonistic effect of CD105 on TGF-ß signaling. However, neither soluble nor plate-bound anti-CD105 stimulation decreased the activity of SMAD-responsive reporter systems, suggesting that CD105 over-expression interferes with the normal TGF-ßR complex, possibly by rendering it inaccessible to SMAD2/3. The current finding shows that although anti-CD105 cross-linking clearly affects T cells, it does not significantly affect the transcriptional activity of SMAD molecules. Further studies are required to analyze whether CD105 affects SMAD2 and SMAD3 differentially, since the reporter assays applied here are not selective for these two transcription factors.
The lacking SMAD impact contradicts the effect of CD105 on T cell proliferation. However, the SMAD response measured in the reporter gene assays does not reflect the TGF-ß-induced signaling repertoire. Although SMADs are the best described signaling targets of TGF-ß, there are at least five SMAD-independent signaling pathways (TAK-1/MEKK, PI3K, RhoA, PP2A and RAS) (8). The present study demonstrates that CD105 cross-linking is sufficient to induce ERK 1/2 phosphorylation and further enhances CD3-induced ERK phosphorylation. In fact, recent studies suggest that a SMAD-independent pathway acts on T cell differentiation, by inhibiting the MAPK ERK 1/2 (9). It is also known that TGF-ß itself can induce MAPKs, since the TAK-I is also a MAPK and the ERK kinase has also been described as signaling target of TGF-ß in non-lymphoid cells, such as chondrocytes (41). The ERK kinases can phosphorylate SMAD2 and SMAD3 at specific sites in the region linking the DNA-binding domain and the transcriptional activation domain (42), which are separate from the TGF-ßR phosphorylation sites that activate SMAD nuclear translocation. CD105 cross-linking induced to some degree ERK 1/2 phosphorylation and strongly enhanced CD3-induced ERK phosphorylation. Since the MAPK pathway is known to be an activatory pathway, it is likely that CD105-mediated phosphorylation of ERK underlies the CD105 enhancement of T cell proliferation. In contrast, CD105 did not alter phosphorylation of AKT, which is a target of the PI3K and has also been described as the target of TGF-ß signaling (43, 44). Although more research is required to define the role of TGF-ß-induced and SMAD-independent signal transduction, the current study supports the previously formulated hypothesis that engagement of the MAPK pathway may serve to finely regulate the TGF-ß response (8).
In conclusion the present study shows that preformed CD105 is delivered to the cell surface upon T cell activation and antagonizes the suppressive capacity of TGF-ß most likely by triggering the SMAD-independent MAPK pathway. Therefore, CD105 modulates TGF-ß responsiveness in a MAPK-dependent manner, when T cells are activated via TCR. Current studies are focusing on the role of CD105 during antigen presentation and the concomitant suppression of T cells. The molecular contribution and composition of TGF-ßRs are important to define the requirements for effective suppression of T cells and thus for induction and maintenance of peripheral tolerance. Moreover they represent molecular targets in therapy of T cell-related immune disorders such as allergy, autoimmunity and graft versus host disease.
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Supplementary data
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Supplementary data are available at International Immunology Online.
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Acknowledgements
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This work was supported by the Swiss National Foundation Grant no. 31.52986.97, no. 31-65436.01 and no. 3100A0-100164; the Roche Research Foundation (Mkl/stm 115-2001); Saurer Foundation, Zurich; EMDO Foundation, Zurich; OPO Foundation, Zurich and the Canadian Institute of Health Research (M.L.). S.K. was fellow of the Deutsche Forschungsgemeinschaft (KU 1403/1-1).
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Abbreviations
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BrdU | 5-bromo-2-deoxyuridine |
DTT | dithiothreitol |
MAPK | mitogen-activated protein kinase |
MEKK | mitogen-activated protein kinase kinase kinase |
PKC | protein kinase C |
PMA | phorbol myristate acetate |
PP2A | protein phosphatase A2 |
SMAD | SMA- and MAD-related protein |
TAK | transforming growth factor-ß-activated kinase |
TGF-ß | transforming growth factor beta |
TßRI | TGF-ßR type I |
TßRII | TGF-ßR type II |
TRIP-1 | TGFß interacting protein |
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Notes
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Transmitting editor: S. Kaufmann
Received 19 January 2005,
accepted 22 April 2005.
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