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The Journal of Immunology, 2001, 167: 6431-6440.
Copyright © 2001 by The American Association of Immunologists

The Alternatively Spliced Domain TnFnIII A1A2 of the Extracellular Matrix Protein Tenascin-C Suppresses Activation-Induced T Lymphocyte Proliferation and Cytokine Production1

Marta D. Puente Navazo*, Danila Valmori{dagger} and Curzio Rüegg2,*

* Centre Pluridisciplinaire d’Oncologie, University of Lausanne Medical School, Lausanne, Switzerland; and {dagger} Division of Onco-Immunology, Ludwig Institute for Cancer Research, Lausanne Branch, Lausanne University Hospital, Lausanne, Switzerland


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Several lines of evidences have suggested that T cell activation could be impaired in the tumor environment, a condition referred to as tumor-induced immunosuppression. We have previously shown that tenascin-C, an extracellular matrix protein highly expressed in the tumor stroma, inhibits T lymphocyte activation in vitro, raising the possibility that this molecule might contribute to tumor-induced immunosuppression in vivo. However, the region of the protein mediating this effect has remained elusive. Here we report the identification of the minimal region of tenascin-C that can inhibit T cell activation. Recombinant fragments corresponding to defined regions of the molecule were tested for their ability to inhibit in vitro activation of human peripheral blood T cells induced by anti-CD3 mAbs in combination with fibronectin or IL-2. A recombinant protein encompassing the alternatively spliced fibronectin type III domains of tenascin-C (TnFnIII A–D) vigorously inhibited both early and late lymphocyte activation events including activation-induced TCR/CD8 down-modulation, cytokine production, and DNA synthesis. In agreement with this, full length recombinant tenascin-C containing the alternatively spliced region suppressed T cell activation, whereas tenascin-C lacking this region did not. Using a series of smaller fragments and deletion mutants issued from this region, we have identified the TnFnIII A1A2 domain as the minimal region suppressing T cell activation. Single TnFnIII A1 or A2 domains were no longer inhibitory, while maximal inhibition required the presence of the TnFnIII A3 domain. Altogether, these data demonstrate that the TnFnIII A1A2 domain mediate the ability of tenascin-C to inhibit in vitro T cell activation and provide insights into the immunosuppressive activity of tenascin-C in vivo.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
It is generally accepted that the immune system is capable of eliminating cancer cells from the organism. The concept of antitumor immunity was originally fostered by the observation that tumor cells transplanted into immunodeficient mice grow to form large tumors whereas the same cells transplanted to immunocompetent animals are rejected (1, 2). This concept was recently reinforced by the identification and characterization of tumor-associated Ags (TAA)3 and TAA-specific CTLs present in cancer patients and healthy individuals (3). Despite these observations, however, it is clear that lymphocytes present in the tumor stroma are often unable to completely eliminate tumor cells possibly due to tumor-induced immunosuppression (4, 5). Among other mechanisms, we have reported that an increased expression of tenascin-C in the tumor stroma might play a role in tumor-induced immunosuppression (6).

Tenascin-C is a hexameric protein member of the tenascin family of extracellular matrix (ECM) proteins which also includes tenascin-X, -Y, -R, and -W (7). Each human tenascin-C subunit consists of an N-terminal region forming coiled coil structures and interchain disulfide bonds essential for subunit oligomerization; 14 and half epidermal growth factor (EGF)-like domains, a variable number of fibronectin type III (TnFnIII) repeats, and a C-terminal fibrinogen-related domain (FRED) (7, 8, 9, 10). Alternative mRNA splicing within the TnFnIII repeats region can generate different tenascin-C isoforms. There are eight conserved TnFnIII repeats (designated by numbers 1–8) and up to seven alternatively spliced TnFnIII repeats (designated by letters A–D) inserted between the conserved repeats 5 and 6 (11). The small tenascin-C variant lacking the alternative spliced TnFnIII domains is present in static tissues such as cartilage (12), whereas the large isoforms containing the alternatively spliced TnFnIII domains in various combinations are found in tissues containing migrating cells (13) or undergoing remodeling, such as healing wounds and tumor stroma (14, 15, 16). Tenascin-C is strongly expressed during embryonic development, in particular during morphogenesis of the myoskeletal system, connective tissue, and the vasculature (17) and at the borders of epithelial-mesenchymal transition (18). Tenascin-C is expressed by glial cells in the developing central nervous system (19) and by Schwann cells in the peripheral nervous system (7). In normal adult tissues, tenascin-C expression is mostly restricted to the bone marrow (20), thymus, spleen, and lymph nodes, in particular in T lymphocyte-dependent zones (21). Increased tenascin-C expression is observed in chronically inflamed tissues, mainly in areas rich in CD4+ memory T cells (22). Highly increased tenascin-C deposition is found at sites of angiogenesis, in healing wounds and in the stroma of malignant tumors (23), including breast (16), glioma (24), melanoma (25), endometrial adenocarcinoma (26), ovarian cancers (27), and Hodgkin’s lymphoma (28). Tenascin-C is present in the serum of healthy individuals and, at elevated concentrations, in cancer patients and in acute inflammation (29, 30).

Several functions have been attributed to tenascin-C through in vitro experiments. In particular, promotion or suppression of cell adhesion, migration, and proliferation (as reviewed by Crossin (31)). Functional-structural analysis have revealed that many of these functions are mediated by defined tenascin-C domains interacting with ECM proteins or cell surface receptors (32). For example, the EGF type repeats prevent adhesion of L929 mouse fibroblasts (33) and induce neurite outgrowth (34). The TnFnIII 2–6 repeats promote migration of C6 glioma cells, and the TnFnIII 3 repeat induces glioma attachment and spreading (35). The TnFnIII domains 1–5 and 6–8 support adhesion and induce proliferation of human hemopoietic cells (36). One or more of the TnFnIII 1–5 repeats bind to fibronectin (37) and thereby inhibit attachment of primary fibroblasts and T lymphocytes to immobilized fibronectin (37, 38). The alternatively spliced region, TnFnIII A–D, induces loss of focal adhesions and inhibits proliferation of endothelial cells through interaction with annexin II (39, 40, 41), supports attachment of mouse embryonic neurones (34), mediates neurite outgrowth and guidance (42), and promotes proliferation of hemopoietic precursors cells in the bone marrow (36). The FRED of tenascin-C promotes endothelial cell adhesion and sprouting through interaction with {alpha}2{beta}1 and {alpha}V{beta}3 integrins (43, 44). We and other have reported that tenascin-C inhibits in vitro T cell activation induced by natural Ags (i.e., tetanus toxoid and purified protein derivative of tuberculin) or alloantigens (6), immobilized anti-CD3 mAb, and fibronectin (45) or by anti-CD28, ICAM-1, or laminin (46). The putative role of tenascin-C in inhibiting T cell activation in vivo is supported by the observation that tenascin-C-deficient mice develop severe dermatitis in response to hapten sensitization (47) as well as progressive inflammation and glomerular damage in an experimental model of venom-induced glomerulonephritis (48).

The tenascin-C region that suppresses T lymphocyte activation has not been identified yet. Here we report that the first two TnFnIII repeats of the alternative spliced region of tenascin-C inhibits in vitro T cell activation induced by anti-CD3 mAbs and immobilized fibronectin or soluble IL-2.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents

Human natural tenascin-C purified from U251-MG glioma cells, consisting mostly of the large isoform, was purchased from Life Technologies (Basle, Switzerland). Human recombinant large and small tenascin-C isoforms were kindly provided by Dr. L. Zardi (Istituto Nazionale dei Tumori, Genoa, Italy) (49). Recombinant chicken tenascin-C variants consisting of EGF repeats or FRED only were kindly provided by Dr. R. Chiquet-Ehrismann (Friedrich Miescher Institute, Basel, Switzerland) (34). The bacterial expression vector pET11 containing the cDNAs encoding for these fragments were kindly provided by Dr. H. Erikson (University of North Carolina, Chapel Hill, NC). Human plasma fibronectin was from Sigma (St. Louis, MO). Purified anti-CD3 mAb (clone UCHT1) was from Beckman Coulter (Fullerton, CA). PE-labeled anti-CD3 mAb (clone HIT3a), FITC anti-IFN-{gamma} mAb (clone 4S.B3), and PerCP-labeled anti-CD8 mAb (clone HIT8a) were obtained from BD PharMingen (BD Biosciences, Basle, Switzerland). IMDM and RPMI 1640 cell culture medium and FCS were obtained from Life Technologies. The medium supplement Nutridoma NS was from Roche Molecular Biochemicals (Rotkreuz, Switzerland). Human recombinant IL-2 (hrIL-2) was kindly provided by Dr. M. Nabholz (Swiss Institute for Experimental Cancer Research, Epalinges, Switzerland). Tissue culture plastic-ware were from Falcon (BD Biosciences). Nylon wool was from Polysciences (Eppelheim, Germany). Anti-6His mAb and Ni-NTA resin were purchased from Qiagen (Basle, Switzerland). The bacterial expression vector pRSET-A was from Invitrogen (Leek, The Netherlands), and pGEX-2T was from Amersham Pharmacia Biotech (Düberdorf, Switzerland). pGEM-Teasy vector was from Promega (Madison, WI). Isopropyl {beta}-D-thiogalactoside (IPTG) was from Eurogentec (Seraing, Belgium). 4',6'-Diamidino-2-phenylindole, dihydrochloride (DAPI) was obtained from Molecular Probes (Leiden, The Netherlands). Orthopermeafix was from Ortho (Raritan, NJ). Lysozyme, imidazole, brefeldin A, DNase I, Histopaque, and chloramphenicol were obtained from Sigma. [methyl-3H]Thymidine was from Hartmann Analytic (Zurich, Switzerland). 51Cr was from NEN (Zaventem, Belgium).

Production of recombinant human tenascin-C fragments

The human recombinant tenascin-C fragments used in initial experiments were previously described by Aukhil et al. (11). TnFnIII 1–8 contains all the constant TnFnIII repeats. TnFnIII All includes eight constant TnFnIII repeats and seven alternatively spliced repeats (A1, A2, A3, A4, B, C, and D) inserted between TnFnIII repeats 5 and 6. TnFnIII 1–5 consists of the first five constant TnFnIII repeats. TnFnIII 6–8 consists of the last three constant TnFnIII repeats. TnFnIII A–D contains the seven alternatively spliced domains. Expression and purification of recombinant proteins were conducted as described elsewhere (11, 38). To produce recombinant proteins containing consecutive TnFnIII repeats of the alternative spliced region, we generated the corresponding cDNAs by PCR using pET11-TnFnIII A–D as template and the primers shown in Table IGo. Restriction sites were included in the primers to facilitate directional cloning. PCR products were first cloned into the pGEM-Teasy vector for confirmatory sequencing (Swiss Institute for Experimental Cancer Research sequencing core facility) and subcloned into the BamHI and HindIII sites of the pRSET-A expression vector. For protein expression, single colonies of BL21(DE3)LysS cells transformed with the different expression constructs were grown at 37°C in 5 ml LB medium containing 200 µg/ml ampicillin and 35 µg/ml chloramphenicol for 8 h. Bacteria were then centrifuged, resuspended in 1 l LB medium (containing 200 µg/ml ampicilin), and cultured at 37°C until the OD600 reached 0.5–0.6, and induction was started by adding 1 mM IPTG. When OD600 reached 0.8–1.0, bacteria were collected by centrifugation and resuspended in 12 ml lysis buffer (50 mM NaH2PO4 (pH 8.0), 300 mM NaCl, 20 mM imidazole). Lysis of bacteria was conducted by treatment with 10 µg/ml lysozyme, 10 cycles of sonication (10 s/cycle), and 5 cycles of freezing in dry ice and thawing at 37°C. This suspension was centrifuged at 15,000 x g for 30 min at 4°C, and the supernatant was mixed with Ni-NTA slurry (5 ml resin/1 ml lysate) and incubated under agitation for 1 h at 4°C. The cell lysate/Ni-NTA slurry mixture was packed into a column and washed with 2 x 15 ml washing buffer (50 mM NaH2PO4 (pH 8.0), 300 mM NaCl, 20 mM imidazole). Recombinant proteins were eluted using elution buffer (50 mM NaH2PO4 (pH 8.0), 300 mM NaCl, 250 mM imidazole). The fractions containing the purified proteins (as determined by SDS-PAGE) were dialyzed against 1000 volumes of PBS. All recombinant proteins except TnFnIII A2A3A4 were purified under native conditions. TnFnIII A2A3A4 was purified under denaturing conditions by resuspending the induced bacteria in 12 ml 8 M urea, 0.1 M NaH2PO4, 10 mM Tris (pH 8.0) (lysis buffer II). Cells were then lysed by 10 sonication cycles and 5 freezing and thawing cycles. The soluble fraction was then mixed with the Ni-NTA resin (5 ml resin/1 ml lysate) for 1 h at 4°C. The resin was packed into a column, washed twice with 15 ml washing buffer II (8 M urea, 0.1 M NaH2PO4, 10 mM Tris (pH 6.3)), and eluted with 4 x 2 ml elution buffer II (8 M urea, 0.1 M NaH2PO4, 10 mM Tris (pH 5.9)) and 4 x 2 ml elution buffer III (8 M urea, 0.1 M NaH2PO4, 10 mM Tris (pH 4.5)). Eluted proteins were refolded by dilution into a folding buffer (50 mM Tris (pH 7.5), 500 mM NaCl, 10 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, and 2 mM DTT), concentrated using a membrane concentrator, and subsequently dialyzed against PBS. These refolding conditions have been successfully used to refold TnFnIII repeats to their native conformation (50).


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  		Table I. PCR primers used for the generation of recombinant proteins issued from the alternatively spliced domain of human tenascin-C

 
Single TnFnIII repeats were expressed as GST fusion proteins to gain stability. Inserts coding for the single TnFnIII repeats were generated by PCR using specific primers (Table IGo). PCR products were subcloned into pGEM-Teasy, sequenced, and subcloned into the BamHI and HindIII sites of a modified pGEX-2T vector containing a sequence coding for an N-terminal GST and a C-terminal 6His tag (pGEXT-6His). These constructs were then used to transform XL1B cells. Expression of recombinant proteins was conducted as above except that induction was conducted in 200 µM IPTG overnight at 30°C. These recombinant proteins were purified under denaturing conditions and subsequently refolded by dilution as described above for fragment TnFnIII A2A3A4.

Purified recombinant proteins were analyzed for relative molecular mass, purity, and the absence of degradation by SDS-PAGE/Coomassie blue staining and Western blotting analysis.

T cell isolation and activation

Venous blood from healthy donors was collected using a Vacutainer (BD Biosciences) and anticoagulated with lithium heparin. PBMC were isolated by density gradient centrifugation on Histopaque 1.077. PBMC were resuspended in RPMI 1640 with 10% FCS and incubated on plastic 25-cm2 culture flasks for 1 h at 37°C to remove monocytes by adherence. T lymphocytes were enriched using nylon wool columns as described (51). Purified T lymphocytes were >90% CD3+ as assessed by flow cytometry. T cells were washed three times with RMPI 1640 with 10% FCS and resuspended in IMDM supplemented with 1% Nutridoma NS.

For anti-CD3 mAb/fibronectin-mediated T cell activation, 96-well tissue culture plates were coated with the anti-CD3 mAb UCHT1 (1 µg/ml in PBS) overnight at 4°C. Wells were then washed once with PBS and incubated with plasma fibronectin (10 µg/ml in PBS) for 3 h at 37°C. For anti-CD3 mAb/IL-2-mediated T cell activation, wells were coated only with anti-CD3 mAb. Wells were then washed twice before T lymphocytes were added (1.5 x 105 cells/well in IMDM containing 1% Nutridoma NS) alone or together with natural tenascin-C or recombinant tenascin-C fragments. For anti-CD3 mAb/IL-2-mediated T cell activation, 20 U/ml hrIL-2 were added with the cells. After 3 days of culture at 37°C under 5% CO2, 1 µCi [methyl-3H]thymidine was added to each well during the last 16 h of culture to detect DNA synthesis.

For alloantigen-induced activation (MLR), 105 PBMC from two nonmatched donors were mixed in each well. T cell proliferation was quantified at day 4 by adding 1 µCi [methyl-3H]thymidine to each well during the last 8 h of culture. [methyl-3H]Thymidine incorporation was quantified by harvesting cells onto glass fiber filters (Wallac, Turku, Finland) and counting using a scintillation counter (Wallac).

Flow cytometry analysis

T lymphocytes were cultured for 24 h in the presence or absence of recombinant tenascin-C fragments in 24-well tissue culture plates previously coated with anti-CD3 mAb UCHT1 (1 µg/ml in PBS) and fibronectin (10 µg/ml in PBS). Cells were then collected and incubated with PE-labeled anti-CD3 (HIT3a, 20 µg/ml) and PerCP-labeled anti-CD8 (HIT3b, 20 µg/ml) mAbs for 20 min at 4°C. Cells were then washed twice with PBS, 1% BSA and resuspended in PBS. DAPI (10 µg/ml) was added to cell suspension 10 min before acquisition with a FACSCalibur cytofluorometer and CellQuest software (BD Biosciences). Typically, 105 events were acquired.

Cytokine production

TNF secretion by activated T cells (52) was measured in the culture supernatants after 72 h of culture by a bioassay using the TNF-sensitive cell line WEHI-164 as described (53). Production of intracellular IFN-{gamma} was measured by flow cytometry. T cells were activated for 6 h with immobilized anti-CD3 mAbs and fibronectin in the presence of 10 µg/ml brefeldin A to inhibit cytokine secretion. Cells were then collected and fixed/permeabilized for 40 min at room temperature using Orthopermeafix solution. After three washes with PBS, 1% BSA, the cell suspension was incubated with for 30 min at 4°C with 10 µg/ml FITC-conjugated anti-IFN-{gamma} mAb. After two washes with PBS, 1% BSA, cells were resuspended in PBS and analyzed on a FACScan using CellQuest software (BD Biosciences).

SDS-PAGE and Western blotting

For protein analysis, 50-µl samples were resolved by 7.5–12.5% SDS-PAGE following standard protocols and stained with Coomassie brilliant blue R250 (Bio-Rad, Glattbrugg, Switzerland). For Western blotting, material resolved by SDS-PAGE was blotted onto Immobilon-P membranes (Millipore, Volketswil, Switzerland). Membranes were sequentially incubated in 5% dry milk for 1 h at room temperature, with anti-6His mAb (1/1000; Clontech, Palo Alto, CA) for 2 h at room temperature, and with 1 µg/ml HRP-labeled secondary Ab (DAKO, Zug, Switzerland) for 1 h at room temperature. The ECL system was used for detection (Amersham Pharmacia Biotech).

CTL assay

Human CD8+ T cell clones specific for peptides Melan-A 26–35 (EAAGIGILTV) and tyrosinase 368–376 (YMDGTMSQV) were derived from melanoma patients as previously described (54, 55). Ag-specific cytolytic activity of T cell clones in the presence of tenascin-C recombinant fragments was measured using the chromium release assay as described (56). Briefly, target cells (T2, a lymphoblastoid cell line) were labeled with 51Cr in Tris-Dulbecco buffer supplemented with 2 mg/ml BSA for 1 h at 37°C. Labeled target cells (1000 cells in 50 µl) were then incubated with various concentrations of the antigenic peptide (50 µl) for 15 min at room temperature before the addition of CTL clones at a E:T ratio of 10:1. 51Cr release into the culture supernatant was measured after incubation for 4 h at 37°C using a Packard scintillation counter (Packard BioScience, Zurich, Switzeland). The percent specific lysis was calculated as: 100 x [(experimental - spontaneous release)/(total - spontaneous release)].


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
A recombinant tenascin-C fragment encompassing the alternatively spliced domain TnFnIII A–D inhibits T lymphocyte activation

Natural tenascin-C subjected to plasmin-mediated proteolysis retains its ability to inhibit T lymphocyte activation elicited by tetanus toxoid or alloantigens (57), raising the possibility that the immunosuppressive activity may be ascribed to a well-defined region of the molecule. On the basis of these considerations, we initiated experiments aimed at mapping the immunosuppressive region on tenascin-C by using recombinant fragments encompassing various regions of the molecule. To this purpose, we tested the effect of recombinant tenascin-C fragments on the activation of freshly purified peripheral blood T cells (>90% CD3+) stimulated with plastic-immobilized anti-CD3 mAb and fibronectin or with immobilized anti-CD3 mAb and soluble hrIL-2. T cell activation was determined after 72 h culture in serum-free medium by measuring DNA synthesis. Both T cell activation assays were sensitive to inhibition caused by soluble natural human tenascin-C purified from the U251-MG glioma cell line (Fig. 1Go) at concentrations similar to those reported by other using the same assay (45, 46). This inhibitory effect was specific for tenascin-C, because addition of equivalent amounts of soluble plasma fibronectin had no effect on anti-CD3 mAb/fibronectin-mediated T cell activation (Fig. 1GoA) while it had a costimulatory effect on anti-CD3 mAb/hrIL-2 induced activation (Fig. 1GoB).



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  		FIGURE 1. Natural tenascin-C inhibits T lymphocyte activation. T lymphocytes were activated by immobilized anti-CD3 mAb and fibronectin (A) or by immobilized anti-CD3 and rhIL2 (B) in the presence of the indicated amounts of soluble plasma fibronectin and glioma-derived tenascin-C. T cell activation was evaluated by measuring [methyl-3H]thymidine incorporation at 72 h after activation. Data are representative of three independent experiments, and each point is the mean of quintuplicate determinations (SD <=15%).

 
We initiated the structural-functional analysis of tenascin-C by testing the effects of the EGF repeats and of the FRED on T cell activation. To this purpose, we used recombinant tenascin-C molecules expressed in mammalian cells consisting of the coiled-coils-forming region and the EGF repeats or the FRED only (34). Consistently, these molecules did not inhibit T cell activation (data not shown). The EGF repeats or the FRED were therefore not further investigated, and we concentrated our efforts on the analysis of the TnFnIII domains. A bacterial recombinant protein containing all 15 TnFnIII repeats, i.e., the 8 constant and the 7 alternatively spliced repeats, referred to as TnFnIII All, efficiently inhibited T cell activation elicited by both anti-CD3 mAbs/fibronectin and anti-CD3/hrIL-2 (Fig. 2Go, A and B). In contrast a recombinant protein encompassing the eight constant TnFnIII repeats (TnFnIII 1–8) was ineffective, suggesting that the immunosuppressive activity was contained within the alternatively spliced region of tenascin-C. To collect direct evidence supporting this possibility, we tested a recombinant fragment consisting of the alternatively spliced TnFnIII repeats (referred to as TnFnIII A–D) and recombinant fragments containing the constant TnFnIII repeats 1–5 (TnFnIII 1–5) or 6–8 (TnFnIII 6–8). These experiments demonstrated that TnFnIII A–D efficiently inhibited T cell proliferation elicited by anti-CD3 mAb/fibronectin or anti-CD3/hrIL-2, whereas TnFnIII 1–5 or TnFnIII 6–8 did not have any inhibitory activities (Fig. 2Go, C and D). Taken together, these results indicate that the inhibitory activity on T cell activation previously described for natural tenascin-C is contained within its alternatively spliced region.



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  		FIGURE 2. A recombinant tenascin-C fragment corresponding to the alternatively spliced region of tenascin-C inhibits T lymphocyte activation. T lymphocytes were activated by immobilized anti-CD3 mAb and fibronectin (A and C) or anti-CD3 mAb and rhIL2 (B and D) in the presence of the recombinant tenascin-C fragments encompassing the fibronectin type III domains: TnFnIII All, 1–8, 1–5, A–D, and 6–8. T cell activation was evaluated by measuring [methyl-3H]thymidine incorporation at 72 h after activation. Data are representative of five independent experiments, and each point is the mean of quintuplicate determinations (SD <=15%).

 
TnFnIII A1A2 repeats are essential to inhibit T lymphocyte activation

The alternatively spliced region of tenascin-C consists of seven TnFnIII domains, i.e., A1, A2, A3, A4, B, C, and D. To identify which one of these domains is essential for suppressing T cell activation, we produced recombinant proteins encompassing three consecutive TnFnIII repeats in an overlapping manner (see Fig. 3GoA). These proteins were generated using the pRSET-A expression vector and contained a 6His tag at their N-terminal ends enabling purification by Ni-NTA affinity chromatography (Fig. 3GoB). Recombinant proteins were not toxic for T lymphocytes or the human lymphoblastoid cell line T2 as assessed by trypan blue exclusion, by chromium release assay, and by DAPI staining and flow cytometry analysis (data not shown). When tested in the anti-CD3 mAb/fibronectin T cell activation assay, fragments TnFnIII A1A2A3 and TnFnIII A2A3A4 inhibited T cell activation in a dose-dependent manner, whereas fragments TnFnIII A3A4B, TnFnIII A4BC, and TnFnIII BCD were ineffective (Fig. 3GoC). These data indicate that the TnFnIII domains A1 and A2 play a crucial role in mediating the immunosuppressing activity of tenascin-C.



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  		FIGURE 3. Inhibition of T cell activation by recombinant proteins issued from the alternatively spliced domain of tenascin-C. A, Schematic diagram of the structure of small recombinant proteins encompassing three adjacent domains of the TnFnIII A–D region. B, SDS-PAGE analysis of the purified material to demonstrate the correct size and purity of the recombinant proteins. The gel is stained with Coomassie blue. C, Effect of the small recombinant proteins issued from the TnFnIII A–D region on T cell activation elicited by anti-CD3 mAbs and fibronectin. T cell activation was evaluated by measuring [methyl-3H]thymidine incorporation at 72 h after activation. Data are representative of five independent experiments, and each point is the mean of quintuplicate determinations (SD <=15%).

 
To seek further evidence supporting the role of the repeats A1 and A2 on the immunosuppressive function of tenascin-C, we produced TnFnIII A–D lacking the TnFnIII domains A1 (TnFnIII A–D{Delta} A1), A1, and A2 (TnFnIII A–D{Delta}A1A2) or A1, A2, and A3 (TnFnIII A–D{Delta}A1A2A3), as well as a recombinant fragment consisting of domains A1 and A2 only (termed TnFnIII A1A2; see Fig. 4AGo). The 6His-tagged TnFnIII A–D protein purified by Ni-NTA affinity chromatography inhibited T cell activation (Fig. 4GoC) like the untagged TnFnIIIA–D, purified by ammonium sulfate precipitation and MonoQ chromatography, suggesting that the 6His tag did not contribute to inhibit T cell activation. The deletion mutants TnFnIII A–D{Delta}A1, TnFnIII A–D{Delta}A1A2, and TnFnIII A–D{Delta}A1A2A3 did not inhibit T cell activation. Fragment TnFnIII A1A2 inhibited T cell activation by 60% at 800 nM, whereas fragment TnFnIII A1A2A3 effectively blocked activation by >90% at the same dose (Fig. 4GoC).



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  		FIGURE 4. The TnFnIII A1A2 domain inhibits T cell activation. A, Schematic diagram of the structure of the recombinant deletion mutants and the two-domains protein issued from the TnFnIII A–D region. B, SDS-PAGE analysis of the purified material to demonstrate the correct size and purity of the recombinant proteins. The gel is stained with Coomassie blue. C, Effect of the recombinant TnFnIII A1A2 protein and deletion mutants issued from the TnFnIII A–D region on T cell activation elicited by anti-CD3 mAbs and fibronectin. T cell activation was evaluated by measuring [methyl-3H]thymidine incorporation at 72 h after activation. Data are representative of three independent experiments, and each point is the mean of quintuplicate determinations (SD <=15%).

 
To identify which of these two TnFnIII domains was essential for suppressing T cell activation, we generated recombinant proteins consisting of the single TnFnIII repeat A1, A2, and A3. These domains were expressed as GST fusion proteins to assure optimal expression and stability and carried a 6His tag at their C terminus (Fig. 5GoA). Addition of GST-TnFnIII A1, GST-TnFnIII A2, or GST-TnFnIII A3 fusion proteins, or combinations thereof, to the anti-CD3 mAb/fibronectin T cell activation assays did not have any detectable inhibitory effect (Fig. 5GoC).



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  		FIGURE 5. Single-domain TnFnIII A1 or TnFnIII A2 does not inhibit T lymphocyte activation. A, Schematic diagram of the structure of the GST-TnFnIII single-domain fusion proteins. B, SDS-PAGE analysis of the purified material to demonstrate the correct size and purity of the recombinant proteins. The gel is stained with Coomassie blue. C, Effect of GST-TnFnIII fusion proteins on T cell activation elicited by anti-CD3 mAb and fibronectin. T cell activation was evaluated by measuring [methyl-3H]thymidine incorporation at 72 h after activation. Data are representative of three independent experiments, and each point is the mean of quintuplicate determinations (SD <=15%).

 
Taken together, these results demonstrate that TnFnIII A1A2 is the smallest region of tenascin-C that retains the ability to inhibit in vitro T lymphocyte activation. Maximal inhibition, however, required the presence of the TnFnIII domain A3.

TnFnIII A–D and TnFnIII A1A2A3 inhibit activation-induced down-modulation of the TCR complex

Tenascin-C inhibits T cell activation when present at time of stimulation but not when cells are already proliferating (6), suggesting that it affects early events in the T cell activation cascade. To collect direct evidence supporting this possibility, we studied the effect of recombinant tenascin-C fragments on activation-induced internalization of the TCR/CD3 complex, a phenomenon also known as TCR down-modulation. This is one of the earliest events following TCR/CD3-dependent T cell activation, and it was shown to closely reflect the magnitude of the resulting activation (58). CD3 expression at the T lymphocyte surface was measured by staining cells with FITC-labeled anti-CD3 mAb (clone HIT-3a) and flow cytometry analysis 24 h after stimulation with immobilized anti-CD3 mAb and fibronectin. T cell activation by anti-CD3 mAb and fibronectin caused a complete down-regulation of CD3 from the cell surface (Fig. 6Go). Addition of soluble TnFnIII A–D or TnFnIII A1A2A3 to the T cell activation assays greatly suppressed activation-induced CD3 down-regulation. In contrast, addition of the noninhibitory fragment TnFnIII A–D{Delta}A1A2A3 had only minimal effects on CD3 down-modulation (Fig. 6Go). The addition of TnFnIII A–D and TnFnIII A1A2A3 also affected the activation-induced down-regulation of CD8, whereas TnFnIII A–D{Delta}A1A2A3 had no any effect (Table IIGo).



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  		FIGURE 6. Recombinant TnFnIII A–D and TnFnIII A1A2A3 inhibit TNF (A) and IFN-{gamma} (B) production by T lymphocytes activated with immobilized anti-CD3 mAb and fibronectin. TNF was measured in the cell culture supernatant 72 h after activation using the WEHI-164 bioassay. IFN-{gamma} was detected intracellularly (iIFN{gamma}) by staining permeabilized T cells with a direct labeled anti-IFN-{gamma} mAbs 6 h after activation, analysis by flow cytometry. Values are expressed as mean fluorescent intensity (MFI). Data are representative of three independent experiments.

 

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  		Table II. Flow cytometry analysis of CD3 and CD8 expression by T lymphocytes activated in the presence of TnFnIII recombinant proteins1

 
The alternatively spliced domain of tenascin-C inhibits activation-induced cytokine production by activated T lymphocytes

TNF and IFN-{gamma} are two major cytokines produced by activated T lymphocytes which play a critical role in the development of cellular immunity and in the activation of CTLs (59). We therefore measured the effect of the inhibitory tenascin-C fragments on the release of TNF and IFN-{gamma} by activated T cells. T lymphocyte activation by immobilized anti-CD3 mAb/fibronectin in the presence of the recombinant fragments TnFnIII A–D and TnFnIII A1A2A3 resulted in the complete inhibition of TNF release, whereas fragment TnFnIII A–D{Delta}A1A2A3 had no effect. The inhibition was complete, because the levels of TNF released by T cells activated in the presence of these fragments were identical with those found in the supernatant of nonactivated cells (Fig. 7GoA). To assess the effect of these tenascin-C fragments on IFN-{gamma} production, we measured the expression of intracellular IFN-{gamma} (iIFN-{gamma}) in resting and activated T cells. The expression levels of iIFN-{gamma} in T cells activated with anti-CD3 mAb/fibronectin in the presence of TnFnIII A–D or TnFnIII A1A2A3 were lower than iIFN-{gamma} levels in T cells activated in the absence of tenascin-C fragments or in the presence of TnFnIII A–D{Delta}A1A2A3 (Fig. 7GoB).



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  		FIGURE 7. Recombinant TnFnIII A–D and TnFnIII A1A2A3 prevent activation-induced TCR/CD3 complex down-modulation. T lymphocytes were stimulated with immobilized anti-CD3 mAbs and fibronectin in medium alone or in the presence of 800 nM TnFnIII A–D, TnFnIII A1A2A3 or TnFnIII A–D{Delta}A1A2A3. After 24 h of culture cells were collected, stained with direct-labeled anti-CD3 mAbs, and analyzed by flow cytometry. , Fluorescence in the absence of Abs. Data are representative of three independent experiments.

 
These data indicate that the alternatively spliced region of tenascin-C and the smaller fragment TnFnIII A1A2A3 efficiently suppress the expression of TNF and IFN-{gamma} cytokines

The alternatively spliced domain of tenascin-C does not affect cytolytic activity

We have previously reported that natural tenascin-C does not affect the cytolytic effector function of T cells (6). We therefore assessed the effect of the above characterized recombinant tenascin-C fragment on the lysis of target cells by CTL clones specific for the TAA Melan-A or anti-tyrosinase using the chromium release assay. None of the recombinant fragments tested (i.e., TnFnIII A–D, TnFnIII A1A2A3, and TnFnIII A–D{Delta}A1A2A3) had any effects on the lysis of T2 target cells by Melan-A or tyrosinase-specific CTL clones (Fig. 8Go). These results demonstrate that the tenascin-C fragments do not inhibit cytotoxic activity of T cells and confirm our original data obtained with natural tenascin-C.



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  		FIGURE 8. The recombinant tenascin-C fragments TnFnIII A–D and TnFnIII A1A2A3 do not inhibit the cytolytic activity of CTL clones. The cytolytic activity of a CTL clone specific for tyrosinase (clone 34; A) or Melan-A (clone 0.3/3; B) was tested against T2 target cells labeled with tyrosinase peptide at increasing concentrations in the absence of presence of recombinant TnFnIII fragments as indicated. Cytolytic activity was measured using the 51Cr release assay. Results are expressed as percent specific lysis (100 x (experimental - spontaneous release/total - spontaneous release)). Data are representative of two independent experiments, and each point is the mean of triplicate determinations (SD <10%).

 
In addition, neither TnFnIII A–D nor TnFnIII A1A2A3 affected down-regulation of CD3/CD8 in tyrosinase specific CTL clones activated with specific peptides, or in Melan-A and flu-specific CTL clones incubated on immobilized anti-CD3 mAbs and fibronectin (data not shown). This suggested that TnFnIII A–D inhibits activation only of ex vivo peripheral blood T lymphocytes but not of preactivated Ag-specific CTL clones, consistent with the hypothesis that tenascin-C affects the earliest events of T cell activation.

The large but not the small tenascin-C isoform inhibits T lymphocytes activation

To confirm that the TnFnIII A–D region suppresses T cell activation in the context of the whole tenascin-C molecule, we tested the effects of two full length recombinant tenascin-C molecules produced in mammalian cells, the first containing the TnFnIII A–D domain (tenascin-C large isoform) and the second lacking it (tenascin-C small isoform) (49) on alloantigen-induced T cell activation. In these experiments, the large but not the small isoform suppressed T cell activation in a dose-dependent manner (Fig. 9Go).



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  		FIGURE 9. The large but not the small tenascin-C isoform inhibits T lymphocyte activation. In this assay, T cell activation was induced in a mixed leukocyte reaction (alloantigen-induced activation) using PBMC from two nonmatched donors, in the absence or presence of full length recombinant large or small tenascin-C isoforms. T cell activation was evaluated by measuring [methyl-3H]thymidine incorporation at 96 h after activation. Data are representative of two independent experiments.

 
Thus, these results confirm the results obtained with glioma-derived natural tenascin-C (consisting mostly of the large isoform) and are consistent with the essential role of TnFnIII A–D in the inhibition of T cell activation.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This work was initiated with the purpose of identifying the region of tenascin-C responsible for the inhibition of T cell activation previously reported by us and others (6, 45, 46). Here we report that: 1) the alternatively spliced region of tenascin-C (i.e., TnFnIII A–D) is essential for inhibiting T cell activation; 2) within TnFnIII A–D, TnFnIII A1A2 is the minimal region retaining the ability to inhibit T cell activation; and 3) the TnFnIII A–D region and derived fragments also suppress activation-induced TCR down-regulation and TNF and IFN-{gamma} secretion by freshly isolated peripheral blood T cells but do not inhibit neither activation-induced CD3 and CD8 down-regulation nor cytolytic activity of Ag-specific CTLs clones.

The fibronectin type III repeats region is one of the best studied regions of tenascin-C, and a number of activities have been attributed to it. In general, the TnFnIII domains 1–5 and 6–8 promote cell adhesion, spreading, and migration of many different cells, including glioma (35), hemopoietic cells (36), endothelial cells (43), neurones (60), and mammary epithelial cells (61). The third TnFnIII repeat bears binding sites for integrin {alpha}V{beta}3, {alpha}V{beta}6, and {alpha}9{beta}1 (62) and thereby plays a key role in promoting integrin-dependent cell adhesion and migration. The alternatively spliced region, TnFnIII A–D, supports attachment of neurons (34), induces neurite outgrowth and guidance (42), and promotes proliferation of hemopoietic precursor bone marrow cells (36). TnFnIII A–D was also reported to promote the dissolution of focal adhesions and to inhibit proliferation of endothelial cells by binding to annexin II (39, 40, 41). Within the TnFnIII A–D, TnFnIII A1–A4 promotes neurite outgrowth, whereas TnFnIII D may play a role in neurite guidance (63).

The main contribution of this work is the identification of the TnFnIII domains A1A2 as the smallest region of tenascin-C able to inhibit T cell activation. Although this result implicates for the first time a specific region of tenascin-C in its immunosuppressive activity, it also raises new questions about the role of the individual TnFnIII domains in mediating this effect. The fact that TnFnIII domains A1A2 are active when expressed together but not when expressed individually (and tested either alone or together) suggests that the formation of the inhibitory domain requires the concerted action of at least two contiguous TnFnIII repeats. Strikingly, TnFnIII A1 is essential for the inhibitory activity of the seven-domain fragment TnFnIII A–D, but not of a shorter fragment consisting of three domains only (i.e., TnFnIII A2A3A4), suggesting that the physical context in which the TnFnIII repeats are present may also have a profound influence on the formation of this activity. This concept is further supported by the observation that addition of domain A3 substantially enhances the inhibitory activity of TnFnIII A1A2. Also, it has been reported that TnFnIII repeats are highly elastic (64) and can undergo conformational changes in response to tensional forces transduced by adjacent domains, resulting in altered affinity for the cognate receptors (65, 66). Also, the A1–A4 domains in human tenascin-C are >90% homologous to each other as are generated by a recent reduplication of one single fibronectin type III repeat (67). This raises the possibility that some redundant or overlapping activities may exist within these highly homologous domains.

The mapping of the immunosuppressive domain of tenascin-C within TnFnIII A–D is of considerable interest in the context of tumor stroma and tissue remodeling. Indeed, the tenascin-C large variant is highly expressed in the stroma of malignant tumors, a site in which T cells have been shown to be immunosuppressed (5). Strikingly, the alternatively spliced region of tenascin-C is highly sensitivity to degradation by matrix metalloproteinases and serine proteinases (68). This may represent a mechanism by which to modulate the immunosuppressive activity of tenascin-C at tissue sites. This possibility is also consistent with the presence of numerous potential sites for N-glycosylation within the TnFnIII repeats A1, A2, A3, and A4 (69). Carbohydrate side chains may protect this region against proteolytic processing and thereby promote its immunosuppressive activity. Thus, changes in tenascin-C glycosylation and proteolytic activities might contribute to the control of tenascin-C degradation and thereby to the regulation of its immunosuppressive activity at tissue sites. In addition, one important conclusion from these findings is that the previously described ability of TnFnIII 1–5 to inhibit T cell adhesion to fibronectin (38) does not interfere with T cell activation (70).

Although we present here evidence showing that the TnFnIII domains A1 and A2 are critically involved in inhibiting T cell activation, we still have little knowledge on how this effect is mediated. To further understand the molecular mechanism by which tenascin-C inhibits T cell activation, it will be essential to identify the receptor and signaling pathway that are specifically engaged or inhibited by tenascin-C. The observation that recombinant tenascin-C fragments inhibit activation-induced TCR down-regulation may provide useful insights into this question. It has been proposed that the extent of TCR down-regulation is determined by the number of TCR triggered in response to activatory stimuli and closely reflects the magnitude of the induced T cell effector functions such as cytokine production and proliferation (71). Two distinct early signaling events are involved in T cell activation and TCR down-modulation: the tyrosine kinases p59fyn and p56lck; and the protein kinase C pathways (72). Because tenascin-C does not prevent T cell activation induced by the synthetic protein kinase C agonist PMA (6, 45), the possibility that tenascin-C interferes with p59fyn and/or p56lck-dependent signaling events should be explored. Because both p59fyn and p56lck also promote CD8 association with the TCR complex (73), this hypothesis would be consistent with the suppressed CD8 down-regulation by tenascin-C. Intriguingly, there is direct experimental evidence indicating that p59fyn and p56lck function is abrogated in T lymphocytes of tumor-bearing mice (74). Alternatively, the small GTP-binding protein Rho should also be considered as a putative target of the tenascin-C effect. Inhibition of Rho in T lymphocytes suppresses T cell proliferation induced by anti-CD3 mAbs and fibronectin (75), and tenascin-C was shown to suppress Rho activation in fibroblasts (76).

In conclusion, we have identified the TnFnIII A1A2 domain within the alternatively spliced region of tenascin-C as the smallest region of the molecule able to suppress activation-induced T cell proliferation and cytokine secretion. This results will be helpful to further study the immunosuppressive activity of tenascin-C, in particular to identify the receptor and signaling pathway affected by tenascin-C, as well as to design and generate antagonistic molecules to neutralize tenascin-C immunosuppressive activity in malignant tumors. Such a reagent may prove helpful to increase the effectivity of anticancer immunotherapy approaches that are currently being developed and tested in the clinics.


   Acknowledgments
 
We thank Dr. F. J. Lejeune for continuous support, Drs. L. Zardi and R. Chiquet-Ehrismann for providing recombinant tenascin-C protein, Dr. H. P. Erickson for providing tenascin-C expression plasmids, P. Batard for help in flow cytometry analysis, and N. Montandon for help with the CTL assay and TNF bioassay.


   Footnotes
 
1 This work was supported by grants from the Swiss National Science Foundation (31-52946.97) and the Swiss Cancer League (943-09). C.R. is recipient of a SCORE-A award (32-41611.94) from the Swiss National Science Foundation. Back

2 Address correspondence and reprint requests to Dr. Curzio Rüegg, Laboratory of the Centre Pluridisciplinaire d’Oncologie, c/o Swiss Institute for Experimental Cancer Research, 155 Chemin des Boveresses, CH-1066 Epalinges, Switzerland. E-mail address: curzio.ruegg{at}isrec.unil.ch Back

3 Abbreviations used in this paper: TAA, tumor-associated Ag; DAPI, 4',6'-diamidino-2-phenylindole, dihydrochloride; ECM, extracellular matrix; EGF, epidermal growth factor; FRED, fibrinogen-related domain; TnFnIII, fibronectin type III repeat of tenascin-C; IPTG, isopropyl {beta}-D-thiogalactoside; hrIL-2, human recombinant IL-2; iIFN-{gamma}, intracellular IFN-{gamma}. Back

Received for publication June 14, 2001. Accepted for publication October 3, 2001.


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