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IL-10 Induces Regulatory T Cell Apoptosis by Up-Regulation of the Membrane Form of TNF- ¹

Luciano E. Marra^*, Zhu X. Zhang^*, Betty Joe^*, Jon Campbell^*, Gary A. Levy^*, Josef Penninger and Li Zhang^2,*

^* Departments of Laboratory Medicine and Pathobiology, Immunology and Multi Organ Transplantation Program, Toronto General Research Institute, and Ontario Cancer Institute, University Health Network, University of Toronto, Toronto, Ontario, Canada

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Numerous studies have demonstrated the role of regulatory T (Treg) cells in peripheral tolerance. Nevertheless, how the survival and death of Treg cells is controlled is largely unknown. In this study, we investigated the mechanisms involved in regulating the homeostasis of a subset of Ag-specific TCR⁺ CD4^-CD8^- double negative (DN) Treg cells. We demonstrate that DN Treg cells are naturally resistant to TCR cross-linking-induced apoptosis. Administration of exogenous IL-10 renders DN Treg cells susceptible to apoptosis, and abolishes their suppressive function. Furthermore, TCR cross-linking of DN Treg cells in the presence of IL-10 leads to the up-regulation of the membrane-bound but not the soluble form of TNF-. Interaction of membrane bound TNF- with TNFR2 sends death signals to DN Treg cells. Blocking their interaction can reverse the effects of IL-10 on DN Treg cells. These results provide insights into the mechanisms that regulate the function and homeostasis of DN Treg cells.

	Introduction

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A growing body of evidence indicates that regulatory T (Treg)³ cells play an important role in controlling immune responses to autoantigens, alloantigens, tumor Ags, and infectious agents. Many results obtained from animal studies have clearly indicated the beneficial role of Treg cells in preventing the development of organ-specific autoimmune diseases, graft rejection and allergy (1, 2, 3, 4, 5). However, an increase in the number and function of Treg cells can hamper the immunity to tumor Ags and infectious agents (1, 4). It is therefore very important to understand how to control the number and the function of Treg cells. Studies on Treg cells have been focused on their function and mechanisms of action, and little is understood about the mechanisms involved in controlling their homeostasis.

A variety of Treg cells have been described, including CD4⁺, CD8⁺, CD4^-CD8^- double negative (DN) -TCR⁺ cells, -TCR⁺ cells, NK cells, and NKT cells (6, 7). The most extensively studied Treg cells are CD4⁺CD25⁺ T cells, which play an important role in preventing the development of autoimmune diseases and allograft rejection (1, 2, 3, 4, 5). Recently we have identified an TCR⁺NK1.1^-CD4^-CD8^- DN T cell population in mice that exhibits immune regulatory capacity both in vitro and in vivo (8). These peripheral DN Treg cells are able to specifically suppress the activity of both CD8⁺ and CD4⁺ T cells, prevent allograft and xenograft rejection, and attenuate graft-vs-host disease (8, 9, 10, 11, 12). Furthermore, DN T cells have been shown to regulate immune responses in autoimmune disease models (13, 14) and in infectious disease models (15, 16). Unlike CD4⁺CD25⁺ Treg cells that execute their function in an Ag nonspecific manner (1, 2, 3, 4, 5) and are susceptible to apoptosis (17), the DN Treg cells can down-regulate allogeneic immune responses in an Ag-specific manner both in vitro and in vivo (8, 9, 10, 11). Furthermore, following the infusion of allogeneic splenic cells, we found that although the CD8⁺ T cells underwent activation and vigorous clonal expansion followed by activation-induced cell death (AICD), the number of DN Treg cells increased slowly but steadily after encountering alloantigens in vivo (8, 9, 10, 11, 12). Moreover, we have demonstrated that the DN Treg clones generated from mice that permanently accepted allogeneic skin grafts after pretransplant donor lymphocyte infusion were resistant to TCR cross-linking-induced apoptosis in the presence of exogenous IL-4 (18).

Although much is known about the factors that regulate apoptosis of CD4⁺ and CD8⁺ T cells, relatively little is understood about the mechanisms that regulate apoptosis of DN Treg cells. Numerous studies have indicated that cytokines play a critical role in the regulation of the function, growth and differentiation, maintenance, and demise of T lymphocytes (19, 20, 21). For instance, IL-2 is critical for T cell survival and proliferation. However, high concentrations of IL-2 may induce apoptosis of activated T cells (20, 22). Furthermore, many studies have demonstrated that IL-4 protects different cells from a variety of apoptotic stimuli (18, 23, 24, 25, 26). We have demonstrated that DN Treg cells require the presence of exogenous IL-2 and IL-4 to survive and proliferate in vitro and that IL-4 protects these cells from TCR cross-linking-induced apoptosis (18). Our recent studies indicate that unlike some CD4⁺ Treg cells, which produce and require IL-10 for their regulatory function (27, 28, 29, 30, 31, 32), DN Treg cells do not express IL-10 mRNA (8). Furthermore, preincubation of DN Treg cells with exogenous IL-10 can abolish their suppressive function in vitro (10).

IL-10 is a pleiotropic cytokine that can be produced by various types of cells, including Th0, Th1, Th2, CD8⁺ T lymphocytes, B lymphocytes, monocytes and keratinocytes (33). It has been well documented that IL-10 mediates its action on a wide variety of cell types, and can have both stimulatory and inhibitory effects on the immune responses (33). For instance, IL-10 can be a potent suppressor of macrophages, Th1 cells, B cells, and NK cells (33). Furthermore, IL-10 has been demonstrated to play an important role in controlling autoimmune diseases (34, 35, 36). In contrast, IL-10 has been shown to stimulate the growth of mast cells, B and T lymphocytes, enhance the ability of CD4⁺ T cells to produce IL-2, IL-4, IFN-, and TNF-, and promote adaptive immunity (33). Moreover, many studies indicate that IL-10 can aggravate graft rejection (37, 38, 39). In addition, over-expression of IL-10 associated with aberrant DN T cell function is strongly associated with manifestations of some autoimmune lymphoproliferative disorders in both patients and genetically altered mouse models of autoimmunity (40, 41, 42). Despite numerous researches it remains unclear as to why the same cytokine can have apparent opposite effects on immune responses.

The specific objectives of this study were to determine the nature of resistance to apoptosis by DN Treg cells, and to delineate the molecular mechanism by which IL-10 inhibits the function of DN Treg cells. Our data demonstrate that DN Treg cells obtained from both tolerant and naive mice are resistant to TCR cross-linking-induced apoptosis. IL-10 abrogates the suppressive function of DN Treg cells by enhancing their susceptibility to TCR cross-linking-induced cell death, which involves up-regulation of the membrane form of TNF- (mTNF-) expression. The interaction between the mTNF- and TNFR2 sends death signals to DN Treg cells. These findings provide insights into the mechanism by which IL-10 regulates the function and homeostasis of Ag-specific DN Treg cells. Furthermore, the apoptotic resistant nature of these cells makes the DN Treg cells a practical candidate for long-term cellular therapy of immune-mediated diseases.

	Materials and Methods

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Mice

C57BL/6 (B6, H-2^b), (B6 x BALB/c)F₁ (B6, H-2^b/d L^d+), and BALB/c H-2^dm2 (dm2, a BALB/c L^d loss mutant, H-2D^d+, K^d+, L^d-) mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Breeding stock of 2C transgenic mice (on B6 background) was kindly provided by Dr. D. Y. Loh (Nippon Research Center, Kanagawa, Japan). A large fraction of T cells in the periphery of the 2C mice express a transgenic -TCR reactive against the MHC class I L^d (8). These transgenic T cells can be detected by the clonotypic mAb 1B2 (8). All animals were maintained in the animal facility at the Ontario Cancer Institute (Toronto, Ontario, Canada).

Antibodies

1B2 mAb was produced in our laboratory from the 1B2 hybridoma, which was kindly provided by Dr. H. Eilson (Massachusetts Institute of Technology, Cambridge, MA). The anti-mouse CD3 mAb was produced in our laboratory by using the 145-2c11 hybridoma. CD3-FITC, CD4-PE, CD8-CyChrome (all from eBioscience, San Diego, CA), anti-mouse TNF- (Pierce, Rockford, IL), TNFR1 (p55) (T55-593.4), TNFR2 (p75) (T75-54.7.14), Fas ligand (BD Biosciences, San Jose, CA), and -actin (Sigma-Aldrich, St. Louis, MO) were purchased and used according to the manufacturers’ instructions. HRP-conjugated anti-rabbit and anti-mouse Abs were purchased from Bio-Rad (Hercules, CA).

Purification of naive DN and CD8⁺ T cells

Spleen cells collected from C57BL/6 mice were stained with FITC-conjugated anti-CD3, PE-conjugated anti-CD4, and CyChrome-conjugated anti-CD8 mAbs. CD3⁺CD4^-CD8^- and CD3⁺CD4^-CD8⁺ T cells were purified using a cell sorter (Coulter Epicx V; Corixa, Seattle, WA). The purity and viability of the cells after sorting was >95%.

Generation and maintenance of DN and CD8⁺ T cells and clones

To generate DN and CD8⁺ T cell clones, DN and CD8⁺ T cells were purified from the spleen of naive (2C x dm2)F₁ or the (2C x dm2)F₁ mice that permanently accepted L^d mismatched skin allografts from (B6 x BALB/c)F₁ donor mice and standard cloning and subcloning procedures were used as previously described (8). To maintain the 1B2⁺DN Treg and 1B2⁺CD8 clones, 2 x 10⁵ clone cells/ml were cultured in flasks containing 2 x 10⁶/ml irradiated (20 Gy) L^d+ cells in -MEM supplemented with 10% FCS and 50 U/ml rIL-2. The cells were incubated at 37°C with 5% CO₂. The T cell clones were stimulated in the manner previously described every 3–4 days. To generate activated CD8⁺ T cells, 7.5 x 10⁵ cells/ml (2C x dm2)F₁ splenocytes were cocultured with 7.5 x 10⁵ cells/ml irradiated (20 Gy) splenocytes from (B6 x BALB/c)F₁ for 5 days at 37°C and 5% CO₂ in -MEM supplemented with 10% FCS and 50 U/ml rIL-2 and 25 U/ml rIL-4. Viable cells, of which >95% were CD8⁺, were isolated using Lympholyte M (Cedarlane Laboratories, Hornby, Ontario, Canada) for further analysis.

Suppression assay

Naive splenic (2C x dm2)F₁ 1B2⁺CD8⁺ T cells (1000 cells/well) were cocultured in 96-well plates with irradiated (20 Gy) sex-matched splenocytes (3 x 10⁵ cells/well) from (B6 x BALB/c)F₁ mice in -MEM supplemented with 10% FCS and 50 U/ml of rIL-2 and 25 U/ml rIL-4. 1B2⁺DN Treg clones were either preincubated with 10 or 100 ng/ml rIL-10 (kindly provided by Dr. D. Spaner, University of Toronto, Toronto, Ontario, Canada) for 5 days, washed, or left untreated before being added to MLR as suppressor cells. After a 3-day incubation, 1 µCi [³H]TdR was added to each well. Eighteen hours later, cells were harvested and cell proliferation was measured by [³H]TdR incorporation. Cultures to which no DN T cells were added were used as controls.

Induction of apoptosis by TCR cross-linking

TCR cross-linking was performed as previously described (18). Briefly, 1B2⁺DN Treg clones, 1B2⁺CD8⁺ T cell clones, and primary activated DN Treg or CD8⁺ T cells were plated in 24-well culture plates (1.25 x 10⁵ cells/well), which were precoated with either 1B2 or anti-CD3 mAb (65 µg/ml) that specifically recognizes the TCR on these cells, or with isotype-matched control Abs. All cells were supplemented with rIL-2 (50 U/ml) and rIL-4 (25 U/ml) during TCR cross-linking in the presence or absence of exogenous recombinant mouse IL-10. Numbers of viable and dead cells were measured using trypan blue exclusion at various time points after cross-linking. Apoptotic cell death was further confirmed by flow cytometric analysis using propidium iodide and annexin V surface staining, as further described.

Flow cytometric analysis

Following TCR cross-linking, DN Treg cells or CD8⁺ cells were either dual stained with FITC-conjugated recombinant annexin V in combination with propidium iodide or FITC-conjugated recombinant annexin V alone (both from BioSource International, Camarillo, CA) and the percentage of apoptotic cells was determined by flow cytometry. To examine surface TNF- expression, DN Treg cells were stained with biotinylated anti-mouse TNF- followed by PE-conjugated streptavidin (both by Pierce). Hamster anti-mouse TNFR1 and TNFR2 Abs followed by anti-hamster FITC-conjugated Ab were used to examine surface expression of TNF receptors. Data were acquired and analyzed on an EPICS XL-MCL flow cytometry machine (Coulter).

Detection of TNF- mRNA by Northern blot analysis

The PCR-Select cDNA subtraction kit (Clontech Laboratories, Palo Alto, CA) was used to subtract mRNA isolated from DN Treg cells cross-linked in the presence of IL-10 from the mRNA of DN Treg cells cross-linked in the absence of IL-10. After subcloning, the differentially expressed cDNA was sequenced on an ABI Prism 377 DNA Sequencer (Applied Biosystems, Foster City, CA) and subjected to homology searches using BLAST software. The TNF- probe was synthesized using inserts isolated from cloned plasmids. After isolation on agarose gels, the probes were labeled with [³²P]dCTP using the T7 Quick Prime kit (Pharmacia Biotech, Uppsala, Sweden). For Northern blot analysis total RNA (15 µg) was extracted with TRIzol reagent (Life Technologies, Grand Island, NY) from the DN Treg cells (5 x 10⁶ cells) that were TCR cross-linked in the presence or absence of IL-10 according to the manufacturer’s instructions. The RNA samples were denatured with formaldehyde, resolved by electrophoresis on a 1.2% agarose gel, transferred to Hybond-N membrane (Amersham Pharmacia Biotech, Piscataway, NJ), and covalently linked by UV irradiation. Hybridizations were performed at 65°C overnight using standard protocols. Hybridization signals were analyzed by autoradiography performed with intensifying screens at -70°C overnight. As a loading control the membrane was stripped and reprobed with a GAPDH cDNA. Densitometry was performed using gel analysis software from the Computational Molecular Biology Resources Center at the National Institutes of Health (Bethesda, MD).

Western blot analysis

DN Treg cells were collected after TCR cross-linking with or without IL-10, washed with PBS, and lysed using RIPA buffer (1% Nonidet P-40, 150 mM NaCl, 50 mM Tris-HCl, pH 8.0, 50 mM NaF, 2 mM EDTA, 1 mM sodium orthovanadate and 0.05% NaN₃) containing 0.1% aprotinin, 0.1% leupeptin, and 1 mM PMSF as protease inhibitors. Total protein (20 µg) from each sample was denatured in SDS sample loading buffer containing 2-ME, resolved by SDS-PAGE on 12% polyacrylamide gels and transferred (2 h, 230 mA) to Hybond-P polyvinylidene difluoride membranes (Amersham Pharmacia Biotech). The membranes were blocked (1 h, room temperature) with 5% milk and Tween 20 in TBS-25 mM Tris, pH 7.5, 0.15 M NaCl, 0.05% Tween 20. After blocking, the blots were incubated (4°C, overnight) with the appropriate primary Abs, washed and subsequently incubated (1 h at room temperature) with appropriate secondary Abs. Membrane fractions were similarly treated but prepared by sonication using a B. Braun Melsungen AG Braun-sonic 1510 sonicator (400 watts, 3 bursts, 20 s). Hybridization signals were visualized using the Western Lightning Chemiluminescence Reagent Plus kit (PerkinElmer, Wellesley, MA) after exposure to Kodak X-Omat Blue x-ray film (Rochester, NY).

TNF- antisense blockade

Phosphorothioate antisense (5'-CATGCTTTCTGTGCTCATGGTGTC-3') and nonsense oligomers (5'-GAGCGCCTATGAGTTGACTCCG-3') were generated at the DNA Synthesis Facility, Center for Applied Genomics, (Toronto, Ontario, Canada). DN Treg cells (5 x 10⁶) were electroporated (260 kV, 960 µF) with 50 µg of TNF- antisense oligomers or nonsense oligomers. Immediately after electroporation the cells were incubated in prewarmed (37°C) -MEM supplemented with 10% FCS and 50 U/ml rIL-2 and allowed to recover for 30 min before TCR cross-linking. Soluble and membrane fractions were examined by Western blot analysis.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Ag-specific DN Treg clones are resistant to TCR cross-linking-induced apoptosis

Previously we have shown that Ag-specific DN Treg cell clones, generated from tolerant mice that permanently accepted an allograft after donor lymphocyte infusion, are highly resistant to apoptosis induced by cross-linking of their TCR (18). To explore whether the apoptotic resistant phenotype is a feature intrinsic to DN Treg cells, or acquired as a consequence of encountering Ag in vivo, DN Treg cell clones were generated from naive (CN04) and tolerant (TN02, TN12) mice, and subjected to TCR cross-linking in 24-well culture plates containing immobilized anti-TCR mAb. Primary activated CD8⁺ T cells and the CD8⁺ T cell clone (T01) were used as controls. At 24, 48, 72, and 96 h after cross-linking of the TCR, the cells were harvested and cell viability was determined by trypan blue exclusion. As shown in Fig. 1a, both primary activated CD8⁺ T cells and CD8⁺ T cell clones were susceptible to TCR cross-linking-induced death as >70% cell death was found by 96 h. In contrast, <30% death was observed in all the DN Treg cells from both tolerant and naive mice (Fig. 1a). At all time points DN Treg cell death induced by TCR cross-linking was significantly less than that observed in CD8⁺ cells similarly treated (Fig. 1).

View larger version (26K): [in this window] [in a new window]   FIGURE 1. DN Treg cells are resistant to TCR cross-linking-induced apoptosis. a, Primary activated CD8+ T cells, CD8+ T cell clone TO1 (dotted line), and various DN Treg clones TN02, TN12, CN04 (solid line) from (2C x dm2)F1 mice were TCR cross-linked in 24-well tissue culture plates precoated with the clonotypic anti-TCR mAb, 1B2. The cells were collected after 24, 48, 72, and 96 h of cross-linking (CxL), and viability was analyzed using trypan blue exclusion. Noncross-linked CD8+ T and DN Treg cells served as controls. b, (2C x dm2)F1 CD8+ T cells (left panel) and the TN12 DN Treg clone (right panel) were cross-linked as in a, collected after 24 h of cross-linking, stained with FITC-labeled annexin V and analyzed using flow cytometry. The percentage of dead cells was compared with noncross-linked CD8+ and TN12 cells, respectively. Values are given as mean percentage of death ± SD from five independent experiments.

IL-10 reduces the suppressive function of DN Treg cells by rendering them susceptible to TCR cross-linking-induced apoptosis

IL-10 has been shown to be important for the activation and function of some Treg cells (27, 28, 29, 30, 31, 32). We have shown that DN Treg cells do not produce IL-10 (8). To study the effect of exogenous IL-10 on DN Treg cells, the DN Treg cell clone TN12 was pretreated with IL-10 before being used as suppressor cells in a standard suppression assay. Fig. 2a demonstrates that the ability of the DN Treg cells to suppress the proliferation of CD8⁺ T cells was significantly diminished after IL-10 treatment compared with nontreated DN Treg cells (p < 0.01). Similar results were obtained with various DN Treg cell clones (data not shown). These data indicate that IL-10 can inhibit the regulatory function of DN Treg cells.

View larger version (21K): [in this window] [in a new window]   FIGURE 2. IL-10 abrogates DN Treg cell-mediated suppression of CD8+ cells by rendering them susceptible to TCR cross-linking-induced apoptosis. a, 1B2+CD8+ T cells were purified from the spleen of (2C x dm2)F1 mice and stimulated with irradiated (B6 x BALB/c)F1 splenocytes in an MLR. Varying numbers of DN Treg clone TN12 cells were either left untreated () or incubated with IL-10 and washed () before being used as suppressor cells in the MLR. Cell proliferation was measured by [3H]TdR incorporation. The results represent three independent experiments each with three replicates. The suppressive ability of DN Treg cells was significantly inhibited in the presence of IL-10 when compared with those in the absence of IL-10 (p < 0.01). Similar results were obtained when other DN Treg cell clones were used as suppressors. b, Activated DN Treg clone TN12 cells () were left untreated, TCR cross-linked, cultured in the presence of IL-10, or both as indicated. CD8+ T cells () were either left untreated or TCR cross-linked and served as controls. The cells were collected after 96 h of cross-linking, and viability was analyzed using trypan blue exclusion. Values are given as mean percentage of death ± SD from at least three independent experiments. c, Primary activated DN Treg cells isolated from C57BL/6 spleens were cross-linked using anti-CD3 mAb in the presence () or absence () of IL-10. Cell viability was determined by trypan blue exclusion at 24 h intervals and values are given as mean percentage of death ± SD from at least three independent experiments.

To eliminate the possibility that the observed phenomenon was specific to T cell clones, similar experiments were conducted using primary activated DN splenocytes. Fresh DN T cells were purified from the spleens of B6 mice and stimulated with irradiated BALB/c splenocytes. The primary activated DN Treg cells were subjected to TCR cross-linking in the presence or absence of IL-10, or incubated with IL-10 without TCR cross-linking. When cell viability was assessed at various time points it was found that primary activated DN Treg cells were also resistant to TCR cross-linking-induced cell death (Fig. 2c). By 96 h after cross-linking <32% of the primary activated DN Treg cells underwent apoptosis compared with 60% death (p < 0.05) of primary DN Treg cells that were cross-linked in the presence of IL-10 (Fig. 2c), which is similar to that observed in the DN Treg cell clones (Fig. 2c). Together these data demonstrate that DN Treg cell clones generated from both tolerant and naive mice as well as primary DN Treg cells are resistant to TCR cross-linking-induced cell death, and IL-10 enhances the susceptibility of DN Treg cells to TCR cross-linking-induced cell death.

IL-10 up-regulates TNF- expression in cross-linked DN Treg cells

To identify genes that are up-regulated in DN Treg cells by IL-10 during TCR cross-linking, we used subtractive hybridization technologies. DN Treg cells were subjected to TCR cross-linking for 5 h in the presence or absence of IL-10 and the mRNAs were purified. After subtraction of the cross-linked DN Treg cell cDNA in the absence of IL-10 from those in the presence of IL-10, differentially expressed genes were subcloned and sequenced. cDNA subtraction and differential screening indicated that TNF- was up-regulated when DN Treg cells were cross-linked in the presence of IL-10. Because the TNF- pathway is involved in T cell death (43), we examined by Northern blot analysis whether IL-10 could sensitize the DN Treg cells to apoptosis by altering the TNF- expression. When the DN Treg cells were cross-linked in the presence of IL-10 the level of TNF- mRNA was up-regulated by 4.2-fold as determined by densitometry compared with the cells that were cross-linked in the absence of IL-10 (Fig. 3). This finding suggests that IL-10 may sensitize DN Treg cells to apoptosis through up-regulation of TNF-.

View larger version (16K): [in this window] [in a new window]   FIGURE 3. IL-10 up-regulates TNF- RNA expression in cross-linked DN Treg cells. Total RNA (15 µg/lane) from the DN Treg clone TN12 was collected after 5 h cross-linked with immobilized anti-TCR mAb in the presence or absence of IL-10 and resolved on 1% agarose gels and transferred to nitrocellulose. TNF- mRNA expression was demonstrated using 32P-labeled TNF- oligonucleotide probes and visualized after overnight exposure to x-ray film. GAPDH hybridization confirms equal loading. Densitometry demonstrates that TNF- mRNA expression was up-regulated in the DN Treg cells by 4.2-fold in the presence of IL-10 compared with control cells cross-linked in the absence of IL-10.

Treatment with antisense TNF- oligonucleotides reduces IL-10-induced DN Treg cell apoptosis

TNF- is expressed as a transmembrane protein mTNF-, which is released as a soluble cytokine sTNF- by proteolysis. To further confirm that IL-10 sensitizes DN Treg cells to apoptosis through up-regulation of TNF-, we attempted to block the synthesis of TNF- at the transcriptional level. DN Treg cells were pre-exposed to antisense TNF- oligonucleotides before TCR cross-linking in the presence of IL-10. Nonsense oligonucleotide-treated DN Treg cells served as controls. Western blot analysis of DN Treg cells demonstrated that both sTNF- and mTNF- proteins were significantly down-regulated by antisense TNF- compared with controls (Fig. 4a). Furthermore, cell death was reduced significantly (p < 0.001) in antisense-treated DN Treg cells compared with nonsense-treated controls (Fig. 4b). These data further support the hypothesis that IL-10 induced DN Treg cell death involves up-regulation of TNF-.

View larger version (37K): [in this window] [in a new window]   FIGURE 4. IL-10 induced DN Treg cell death can be blocked with antisense TNF- oligonucleotides. a, Activated DN Treg cells were pretreated with 5 µM antisense TNF- oligonucleotides (right) or nonsense oligomers (left) and then TCR cross-linked in the presence of IL-10 for 2 h. Soluble and membrane protein fractions (20 µg/lane) from the above treated samples were collected and resolved on 12% SDS-PAGE gels. TNF- expression was demonstrated by immunoblotting with murine anti-TNF- Ab. -actin and the transgenic TCR (detected by 1B2 mAb) served as a loading control for the soluble and membrane fractions, respectively. b, Blocking TNF- reverses the effect of IL-10 on DN Treg cells. Activated DN Treg cells cross-linked in the presence of IL-10 were pretreated with antisense TNF- ( ) or nonsense oligonucleotides () and cell viability was determined at 96 h using trypan blue exclusion. Values are given as mean percentage of death ± SD from at least three independent experiments.

IL-10 increases mTNF- but not sTNF- expression on DN Treg cells during cross-linking

Previously we have shown that DN Treg cells produce significant levels of sTNF-, which appears to have little effect on their own demise (8, 18). Moreover, the addition of exogenous sTNF- during cross-linking has no effect on DN Treg cell death (18). We therefore hypothesized that IL-10 may increase the susceptibility of DN Treg cell to TCR cross-linking induced apoptosis by up-regulating the expression of mTNF-. To this end, DN Treg cells were cross-linked in the presence or absence of IL-10. Both membrane and soluble fractions of DN Treg cells were collected at 24 h after TCR-cross-linking. The expression of TNF- was examined by Western blot analysis. Fas ligand on the membrane and -actin in the soluble fraction were used as loading controls. Immunoblotting with anti-TNF- mAb showed that mTNF- was up-regulated which correlated with a concomitant decrease in sTNF- in the DN Treg cells that were cross-linked in the presence of exogenous IL-10 (Fig. 5a). Flow cytometric analysis confirmed that the cell surface expression of TNF- was increased on DN Treg cells cross-linked in the presence of IL-10 compared with those cells cross-linked in the absence of IL-10 (Fig. 5b). These results demonstrate that IL-10 can increase membrane bound, but not sTNF- in DN Treg cells during TCR cross-linking.

View larger version (33K): [in this window] [in a new window]   FIGURE 5. IL-10 increases the expression of membrane but not the soluble form of TNF- by DN Treg cells. a, The DN Treg clone TN12 was cross-linked for 24 h supplemented with IL-10. Both soluble and membrane fractions were collected and subjected to Western blot analysis. Immunoblotting with anti-TNF- mAb confirms that IL-10 promotes the up-regulation of mTNF- during TCR cross-linking. -actin and Fas ligand (FasL) served as loading controls for the soluble and the membrane fractions, respectively. b, The surface expression of TNF- was confirmed using PE-labeled murine anti-TNF- and flow cytometric analysis.

TNF- has been shown to be a potent inducer of death in various cell types, including CD4⁺ and CD8⁺ T cells, through its interaction with the cell surface TNFR1 and TNFR2. Both mTNF- and sTNF- can bind to both TNFR1 and TNFR2 membrane receptors. Because the DN Treg cells produce high level of TNF- yet are highly resistant to TCR cross-linking-induced apoptosis, we first wanted to see whether their apoptotic resistant phenotype was due to an impairment of the TNF/TNFR pathway. Apoptosis-resistant DN Treg cells were collected at 1, 2, 3, and 4 days after TCR cross-linking and their expression of TNFR1 and TNFR2 was compared with L929 cells, which are known to express both of the receptors, by flow cytometric analysis. Fig. 6 shows that apoptosis-resistant DN Treg cells express a high level of TNFR2 3–4 days after TCR-cross linking. However no TNFR1 could be detected 1–3 days after TCR-cross linking, and only minimal levels of TNFR1 were detected 4 days after TCR cross-linking. Furthermore, we confirmed that the addition of exogenous sTNF- during TCR cross-linking did not alter the number of dead cells at any time point compared with DN Treg control cells cross-linked in the absence of sTNF- (data not shown). These data suggest that neither sTNF- nor the TNFR1 play a significant role in regulating DN Treg cell death. Furthermore, the observed high-level expression of TNFR2 suggests that TNFR2 may play a predominant role in the IL-10-induced cell death of DN Treg cells.

View larger version (26K): [in this window] [in a new window]   FIGURE 6. DN Treg cells express high levels of TNFR2 during TCR cross-linking. The apoptosis-resistant DN Treg clone TN12 were collected at 1, 2, 3, and 4 days after TCR cross-linking and stained with hamster anti-mouse TNFR1 and TNFR2 mAb followed by goat anti-hamster FITC. L929 cells that express both TNFR1 and TNFR2 and TN12 cells that were stained with goat anti-hamster FITC alone were used as positive and negative controls, respectively. Data were analyzed by flow cytometry. Data shown are the cells collected at 4 days after TCR cross-linking and are representative of two independent experiments.

Interaction between mTNF- and TNFR2 is critical for IL-10-induced apoptosis of DN Treg cells

To investigate whether blocking the interaction of mTNF- with TNFR2 could prevent IL-10-induced DN Treg cell apoptosis, neutralizing anti-TNFR2 mAb was added to DN Treg cells during cross-linking in the presence or absence of IL-10. At 24, 48, 72, and 96 h after cross-linking of the TCR, the DN Treg cells were harvested and cell viability was determined. DN Treg cells cross-linked either in the presence of IL-10 or the anti-TNFR2 mAb served as controls. Fig. 7 shows that TNFR2 blockade during TCR cross-linking effectively reversed the IL-10-induced apoptosis of the DN Treg cells. These data demonstrate that blocking the interaction of mTNF- with TNFR2 prevents IL-10-induced DN Treg cell apoptosis, and suggests that mTNF- promotes DN Treg cell death in an autocrine suicide or paracrine fratricide manner through TNFR2.

View larger version (12K): [in this window] [in a new window]   FIGURE 7. Membrane-bound mTNF-, not soluble sTNF-, promotes IL-10-induced apoptosis of DN Treg cells. Blocking the interaction between mTNF- and TNFR2 abrogates IL-10-induced DN Treg cell death during TCR cross-linking. Activated DN Treg cells were pretreated with neutralizing TNFR2 Ab (10 µg/ml), supplemented with IL-10, followed by TCR cross-linking (). Cross-linked DN Treg cells supplemented with IL-10 alone () or neutralizing TNFR2 Ab alone () served as controls. Cell viability was determined by trypan blue exclusion at 24 h intervals, and values are given as mean percentage of death ± SD from at least three independent experiments.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

It is well known that the effects of IL-10 on immune responses can be bidirectional (33). As reported, IL-10 can function as a potent immunosuppressive cytokine to down-regulate inflammatory and autoimmune responses (33). Several studies have shown that IL-10 can enhance the function of CD4⁺ Treg cells (28, 29, 30) and suppress organ-specific autoimmune diseases (1, 2, 4). In contrast, many studies have clearly shown that elevated levels of IL-10 result in autoimmunity (41, 42) and injection of IL-10 accelerates islet (44) and cardiac (37, 39) allograft rejection. Administration of IL-10 to cardiac transplant recipients exacerbated parenchymal rejection and graft arterial disease, which was associated with a concomitant rise in the number of CD8⁺ lymphocytes (39). Furthermore, IL-10 blockade improved heart allograft survival (38, 39). However, the mechanism by which IL-10 promotes graft rejection in these studies was not clear. We have demonstrated that DN Treg cells can enhance allograft and xenograft skin and cardiac graft survival by suppressing and killing anti-donor CD4⁺ and CD8⁺ T cells (8, 9, 10, 11, 12). In this study we have shown that IL-10 can inhibit the regulatory function of DN Treg cells by rendering them susceptible to TCR cross-linking-induced cell death (Fig. 2). Together, these data suggest that IL-10 may exacerbate allograft rejection by rendering DN Treg cells susceptible to apoptosis so that they cannot suppress anti-donor T cells. Our findings may help to explain the contradictory results reported in studies attempting to manipulate IL-10 levels during various in vivo experimental paradigms (37, 38, 39). They also suggest that the resulting effect of IL-10 on immune responses is cell type- and milieu-dependent.

Studies have shown that IL-10 can induce or potentiate apoptosis in various cell types, including human monocytes, B chronic lymphocytic leukemia cells, Langerhans cells, Th1 cells, and dendritic cells (33). The mechanisms by which IL-10 promotes apoptosis have not been clearly identified. Several studies on various cell types suggest that IL-10 acts as a negative regulator of TNF- (45, 46, 47, 48). TNF- is a pleiotropic cytokine that is involved in the death of a variety of cell types (49). TNF- is first produced as a 26-kDa mTNF-, which is cleaved by TNF-converting enzyme to generate 17-kDa sTNF- monomers that are then secreted and form bioactive homotrimers (50, 51). Studies have shown that both the soluble and membrane portions of TNF- are biologically functional (52, 53). Furthermore, studies indicate that mTNF- plays an important role in various immune responses (54, 55). Although much is known about the role of sTNF- in apoptosis, an understanding of the role of mTNF- in causing cell death remains limited. Monastra et al. (55) showed that mTNF- on CTL is involved in killing tumor cells through direct cell-cell contact. Whether mTNF- is involved in the cell death of lymphocytes was unknown. In this study, we demonstrate that the addition of exogenous IL-10 during cross-linking only up-regulated mTNF- but not sTNF- (Fig. 5), suggesting that mTNF- may be an important mechanism by which DN Treg cell homeostasis is controlled.

Both TNFR1 and TNFR2 have been ascribed critical proapoptotic activity (43, 56). Whereas the sTNF- preferentially binds to TNFR1, mTNF- preferentially binds to TNFR2 (56). Because DN Treg cells express high levels of TNFR2 but not TNFR1 (Fig. 6) and IL-10 up-regulates mTNF- expression (Fig. 5), DN Treg cells may be sensitized to cross-linking-induced cell death by the up-regulation of mTNF- via an autocrine suicide/paracrine fratricide signaling loop. The following observations support this hypothesis: 1) DN Treg cells produce high levels of sTNF- but are resistant to apoptosis (Figs. 3, 5a, and 2, b and c); 2) the administration of sTNF- during cross-linking did not increase DN Treg cell death (data not shown); 3) flow cytometry and Western blot analysis indicate that by 24 h after cross-linking in the presence of exogenous IL-10, the mTNF- on the surface of DN Treg cells was up-regulated compared with controls (Fig. 5); 4) antisense TNF- oligomers, which inhibit the expression of TNF-, completely abolished the death promoting effects of exogenous IL-10 (Fig. 4b); and 5) anti-TNFR2 mAb abrogated IL-10 induced DN Treg cell death (Fig. 7). Taken together, these results strongly suggest that IL-10 sensitizes the DN Treg cells to TCR cross-linking-induced cell death via mechanisms involving mTNF-. To the best of our knowledge this is the first report suggesting that IL-10 can promote apoptosis by up-regulation of mTNF- expression. This observation could help explain one aspect of the paradoxical roles of IL-10 in various cell types and microenvironments. For example, in cell types that are capable of expressing increased levels of mTNF- in response to elevated levels of IL-10, then the IL-10 may function as an immunosuppressive cytokine to these cells. If these cells are Ag-reactive cells, an immune response would be suppressed. Conversely, if the affected cells are immune regulatory cells, such as DN Treg cells, IL-10 might promote an immune response by inhibiting the function of regulatory cells. Whether other cell types respond to IL-10 by up-regulating mTNF- remains to be determined. Conversely, whether IL-10 acts as an immunostimulatory cytokine in those cell types that do not express high levels of mTNF- requires further study.

In summary, this report provides direct evidence indicating that DN Treg cells are resistant to TCR cross-linking-induced apoptosis, and that exogenous IL-10 inhibits the function of DN Treg cells by enhancing their susceptibility to apoptosis. Furthermore, we present compelling data indicating that IL-10 increases expression of the membrane form of TNF-. The interaction of mTNF- with TNFR2 may ultimately provide the autocrine/paracrine signaling loop that promotes the apoptosis of DN Treg cells. These results provide insights into the mechanisms involved in regulating the homeostasis of DN Treg cells.

	Acknowledgments

 
We thank Dr. Kevin Young for critically reading the manuscript and providing useful comments.

	Footnotes

 
¹ This work was supported by Grants from the National Cancer Institutes of Canada 12160 (to L.Z.), and Canadian Institutes of Health Research MT14431. L.Z. is a Clinical and Research Chair in Transplantation cosponsored by the Canadian Institutes of Health Research and Wyeth-Ayerst.

² Address correspondence and reprint requests to Dr. Li Zhang, The Toronto General Hospital, University Health Network, 621 University Avenue, NU-G001, Toronto, M5G 2C4 Ontario, Canada. E-mail address: lzhang{at}transplantunit.org

³ Abbreviations used in this paper: Treg, T regulatory; DN, double negative; mTNF, transmembrane TNF; sTNF, soluble TNF; AICD, activation-induced cell death.

Received for publication July 30, 2003. Accepted for publication November 7, 2003.

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