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FASEB J. Author manuscript; available in PMC 2011 February 24.
Published in final edited form as:
Published online 2010 September 8. doi:  10.1096/fj.10-166843
PMCID: PMC3005434
EMSID: UKMS31958

Naive and activated T cells display differential responsiveness to TL1A that affects Th17 generation, maintenance, and proliferation

Abstract

Tumor necrosis factor (TNF)-like cytokine (TL1A) is a T-cell costimulator that bolsters cytokine-induced activation through death receptor 3 (DR3). To explore the relationship between T-cell activation and TL1A responsiveness, flow cytometry profiled DR3 expression in resting and activated T cells. In human CD4+ T cells, DR3 was induced rapidly following activation and expressed prominently by interleukin (IL)-17-secreting T cells (Th17). Splenic T cells from wild-type and DR3-deficient mice showed that TL1A activation of DR3 inhibits Th17 generation (81±2.6% at 100 ng/ml TL1A) from naive T cells. This response was not associated with suppression of T-cell proliferation. Using neutralizing antibodies or T cells derived from genetically modified mice, TL1A inhibition of Th17 development was found to be independent of IL-2, IL-27, γIFN, IFNAR1, and STAT1. Under suboptimal TCR activation, TL1A continued to block IL-17A secretion, however, the reduced threshold of TCR engagement was now linked with an increase in TL1A-driven proliferation. In contrast, fully committed Th17 cells displayed an altered TL1A responsiveness and in the absence of TCR costimulation supported the maintenance of T cell IL-17A expression. Consequently, TL1A orchestrates unique outcomes in naive and effector T-helper cells, which may affect the proliferation, differentiation and maintenance of Th17 cells in peripheral compartments and inflamed tissues.—Jones, G. W., Stumhofer, J. S., Foster, T., Twohig, J.P., Hertzog, P., Topley, N., Williams, A. S., Hunter, C. A., Jenkins, B. J., Wang, E. C. Y., Jones, S. A. Naive and activated T cells display differential responsiveness to TL1A that affects Th17 generation, maintenance, and proliferation.

Keywords: cytokine, T-cell regulation, inflammation

Tumor necrosis factor (TNF)-like cytokine (TL1A; TNFSF15) has primarily been linked with a capacity to direct cytokine-mediated T-cell activation (1-5). Regulation of these TL1A-mediated responses occurs via death receptor 3 (DR3), a death-domain-containing receptor (TNFRSF25) that is expressed on activated T cells (3). T-cell DR3 expression coincides with the rapid induction of TL1A expression by activated dendritic cells, which implies that TL1A regulates T-cell activation and expansion during antigen presentation (6-8). Indeed, TL1A activation of DR3 results in enhanced NF-κB signaling capacity and is associated with altered apoptosis, increased GM-CSF expression, and regulation of γIFN production in response to IL-12 and IL-18 (2-5). The coordination of T-cell responses by TL1A is illustrated further by its capacity to regulate IL-2 responsiveness in activated T cells (3). In this respect, TL1A has been shown to increase IL-2-dependent T-cell proliferation through enhanced expression of IL-2 and its receptor subunits CD25 and CD122 (3). While TL1A was originally proposed to promote Th1-effector responses, more recent reports show that TL1A affects T-cell-mediated inflammatory diseases by regulating the proliferation and effector characteristics of both Th1 and Th17 cells (9, 10). DR3−/− mice show resistance to experimental autoimmune encephalomyelitis (EAE), OVA-induced lung inflammation, Crohn's disease, and experimental arthritis, while TL1A influences murine models of ileitis and colitis (2, 7, 11). Consistent with these observations, TL1A−/− mice have decreased clinical severity in EAE, and this might correlate with the ability of TL1A to promote the proliferation of effector Th17 cells (9). TL1A therefore represents a prominent T-cell costimulator with the capacity to steer T-cell expansion and commitment.

The differentiation of CD4+ T-helper cells into type 1 (Th1) and type 2 (Th2) subsets has classically been considered the primary mechanism for governing adaptive immunity during infection, inflammation, and allergic responses (12). However, this paradigm has been revised recently through characterization of a unique T-helper population, which is best defined by expression of the transcription factors RORγt and RORα, the receptors IL-23R and CCR6, and secretion of the cytokines IL-17A, IL-17F, and IL-22 (13-17). Experimental evidence suggests that these IL-17-producing T-helper cells (termed Th17 cells) play a central role in autoimmune conditions (18, 19). Considerable emphasis has therefore been placed on identifying factors responsible for directing Th17 expansion. Although IL-23 was initially thought to direct Th17 differentiation (13, 20), compelling evidence from murine T-cell studies endorses a role for IL-6 and IL-21 in steering the TGFβ-mediated differentiation of Th17 cells (21-26). From initial studies relating to the IL-23-mediated commitment of Th17 cells, it is evident that cytokines promoting Th1 (γIFN) and Th2 (IL-4) expansion not only antagonize each other but also suppress Th17 development (13, 16). Further studies have assigned IL-2, IL-27, type 1 interferons, and retinoic acid as negative regulators of Th17 polarization (27-31).

Costimulatory receptors for T cells provide necessary signals that enable activation or attenuation of T-cell responses following engagement by antigen-presenting cells. These costimulatory receptors typically belong to either the Ig (CD28-like) or TNF receptor (TNFR) superfamilies (32). Although CD28 and inducible T-cell costimulator have been linked to Th17 differentiation (16), the involvement of TNF family members remains unclear. The significance of these findings is highlighted further by an observed crosstalk among these costimulatory superfamilies, which influence effector function (33). Prior investigations have shown that TL1A can promote the expansion of Th17 cells, which might account for the protection provided by TL1A/DR3-deficiency in experimental disease models (7, 9, 11). However the inclusion of blocking anti-IL-2 antibodies in ex vivo studies and the apparent effect of TL1A on γIFN-, γIFN/IL-17A-, and IL-17A-secreting effector subsets, suggests a greater complexity to the action of TL1A in controlling T-cell responses. In this respect, we now build on these initial reports by demonstrating that TL1A differentially regulates distinct outcomes in naive (or resting) and activated T cells, which affect Th17 generation, maintenance and proliferation.

MATERIALS AND METHODS

Mice

Experiments were performed using age-matched 7- to 12-wk-old DR3−/− (5), IL-6−/−, γIFN−/−, IL-27R−/− (WSX-1 deficient), and STAT1−/− mice and genetically matched wild-type (WT) controls. DR3−/− mice were originally obtained from Cancer Research UK (London, UK). Mice deficient for γIFN were housed and bred at Charles River Laboratories (Margate, UK). IL-27R−/− (WSX-1 deficient) mice were originally from C. Saris (Amgen, Thousand Oaks, CA, USA).

Cell purification

Human peripheral blood mononuclear cells (PBMCs) were isolated from fresh whole blood by density gradient centrifugation using Lymphoprep (Axis-Shield, Kimbolton, UK), and the Naive CD4+ T-cell isolation kit (Miltenyi Biotec, Surrey, UK) was utilized to enrich for CD4+CD45RA+ T cells by magnetic cell sorting. Single-cell suspensions of splenocytes were prepared from homogenized spleens, and contaminating erythrocytes were removed by washing in lysis buffer (155 mM NH4Cl, 12 mM NaHCO3, and 1 mM EDTA, pH 7.3). Total CD4+ T cells were purified from splenocyte preparations by magnetic beads (CD4+ isolation kit; Miltenyi Biotec), and naive CD4+ T cells were purified using a MoFlo cell sorter (DakoCytomation, Glostrup, Denmark) to isolate CD4+CD25CD44loCD62Lhi cells.

In vitro expansion of murine Th17 cells

Cells were cultured in RPMI 1640, except where indicated that Iscove's modified Dulbecco medium (IMDM) was used. Media were supplemented with 10% (v/v) FCS, 2 mM l-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, 1 mM sodium pyruvate, and 50 μM β-mercaptoethanol (all from Invitrogen, Carlsbad, CA, USA). A total of 1 × 105 cells/well was cultured in 96-well plates (or 7×105 cells/well in 48-well plates) coated with anti-CD3 (1 μg/ml; R&D Systems, Minneapolis, MN, USA) and soluble anti-CD28 (5 μg/ml; BD Biosciences, San Jose, CA, USA) for 4 d. Where indicated, cultures were supplemented with the cytokines TGFβ1 (1 ng/ml, R&D Systems), IL-6 (20 ng/ml, R&D Systems), IL-23 (10 ng/ml, R&D Systems), IL-21 (100 ng/ml, R&D Systems), IL-27 (1–100 ng/ml, Amgen), and TL1A (1–100 ng/ml, R&D Systems). γIFN and IL-2 were blocked by addition of anti-γIFN (10 μg/ml, clone 37895; R&D Systems) and anti-IL-2 (10 μg/ml, clone JES6–1A12; R&D Systems) neutralizing antibodies. Type I interferon signaling was neutralized using a blocking anti-IFNAR antibody (10 μg/ml, clone MAR1–5A3). Cultures were split on d 3 and supplemented with fresh neutralizing antibodies.

Human T-cell cultures

Cells were cultured in RPMI 1640 supplemented with 10% fetal calf serum, 2 mM l-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, and 1 mM sodium pyruvate (all from Invitrogen). Cells (5 × 104/well) were cultured in 96-well plates containing plate-bound anti-CD3 (1 μg/ml; R&D Systems) and soluble anti-CD28 (5 μg/ml; BD Biosciences). Where indicated, cultures were supplemented with the cytokines IL-6 (50 ng/ml; R&D Systems), IL-1β (10 ng/ml; R&D Systems), and TL1A (1–100 ng/ml). All human participants gave written informed consent.

CD4+ T-cell proliferation studies

Purified CD4+ T cells were labeled with 10 μM 5 (6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) in PBS for 10 min at 37°C. An excess of ice-cold RMPI 1640 with 10% FCS was added to the cells to quench the reaction, and the cells were washed extensively. CFSE-labeled cells (1×105 cells/well) were cultured in IMDM containing TGFβ (1 ng/ml) and IL-6 (20 ng/ml) in 96-well plates coated with anti-CD3 (0.1 or 1 μg/ml as indicated) and soluble anti-CD28 (1 μg/ml). For detection of cytokine production, intracellular staining was performed as described below.

Flow cytometry

For use in flow cytometric analysis of murine T-cell cultures, anti-CD4 FITC (RM4-5), anti-IL-17A PE (TC11-18H10.1), and anti-IL-2 PE (JES6-5H4) were purchased from BD Biosciences; anti-CD4 eFluor 450 (RM4-5) was from eBioscience (Hatfield, UK); anti-IL-17A conjugated to FITC and Alexa Fluor 647 (TC11-18H10.1) were from BioLegend (Cambridge, UK); and anti-γIFN FITC (XMG1.2) and anti-CD4 APC (RM4-5) were from Invitrogen. Biotinylated goat anti-mouse IL-17F and Streptavidin PE were obtained from R&D Systems and Serotec (Kidlington, UK), respectively. Mouse cells were treated with mouse Fc block (BD Biosciences) prior to staining.

Anti-human CD4 PE (M-T477) was purchased from BD Biosciences; anti-human IL-17 Alexa Fluor 488 (eBio64DEC17), from eBiosciences, biotinylated anti-human DR3 (JD3), from Abcam; and Streptavidin-APC, from Beckman Coulter (Brea, CA, USA).

For detection of intracellular cytokine production, cells were treated with 50 ng/ml PMA and 500 ng/ml ionomycin, in the presence of 3 μM monensin (all Sigma, Dorset, UK) for 4 h at 37°C. Cells were stained for cell surface markers before fixation, and permeabilization in BD Cytofix/Cytoperm (BD Biosciences) was followed by intracellular staining for cytokines. Cells were collected on a FACSCalibur (Becton Dickinson, Franklin Lakes, NJ, USA) or CyAn ADP (Beckman Coulter) and further analyzed using FlowJo software (Tree Star, Ashland, OR, USA) or Summit (Beckman Coulter). For ex vivo cytokine analysis of T cells from inguinal lymph nodes, cells were harvested directly by homogenization and counted (Coulter Z2; Beckman Coulter). Intracellular cytokine staining was performed as above.

Cell culture supernatant measurement of cytokines

Cytokine production in cell culture supernatants was analyzed by ELISA using mouse IL-17A and human IL-17A DuoSet kits (all R&D Systems).

Statistics

Data are expressed as means ± se, and statistical analysis was performed using the Student's t test or 1-way analysis of variance (ANOVA) using Graph-Pad Prism software (Graph-Pad, San Diego, CA, USA). P < 0.05 was considered significantly different.

RESULTS

TL1A is a negative regulator of human IL-17-secreting T-helper cells

The novel TNF-like factor TL1A is best described for its ability to affect cytokine-mediated T-cell responses (3). Although TL1A has been shown to support IL-12-, IL-18-, and γIFN-mediated events (4), these activities appear only to complement the properties already assigned to these T-cell activating factors. To assign a direct function to TL1A, initial studies used flow cytometry to define the cellular expression of the TL1A receptor DR3 on human T cells. Although DR3 expression was not detected on resting CD4+ T cells, activation with stimulatory anti-CD3 antibodies led to a marked up-regulation in DR3 (Fig. 1A). Subsequent analysis highlighted that DR3 expression was most prominent on human CD4+ T cells actively secreting IL-17A (Fig. 1B). To evaluate whether DR3 signaling affects the development of IL-17-secreting T cells, changes in IL-17A expression in response to TL1A was monitored in naive human CD4+ T cells from 4 independent healthy donors (Fig. 1C). In all cases, the proportion of IL-17-secreting T cells observed following anti-CD3/anti-CD28 antibody costimulation was markedly suppressed by the action of human TL1A (Fig. 1C). Furthermore, the modest induction of human Th17 cell expansion observed following activation with IL-1β and IL-6 was again susceptible to inhibition by TL1A. Quantification of human IL-17A in culture supernatants further illustrated the potential of TL1A to block IL-17A secretion (Fig. 1C). Given the suppressive effects observed for TL1A on human Th17 cells, we predicted that mice deficient in DR3 would have an increased proportion of their T-helper cells displaying a Th17 phenotype. Initial in vitro studies with whole CD4+ cultures from WT and DR3−/− mice showed that cells from both strains can differentiate to Th17 cells under appropriate cytokine stimulation (Fig. 1D). However, consistent with the human data, T cells analyzed directly from the inguinal lymph nodes of unchallenged DR3-deficient mice identified a greater number of Th17 cells than WT mice (Fig. 1E). These data suggest that TL1A acts as a regulator of Th17 cells.

Figure 1
TL1A inhibits IL-17A secretion by human and mouse T-helper cells. A) Flow cytometry of DR3 expression on CD4+ T-cells from human PBMCs, cultured in the absence of TCR stimulation and with plate-bound anti-CD3 for 3 d. B) Flow cytometric analysis of DR3 ...

DR3 signaling suppresses murine Th17 and Tc17 development

To evaluate the role of DR3 signaling in Th17 development, splenic T-cell cultures were derived from DR3−/− mice and WT littermates (Fig. 2A). Splenocytes were cultured for 4 d in Th17-polarizing conditions with stimulatory anti-CD3 and anti-CD28 antibodies. As illustrated in Fig. 1D, Th17 commitment in response to IL-6 and TGFβ was comparable in cultures prepared from both genotypes. Supplementation of these cultures with TL1A, however, resulted in dose-dependent inhibition of Th17 polarization in cells derived from WT but not DR3-deficient mice (Fig. 2A, B). To determine whether TL1A affects other Th17-specific effector cytokines, T-cell IL-17F production was also examined (Fig. 2C). Again, TL1A was found to be equally effective at blocking development of both CD4+IL-17A+ and CD4+IL-17F+ cells. The inhibitory action of TL1A not only was evident in CD4+ Th17 cells, but also blocked IL-17A secretion by CD8+ T cells (Fig. 2D). Next we tested whether TL1A could act at the level of IL-6 or TGFβ. To bypass IL-6R/gp130 signaling, experiments examined the activity of TL1A on development of Th17 cells following costimulation with IL-21 and TGFβ. To eliminate the contribution of endogenous IL-6 splenic T cells from IL-6−/−, mice were maintained under Th17-polarizing conditions with IL-21 and TGFβ. As shown in Fig. 2E, TL1A dose-dependently inhibited the IL-21-mediated development of Th17 cells. To investigate the effect of TL1A on TGFβ signaling, we monitored generation of FoxP3+-regulatory CD4+ T cells in response to TGFβ (Fig. 2F). Inclusion of TL1A in the T-cell cultures had no effect on the TGFβ-mediated generation of FoxP3+ CD4+ T cells.

Figure 2
TL1A inhibits development of murine Th17 cells. A) Flow cytometry of CD4+ T cells from WT and DR3−/− splenocytes activated (anti-CD3/anti-CD28) under neutral (Th0) and Th17-polarizing conditions (TGFβ and IL-6). Cultures contained ...

TL1A inhibits de novo Th17 differentiation, but is independent of STAT1 signaling

To see whether TL1A inhibits de novo Th17 differentiation, MoFlo-sorted naive CD4+ T cells were cultured with TGFβ and IL-6, or TGFβ and IL-6 in combination with IL-23 (Fig. 3). Consistent with our observations using splenic cultures, addition of TL1A significantly blocked the generation of Th17 cells.

Figure 3
TL1A inhibits differentiation of Th17 cells from naive CD4+ T cells. Flow cytometry of naive CD4+ T cells from WT mice activated (anti-CD3/anti-CD28) under Th17-polarizing conditions in the presence or absence of TL1A (100 ng/ml) for 4 d. Differentiation ...

Enhanced STAT1 activity in T cells represents a suppressor signal of Th17 differentiation, with inflammatory mediators, including IL-27, type I interferons, and γIFN, acting as negative regulators of Th17 development (13, 16, 27, 28, 31). We therefore determined whether the inhibition of Th17 cells by TL1A was mediated indirectly through a STAT1 mechanism. Initial flow cytometric analysis of CD4+ T cells showed that TL1A inhibition of IL-17A secretion was not associated with a proportionate increase in γIFN-secreting T cells, indicating that TL1A does not redirect Th17 cells toward a Th1 phenotype (Fig. 4A). Furthermore, antibody blockade of γIFN activity had no effect on the TL1A-mediated suppression of Th17 cell development (Fig. 4B). Comparable data were also obtained in splenic T-cell cultures derived from γIFN−/− mice (Fig. 4B). Similarly, inclusion of the anti-mouse IFNAR1 blocking monoclonal antibody (MAR1-5A3) had no effect on the inhibitory TL1A response (Fig. 4C). To assess the role of IL-27, Th17 differentiation was recorded in CD4+ T cells from IL-27R−/− mice (Fig. 4D). Again, TL1A was found to block Th17 polarization. The inhibitory action of TL1A is therefore independent of known suppressors of Th17 development, which function through activation of STAT1. This conclusion is supported further by studies using T cells from STAT1−/− mice, which show that TL1A still retains the capacity to block Th17 generation in the absence of STAT1 signaling (Fig. 4E).

Figure 4
TL1A inhibition of Th17 development is independent of STAT1-mediated inhibitors of Th17 cells. A) Flow cytometry of CD4+ T cells from WT splenocytes activated in the presence of IL-6 and TGFβ and supplemented with 0, 1, 10, or 100 ng/ml rmTL1A. ...

TL1A induction of T-cell IL-2 expression does not affect Th17 development

Studies have illustrated a link between TL1A and the induction of IL-2, which is known to affect development of both Th17 and regulatory T cells (7, 29, 34). Intracellular flow cytometric staining of CD4+ T cells showed that TL1A inhibition of Th17 differentiation resulted in an associated dose-dependent increase in T-cell IL-2 secretion (Fig. 5A). To assess whether these increases in IL-2 could account for the TL1A suppression of Th17 cell differentiation, sorted naive T cells were cultured under Th17-polarizing conditions in the presence of a blocking anti-IL-2 antibody (Fig. 5B). Neutralization of IL-2 led to a more pronounced differentiation of Th17 cells (Figs. (Figs.33 and and5B).5B). However, the generation of Th17 cells under these optimal conditions was still inhibited by the action of TL1A (Fig. 5B, C).

Figure 5
TL1A inhibition of Th17 differentiation is independent of its ability to induce IL-2 secretion by CD4+ T cells. A) WT CD4+ T cells were assessed by intracellular cytokine staining for IL-17A and IL-2 production after activation under Th17-polarizing conditions ...

T-cell proliferation and Th17 development are controlled differentially by TL1A

TL1A is a costimulator of lymphocytes that triggers both cytokine production and proliferative responses following TCR activation (1-5, 7). We therefore examined whether the inhibition of Th17 development was attributable to TL1A control of T-cell proliferation. Purified naive CD4+ T cells were loaded with the cytosolic dye CFSE and cultured for 4 d with IL-6 and TGFβ under optimal anti-CD3/anti-CD28 costimulation in the presence of TL1A (Fig. 6A). Addition of TL1A significantly inhibited IL-17A secretion but did not alter T-cell proliferation. However, previous reports have shown that TL1A activity is dependent on the level of TCR engagement, which affects the nature of the cytokine response elicited (2). Analysis of IL-17A generation by naive CD4+ T cells conditioned with IL-6 and TGFβ in suboptimal doses of anti-CD3 stimulation, showed that TL1A enhanced T cell proliferation but did not promote the induction of IL-17A by these actively proliferating cells (Fig. 6B). Consequently TL1A could bolster T-cell proliferation under suboptimal TCR activation, but this does not correlate with an induction of IL-17A secretion, suggesting that TL1A does not drive a proliferative expansion of Th17 cells.

Figure 6
TL1A inhibits Th17 development but promotes T-helper cell proliferation. CFSE dilution analysis of WT CD4+ T cells activated under optimal (1 μg/ml anti-CD3/anti-CD28 costimulation; A) and suboptimal (0.1 and 1 μg/ml anti-CD3 only; B) ...

TL1A maintains the phenotypic characteristics of Th17 cells

Initial observations (Fig. 1A, B) emphasized that the TL1A receptor DR3 is up-regulated following TCR activation and was expressed most prominently by CD4+ T-cells that secrete IL-17A. Consequently, TL1A would be predicted to regulate the effector properties of activated Th17 cells. The studies conducted so far suggest that TL1A promotes neither the differentiation nor the proliferation of Th17 cells derived from naive CD4+ T cells. Experimental models of T-cell-mediated pathology demonstrate that DR3−/− mice are protected from disease (7, 9-11). This would contradict an inhibitory role for TL1A in Th17 development and might point to a contrasting role for TL1A in Th17 development vs. effector function. In this respect, the activation-induced expression of DR3 (Fig. 1A) suggests a potential role for TL1A in regulating the effector phenotype of precommitted Th17 cells. To test this, purified CD4+ T cells were first activated for 4 d under Th17-polarizing conditions in IMDM containing an anti-IL-2 blocking antibody to ensure development of a robust Th17 response (Fig. 7 A). These primary CD4+ T cells were transferred subsequently to fresh IMDM containing IL-6 and TGFβ or IL-6, TGFβ, and IL-23, but in the absence of TCR activation (no anti-CD3/ anti-CD28). Cells cultured under these conditions resulted in a substantially diminished proportion of Th17 cells (Fig. 7B). However, when TL1A was included in these secondary cultures, the Th17 phenotype was almost fully maintained (Fig. 7B). The manner by which TL1A affects T-cell proliferation, differentiation, and effector characteristics might ultimately depend on the nature or activation status of the T-cell population.

Figure 7
TL1A inhibits Th17 differentiation but promotes IL-17 production in effector CD4+ T cells. A) Primary cultures were prepared from WT CD4+ T cells activated (anti-CD3/anti-CD28) under robust Th17-polarizing conditions (IMDM; TGFβ, IL-6, anti-IL-2) ...

DISCUSSION

TL1A represents a member of the TNF/TNFR super-family specifically implicated in the control of T-cell activation events that affect both central tolerance and T-helper responses triggered during pathology. In this respect, TL1A enhances costimulatory events that regulate T-cell activation. Our studies build on these prior findings and suggest that TL1A signaling in naive and preactivated effector cells has the ability to suppress the initial commitment of naive T cells into Th17 cells but can serve to maintain the effector characteristics of preconditioned Th17 cells. These data suggest that TL1A-driven T-cell responses are based on mechanistic differences between the signaling events required for the initial differentiation, maintenance, and proliferation of effector cells.

Prior investigations have shown that TL1A is associated with the expansion of T-helper subsets and can synergize with IL-12 and IL-18 to affect development of γIFN-producing T cells (3, 4). This finding led to the view that TL1A, signaling via its receptor DR3, complements the expansion of Th1 cells (1, 2, 4). More recent investigations of TL1A/DR3 signaling in experimental models of encephalitis have shown that DR3−/− mice and mice lacking TL1A display reduced disease severity, which is associated with a reduced number of CD4+ T cells, including Th17 cells, in the CNS (7, 9). As proposed by Pappu et al. (9), this finding suggests that TL1A might promote the development of Th17 responses, which promote disease pathology. However, it is unclear whether this impaired T-cell infiltrate arises due to a defect in T-cell trafficking, survival, or maintenance of effector function. Further proinflammatory roles for TL1A have also been described in Th2-mediated Ova-induced lung disease (7), suggesting that TL1A might have a more universal role in regulating effector outcomes. Based on models of colitis (1, 2, 10) and rheumatoid arthritis (6, 11, 35), it is likely that TL1A predominantly bolsters development of Th1 and Th17 responses. However, it is currently unclear how TL1A coordinates these effector functions. In this respect, we now report that TL1A/DR3 signaling in newly challenged naive T cells inhibits Th17 (based on IL-17A and IL-17F measurements) and Tc17 development; the level of TCR activation influences the ability of TL1A to drive T-cell proliferation; TL1A control of IL-17A expression is independent of its ability to govern proliferative responses; and TL1A can maintain the effector characteristics of precommitted Th17 cells. These data indicate that TL1A might differentially affect the outcome of T-cell responses elicited by naive and activated/memory T-cell subsets.

Previously, T-cell expression of receptors for TNF superfamily members has been linked with T-cell survival, memory expansion, and costimulatory events (36, 37). The ability of TL1A to elicit T-cell responses relates to an activation-induced regulation of DR3 expression. Specifically, DR3 is expressed universally by activated T cells; however, a composite analysis of the CD4+ population show that DR3 is highly expressed by human IL-17-secreting T cells. A similar expression pattern has also been observed in murine T cells with TL1A predominantly associated with the activation of CD4+CD45RBlo memory subsets (2). An activation-induced control of T-cell DR3 expression would therefore suggest that signaling via this receptor might occur during antigen presentation by dendritic cells. Indeed, CD11chi dendritic cells display TL1A as a membrane-bound ligand, which could be presented to DR3+ T cells (2). A primary function of DR3 signaling might therefore be to steer the nature of the T-cell response following TCR activation. In this regard, we noted that TL1A dose-dependently inhibited the differentiation of Th17 cells from naive T cells and an increased number of Th17 cells was observed in draining lymph nodes isolated from DR3−/− mice. An inhibitory role for TL1A in Th17 differentiation is somewhat unexpected, considering the proinflammatory nature of its outcomes in T-cell-mediated inflammatory disease (7, 9, 10). This finding might point to another level of complexity for TL1A/DR3 signaling in governing T-cell responses. In addition to these centrally regulated immunological processes, TL1A/DR3 is also likely to influence inflammatory events at sites of disease activity. Endothelial cells, for example, secrete TL1A, while gene analysis of DR3 expression in human osteoblasts and synovial cells from rheumatoid arthritis patients suggest a more localized role for TL1A in degenerative and inflamma-tory arthritis (11, 38-40). When compared with WT littermates, mice deficient in TL1A or DR3 display an overall reduction in the number of effector T cells at sites of inflammation (2, 7, 9). This provides a potential link between TL1A/DR3 and the regulation of T-cell recruitment or the retention of activated cells at sites of inflammation. Our results show that TL1A signaling in preactivated T cells supported maintenance of IL-17A expression. This finding might explain the apparent paradox between the inhibitory action of TL1A in Th17 differentiation and the protection provided by TL1A/DR3-signaling-deficient mice in experimental disease models where Th17 cells have been implicated in the pathology. In this regard, the ability of TL1A to inhibit Th17 differentiation might be secondary to its proinflammatory role in the local maintenance of Th17-effector function. This observation builds on studies that demonstrate the importance of TGFβ and the role of IL-6 trans-signaling in maintaining a Th17 phenotype at inflammatory lesions (41, 42). Consistent with our study, none of these Th17 “maintenance factors” skew or influence the proportion of γIFN-secreting T cells, which suggests a selective activity on Th17 cells.

The negative regulation of Th17 cells by TL1A implies that DR3 activation antagonizes the commitment signals governing development of this population from naive T cells. The TL1A control of Th17 differentiation appears to be independent from its capacity to regulate proliferative responses. Consistent with previous reports, TL1A only promoted T-cell proliferation in suboptimal conditions of TCR activation (2, 3, 7, 9). This condition, however, was not associated with an induction of IL-17A. Instead, optimal TCR engagement was required for maximal IL-17A secretion. In this respect, TL1A was found to block Th17 generation but not T-cell proliferation. These findings point to an inverse relationship between TL1A/DR3 signaling and TCR activation that differentially affects T-cell proliferation vs. Th17 commitment. Coordination of these events could relate to the known TL1A regulation of T-cell IL-2 expression, which acts as a negative regulator of Th17 generation and a survival factor for regulatory T cells (29, 34). Although an anti-IL-2 blocking antibody enhanced IL-6/TGFβ and IL-6/TGFβ/IL-23 differentiation of Th17 cells, the negative regulation provided by TL1A appeared independent of IL-2 activity. Similar to other cytokines of the TNF superfamily, TL1A primarily triggers NF-κB and MAP-kinase signaling events (3, 43). Although it is unclear how these signaling cascades might feed into Th17 differentiation pathways, activation of DR3 by TL1A affects the expression of T-cell activating cytokines, including γIFN, which triggers STAT1 mediated responses (1, 4). Factors that enhance STAT1 activity in T cells are characterized inhibitors of Th17 differentiation, and αIFN, βIFN, γIFN, IL-27, and retinoic acid all inhibit STAT3-driven Th17 generation (13, 16, 27, 28, 30, 31, 44). Utilizing cytokine-deficient mouse strains and anticytokine neutralizing antibodies, studies excluded a role for STAT1 in the TL1A-driven suppression of Th17 commitment. The mostly likely explanation for TL1A control of Th17 development is therefore a potential signaling interplay between a signaling event associated with the TNFRSF and the latent transcription factor STAT3, which is activated by IL-6, IL-21, and IL-23 to steer Th17 commitment. In this respect, CD4+ T cells lacking the molecular adaptor TNF receptor-associated factor-6 (TRAF-6) display increased Th17 differentiation (45). Although TRAF6 plays an intrinsic role in the maintenance of peripheral tolerance and anergy induction, studies have not examined activation of TRAF6 by TL1A. Our data further emphasize that while STAT3 activity is integral to Th17 differentiation, it is insufficient to maintain the Th17 phenotype in the absence of TCR stimulation. In this regard, we noted that TL1A stimulation of precommitted Th17 cells helped to retain IL-17A expression.

Given an ability of TL1A to differentially control Th17 responses in naive vs. activated T cells, it is conceivable that the signals required for the long-term maintenance of Th17 cells are distinct from those affecting differentiation. One possible explanation could relate to a TL1A modulation of pro- or antiapoptotic regulators, including members of the Bcl or IAP families. Indeed, activation-induced changes in T-cell X-linked inhibitor of apoptosis (XIAP) expression have been associated with the development of EAE, which is considered a Th17-driven model (46). Notably, DR3−/− mice are resistant to EAE pathology (7, 9). However these activities might need to be placed in the context of STAT3 activation, which drives Th17 differentiation but also rescues T cells from entering apoptosis (47-50). An alternative hypothesis would suggest a modification in T-cell signaling capacity following activation. When compared to resting T cells, activated memory T cells display altered IL-6 signaling, which is attributable to both an activation-induced loss of IL-6R and dramatically impaired STAT1, but not STAT3, activity (41, 51, 52). Our findings echo these findings and suggest that activation-induced expression of DR3 contributes to an alteration in TL1A responsiveness. Although the molecular basis for this regulation is unclear, the observed relationship between TL1A activity and TCR signaling remains an intriguing aspect of this study. Collectively, these results support the view that TL1A activities depend on the activation status or activation history of the T-cell population.

Acknowledgments

This work was funded through grant support provided by Arthritis Research UK (project grant 18286 to S.A.J.; traveling fellowship ARC19234 to G.J.), the Medical Research Council (grants G0300180 and G0901119 to E.W.), the Wellcome Trust, the National Institutes of Health (grant AI42334 to C.H.), and the Mari Lowe Centre (to C.H.). The authors also acknowledge the help and advice provided by Drs. Philip Taylor and Awen Gallimore.

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