Role of TL1A and its receptor DR3 in two modelsof chronic murine ileitisGiorgos Bamias*, Margarita Mishina*, Mark Nyce*, William G. Ross*, Giorgos Kollias†, Jesus Rivera-Nieves*,Theresa T. Pizarro*, and Fabio Cominelli*‡

*Digestive Health Center of Excellence, University of Virginia Health Sciences Center, Charlottesville, VA 22908; and †Institute of Immunology,Alexander Fleming Biomedical Sciences Research Center, 16672 Vari, Greece

Edited by Charles A. Dinarello, University of Colorado Health Sciences Center, Denver, CO, and approved March 31, 2006 (received for reviewDecember 16, 2005)

TL1A is a TNF-like cytokine that binds to the death-domain receptor(DR)3 and provides costimulatory signals to activated lymphocytes.Through this interaction, TL1A induces secretion of IFN-� and may,therefore, participate in the development of T helper-1-type ef-fector responses. In this study, we investigated whether interac-tions between TL1A and DR3 are involved in the pathogenesis ofchronic murine ileitis. We demonstrate that alternative splicing ofDR3 mRNA takes place during the activation of lymphocytes, whichresults in up-regulation of the complete�transmembrane (tm) formof DR3. Using two immunogenetically distinct animal models ofCrohn’s disease, we demonstrate that induction of intestinal in-flammation is associated with significant up-regulation of TL1Aand tm DR3 in the inflamed mucosa. In addition, within isolatedlamina propria mononuclear cells from mice with inflammation,TL1A is primarily expressed on CD11chigh dendritic cells. We alsoreport that TL1A acts preferentially on memory CD4��CD45RBlo

murine lymphocytes by significantly inducing their proliferation,whereas it does not affect the proliferation of the naı̈ve CD4��CD45RBhi T helper cell subpopulation. Finally, we demonstrate thatTL1A synergizes with both the cytokine-dependent IL-12�IL-18pathway and with low-dose stimulation of the T cell receptor tosignificantly induce the secretion of IFN-� via an IL-18-independentpathway. Our results raise the possibility that interaction(s) be-tween TL1A expressed on antigen-presenting cells and tm DR3 onlymphocytes may be of particular importance for the pathogenesisof chronic inflammatory conditions that depend on IFN-� secretion,including inflammatory bowel disease. Blockade of the TL1A�DR3pathway may, therefore, offer therapeutic opportunities inCrohn’s disease.

Crohn’s disease � cytokines � mucosal inflammation

The differentiation of naı̈ve CD4� lymphocytes into IFN-�-secreting Th1 ‘‘effector’’ cells is a multistep process that

involves several cell types, costimulatory molecules, transcrip-tion factors, and secreted cytokines (1). Antigen-presenting cell(APC)-derived IL-12 is essential for the induction of IFN-�, aneffect that is greatly enhanced by IL-18 (2). IL-12 up-regulatesT-bet, a transcription factor that is critical for the stabilization ofa T helper (Th)1-polarized phenotype (3). Recently, additionalcytokines that play prominent roles during Th1 responses havebeen described, such as IL-27 and IL-23 (4). Engagement of theT cell receptor (TCR) provides further signals for the inductionof IFN-�, both in parallel to and independently of cytokine-mediated pathways (1).

Members of the TNF and TNF-receptor superfamilies ofproteins (TNFSFPs and TNFRSFPs, respectively) are abun-dantly expressed in the immune system, and are critically in-volved in the differentiation, proliferation, and apoptosis ofimmune cells (5). Several members of these families inducesecretion of IFN-� upon ligand�receptor binding, thereby en-hancing Th1-type responses (6–8). TL1A (TNFSPF15) is arecently identified, TNF-like factor that is currently the only

known ligand for death-domain receptor (DR)3 (9), which isprimarily expressed on activated lymphocytes (10, 11).

Binding of TL1A to DR3 triggers proliferative�activationsignals, most likely through activation of NF-�B-mediated path-ways (9, 12). TL1A specifically induces secretion of IFN-� byhuman T cells (9), raising the possibility that TL1A�DR3 mayparticipate in Th1-mediated responses. Indeed, we and othershave recently reported up-regulation of both TL1A and DR3 ininflammatory bowel disease (IBD), particularly Crohn’s disease(CD) (13–15). Whereas original reports indicated that TL1Aexpression was confined to endothelial cells (9), subsequentstudies demonstrated that in involved intestinal tissue frompatients with IBD, TL1A was also expressed on lymphocytes,plasma cells, and monocytes. TL1A may participate in thepathogenesis of IBD, likely by inducing secretion of IFN-� fromlamina propria mononuclear cells (LPMCs) and the subsequentgeneration of proinflammatory responses (13–15).

In contrast to the aforementioned human studies, limited datahave been published describing the function of TL1A and DR3in mice. In this study, we investigated the hypothesis thatinteraction(s) between TL1A and DR3 participate in the patho-genesis of murine chronic small intestinal inflammation. Usingtwo mouse models of CD (16–18), we have demonstrated thatexpression of DR3 is significantly up-regulated during chronicileitis in an inflammation-specific manner. This up-regulationinvolves alternative splicing of the mRNA that encodes DR3,which results in predominant expression of the transmembrane(tm) form of the receptor in preference to the soluble form. Inaddition, we show that there is abundant expression of TL1A onthe surface of CD11chigh dendritic cells, raising the possibilitythat there is an interaction between lymphocytic tmDR3 andAPC-derived TL1A. We also show that TL1A acts as a costimu-lator for lymphocytes, enhancing IFN-� production in synergywith low-level stimulation of the TCR or with IL-12, whereas itsactions are independent of IL-18. Finally, we report that TL1Ainduces the proliferation of memory but not naı̈ve CD4� lym-phocytes, a finding that supports a role for TL1A during theeffector phase of Th1 responses.

ResultsTL1A Enhances TCR-Mediated Secretion of IFN-� by Murine Lympho-cytes. We initially investigated whether TL1A affects TCR-mediated secretion of IFN-� by murine lymphocytes. Addition of

Conflict of interest statement: No conflicts declared.

This paper was submitted directly (Track II) to the PNAS office.

Abbreviations: APC, antigen-presenting cell; CD, Crohn’s disease; DR, death-domain re-ceptor; IBD, inflammatory bowel disease; LPMC, lamina propria mononuclear cell; rm,recombinant murine; tm, transmembrane; TCR, T cell antigen receptor; Th, T helper;TNFSFPs, TNF superfamily of proteins; TNFRSFPs, TNF receptor superfamily of proteins.

See Commentary on page 8303.

‡To whom correspondence should be addressed at: Digestive Health Center of Excellence,University of Virginia Health Sciences Center, P.O. Box 800708, Charlottesville, VA 22908.E-mail: fc4q@virginia.edu.

© 2006 by The National Academy of Sciences of the USA

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recombinant murine (rm)TL1A to cultured CD4� cells, in thepresence of immobilized anti-CD3 and soluble anti-CD28 Abs,significantly increased IFN-� secretion in a dose-dependentfashion (Fig. 1 A and B). In addition, we found that low-dose (0.2�g�ml) anti-CD3 stimulation provided optimal conditions forTL1A function (average increase in IFN-� secretion, 223%;range, 143–349%) (Fig. 1C). In contrast, the effect was greatlydiminished when 1 �g�ml anti-CD3 was used (122%; range,112–136%), and there was no effect with high-dose (5–10 �g�ml)stimulation (100%; range, 78–117%).

TL1A Acts in Synergy with IL-12 and Independently of IL-18 for theInduction of IFN-�. It has been demonstrated that a combinationof IL-12 and IL-18 can up-regulate IFN-� in the absence of

Ag�TCR stimulation (19). We investigated whether TL1A couldalso synergize with this cytokine pathway for IFN-� secretion.Our data demonstrate that synergy between TL1A and theIL-12�IL-18 pathway does indeed occur. Addition of TL1A tocultures of IL-12�IL-18-stimulated CD4� cells caused a dose-dependent increase in the levels of IFN-� secreted, in thecomplete absence of TCR-mediated signals (Fig. 2A).

To further explore the role played by TL1A in the pathways thatlead to secretion of IFN-�, we studied the effect of combining TL1Awith IL-12 or IL-18, in the presence of TCR stimulation. In culturedCD4� cells, TL1A synergized with the combination of TCR stim-ulation and IL-12; however, when used in combination with IL-18and TCR stimulation, TL1A failed to induce higher levels ofsecretion of IFN-� than were induced by TCR stimulation andIL-18 alone (Fig. 2B). These data indicate that there is a potentsynergy between TL1A and IL-12 that is independent of IL-18signaling. This finding was further confirmed in experiments thattested the synergy between TL1A and IL-12 in the absence of TCRengagement. Addition of TL1A to IL-12-stimulated CD4� cells had

Fig. 1. Effects of TL1A stimulation on TCR-mediated IFN-� secretion. CD4�

splenocytes were purified and cultured as described in Materials and Methodsand indicated on the axes of the graphs. (A) Cells were stimulated withanti-CD3 (0.5 �g�ml) and anti-CD28 (1 �g�ml) Abs, then rmTL1A (100 ng�ml)was added to the cultures. The concentration of IFN-� was measured at 72 h.A representative of four experiments is shown. (B) Cells were stimulated withanti-CD3�anti-CD28, then rmTL1A was added to the cultures. The concentra-tion of IFN-� was measured at 24–96 h. A representative of three experimentsis shown. Asterisks indicate values of P � 0.05 for comparison between IFN-�secretion with or without rmTL1A for each time point. (C) Cells were stimu-lated with different doses of anti-CD3 (0.5–10 �g�ml). The concentration ofIFN-� was measured at 72 h. Pooled data from four independent experimentsare shown. All data are presented as mean � SEM.

Fig. 2. TL1A synergizes with IL-12. (A) CD4� splenocytes were stimulatedwith the combination of IL-12�IL-18, then rmTL1A was added to the cultures.A representative of four experiments is shown. (B) CD4� splenocytes werestimulated with anti-CD3 (0.5 �g�ml), anti-CD28 (1 �g�ml), rmIL-12 (10 ng�ml), rmIL-18 (100 ng�ml), and rmTL1A (100 ng�ml), as indicated. The concen-tration of IFN-� in the supernatant was measured after 72 h. Pooled data fromsix experiments are shown. All data are presented as mean � SEM. (C) CD4�

splenocytes were stimulated with rmIL-12, with or without the addition ofTL1A (200 ng�ml). The concentration of IFN-� in the supernatant was mea-sured after 72 h. Six individual experiments are shown. Horizontal linesindicate the average for each group.

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a significant costimulatory effect, leading to a 3- to 17-fold increasein IFN-� secretion (Fig. 2C).

TL1A Acts on Memory Th Cells. Next, we tested whether TL1Astimulates the naı̈ve or memory lymphocytic subpopulations. Wetherefore studied the proliferative responses of highly purifiedCD4��CD45RBhi (naı̈ve) and CD4��CD45RBlo (memory) cellsisolated from the spleens of C57�Bl6 mice. As expected, naı̈vesplenocytes were highly responsive to stimulation with IL-12 (Fig.3A). However, no increase in proliferation was observed afteraddition of TL1A to cultured naı̈ve lymphocytes, nor was there anysynergistic effect when IL-12 and TL1A were combined (Fig. 3A).By contrast, CD4��CD45RBlo cells did not respond to stimulationwith IL-12 (Fig. 3B). However, when TL1A was added to culturesof memory lymphocytes, the proliferation rate was greatly en-hanced (Fig. 3B). Indeed, whereas TL1A induced only a modestproliferative response from naı̈ve lymphocytes (148% � 88% overbaseline), it exerted a far greater stimulatory effect on memory cells(380% � 141%) (Fig. 3C).

Activation of Lymphocytes Is Associated with Alternative Splicing ofDR3 and Up-Regulation of the tm Form of DR3. Three splice variantsof DR3 have been described in the mouse (20). The first encodes

the full protein, including both the tm and extracellular regions. Thesecond lacks the tm region and is, therefore, predicted to encode asoluble protein. Finally, the third variant lacks one of the cysteine-rich regions in the extracellular domain. Because expression of DR3is limited to activated lymphocytes (10, 11), we hypothesized thatlymphocytic activation might be associated with up-regulation ofthe splice variant that encodes the full-length DR3 protein, resultingin the expression of a fully functional tm receptor and increasedTL1A signaling.

To test our hypothesis, we designed a dual amplificationsystem for the detection of DR3 mRNA by real-time PCR. A firstset of primers and probe, referred to as tm, was designed toamplify a region that is present only in the full�tm form of DR3mRNA. The second set, referred to as total, amplifies a regionthat is present in all three variants. We then used this system tocompare the relative ratio of the tm to the total mDR3 inlymphocytes at different stages of activation. As shown in Fig.

Fig. 4. Expression of tm DR3 mRNA during activation of lymphocytes and inchronic ileitis. Specific amplification of tm and total DR3 mRNA was performedas described in Materials and Methods. (A) Lymphocytes (CD4� and CD8�)were isolated from mouse spleens, and the ratio of the relative expression oftm vs. total DR3 mRNA was measured in freshly isolated, overnight resting orovernight stimulated (aCD3 � aCD28) cells (n � 4 per group). (B) The relativeexpression of tmDR3 mRNA was measured by quantitative real-time RT-PCR intotal tissue RNA extracted from the terminal ileum of SAMP1�YitFc mice withileitis (�20-wk-old) or before the development of ileitis (4-wk-old) and age-matched normal AKR control mice (n � 6–7 mice per group). (C) Relativeexpression of tmDR3 mRNA was measured in total tissue RNA extracted fromthe terminal ilea of TNF�ARE mice with ileitis (24-wk-old) or before the devel-opment of ileitis (4-wk-old) and age-matched wild-type mice (n � 5–6 mice pergroup). All data are presented as mean � SEM.

Fig. 3. TL1A acts on memory lymphocytes. Naı̈ve (CD4��CD45RBhi) andmemory (CD4��CD45RBhi) splenocytes were purified and cultured as de-scribed in Materials and Methods. Stimulatory conditions are indicated on theaxes of the graphs. Proliferation of naı̈ve (A) and memory (B) lymphocytes wasestimated by measuring thymidine incorporation. Representatives of twoexperiments are shown. (C) Average proliferative responses for naı̈ve andmemory lymphocytes from four independent experiments are shown. All dataare presented as mean � SEM.

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4A, the ratio of tm to total mDR3 was relatively low in freshlyisolated or resting T cells, whereas activation for 24 h resulted ina definitive increase in the relative amount of tmDR3 (�5-foldincrease over baseline). In fact, rather than altering the totallevels of mRNA for DR3, activation of the cells increased thelevel of tmDR3 (data not shown). This result indicates that theincreased ratio of tm to total mDR3 after cell activation is adirect result of an increased proportion of the tm splice variants.

Chronic Small Intestinal Inflammation Is Associated with MucosalUp-Regulation of tmDR3. To explore the importance of the TL1A�DR3 pair under inflammatory conditions in vivo, we investigatedthe hypothesis that the expression of tmDR3 should be increasedin IBD, because this immunological condition is characterized byheavy infiltration of the intestinal lamina propria by activatedlymphocytes (21). We tested our hypothesis in two immunoge-netically diverse models of chronic ileitis, namely SAMP1�YitFcand TNF�ARE mice. Both models share clinical, pathological,and immunological characteristics with CD, and are consideredto be representative of the human condition (16, 17). Wemeasured the relative expression of the mRNA for tmDR3 in theterminal ileum of inflamed SAMP1�YitFc mice and comparedit to the levels in both uninflamed control AKR and youngSAMP1�YitFc mice before the development of ileitis, with thelatter serving as the internal, strain-specific control.

As shown in Fig. 4B, there was significantly increased expres-sion of tmDR3 in inflamed SAMP1�YitFc mice compared withage-matched AKR controls (230% increase). More importantly,the up-regulated expression of tmDR3 was clearly related toinflammation, because it was absent in the young, noninflamedSAMP1�YitFc mice (350% increase after the development ofileitis). On the other hand, no difference was observed between

young and old AKR mice. A similar, ileitis-specific up-regulationof tmDR3 was observed in TNF�ARE mice, with older mice againdemonstrating significantly higher expression of tmDR3 (270%increase vs. age-matched wild-type and 230% vs. young, prein-f lamed TNF�ARE mice) (Fig. 4C).

TL1A Is Up-Regulated in Chronic Ileitis and Is Primarily Expressed onAPCs. Next, we measured the expression of TL1A in the terminalileum of TNF�ARE and wild-type mice. As shown in Fig. 5A,there was significantly increased expression of TL1A in micewith ileitis as compared with uninflamed control mice (3.4-foldincrease, P � 0.001). Similar to our studies in CD patients (13),immunostaining for TL1A protein showed increased TL1Aexpression in the lamina propria of mice with ileitis comparedwith wild type controls (data not shown). The significant up-regulation of both TL1A and the active�tm form of its receptorDR3 during chronic ileitis indicates that binding of TL1A anddownstream signaling takes place within the inflamed mucosa.It is, therefore, critically important to identify the cellularsource(s) of TL1A. To achieve this end, we used flow cytometryto study the expression of TL1A in small intestinal LPMCsisolated from inflamed SAMP1�YitFc and TNF�ARE mice.Using a monoclonal antibody against TL1A, we demonstratedthat expression of TL1A was confined to CD11c-positive cells(Fig. 5B). We further confirmed that CD11c and TL1A colo-calize by using confocal microscopy of stained LPMCs (Fig. 5C).To determine whether the major source of TL1A is true den-dritic cells, or nondendritic CD11c-positive cells, we furtherstained for expression of MHC-II. By applying sequential gating,we clearly identified two major populations of cells that expressTL1A (Fig. 5D). Firstly, TL1A is highly expressed on CD11chigh�MHC-IIpos cells. Secondly, TL1A was also expressed on a

Fig. 5. TL1A is up-regulated during chronic ileitis and is expressed on mucosal dendritic cells. (A) Relative expression of TL1A mRNA was measured in total tissueRNA extracted from the terminal ilea of TNF�ARE mice (�8-wk-old, n � 13) and age-matched wild-type mice (n � 14). All data are presented as mean � SEM. (B)Flow-cytometric analysis of LPMCs from inflamed SAMP1�YitFc mice after staining with a specific anti-TL1A Ab demonstrated that expression of TL1A is confinedto the CD11c-positive fraction. (C) Confocal microscopy of LPMCs incubated with antibodies against mouse TL1A (red) and CD11c (green) demonstrated that thetwo markers were colocalized. (D) LPMCs were incubated with mAbs against mouse TL1A, CD11c, and MHC-II. Gates were set as indicated in the figure. Histogramplots indicate the expression of TL1A (green) or negative control (red) in each subpopulation. Negative control indicates staining with secondary Ab alone.

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population of CD11clow�MHC-IIneg cells. On the other hand,only very low expression of TL1A was observed on CD11clow�MHC-IIpos cells, and no expression was detected on CD11c-negative cells. Taken together these results indicate that laminapropria dendritic cells are the major source of TL1A in theintestinal mucosa of mice with spontaneous ileitis.

DiscussionMembers of the TNF�TNFR superfamilies of proteins areabundantly expressed throughout the immune system and exerta wide array of effects on immune cells (5). An importantproperty of many of these proteins is their ability to providecostimulatory signals for the enhancement of immune responses(6–8, 22). In the present study, we provide evidence that theTNF-like cytokine TL1A and its functional receptor DR3 alsoact as costimulators for T cells. In addition we show thatinteractions between lymphocytic tmDR3 and APC-derivedTL1A occur and may be of pathophysiological importanceduring chronic CD-like ileitis in mice.

Our studies clearly demonstrate that one of the main functionsof TL1A is to induce production of IFN-� by murine lymphocytes.Similar effects were recently reported in studies of human lympho-cytes (9, 13, 15, 23). This study further explored the specific role thatTL1A plays in the pathways that lead to production of IFN-�. First,the effects of TL1A are more pronounced in the presence ofsuboptimal (i.e., low-dose) stimulation through the TCR, whichmay indicate that TL1A is involved in the preservation or ampli-fication of IFN-� responses when the antigenic load is low. Second,the in vitro experiments we carried out demonstrated that TL1Asynergizes with IL-12 and that this synergy occurs independently ofIL-18. Combining IL-12 and TL1A, therefore, provides an alter-native route for the cytokine-mediated induction of IFN�, whichmay be of particular importance when IL-18 signaling is absent ordefective. Finally, TL1A preferentially acts on memory cells, whichindicates that TL1A may be particularly important during thelate�effector phase of Th1 immunity, increasing the amount ofIFN-� that is available and, thereby, increasing the magnitude of theimmune response.

The majority of the TNFRSFPs are produced as tm proteinsunder normal circumstances, with the soluble forms being gener-ated by proteolytic cleavage (24). It is therefore of particular interestthat a soluble form of DR3 can be directly generated by means ofalternative splicing (20). Our study clearly demonstrates thattmDR3 predominates only after T cell activation and that thispredominance is a result of mRNA rearrangement. This phenom-enon may be indicative of a mechanism that prevents inappropriatefunctional ligation by TL1A under physiological conditions. Thetight regulation of TL1A is further strengthened by the existence ofan inhibitory receptor, decoy receptor 3 (DcR3)�TR6, which occursonly as a soluble protein (9). DcR3 competes with DR3 for thebinding of TL1A and abrogates its stimulatory effects on T cells (9),as was recently shown in studies with DcR3-deficient mice. TL1Asignaling appears to be compromised in these mice, resulting indefective IFN-� production and a bias toward Th2 polarization ofthe immune response, resulting in increased susceptibility to infec-tion (25). Similar control mechanisms also exist for other membersof the TNF�TNFRSFPs, such as Fas (26, 27). It is possible that,under conditions of chronic inflammation, these regulatory mech-anisms are compromised, as indicated for IBD by the up-regulationof tmDR3 expression reported in our study. This up-regulationcould then lead to unrestricted TL1A signaling and deleteriousproinflammatory effects mediated by the increased levels of IFN-�.

CD results from a dysregulated activation of the gut-associated mucosal immune system in genetically predisposedindividuals (21). Many of the findings presented herein indicatethat TL1A�DR3 may participate in the pathogenesis of CD. Ourdata show that chronic mucosal inflammation is associated withalternative splicing of DR3 and up-regulated expression of the

tm form of the receptor. At the same time, there is overexpres-sion of TL1A primarily on mucosal APCs. A central pathogenicmechanism in CD involves aberrant presentation of lumenalbacterial antigens by APCs to lamina propria T cells (21). Thelocalization of TL1A and DR3 in APCs and T cells, respectively,therefore raises the possibility that a functional association takesplace between these proteins during chronic ileitis. Our in vitrofindings indicate that such an association would lead to prolif-eration of effector lymphocytes in the inflamed mucosa. Expan-sion of lamina propria T cells is a central characteristic of bothexperimental ileitis (including SAMP1�YitFc and TNF�ARE

mice) and the human condition (16, 17, 21). Binding of TL1A toDR3 on mucosal lymphocytes would lead to increased secretionof IFN-� from the lymphocytes. This increased secretion may beof particular importance, because CD is considered a prototypicTh1-mediated condition in which IFN-� plays a central patho-genetic role. In fact, as we and others have shown, the TL1A�DR3 system is up-regulated during CD (13–15). Interestingly, itwas recently reported that single nucleotide polymorphismsconferred susceptibility to CD in one Japanese and two Euro-pean cohorts (28). In addition to our findings, recent studies byother research groups have implicated TL1A�DR3 in the patho-genesis of other inflammatory conditions. An association withrheumatoid arthritis has recently been reported for a destabi-lizing mutation in the dr3 gene (29). Furthermore, TL1A andDR3 may be involved in the development of atherogenesisthrough the induction of proinflammatory cytokines and matrixmetalloproteinases (30).

The data presented herein lead us to propose that, underphysiological conditions, expression of tmDR3 on lymphocytes isminimal, and functional signaling is inhibited. However, whenchronic inflammation develops, T cells up-regulate the expressionof the active, tm form of DR3. In addition, TL1A expressed onAPCs binds to tmDR3 on lymphocytes, which triggers costimula-tory signals that significantly amplify production of IFN-�. Thisinteraction and the subsequent functional outcomes may be ofparticular importance during disease phases when there is onlylow-level stimulation with antigens, such as during the early�induction or low-activity�maintenance stages. We therefore con-clude that interactions between TL1A and DR3 participate in thepathogenesis of Th1-mediated inflammation, including the inflam-mation observed in patients with CD. Manipulation of theseinteractions may have therapeutic potential for these conditions.

Materials and MethodsRecombinant Proteins and Antibodies. A DNA sequence that en-coded the C-terminal extracellular domain of mouse TL1A(Ser-81 to Leu-252) was cloned into the pET 28a(�) expressionvector (pET System; Novagen) and expressed in Escherichia coli.The resulting rmTL1A was purified via its His-tag under nativeconditions by metal chelation chromatography (Ni-NTA HisBind Resins; Novagen). Lipopolysaccharide was removed byusing Detoxi-Gel Endotoxin Removing Gel (Pierce). A humananti-mouse anti-TL1A mAb (clone MT101) was kindly providedby Human Genome Sciences, Rockville, MD. Anti-CD3e (145-2C11) and anti-CD28 (37.51) mAbs were purchased from BDBiosciences, and rm IL-12 and IL-18 from R & D Systems.

Mice. SAMP1�YitFc, control AKR, heterozygous TNF�ARE/�,and wild-type TNF�/� mice were maintained under specificpathogen-free conditions in the Animal Facility at the Universityof Virginia. All protocols were approved by the Animal Care andUse Committee of the University of Virginia.

Cell Isolation and Culture. Highly enriched suspensions of CD4�

lymphocytes (�90%) were obtained by positive selection usingan immunomagnetic cell-sorting system (Miltenyi Biotec). Lym-phocytes were cultured in 96-well round-bottom plates at 106

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cells per ml of complete medium (RPMI medium 1640, 10%FBS, 2 mM L-glutamine, 1 � 10�5 mol�l �-mercaptoethanol, and1% penicillin�streptomycin) under the conditions indicated inthe figure legends. After 24–72 h, supernatants were collectedand stored at �80°C until further use. The concentration ofIFN-� in the supernatants was measured by using a commerciallyavailable ELISA kit (BD Biosciences Pharmingen). For mea-surement of DR3 mRNA content, cells were cultured overnightin 24-well plates at 2 � 106 cells per ml and then recovered fromthe wells and stored as pellets at �80°C.

Proliferation Assay for Naı̈ve and Memory Lymphocytes. Naı̈ve andmemory lymphocytes were purified from the magnetically sortedCD4� splenocytes by incubation with fluorochrome-conjugatedantibodies against CD4� and CD45RB. Separation of CD4��CD45RBhi (naı̈ve) and CD4��CD45RBlo (memory) cells wasperformed on a FACScalibur flow cytometer (Becton Dickin-son). To determine the levels of proliferation, cells (105 percondition) were cultured in triplicate for 96 h and then pulsedwith [3H]thymidine [1 �Ci (1 Ci � 37 GBq) per well] (MPBiomedicals, Irvine, CA) overnight. Proliferation was estimatedby measuring incorporation of the thymidine.

Real-Time PCR. Total RNA was isolated from homogenized tissue orcell pellets by using the RNeasy Mini kit (Qiagen, Valencia, CA)and converted to cDNA with the GeneAmp RNA PCR kit (Ap-plied Biosystems) by using random hexamers (0.75 �g of total RNAin a final reaction volume of 20 �l). cDNA was quantified byreal-time PCR using an iCycler detection system (Bio-Rad). Theprimers and TaqMan probes were designed with the assistance ofBeacon Designer software (PREMIER; Biosoft). To specificallyquantify the full�tm DR3 splice variants, the following primers wereused: forward 5-TGGCTTCTATATACGTGGCAATGA-3; re-verse 5-GCACCTGGACCCAAAACATCT-3; probe 5-AGC-CACAGACAGCAGTGCAAGCCT-3. For total DR3 (all splicevariants), the primers were: forward 5-AAGAGGCCCT-TCAAGTGACC-3; reverse 5-AGTCAACACACCAGCCT-GAC-3; probe 5-CTCGGCAAAGTCGGACACCCACTG-3.The real-time PCR was performed with iTaq DNA polymerase(Bio-Rad) as per the manufacturer’s recommendations, in a reac-tion mix consisting of 3 mM MgCl2, 200 �M solutions of eachdNTP, 400 nM solutions of each primer, and a 200 nM solution ofthe probe. Five percent of the volume of the first-strand synthesis

was added and the total volume adjusted to 25 �l. TL1A mRNAdetection was quantified by real-time RT-PCR using the iQTM

SYBR green Supermix (Bio-Rad). The following primers wereused: forward, GCTGCCTGTTGTCATTTCC; reverse, TCTGG-GAGGTGAGTAAACTTG. A separate reaction mix was set upwith primers for �-actin mRNA as the reference standard: forward5-CAGGGTGTGATGGTGGGAATG-3, reverse 5-GTA-GAAGGTGTGGTGCCAGATC-3. For detection of �-actin byreal-time PCR, a 400 nM solution of each primer and 5% of thevolume of the first-strand synthesis were used in a total volume of25 �l that included iQ SYBR green Supermix (Bio-Rad) accordingto manufacturer’s directions.

Thermocycling conditions for all targets were as follows: 95°C for3 min (to activate the iTaq DNA polymerase), then 40 cycles of 95°Cfor 15 sec, 60°C for 15 sec, and 72°C for 15 sec. The standard curvemethod was used to quantify the relative mRNA level of mDR3, asdescribed in Essentials of Real-Time PCR (Applied Biosystems).Results were expressed as a relative ratio to the lowest controlsample. All samples were assayed in duplicate.

Flow Cytometry. LPMCs were isolated as described in ref. 18. Theexpression of TL1A on the surface of LPMCs was then analyzedby FACS, using a mAb specific for murine TL1A (MT101).APC-conjugated mouse anti-human IgG was used as the sec-ondary Ab (BD Pharmingen). Antibodies against murine CD11c(HL3), MHC-II (IA�IE, clone 114.15.2), and CD16�CD32(clone 2.4G2, to block nonspecific FcR binding) were all pur-chased from BD Pharmingen.

Statistical Analysis. The Student t test was used for statisticalanalysis, with an � level of 0.05 considered to be significant(P � 0.05).

We thank the Histology�Imaging Core and the Immunology�Cell Iso-lation Core of the National Institutes of Health (NIH)�National Instituteof Diabetes and Digestive and Kidney Diseases, University of Virginia(UVA) Digestive Health Research Center; Paul Moore of HumanGenomic Sciences for the kind gift of the human anti-mouse TL1Aantibody; and Dr. Sarah A. De La Rue for critically reviewing themanuscript. This work was supported by U.S. Public Health Service�NIHGrants DK-42191, DK-44540, and DK-55812 (to F.C.); the UVADigestive Health Research Center (1P30DK67629); and a ResearchFellowship Award from the Crohn’s and Colitis Foundation of America(to G.B.).

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